I. BIOLOGICAL SAFETY. A. Biological Risk Assessment. Risk assessment is a process used to identify the hazardous characteristics of a known

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1 I. BIOLOGICAL SAFETY Microbiological and biomedical laboratories are special, often unique, work environments that may pose special infectious disease risks to persons in or near them. Personnel have contracted infections in the laboratory throughout the history of microbiological and biomedical research. A number of cases have been attributed to carelessness or poor technique in the handling of infectious materials. The most important element of safety is strict adherence to standard microbiological practices and techniques. The Centers for Disease Control and Prevention (CDC) and the National Institutes of Health (NIH) have established four biosafety levels which consist of combinations of laboratory practices and techniques, safety equipment, and laboratory facilities appropriate for the operations performed and the hazard posed by infectious agents or recombinant DNA research. The Principle Investigator is responsible for making a determination of the required levels of physical and biological containment required for the work to be performed through the risk assessment process. When standard laboratory practices are not sufficient to control the hazard associated with a particular agent or laboratory procedure, additional measures may be needed. Vertebrate animal biosafety level criteria are also provided which describe four combinations (designated Animal Biosafety Levels 1-4) of practices, safety equipment, and facilities for experiments on animals infected with agents which produce, or may produce, human infection. These guidelines are also useful in the maintenance of laboratory animals that may naturally harbor zoonotic infectious diseases. Physical and biological containment conditions and practices for recombinant or synthetic nucleic acid molecule-containing plants, plant-associated microorganisms, and small animals that are of a size, number, or have growth requirements that preclude the use of general biosafety containment conditions are provided. Additional arthropod containment guidelines are provided including for arthropods that transmit pathogens; however, those arthropods that cause myiasis, infestation, biting, and stinging are not included. Finally, guidelines for work with toxins of biological origin are presented. Toxins are unique in that they do not replicate, are not infectious, and are difficult to transmit mechanically or manually from person to person. Therefore, toxins are usually handled using established general guidelines for toxic or highly toxic chemicals with the incorporation of additional safety and security measures based upon a risk assessment for each specific laboratory operation. Each laboratory should develop standard operating procedures (SOPs) which identify the hazards that will or may be encountered and which specify practices and procedures designed to minimize or eliminate risks. Guidelines for developing these SOPs are provided here. A. Biological Risk Assessment Risk assessment is a process used to identify the hazardous characteristics of a known VI-1

2 or potentially infectious agent or material, the activities that can result in a person s exposure to an agent, the likelihood that such exposure will cause a laboratory acquired infection, and the probable consequences of such an infection. The information identified by risk assessment will provide a guide for the selection of appropriate biosafety levels and microbiological practices, safety equipment, and facility safeguards that can prevent laboratory acquired infections. Biological risk assessment is a subjective process requiring consideration of many hazardous characteristics of agents and procedures. Below is an outline of some of the considerations that should be evaluated. 1. In deciding on the appropriate containment for an experiment, the first step is to assess the risk of the agent itself. Consider the principal hazardous characteristics of the agent, which include its capability to infect and cause disease in a susceptible human host, severity of disease, and the availability of preventive measures and effective treatments. According to the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines), classifications for etiological agents based on the agent s relative pathogenicity for healthy adult humans are as follows: a. Risk Group 1 (RG1) agents are not associated with disease in healthy adult humans. b. Risk Group 2 (RG2) agents are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available. c. Risk Group 3 (RG3) agents are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available. d. Risk Group 4 (RG4) agents are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available. 2. Make a preliminary determination of the biosafety level that best correlates with the initial risk assessment based on the identification and evaluation of the agent hazards. a. Factors to be considered in determining the level of containment include agent factors such as virulence, pathogenicity, infectious dose, environmental stability, route of spread, communicability, operations, quantity, availability of vaccine or treatment, and gene product effects such as toxicity, physiological activity, and allergenicity. b. Any strain that is known to be more hazardous than the parent (wild-type) strain should be considered for handling at a higher containment level. VI-2

