Biosafety Manual 2016

Transcription

1 2016

2 Policy Statement It is the policy of Northern Arizona University (NAU) to provide a safe working environment. The primary responsibility for insuring safe conduct and conditions in the laboratory resides with the principal investigator. The Biological Safety Office is committed to providing up-to-date information, training, and monitoring to the research and biomedical community concerning the safe conduct of biological, recombinant, and acute toxin research and the handling of biological materials in accordance with all pertinent local, state and federal regulations, guidelines, and laws. To that end, this manual is a resource, to be used in conjunction with the CDC and NIH guidelines, the NAU Select Agent Program, Biosafety in Microbiological and Biomedical Laboratories (BMBL), and other resource materials. Introduction This Biological Safety Manual is intended for use as a guidance document for researchers and clinicians who work with biological materials. It should be used in conjunction with the Laboratory- Specific Safety Manual, which provides more general safety information. These manuals describe policies and procedures that are required for the safe conduct of research at NAU. The NAU Personnel Policy on Safety 5.03 also provides guidance for safety in the workplace. Responsibilities In the academic research/teaching setting, the principal investigator (PI) is responsible for ensuring that all members of the laboratory are familiar with safe research practices. In the clinical laboratory setting, the faculty member who supervises the laboratory is responsible for safety practices. Lab managers, supervisors, technicians and others who provide supervisory roles in laboratories and clinical settings are responsible for overseeing the safety practices in laboratories and reporting any problems, accidents, and spills to the appropriate faculty member. Employees who work with biological materials are responsible for reading this manual and carrying out the safety practices outlined here. The Biosafety Office will provide guidance, information, review, monitoring, and training regarding biological safety programs, when appropriate. This includes implementing registration activities for certain research projects, acting as a consultant for departments regarding implementation and enforcement of biological safety programs, evaluating work practices and personal protective equipment, providing educational materials, tracking employee training, and medical monitoring. The mission of the EH&S Biosafety Office is to minimize injury to faculty, staff, students and visitors and to minimize damage to University property. Inherent in this mission is the charge to provide a safe and healthy environment in which the University s activities can be pursued. All applicable federal and state safety laws, rules and regulations are adopted by the University. In order to carry out its duties and responsibilities, the EH&S Biosafety Office will adhere to standards or codes related to biosafety which have been adopted and promulgated by nationally recognized standards-setting organizations. The interpretation of biosafety regulations and guidelines is the responsibility of the EH&S Biosafety Office. ii

3 In order to assure an effective Biosafety Program for the Northern Arizona University, it is imperative that all individuals associated with the University comply fully with the policies and procedures set forth in the manual. iii

4 Emergency Phone Numbers General University Police Department Flagstaff Fire Department 911 Gas leak 911 Spills/Accidents Biological or Recombinant Materials Chemicals (laboratory) Radioactive Materials Select Agents Incidents involving Air Monitoring Medical Emergency Campus Health Services Flagstaff Medical Center Emergency iv

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6 Table of Contents 1 Biological Safety Principles of Biological Safety Laboratory Practice and Technique Safety Equipment (Primary Barrier) Facility Design (Secondary Barrier) Biosafety Levels Animal Facilities Clinical Laboratories Importation and Interstate Shipment of Certain Biomedical Materials Biological Safety Levels Biosafety Level Biosafety Level Biosafety Level Animal Biosafety Level 1 (ABSL-1) Animal Biosafety Level 2 (ABSL-2) Animal Biosafety Level 3 (ABSL-3) Agents List Biological Safety Level 1 (BSL-1) Biological Safety Level 2 (BSL-2) BSL-2 - Bacterial Agents Including Chlamydia BSL-2 - Fungal Agents BSL-2 - Parasitic Agents BSL-2 - Viruses Biological Safety Level 3 (BSL-3) BSL-3 - Bacterial Agents Including Rickettsia BSL-3 - Fungal Agents BSL-3 - Parasitic Agents BSL-3 - Viruses and Prions Biological Safety Level 4 (BSL-4) BSL-4 - Bacterial Agents BSL-4 - Fungal Agents BSL-4 - Parasitic Agents BSL-4 - Viral Agents Information for Researchers Project Registration Biological Agent (BA) Registration Recombinant DNA (R-DNA) Registration Acute Toxins (AT) Registration Regulated Biological Materials Project Amendment

7 Typical changes include: Table 3: Toxin Table Select Agents Minors in Research Laboratories or Animal Facilities Responsibilities of the Principle Investigator during the Conduct of the Research Reportable Incidents and Violations Institutional Reporting Responsibilities Biological Waste Disposal Policy Categories ) Infectious/potentially infectious/r-dna ) Non-infectious waste ) Mixed radioactive/biohazardous waste ) Mixed chemical/biohazardous waste ) Animal carcasses and materials ) Human remains Packaging ) Biohazard bags ) Sharps ) Containers Labeling ) Date ) Name/Location/Phone Number ) Biohazard sign Transport Training Biological Waste Disposal Containers Bulk Biohazard Receptacles Sharps Boxes Biohazard Bags Autoclave Use and Testing Autoclave Testing Record-Keeping Autoclave Operating Procedures Autoclave Operation and Safety Training Autoclave Guidelines Disinfectants Liquids Alcohols Chlorine compounds Formaldehyde Glutaraldehyde Hydrogen peroxide Iodine and Iodophors

8 Phenol and phenolic compounds Quatenary ammonium compounds Gases Ethylene Oxide (Not currently allowed at NAU) Vapor Phase Hydrogen Peroxide Chlorine Dioxide gas Ozone Formaldehyde Gas (from heating paraformaldehyde) Irradiation Ultraviolet, UV radiation Ionizing radiation Electron Beam Microwaves Disinfectants Bibliography Shipment of Biological Materials Laboratory Spills Spill in the Open Laboratory Spill in a Biological Safety Cabinet Biological Safety Cabinets Biological Safety Cabinets (BSCs) The Class I BSC The Class II BSC The Class II, Type A BSC The Class II, Type B1 BSC The Class II, Type B2 BSC The Class II, Type B3 BSC The Class III BSC Horizontal Laminar Flow Clean Bench Vertical Laminar Flow Clean Bench Operations within a Class II BSC Laboratory Hazards Decontamination Surface Decontamination Gas Decontamination Engineering Requirements Ultraviolet Lamps BSC Placement HEPA Filters Certification of BSCs Programs Animal Contact Medical Monitoring Program NAU Bloodborne Pathogen Program BBP Exposure Information

9 4 -Medical Surveillance Immunoprophylaxis Recommendations for Prophylactic Immunization of Laboratory Personnel Working with Infectious Agents Bacterial agents Rickettsial agents Viral agents Blood Serum Sampling Health Assessments Exposure to Mycobacterium Tuberculosis List of Tables Table 1: Summary of Recommended Biosafety Levels for Infectious Agents Table 2: Summary of Recommended Biosafety Levels for Activities in Which Experimentally or Naturally Infected Vertebrate Animals Are Used Table 3: Toxin Table Table 4: Dilutions of Household Bleach Table 5: Summary and Comparison of Liquid Disinfectants (Page 1) Table 6: Summary of Practical Disinfectants Table 7: Reprocessing Methods for Equipment Used in the Health Care Setting

10 1 Biological Safety Principles of Biological Safety The following is from Biosafety in Microbiological and Biomedical Laboratories, 2009, HHS publication No. (CDC) , Centers for Disease Control & Prevention/National Institutes of Health The term containment is used in describing safe methods for managing infectious agents in the laboratory environment where they are being handled or maintained. The purpose of containment is to reduce or eliminate exposure of laboratory workers, other persons, and the outside environment to potentially hazardous agents. Both good microbiological technique and the use of appropriate safety equipment provide primary containment, the protection of personnel and the immediate laboratory environment from exposure to infectious agents. The use of vaccines may provide an increased level of personal protection. Secondary containment, the protection of the environment external to the laboratory from exposure to infectious materials, is provided by a combination of facility design and operational practices. Therefore, the three elements of containment include laboratory practice and technique, safety equipment, and facility design. The risk assessment of the work to be done with a specific agent will determine the appropriate combination of these elements. Laboratory Practice and Technique The most important element of containment is strict adherence to standard microbiological practices and techniques. Persons working with infectious agents or potentially infectious materials must be aware of potential hazards, and must be trained and proficient in the practices and techniques required for handling such material safely. The director or person in charge of the laboratory is responsible for providing or arranging for appropriate training of personnel. Each laboratory should develop or adopt a biosafety or operations manual that identifies the hazard that will or may be encountered, and which specifies practices and procedures designed to minimize or eliminate risks. Personnel should be advised of special hazards and should be required to read and to follow the required practices and procedures. A scientist trained and knowledgeable in appropriate laboratory techniques, safety procedures, and hazards associated with handling infectious agents must direct laboratory activities. When standard laboratory practices are not sufficient to control the hazard associated with a particular agent or laboratory procedure, additional measures may be needed. The laboratory director is responsible for selecting additional safety practices, which must be in keeping with the hazard associated with the agent or procedure. Laboratory personnel, safety practices, and techniques must be supplemented by appropriate facility design and engineering features, safety equipment, and management practices. 10