3 c. Certain attenuated strains or strains that have been demonstrated to have irreversibly lost known virulence factors may qualify for a reduction of the containment level compared to the Risk Group assigned to the parent strain, if approved by the OU Institutional Biosafety Committee (IBC). d. It is possible to develop an organism containing genetic sequences from multiple sources such that the parent agent may not be obvious. In such cases, the risk assessment should include at least two levels of analysis. The first involves a consideration of the Risk Groups of the source(s) of the sequences and the second involves an assessment of the functions that may be encoded by these sequences (e.g., virulence or transmissibility). It may be prudent to first consider the highest Risk Group classification of all agents that are the source of sequences included in the construct. Other factors to be considered include the percentage of the genome contributed by each parent agent and the predicted function or intended purpose of each contributing sequence. The initial assumption should be that all sequences will function as they did in the original host context. 3. Identify laboratory procedure hazards. The principal laboratory procedure hazards are agent concentration, suspension volume, equipment and procedures that generate small particle aerosols and larger airborne particles (droplets), and use of sharps. There will be situations where the intended use of an agent requires greater precautions. a. It is important to recognize that individuals in the laboratory may differ in their susceptibility to disease. Preexisting diseases, medications, compromised immunity, and pregnancy or breastfeeding that may increase exposure of infants to certain agents, are some of the conditions that may increase the risk of an individual for acquiring a laboratory acquired infection. Consultation with an occupational physician knowledgeable in infectious diseases is advisable in these circumstances. b. Procedures involving animals can present a number of hazards such as bites and scratches, exposure to zoonotic agents, and the handling of experimentally generated infectious aerosols. c. The complexity of a laboratory procedure can also present an increased hazard. d. Aerosol and droplet routes of agent transmission also are important considerations in specification of safety equipment and facility design that result in a given biosafety level. 4. Make a final determination of the appropriate biosafety level and an VI-3

4 appropriate containment level. The final selection of the appropriate biosafety containment level and the selection of any additional laboratory precautions require a comprehensive understanding of the practices, safety equipment, and facility safeguards described the following sections of this manual. The objective of physical containment is to confine microorganisms and organisms containing recombinant or synthetic nucleic acid molecules and to reduce the potential for exposure of the laboratory worker, persons outside of the laboratory, and the environment to these agents. Physical containment is achieved through the use of laboratory practices, containment equipment, and special laboratory design. Emphasis is placed on primary means of physical containment which are provided by laboratory practices and containment equipment. Special laboratory design provides a secondary means of protection against the accidental release of organisms outside the laboratory or to the environment. 5. Evaluate the proficiencies of staff regarding safe practices and the integrity of safety equipment. The protection of laboratory workers, other persons associated with the laboratory, and the public will depend ultimately on the laboratory workers themselves. In conducting a risk assessment, the laboratory director or principal investigator should ensure that laboratory workers have acquired the technical proficiency in the use of microbiological practices and safety equipment required for the safe handling of the agent, and have developed good habits that sustain excellence in the performance of those practices. An evaluation of a person s training, experience in handling infectious agents, proficiency in the use of sterile techniques and biological safety cabinets (BSCs), ability to respond to emergencies, and willingness to accept responsibility for protecting one s self and others is important insurance that a laboratory worker is capable of working safely. The laboratory director or principal investigator should also ensure that the necessary safety equipment is available and operating properly. 6. Review the risk assessment with a biosafety professional or subject matter expert, and ultimately, with the OU Institutional Biosafety Committee (IBC), who must review and approve the biological safety of all research activities involving recombinant or synthetic nucleic acid DNA, gene transfer, microorganisms, viruses, and biological toxins. Sources: CDC/NIH Biosafety in Microbiological and Biomedical Laboratories, U.S. Department of Health and Human Services Public Health Services, Centers for Disease Control and Prevention and National Institutes of Health, HHS Publication No. (CDC) , 4 th Edition, May, 1999, and 5 th Edition, Feb., NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules, March B. General Biosafety Laboratory Practice for All Containment Levels 1. Engineering controls such as biological safety cabinets (BSCs) should be examined and maintained or replaced on a regular schedule to ensure their effectiveness. It is imperative that Class I and II BSCs are tested VI-4

5 and certified in place at the time of installation within the laboratory, at any time the cabinet is moved, and at least annually thereafter. 2. Employees should wash their hands immediately or as soon as possible after removal of gloves or other personal protective equipment and after hand contact with blood or other potentially infectious materials. 3. All personal protective equipment should be removed immediately upon leaving the work area or as soon as possible, if overtly contaminated, and placed in an appropriately designated area or container for storage, washing, decontamination, or disposal. 4. Immediately after use, contaminated sharps should be disposed in closable, puncture-resistant containers which are leak-proof on the sides and bottom and color-coded or labeled with the biohazard symbol. 5. Sharps containers should be easily accessible to personnel and located in the area of use. 6. Used needles and other sharps should not be sheared, bent, broken, recapped, or resheathed by hand. 7. Eating, drinking, smoking, applying cosmetics or lip balm, and handling contact lenses are prohibited in areas where work with biohazardous materials is performed. 8. Food and drink must not be stored in refrigerators, freezers, or cabinets where biohazardous materials are stored. 9. All procedures involving biohazardous materials should be performed in such a manner as to minimize splashing, spraying, and aerosolization of these substances. 10. Mouth pipetting/suctioning is prohibited. 11. Broken glassware which may be contaminated should not be picked up directly with the hands. It should be cleaned up using mechanical means such as a brush and dust pan, tongs, cotton swabs or forceps. 12. All infectious waste should be disposed in accordance with the procedures found in Section VIII., "Biomedical Waste. 13. House vacuum systems should be protected from aspirations of fluids that contain microorganisms or recombinant or synthetic nucleic acid molecules. For Biosafety Level 2 laboratory work, an in-line flask containing a suitable decontamination solution should be used, serving as a fluid overflow collection vessel, connected to the vacuum system. For Biosafety Level 3 laboratory work, two flasks containing a VI-5