11 Safety Equipment (Primary Barrier) Safety equipment includes biological safety cabinets (BSCs), enclosed containers, and other engineering controls designed to remove or minimize exposures to hazardous biological materials. The biological safety cabinet (BSC) is the principal device used to provide containment of infectious splashes or aerosols generated by many microbiological procedures. Three types of BSCs (Class I, II, III) used in microbiological laboratories are described in Section 2. Open-fronted Class I and Class II BSCs are primary barriers which offer significant levels of protection to laboratory personnel and to the environment when used with good microbiological techniques. The Class II BSC also provides protection from external contamination of the materials (e.g., cell cultures, microbiological stocks) being manipulated inside the cabinet. The gas-tight Class III BSC provides the highest attainable level of protection to personnel and the environment. An example of another primary barrier is the safety centrifuge cup, an enclosed container designed to prevent aerosols from being released during centrifugation. To minimize this hazard, containment controls such as BSC's or centrifuge cups must be used for handling infectious agents that can be transmitted through the aerosol route of exposure. Safety equipment also may include items for personal protection such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, or goggles. Personal protective equipment is often used in combination with biological safety cabinets and other devices that contain the agents, animals, or materials being worked with. In some situations in which it is impractical to work in biological safety cabinets, personal protective equipment may form the primary barrier between personnel and the infectious materials. Examples include certain animal studies, animal necropsy, agent production activities, and activities relating to maintenance, service, or support of the laboratory facility. Facility Design (Secondary Barrier) The design of the facility is important in providing a barrier to protect persons working inside and outside of the laboratory within the facility, and to protect persons or animals in the community from infectious agents that may be accidentally released from the laboratory. Laboratory management is responsible for providing facilities commensurate with the laboratory's function and the recommended biosafety level for the agents being manipulated. The recommended secondary barrier(s) will depend on the risk of transmission of specific agents. For example, the exposure risks for most laboratory work in Biosafety Level 1 and 2 facilities will be direct contact with the agents, or inadvertent contact exposures through contaminated work environments. Secondary barriers in these laboratories may include separation of the laboratory work area from public access, availability of a decontamination facility (e.g., autoclave), and hand washing facilities. As the risk for aerosol transmission increases, higher levels of primary containment and multiple secondary barriers may become necessary to prevent infectious agents from escaping into the environment. Such design features could include specialized ventilation systems to assure 11

12 directional air flow, air treatment systems to decontaminate or remove agents from exhaust air, controlled access zones, airlocks as laboratory entrances, or separate buildings or modules for isolation of the laboratory. Design engineers for laboratories may refer to specific ventilation recommendations as found in the Applications Handbook for Heating, Ventilation, and Airconditioning (HVAC) published by the American Society of Heating, Refrigerating, and Airconditioning Engineers (ASHRAE). Biosafety Levels Four biosafety levels (BSLs) are described which consist of combinations of laboratory practices and techniques, safety equipment, and laboratory facilities. Each combination is specifically appropriate for the operations performed, the documented or suspected routes of transmission of the infectious agents, and for the laboratory function or activity. The recommended BSLs for the organisms represent those conditions under which the agent can ordinarily be safely handled. The Principle Investigator (PI) is specifically and primarily responsible for assessing risks and for appropriately applying the recommended biosafety levels. Generally, work with known agents should be conducted at the biosafety level recommended. When specific information is available to suggest that virulence, pathogenicity, antibiotic resistance patterns, vaccine and treatment availability, or other factors are significantly altered, more (or less) stringent practices may be specified. Biosafety Level 1 practices, safety equipment, and facilities are appropriate for undergraduate and secondary educational training and teaching laboratories, and for other facilities in which work is done with defined and characterized strains of viable microorganisms not known to cause disease in healthy adult humans. Bacillus subtilis, Naegleria gruberi, and infectious canine hepatitis virus are representative of those microorganisms meeting these criteria. Many agents not ordinarily associated with disease processes in humans are, however, opportunistic pathogens and may cause infection in the young, the aged, and immunodeficient or immunosuppressed individuals. Vaccine strains which have undergone multiple in vivo passages should not be considered avirulent simply because they are vaccine strains. Biosafety Level 1 represents a basic level of containment that relies on standard microbiological practices with no special primary or secondary barriers recommended, other than a sink for hand washing. Biosafety Level 2 practices, equipment, and facilities are applicable to clinical, diagnostic, teaching, and other facilities in which work is done with the broad spectrum of indigenous moderate-risk agents present in the community and associated with human disease of varying severity. With good microbiological techniques, these agents can be used safely in activities conducted on the open bench, provided the potential for producing splashes or aerosols is low. Salmonellae, clinical samples of Hepatitis B, and Toxoplasma spp. are representative of microorganisms assigned to this containment level. Biosafety Level 2 is appropriate when work is done with any human-derived blood, body fluids, or tissues where the presence of an infectious 12

13 agent may be unknown. (Laboratory personnel working with human-derived materials should refer to the Bloodborne Pathogen Standard for specific, required precautions). Primary hazards to personnel working with these agents relate to accidental percutaneous or mucous membrane exposures, or ingestion of infectious materials. Extreme precaution with contaminated needles or sharp instruments must be emphasized. Even though organisms routinely manipulated at BSL-2 are not known to be transmissible by the aerosol route, procedures with aerosol or high splash potential that may increase the risk of such personnel exposure must be conducted in primary containment equipment, or devices such as a BSC or safety centrifuge cups. Other primary barriers should be used, as appropriate, such as splash shields, face protection, gowns, and gloves. Secondary barriers such as hand washing and waste decontamination facilities must be available to reduce potential environmental contamination. Biosafety Level 3 practices, safety equipment, and facilities are applicable to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents with a potential for respiratory transmission, and which may cause serious and potentially lethal infection. Hepatitis B. (propagation of high concentrations only), Mycobacterium tuberculosis, St. Louis encephalitis virus, and Coxiella burnetii are representative of microorganisms assigned to this level. Primary hazards to personnel working with these agents relate to autoinoculation, ingestion, and exposure to infectious aerosols. At Biosafety Level 3, more emphasis is placed on primary and secondary barriers to protect personnel in contiguous areas, the community, and the environment from exposure to potentially infectious aerosols. For example, all laboratory manipulations should be performed in a BSC or other enclosed equipment, such as a gas-tight aerosol generation chamber. Secondary barriers for this level include controlled access to the laboratory and a specialized ventilation system that minimizes the release of infectious aerosols from the laboratory. Biosafety Level 4 practices, safety equipment, and facilities are applicable for work with dangerous and exotic agents that pose a high individual risk of life-threatening disease, which may be transmitted via the aerosol route, and for which there is no available vaccine or therapy. Additionally, agents with a close or identical antigenic relationship to Biosafety Level 4 agents should also be handled at this level. When sufficient data are obtained, work with these agents may continue at this level or at a lower level. Viruses such as Marburg or Congo-Crimean hemorrhagic fever are manipulated at Biosafety Level 4. The primary hazards to personnel working with Biosafety Level 4 agents are respiratory exposure to infectious aerosols, mucous membrane exposure to infectious droplets, and autoinoculation. All manipulations of potentially infectious diagnostic materials, isolates, and naturally or experimentally infected animals pose a high risk of exposure and infection to laboratory personnel, the community, and the environment. 13