6 decontamination solution prior to the HEPA filter should be used. See Section II.I., House Vacuum System Protection. 14. Biological hazard (biohazard) tags or labels should be used to identify the actual or potential presence of a biological hazard and to identify equipment, containers, rooms, experimental animals, or combinations thereof, that contain or are contaminated with hazardous biological agents. Sources: CDC/NIH Primary Containment for Biohazards: Selection, Installation, and use of Biological Safety Cabinets, U.S. Department of Health and Human Services Public Health Services, Centers for Disease Control and Prevention and National Institutes of Health, September 1995 CDC/NIH Biosafety in Microbiological and Biomedical Laboratories, U.S. Department of Health and Human Services Public Health Services, Centers for Disease Control and Prevention and National Institutes of Health, HHS Publication No. (CDC) , 4 th Edition, May, 1999, and 5 th Edition, Feb., NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules, March OSHA Specifications for Accident Prevention Signs and Tags Standard (29 CFR ) C. Biosafety Level 1 Biosafety Level 1 (BSL-1) is suitable for work involving well-characterized agents not known to cause disease in healthy adult humans, and of minimal potential hazard to laboratory personnel and the environment. BSL-1 laboratories are not necessarily separated from the general traffic patterns in the building. Work is typically conducted on open bench tops using standard microbiological practices, but may be used as determined by appropriate risk assessment. Special containment equipment or facility design is not required nor generally used. Laboratory personnel have specific training in the procedures conducted in the laboratory and are supervised by a scientist with general training in microbiology or a related science. The following standard and special practices, safety equipment and facilities apply to agents assigned to BSL Standard Microbiological Practices a. Access to the laboratory should be limited or restricted at the discretion of the laboratory director when experiments or work with cultures, specimens, or organisms containing recombinant or synthetic nucleic acid molecules are in progress. The Principal Investigator (PI) must enforce the institutional policies that control access to the laboratory. b. Protective eyewear should be worn when conducting procedures that have the potential to create splashes of microorganisms, organisms containing recombinant or synthetic nucleic acid molecules, or other hazardous materials. c. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human consumption must not be permitted in laboratory areas. Food must be stored outside the work area in cabinets or refrigerators designated and used for this purpose only. VI-6

7 d. Mouth pipetting is prohibited; mechanical pipetting devices are used. e. Policies for the safe handling of sharps, such as needles, scalpels, pipettes, and broken glassware are developed and implemented. Whenever practical, PIs should adopt improved engineering and work practice controls that reduce risk of sharps injuries. Precautions, including those listed below, must always be taken with sharp items. These include: (1) Careful management of needles and other sharps are of primary importance. Needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal. (2) Used disposable needles and syringes must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal. (3) Non-disposable sharps must be placed in a hard-walled container for transport to a processing area for decontamination, preferably by autoclaving. (4) Broken glassware must not be handled directly. Instead, it must be removed using a brush and dustpan, tongs, or forceps. Plasticware should be substituted for glassware whenever possible. f. All procedures should be performed carefully to minimize the creation of splashes or aerosols. g. Work surfaces should be decontaminated at least once a day and after any spill of viable or potentially infectious material with an appropriate disinfectant. h. All cultures, stocks, organisms containing recombinant or synthetic nucleic acid molecules, other potentially infectious materials, and other regulated wastes are decontaminated before disposal by an approved decontamination method, such as autoclaving. Materials to be decontaminated outside of the immediate laboratory must be placed in a durable, non-breakable, leakproof container and sealed for transport from the laboratory. Materials to be removed from the facility for decontamination must be packed in accordance with applicable local, state, and federal regulations. i. A sign incorporating the universal biohazard symbol must be posted at the entrance to the laboratory when infectious agents VI-7