14 The laboratory worker's complete isolation of aerosolized infectious materials is accomplished primarily by working in a Class III BSC or a full-body, air-supplied positive-pressure personnel suit. The Biosafety Level 4 facility itself is generally a separate building or completely isolated zone with complex, specialized ventilation and waste management systems to prevent release of viable agents to the environment. The laboratory director is specifically and primarily responsible for the safe operation of the laboratory. His/her knowledge and judgment are critical in assessing risks and appropriately applying these recommendations. The recommended biosafety level represents those conditions under which the agent can ordinarily be safely handled. Special characteristics of the agents used, the training and experience of personnel, and the nature or function of the laboratory may further influence the director in applying these recommendations. Animal Facilities Four biosafety levels are also described for activities involving infectious disease work with experimental mammals. These four combinations of practices, safety equipment, and facilities are designated Animal Biosafety Levels 1, 2, 3, and 4, and provide increasing levels of protection to personnel and the environment. Clinical Laboratories Clinical laboratories, especially those in health care facilities, receive clinical specimens with requests for a variety of diagnostic and clinical support services. Typically, the infectious nature of clinical material is unknown, and specimens are often submitted with a broad request for microbiological examination for multiple agents (e.g., sputa submitted for routine, acid-fast, and fungal cultures). It is the responsibility of the laboratory director to establish standard procedures in the laboratory that realistically address the issue of the infectious hazard of clinical specimens. Except in extraordinary circumstances (e.g., suspected hemorrhagic fever), the initial processing of clinical specimens and identification of isolates can be done safely at Biosafety Level 2, the recommended level for work with bloodborne pathogens such as hepatitis B virus and HIV. The containment elements described in Biosafety Level 2 are consistent with the Occupational Exposure to Bloodborne Pathogens Standard from the Occupational Safety and Health Administration (OSHA), that requires the use of specific precautions with all clinical specimens of blood or other potentially infectious material (Universal Precautions). Additionally, other recommendations specific for clinical laboratories may be obtained from the National Committee for Clinical Laboratory Standards. Biosafety Level 2 recommendations and OSHA requirements focus on the prevention of percutaneous and mucous membrane exposures to clinical material. Primary barriers such as biological safety cabinets (Class I or II) should be used when performing procedures that might cause splashing, spraying, or splattering of droplets. Biological safety cabinets should also be used for the initial processing of clinical specimens when the nature of the test requested or other information is suggestive that an agent readily transmissible by infectious aerosols is likely to be 14

15 present (e.g., M. tuberculosis), or when the use of a biological safety cabinet (Class II) is indicated to protect the integrity of the specimen. The segregation of clinical laboratory functions and limiting or restricting access to such areas is the responsibility of the laboratory director. It is also the director's responsibility to establish standard, written procedures that address the potential hazards and the required precautions to be implemented. Importation and Interstate Shipment of Certain Biomedical Materials The importation of etiologic agents and vectors of human diseases is subject to the requirements of the Public Health Service Foreign Quarantine regulations. Companion regulations of the Public Health Service and the Department of Transportation specify packaging, labeling, and shipping requirements for etiologic agents and diagnostic specimens shipped in interstate commerce. The U. S. Department of Agriculture regulates the importation and interstate shipment of animal pathogens and prohibits the importation, possession, or use of certain exotic animal disease agents that pose a serious disease threat to domestic livestock and poultry. USDA/APHIS permits are required for both importation and within USA transport of many hazardous biological agents. EH&S maintains all permits and shall be contacted for information related to any transport to and from NAU. Please see the section entitled Shipment of Biological Materials for more information. Biological Safety Levels The following is from Biosafety in Microbiological and Biomedical Laboratories, 2009, HHS publication No. (CDC) , Centers for Disease Control & Prevention/National Institutes of Health Biosafety Level 1 Biosafety Level 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. The laboratory is not necessarily separated from the general traffic patterns in the building. Work is generally conducted on open bench tops using standard microbiological practices. 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 the handling of agents assigned to Biosafety Level 1: 15

16 A. Standard Microbiological Practices 1. Access to the laboratory is limited or restricted at the discretion of the laboratory director when experiments or work with cultures and specimens are in progress. 2. Persons wash their hands after they handle viable materials and animals, after removing gloves, and before leaving the laboratory. 3. Eating, drinking, use of tobacco products, handling contact lenses, and applying cosmetics are not permitted in the work areas where there is reasonable likelihood of exposure to potentially infectious materials. Persons who wear contact lenses in laboratories should also wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated and used for that purpose only. 4. Mouth pipetting is prohibited; mechanical pipetting devices are used. 5. All procedures are performed carefully to minimize the creation of splashes or aerosols. 6. Work surfaces are decontaminated at least once a day and after any spill of viable material. 7. All cultures, stocks, and regulated wastes are decontaminated before disposal by an approved decontamination method, such as autoclaving. Materials to be decontaminated outside of the immediate laboratory are to be placed in a durable, leak-proof container and closed for transport from the laboratory. Materials to be decontaminated off-site from the laboratory are packaged in accordance with applicable local, state, and federal regulations, before removal from the facility. 8. An insect and rodent control program is in effect. B. Special Practices: None C. Safety Equipment (Primary Barriers) 1. Special containment devices or equipment such as a biological safety cabinet are generally not required for manipulations of agents assigned to Biosafety Level It is recommended that laboratory coats, gowns, or uniforms are worn to prevent contamination or soiling of street clothes. 3. Gloves should be worn if the skin on the hands is broken or if a rash exists. 4. Protective eyewear should be worn for anticipated splashes of microorganisms or other hazardous materials to the face. D. Laboratory Facilities (Secondary Barriers) 1. Each laboratory contains a sink for hand washing. 2. The laboratory is designed so that it can be easily cleaned. Walls and floors must be constructed of water impervious materials that will stand up to hard disinfectants. Carpeting is not allowed in laboratories. 3. Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat. 16

17 4. Laboratory furniture is sturdy and should not be cloth upholstered. Spaces between benches, cabinets, and equipment are accessible for cleaning. 5. If the laboratory has windows that open, they are fitted with fly screens. Biosafety Level 2 Biosafety Level 2 is similar to Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment. It differs in four ways. 1. Laboratory personnel must have specific training in handling pathogenic agents and should be directed by competent scientists. 2. Access to the laboratory must be limited when work is being conducted. 3. Extreme precautions must be taken with contaminated sharp items. 4. Certain procedures in which infectious aerosols or splashes may be created must be conducted in biological safety cabinets or other physical containment equipment. The following standard and special practices, safety equipment, and facilities apply to the use of agents assigned to Biosafety Level 2: A. Standard Microbiological Practices 1. Access to the laboratory is limited or restricted at the discretion of the laboratory director when experiments are in progress. 2. Persons wash their hands after they handle viable materials and animals, after removing gloves, and before leaving the laboratory. 3. Eating, drinking, the use of tobacco products, handling contact lenses, and applying cosmetics are not permitted in the work areas. Persons who wear contact lenses in laboratories should also wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated for that purpose only. 4. Mouth pipetting is prohibited; mechanical pipetting devices are used. 5. All procedures are performed carefully to minimize the creation of splashes or aerosols. 6. Work surfaces are decontaminated at least once a day and after any spill of viable material. 7. All cultures, stocks, and regulated wastes are decontaminated before disposal by an approved decontamination method, such as autoclaving. Materials to be decontaminated outside of the immediate laboratory are to be placed in a durable, leak-proof container and closed for transport from the laboratory. Materials to be decontaminated off-site from the laboratory are packaged in accordance with applicable local, state, and federal regulations, before removal from the facility. 8. An insect and rodent control program is in effect. B. Special Practices 1. Access to the laboratory is limited or restricted by the laboratory director when work with infectious agents is in progress. In general, persons who are at increased risk of acquiring infection or for whom infection may be unusually 17

18 hazardous are not allowed in the laboratory or animal rooms. For example, persons who are immunocompromised or immunosuppressed may be at risk of acquiring infections. The laboratory director has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory. 2. The laboratory director establishes policies and procedures whereby only persons who have been advised of the potential hazard and meet specific entry requirements (e.g., immunization) enter the laboratory or animal rooms. 3. When the infectious agent(s) in use in the laboratory require special provisions or entry (e.g., immunization), a hazard warning sign incorporating the universal biohazard symbol is posted on the access door to the laboratory work area. The hazard warning sign identifies the infectious agent, lists the name and telephone number of the laboratory director or other responsible person(s), and indicates the special requirement(s) for entering the laboratory. 4. Laboratory personnel receive appropriate immunizations or tests for the agents handled or potentially present in the laboratory (e.g., hepatitis B vaccine or TB skin testing). 5. When appropriate, considering the agent(s) handled, baseline serum samples for laboratory and other at-risk personnel are collected and stored. Additional serum specimens may be collected periodically, depending on the agents handled or the function of the facility. 6. A is prepared or adopted. Personnel are advised of special hazards and are required to read and to follow instructions on practices and procedures. 7. Laboratory personnel receive appropriate training on the potential hazards associated with the work involved, the necessary precautions to prevent exposures, and the exposure evaluation procedures. Personnel receive annual updates, or additional training as necessary for procedural or policy changes. 8. A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels. Needles and syringes or other sharp instruments should be restricted in the laboratory for use only when there is no alternative, such as parenteral injection, phlebotomy, or aspiration of fluids from laboratory animals and diaphragm bottles. Plasticware should be substituted for glassware whenever possible. a. Only needle-locking syringes or disposable syringe-needle units (i.e., needle is integral to the syringe) are used for injection or aspiration of infectious materials. Used disposable needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal; rather, they must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal. Non-disposable sharps must be placed in a hard-walled container for transport to a processing area for decontamination, preferable by autoclaving. b. Syringes that re-sheathe the needle, needle-less systems, and other safe devices should be used when appropriate. c. Broken glassware must not be handled directly by hand, but must be removed by mechanical means such as a brush and dustpan, tongs, or forceps. Containers of 18