8 are present. The sign should indicate the appropriate biosafety level and any personal protective equipment or vaccinations/ immunizations needed to enter the room. j. An integrated pest management program should be in effect. k. The PI must ensure that laboratory personnel receive appropriate training regarding their duties, the necessary precautions to prevent exposures, and exposure evaluation procedures. Personnel must receive annual updates or additional training when procedural or policy changes occur. Personal health status may impact an individual s susceptibility to infection, ability to receive immunizations or prophylactic interventions. Therefore, all laboratory personnel and particularly women of child-bearing age should be provided with information regarding immune competence and conditions that may predispose them to infection. Individuals having these conditions should be encouraged to selfidentify to contact their personal physician or Goddard Health Services for appropriate counseling and guidance. 2. Special Practices: None 3. Safety Equipment (Primary Barriers) a. Special containment devices or equipment such as a BSC are generally not required for manipulations of agents assigned to BSL-1. b. Laboratory coats, gowns, or uniforms are recommended to be worn to prevent contamination of personal clothing. c. Protective eyewear should be worn when conducting procedures that have the potential to create splashes of microorganisms, organisms containing recombinant or synthetic nucleic acid molecules, or other hazardous materials. Persons who wear contact lenses should also wear eye protection. d. Gloves must be worn to protect hands from exposure to hazardous materials. (1) Glove selection should be based on an appropriate risk assessment. (2) Alternatives to latex gloves should be available. (3) Laboratory workers should change gloves when contaminated, integrity has been compromised, or when otherwise necessary. Laboratory workers should remove VI-8

9 gloves and wash hands when work with hazardous materials, materials involving organisms containing recombinant or synthetic nucleic acid molecules, or animals has been completed and before leaving the laboratory. (4) Disposable gloves should not be washed or reused. Used gloves should be disposed with other contaminated laboratory waste. (5) Hand washing protocols must be rigorously followed. 4. Laboratory Facilities (Secondary Barriers) D. Biosafety Level 2 a. Laboratories should have doors for access control. b. Laboratories must have a sink for handwashing. c. The laboratory should be designed so that it can be easily cleaned. Carpets and rugs in laboratories are not appropriate. d. Laboratory furniture must be capable of supporting anticipated loading and uses. Spaces between benches, cabinets, and equipment are accessible for cleaning. e. Bench tops must be impervious to water and are resistant to moderate heat and the organic solvents, acids, alkalis, and other chemicals (such as those used to decontaminate the work surface and equipment). f. Chairs used in laboratory work must be covered with a non-porous material that can be easily cleaned and decontaminated with an appropriate disinfectant. g. Laboratory windows that open to the exterior should be fitted with screens. Biosafety Level 2 (BSL-2) is suitable for work involving agents that pose moderate potential hazard to personnel and the environment. It differs from BSL-1 in that (1) laboratory personnel should have specific training in handling pathogenic agents and be supervised by scientists competent in handling infectious agents and associated procedures; (2) access to the laboratory is limited when work is being conducted; and (3) all procedures in which infectious aerosols or splashes may be created should be conducted in BSCs or other physical containment equipment. The following standard and special practices, safety equipment, and facilities apply to BSL-2. VI-9

10 1. Standard Microbiological Practices a. The PI must enforce the institutional policies and standard operating procedures approved by the OU IBC to limit access to the laboratory. Only persons who have been advised of the potential hazard and meet any specific entry requirements (e.g., immunization) may enter the laboratory or animal rooms. b. Persons must wash hands when work with hazardous materials, materials involving organisms containing recombinant or synthetic nucleic acid molecules, or animals has been completed and before leaving the laboratory. c. Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted in the work areas. Storing food for human consumption is not permitted in the laboratory. Food must be stored outside the laboratory area in cabinets or refrigerators designated for this purpose only. d. Mouth pipetting is prohibited; mechanical pipetting devices must be used. e. Policies for the safe handling of sharps such as needles, scalpels, pipettes, and broken glassware must be developed and implemented. Whenever practical, PIs should adopt improved engineering and work practice controls that reduce risk of sharps injuries. Precautions, including those listed below, must always be taken with sharp items. These include: (1) Hypodermic needles and syringes should only be used for parenteral injection and aspiration of fluids from laboratory animals or diaphragm bottles. Only needle-locking syringes or disposable syringe-needle unites (i.,e., needle is integral to the syringe) may be used for the injection or aspiration of fluids containing microorganisms or viruses. (2) Safe needle devices should be used whenever such a device is available for the task requiring the use of a needle/syringe. (3) Careful management of needles and other sharps are of primary importance. Needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal. (4) Used disposable needles and syringes must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal. VI-10

11 (5) Non-disposable sharps must be placed in a hard-walled container for transport to a processing area for decontamination, preferably by autoclaving. (6) Broken glassware must not be handled directly. Instead, it must be removed using a brush and dustpan, tongs, or forceps. Plasticware should be substituted for glassware whenever possible. f. All procedures should be performed carefully to minimize the creation of splashes or aerosols. g. Work surfaces should be decontaminated after completion of work or after any spill or splash of potentially infectious material with an appropriate disinfectant that is effective against the agent(s) of concern. All spills or potential exposures to potentially infectious material or recombinant/synthetic nucleic acid molecules must be reported to the Principal Investigator and the OU IBC, and the incident must be reported immediately to NIH by the IBC. h. All cultures, stocks, other potentially infectious materials, and other regulated wastes should be decontaminated before disposal by an approved method, such as autoclaving. (1) Materials to be decontaminated outside of the immediate laboratory must be placed in a durable, leakproof container and secured for transport from the facility. (2) Materials to be removed from the facility for decontamination must be packaged in accordance with applicable local, state, and federal regulations. i. A sign incorporating the universal biohazard symbol must be posted at the entrance to the laboratory when infectious agents are present or organisms containing recombinant or synthetic nucleid acid molecules are in use. Posted information must include: the laboratory s biosafety level, the supervisor s name (or other responsible personnel), telephone number, and required procedures for entering and exiting the laboratory. For work with organisms containing recombinant or synthetic nucleic acid molecules, the agent must also be posted. j. An effective integrated pest management program is required. k. The PI must ensure that laboratory personnel receive appropriate training regarding their duties, the necessary precautions to prevent exposures, and exposure evaluation procedures. VI-11

12 Personnel must receive annual updates or additional training when procedural or policy changes occur. l. Personal health status may impact an individual s susceptibility to infection, ability to receive immunizations or prophylactic interventions. Therefore, all laboratory personnel and particularly women of child-bearing age should be provided with information regarding immune competence and conditions that may predispose them to infection. Individuals having these conditions should be encouraged to contact their personal physician or Goddard Health Services for appropriate counseling and guidance. 2. Special Practices a. All persons entering the laboratory must be advised of the potential hazards and meet specific entry/exit requirements. b. Laboratory personnel must be provided medical surveillance and offered appropriate immunizations for agents handled or potentially present in the laboratory. c. A laboratory-specific SOP must be prepared, approved by the OU IBC, and adopted as laboratory policy. The SOP must be available and accessible. d. The PI must ensure that laboratory personnel demonstrate proficiency in standard and special microbiological practices before working with BSL-2 agents. e. Potentially infectious materials must be placed in a durable, leak proof container during collection, handling, processing, storage, or transport within a facility. f. Laboratory equipment should be routinely decontaminated and decontaminated after spills, splashes, or other potential contamination. (1) Spills involving infectious materials must be contained, decontaminated, and cleaned up by staff properly trained and equipped to work with infectious material. (2) Equipment must be decontaminated before repair, maintenance, or removal from the laboratory. g. Incidents that may result in exposure to infectious materials or organisms containing recombinant or synthetic nucleic acid molecules must be immediately evaluated and treated according VI-12

13 to procedures described in the laboratory biosafety manual/sop. All such incidents must be reported to the Principal Investigator and the OU IBC, and the incident must be reported immediately to NIH by the IBC. Medical evaluation, surveillance, and treatment should be provided through Goddard Health Services or the nearest emergency room, and appropriate records maintained. h. Animals and plants not involved in the work being performed are not permitted in the lab. i. All procedures involving the manipulation of infectious materials or organisms containing recombinant or synthetic nucleic acid molecules that may generate an aerosol should be conducted within a BSC or other physical containment devices. 3. Safety Equipment (Primary Barriers) a. Properly maintained BSCs (preferably Class II), other appropriate personal protective equipment, or other physical containment devices must be used whenever manipulation of infectious materials or organisms containing recombinant or synthetic nucleic acid molecules involves: (1) Procedures with a potential for creating aerosols or splashes are conducted. These may include pipetting, centrifuging, grinding, blending, shaking, mixing, sonicating, opening containers of infectious materials, inoculating animals intranasally, and harvesting infected tissues from animals or embryonate eggs. (2) High concentrations or large volumes of infectious materials or organisms containing recombinant or synthetic nucleic acid molecules are used. Such materials may be centrifuged in the open laboratory using sealed rotor heads or centrifuge safety cups are used, and if these rotors or safety cups are opened only in a BSC. b. Protective laboratory coats, gowns, smocks, or uniforms designated for lab use must be worn when working with infectious materials or organisms containing recombinant or synthetic nucleic acid molecules. This protective clothing should be removed and left in the laboratory before leaving for non-laboratory areas (e.g., cafeteria, library, administrative offices). All protective clothing should be either disposed properly or deposited for laundering by the institution. Laboratory protective clothing should not be taken home. c. Eye and face protection (goggles, mask, faceshield or other splatter guards) should be used for anticipated splashes or sprays VI-13