19 contaminated needles, sharp equipment, and broken glass are decontaminated before disposal, according to any local, state, or federal regulations. 9. Cultures, tissues, or specimens of body fluids are placed in a container that prevents leakage during collection, handling, processing, storage, transport, or shipping. 10. Laboratory equipment and work surfaces should be decontaminated with an appropriate disinfectant on a routine basis, after work with infectious materials is finished, and especially after overt spills, splashes, or other contamination by infectious materials. Contaminated equipment must be decontaminated according to any local, state, or federal regulations before it is sent for repair or maintenance. Equipment must also be decontaminated before removal from the facility when it must be packaged for transport. Packaging and shipment shall be in accordance with applicable local, state, or federal regulations. 11. Spills and accidents that result in overt exposures to infectious materials are immediately reported to the laboratory director. Medical evaluation, surveillance, and treatment are provided as appropriate and written records are maintained. 12. Animals not involved in work being performed aren t permitted in the lab. C. Safety Equipment (Primary Barriers) 1. Properly maintained biological safety cabinets, preferably Class II, or other appropriate personal protective equipment or physical containment devices are used under the following conditions. a. Use Biological Safety Cabinets whenever procedures with a potential for creating infectious aerosols or splashes are conducted. These may include centrifuging, grinding, blending, vigorous shaking or mixing, sonic disruption, opening containers of infectious materials whose internal pressures may be different from ambient pressures, inoculating animals intranasally, and harvesting infected tissues from animals or eggs. b. Use Biological Safety Cabinets whenever high concentrations or large volumes of infectious agents are used. Such materials may be centrifuged in the open laboratory if sealed rotor heads or centrifuge safety cups are used, and if these rotors or safety cups are opened only in a biological safety cabinet. 2. Face protection (goggles, mask, face shield or other splatter guards) is required to prevent splashes or sprays of infectious or other hazardous materials to the face, when the microorganisms must be manipulated outside the BSC. 3. Protective laboratory coats, gowns, smocks, or uniforms designated for lab use are worn while in the laboratory. This protective clothing is removed and left in the laboratory before leaving for non-laboratory areas (e.g., cafeteria, library, and administrative offices). All protective clothing is either disposed of in the laboratory or laundered by the institution; personnel should never take it home. 4. Gloves are required when handling infected animals and when hands may contact infectious materials, contaminated surfaces or equipment. Wearing two pairs of gloves may be appropriate; if a spill or splatter occurs, the hand will be protected after the contaminated glove is removed. Gloves are disposed of when 19