14 of infectious materials, organisms containing recombinant or synthetic nucleic acid molecules, or other hazardous materials when the microorganisms must be manipulated outside the BSC or containment device. d. Gloves must be worn to protect hands from exposure to involving infectious materials, or organisms containing recombinant or synthetic nucleic acid molecules, or other hazardous material. (1) Gloves must not be worn outside the laboratory. (2) Gloves selection should be based on an appropriate risk assessment. (3) Alternates to latex gloves should be available. (4) Gloves should be changed when contaminated, integrity has been compromised, or when otherwise necessary. Wearing two pairs of gloves may be appropriate. (5) Gloves should be removed and hands should be washed when work with hazardous materials has been completed and before leaving the laboratory. (6) Disposable gloves should not be washed or reused. Gloves should be disposed with other contaminated laboratory waste. e. Handwashing protocols must be rigorously followed. f. Eye, face, and respiratory protection should be used in rooms containing infected animals as determined by the risk assessment. 4. Laboratory Facilities (Secondary Barriers) a. Laboratory doors should be self-closing and have locks in accordance with the institutional policies. b. Laboratories must have a sink for handwashing. The sink may be manually, hands-free, or automatically operated. It should be located near the exit door. c. The laboratory should be designed so that it can be easily cleaned and decontaminated. Carpets and rugs in laboratories are not permitted. d. Laboratory furniture must be capable of supporting anticipated loading and uses. Spaces between benches, cabinets, and VI-14

15 equipment are accessible for cleaning. (1) Bench tops must be impervious to water and resistant to moderate heat, organic solvents, acids, alkalis, and other chemicals (such as those used to decontaminate the work surfaces and equipment). (2) Chairs and other furniture used in laboratory work should be covered with a non-porous material that can be easily cleaned and decontaminated with appropriate disinfectant. e. Laboratory windows that open to the exterior are not recommended. However, if a laboratory does have windows that open to the exterior, they must be fitted with screens. f. BSCs must be installed in such a manner that fluctuations of the room supply and exhaust air do not interfere with proper operations. BSCs should be located away from doors, windows that can be opened, heavily traveled laboratory areas, and other potentially disruptive equipment. g. Vacuum lines should be protected with High Efficiency Particulate Air (HEPA) filters, or their equivalent. Filters must be replaced as needed. Liquid disinfectant traps may be required. h. An eyewash station must be readily available. i. There are no specific requirements on ventilation systems. However, planning of new facilities should consider mechanical ventilation systems that provide an inward flow of air without recirculation to spaces outside of the laboratory. j. HEPA filtered exhaust air from a Class II BSC can be safely recirculated back into the laboratory environment if the cabinet is tested and certified at least annually and operated according to manufacturer s recommendations. BSCs can also be connected to the laboratory exhaust system by either a thimble (canopy) connection or a direct (hard) connection. Provisions to assure proper safety cabinet performance and air system operation must be verified. k. A method for decontaminating all laboratory wastes should be available in the facility (e.g., autoclave, chemical disinfection, incineration, or other validated decontamination method). E. Biosafety Level 3 and 4 Biosafety Level 3 (BSL-3) is applicable to clinical, diagnostic, teaching, research, or VI-15

16 production facilities in which work is performed with indigenous or exotic agents which may cause serious or potentially lethal disease through inhalation route exposure. Biosafety Level 4 (BSL-4) is required for work with dangerous and exotic agents which pose a high individual risk of aerosol-transmitted laboratory infections and life-threatening disease. At this time, the University of Oklahoma Norman campus does not have a facility designed for work with organisms at BSL-3 or BSL-4. F. Animal Biosafety Level 1 Animal Biosafety Level 1 (ABSL-1) is suitable for work involving well characterized agents that are not known to cause disease in immunocompetent adult humans, and present minimal potential hazard to personnel and the environment. 1. Standard Practices a. The animal facility director establishes policies, procedures, and protocols for emergency situations. Prior to any project s initiation, each protocol must be submitted for review and approval by the Institutional Animal Care and Use Committee (IACUC) and the Institutional Biosafety Committee (IBC). Any special practices are approved at this time. b. A safety manual specific to the animal facility should be prepared and be available and accessible. Personnel are advised of potential hazards, and are required to read and follow instructions on practices and procedures. c. Supervisors must ensure that animal care, laboratory and support personnel receive appropriate training regarding their duties, animal husbandry procedures, potential hazards, manipulations of infectious agents, necessary precautions to prevent hazard or exposures, and hazard/exposure evaluation procedures (physical hazards, splashes, aerosolization, etc.). Personnel must receive annual updates or additional training when procedures or policies change. Records are maintained for all hazard evaluations, employee training sessions and staff attendance. d. Personal health status may impact an individual s susceptibility to infection, ability to receive immunizations or prophylactic interventions. Therefore, all personnel and particularly women of child-bearing age should be provided information regarding immune competence and conditions that may predispose them to infection. Individuals having these conditions should be encouraged to self-identify to the institution s healthcare provider for appropriate counseling and guidance. Personnel using respirators must participate in the OU Respiratory Protection Program. VI-16

17 e. A sign incorporating safety information must be posted at the entrance to the areas where infectious materials and/or animals are housed or are manipulated. The sign must include the animal biosafety level, general occupational health requirements, personal protective equipment requirements, the PI name (or other responsible personnel), telephone number, and required procedures for entering and exiting the animal areas. Identification of specific infectious agents is recommended when more than one agent is being used with in an animal room. A template for this sign is available at f. Access to the animal room is limited. Only those persons required for program or support purposes are authorized to enter the facility. All persons, including facility personnel, service workers, and visitors are advised of the potential hazards (nature of research pathogens, allergens, etc.) and are instructed on the appropriate safeguards. g. Protective laboratory coats, gowns, or uniforms are required to prevent contamination of personal clothing. Gloves are worn to prevent skin contact with contaminated, infectious and hazardous materials, and when handling animals. Gloves and personal protective equipment should be removed in a manner that minimizes transfer of infectious materials outside of the areas where infectious materials and/or animals are housed or are manipulated. Persons must wash hands when work with hazardous materials, materials involving organisms containing recombinant or synthetic nucleic acid molecules, or animals has been completed and before leaving the laboratory. h. Eye and face and respiratory protection should be used in rooms containing infected animals, as dictated by the risk assessment. i. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use should only be done in designated areas and are not permitted in animal or procedure rooms. j. All procedures are carefully performed to minimize the creation of aerosols or splatters of infectious materials and waste. k. Mouth pipetting is prohibited. Mechanical pipetting devices must be used. l. Policies for the safe handling of sharps such as needles, scalpels, pipettes, and broken glassware must be developed and implemented. VI-17

18 (1) When applicable, PIs should adopt improved engineering and work practice controls that reduce the risk of sharps injuries, including the use of safe needle devices wherever possible. (2) Needles and syringes or other sharp instruments should be limited to use in the animal facility when there is no alternative for such procedures as parenteral injection, blood collection, or aspiration of fluids from laboratory animals and diaphragm bottles. (a) (b) (c) Disposable needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal. Used disposable needles must be carefully placed in puncture-resistant containers used for sharps disposal. Sharps containers should be located as close to the work site as possible. Non-disposable sharps must be placed in a hardwalled container for transport to a processing area for decontamination, preferably by autoclaving. (3) Broken glassware must not be handled directly. Instead, it must be removed using a brush and dustpan, tongs, or forceps. (4) Plasticware should be substituted for glassware whenever possible. (5) Equipment containing sharp edges and corners should be avoided. m. Equipment and work surfaces should be routinely decontaminated with an appropriate disinfectant after work with an infectious agent and after spills, splashes, or overt contamination. n. Animals and plants not associated with the work being performed must not be permitted in the areas where infectious materials and/or animals are housed or are manipulated. o. An effective integrated pest management program is required. p. All wastes from the animal room (including animal tissues, carcasses, and contaminated bedding) are transported from the animal room in leak-proof, covered containers for appropriate VI-18

19 2. Special Practices disposal. Incineration is recommended. a. For animal protocol that includes research involving recombinant or synthetic nucleic acid molecules (either associated with or in animals) that are not exempt from the NIH Guidelines, the following procedures are also required: (1) All carcasses are to be disposed through Comparative Medicine and marked for incineration. (2) A permanent record shall be maintained of the experimental use and disposal of each animal or group of animals. (3) All genetically engineered neonates shall be permanently marked within 72 hours after birth, if their size permits. If their size does not permit marking, their containers should be marked. In addition, transgenic animals should contain distinct and biochemically assayable DNA sequences that allow identification of transgenic animals from among nontransgenic animals. (4) A double barrier shall be provided to separate male and female animals unless reproductive studies are part of the experiment or other measures are taken to avoid reproductive transmission. Reproductive incapacitation may be used. 3. Safety Equipment (Primary Barriers and Personal Protective Equipment) a. A risk assessment should be performed by the PI to determine the appropriate type of personal protective equipment to be utilized. The wearing of laboratory coats, gowns, or uniforms are recommended to prevent contamination of personal clothing. Protective outer clothing such as laboratory coats should not be worn outside areas where infectious materials and/or animal are housed or manipulated. Gowns and uniforms are not worn outside the facility. b. Protective eyewear should be worn when conducting procedures that have the potential to create splashes of microorganisms, organisms containing recombinant or synthetic nucleic acid molecules, or other hazardous materials. Persons who wear contact lenses should also wear eye protection when entering areas with potentially high concentrations or airborne particulates. VI-19

20 c. Persons having contact with non-human primates should assess their risk of mucous membrane exposure and wear appropriate eye and face protection. d. Gloves should be worn to protect hands from exposure to hazardous materials. A risk assessment should be performed to identify the appropriate glove for the task, and alternatives to latex gloves should be available. (1) Gloves should be changed when contaminated, the integrity has been compromised, or when otherwise necessary. (2) Gloves must not be worn outside the animal rooms. (3) Gloves and personal protective equipment should be removed in a manner that prohibits transfer of infectious materials. (4) Disposable gloves should not be washed or reused. Dispose of used gloves with other contaminated waste. e. Persons must wash hands when work with hazardous materials, materials involving organisms containing recombinant or synthetic nucleic acid molecules, or animals has been completed and before leaving the laboratory. Hand washing should occur after the removal of gloves. 4. Animal Facilities (Secondary Barriers) a. The animal facility is separated from areas that are open to unrestricted personnel traffic within the building. b. External facility doors are self-closing and self-locking. Access to the animal facility is restricted. Doors to areas where infectious materials and/or animals are housed open inward, are self-closing, are kept closed when experimental animals are present, and should never be propped open. Doors to cubicles inside an animal room may open outward or slide horizontally or vertically. c. The animal facility must have a sink for hand washing. Sink traps are filled with water, and/or appropriate liquid to prevent the migration of vermin and gases. d. The animal facility is designed, constructed, and maintained to facilitate cleaning and housekeeping. The interior surfaces (walls, floors, and ceilings) are water resistant. VI-20

21 e. The animal facility is designed, constructed, and maintained to facilitate cleaning and housekeeping. The interior surfaces (walls, floors, and ceilings) are water resistant. It is recommended that penetrations in floors, walls and ceiling surfaces are sealed, to include openings around ducts, doors and door frames, to facilitate pest control and proper cleaning. Floors must be slip resistant, impervious to liquids, and resistant to chemicals. f. Cabinets and bench tops must be impervious to water and resistant to heat, organic solvents, acids, alkalis, and other chemicals. Spaces between benches, cabinets, and equipment should be accessible for cleaning. Chairs used in animal area must be covered with a non-porous material that can be easily cleaned and decontaminated. Furniture must be capable of supporting anticipated loads and uses. Sharp edges and corners should be avoided. g. External windows are not recommended. Any windows must be resistant to breakage. Where possible, windows should be sealed. If the animal facility has windows that open, they should be fitted with fly screens. h. Ventilation should be provided in accordance with the Guide for Care and Use of Laboratory Animals, latest edition. No recirculation of exhaust air should occur. It is recommended that animal rooms have inward directional airflow. Ventilation system design should consider the heat and high moisture load produced during the cleaning of animal rooms and the cage wash process. i. Internal facility appurtenances, such as light fixtures, air ducts, and utility pipes, should be arranged to minimize horizontal surface areas to facilitate cleaning and minimize the accumulation of debris or fomites. j. If floor drains are provided, the traps must always be filled with water and/or an appropriate disinfectant to prevent the migration of vermin and gases. k. Cages should be washed manually, or preferably, in a mechanical cage washer. The mechanical cage washer should have a final rinse temperature of at least 180 " F. l. Illumination should be adequate for all activities, avoiding reflections and glare that could impede vision. m. Emergency eyewash and shower should be readily available; location is determined by the risk assessment. VI-21

22 G. Animal Biosafety Level 2 Animal Biosafety Level 2 involves practices for work with those agents associated with human disease. It addresses hazards from ingestion as well as from percutaneous and mucous membrane exposure. ABSL-2 builds upon the practices, procedures, containment equipment, and facility requirements of ABSL Standard Practices a. Standard policies, procedures, and protocols for emergency situations should be established by the facility director. Appropriate special standard operating procedures must be developed by the PI and reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) and the Institutional Biosafety Committee (IBC). The form at may be used to facilitate this process. (1) All personnel working in the animal room must be advised of special hazards, and are required to read and follow these standard operating procedures (SOPs). Personnel must be supervised by individuals with adequate knowledge of potential hazards, microbiological agents, animal manipulations, and husbandry procedures. (2) Animal care, laboratory and support personnel receive appropriate training regarding their duties, animal husbandry procedures, potential hazards associated with the work involved, manipulations of infectious agents, the necessary precautions to prevent exposures, and the hazard/exposure evaluation procedures (physical hazards, splashes, aerosolization, etc.). (3) Personnel should receive annual updates, or additional training as necessary for procedural or policy changes. (4) Records of all training provided should be maintained. b. Personal health status may impact an individual s susceptibility to infection, ability to receive immunizations or prophylactic interventions. Therefore, all personnel and particularly women of child-bearing age should be provided information regarding immune competence and conditions that may predispose them to infection. Individuals having these conditions should be encouraged to self-identify to the institution s healthcare provider for appropriate counseling and guidance. Personnel using respirators must be enrolled in the OU Respiratory Protection Program. VI-22