20 contaminated, removed when work with infectious materials is completed, and are never worn outside the laboratory. Disposable gloves are not washed or reused. D. Laboratory Facilities (Secondary Barriers) 1. Each laboratory contains a sink for hand washing. 2. The laboratory is designed so that it can be easily cleaned. Walls and floors should be smooth, hard and constructed of water impervious material that will stand up to harsh disinfectants. Carpeting is not allowed in laboratory facilities. 3. Bench tops should be smooth, hard and impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat. 4. Laboratory furniture is sturdy, and spaces between benches, cabinets, and equipment are accessible for cleaning. No cloth or fabric seating is permitted. 5. If the laboratory has windows that open, they are fitted with fly screens. 6. A method for decontamination of infectious or regulated laboratory wastes is available (e.g., autoclave, chemical disinfection, incinerator, or other approved decontamination system). 7. An eyewash/safety shower facility is readily available. 8. All laboratories require single pass air that is not then recirculated to any other area of the facility. Laboratories should be negative pressure to surrounding areas to prevent accidental spread of potentially infectious or recombinant agents. 9. Install biological safety cabinets in such a manner that fluctuations of the room supply and exhaust air do not cause the biological safety cabinets to operate outside their parameters for containment. Locate biological safety cabinets away from doors, from windows that can be opened, from heavily traveled laboratory areas, and from other potentially disruptive equipment so as to maintain the biological safety cabinets air flow parameters for containment. Biosafety Level 3 Biosafety Level 3 is applicable to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents which may cause serious or potentially lethal disease as a result of exposure by the inhalation route. Laboratory personnel have specific training in handling pathogenic and potentially lethal agents, and are supervised by competent scientists who are experienced in working with these agents. All procedures involving the manipulation of infectious materials are conducted within biological safety cabinets or other physical containment devices, or by personnel wearing appropriate personal protective clothing and equipment. The laboratory has special engineering and design features. It is recognized, however, that many existing facilities may not have all the facility safeguards recommended for Biosafety Level 3 (e.g., access zone, sealed penetrations, directional airflow, etc.). In these circumstances, acceptable safety may be achieved for routine or repetitive operations (e.g. diagnostic procedures involving the propagation of an agent for identification, typing, and susceptibility testing) in Biosafety Level 2 facilities. However, the recommended 20

21 Standard Microbiological Practices, Special Practices, and Safety Equipment for Biosafety Level 3 must be rigorously followed. The decision to implement this modification of Biosafety Level 3 recommendations should be made only by the lab director. The following standard and special safety practices, equipment, and facilities apply to the use of agents assigned to Biosafety Level 3: A. Standard Microbiological Practices 1. Access to the laboratory is limited or restricted at the discretion of the laboratory director when experiments are in progress. 2. Persons wash their hands after handling infectious materials and animals, after removing gloves, and when they leave the laboratory. 3. Eating, drinking, the use of tobacco products, handling contact lenses and applying cosmetics or lip balm are not permitted in the laboratory. Persons who wear contact lenses in laboratories should also wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated for this purpose only. 4. Mouth pipetting is prohibited; mechanical pipetting devices are used. 5. Policies for the safe handling of sharps are instituted. 6. All procedures are performed carefully to minimize the creation of aerosols. 7. Work surfaces are decontaminated at least once a day and immediately after any spill of viable material. 8. All cultures, stocks, and regulated wastes are decontaminated before disposal by an approved decontamination method, such as autoclaving. Materials to be decontaminated outside of the immediate laboratory are to be placed in a durable, leak-proof container and closed for transport from the laboratory. Infectious wastes from BSL-3 laboratories must be decontaminated before removal for offsite disposal. 9. An insect and rodent control program is in effect. 10. Policies for the safe handling of sharps are instituted. B. Special Practices 1. Laboratory doors are kept closed at all times. 2. The laboratory director controls access to the laboratory and restricts access to persons whose presence is required for program or support purposes. For example, persons who are immunocompromised or immunosuppressed, or for whom infection may be unusually hazardous, are not allowed in the laboratory or animal rooms. The director has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory. No minors are allowed in level 3 laboratories. 3. The laboratory director establishes policies and procedures whereby only persons who have been advised of the potential biohazard, who meet any specific entry requirements (e.g., immunizations), and who comply with all entry and exit procedures, enter the laboratory or animal rooms. 21

22 4. When infectious materials or infected animals are present in the laboratory or containment module, a hazard warning sign incorporating the universal biohazard symbol, is posted on all laboratory and animal room access doors. The hazard warning sign identifies the agent, lists the name and telephone number of the laboratory director or other responsible person(s), and indicates any special requirements for entering the laboratory, such as the need for immunizations, respirators, or other personal protective measures. 5. Laboratory personnel receive the appropriate immunizations or tests for the agents handled or potentially present in the laboratory (e.g., hepatitis B vaccine or TB skin testing and periodic testing). 6. Baseline serum samples are collected and stored for all laboratory and other atrisk personnel. Additional serum specimens may be collected periodically, depending on the agents handled or the function of the laboratory. 7. A specific to the laboratory is prepared or adopted. Personnel are advised of special hazards and are required to read and to follow instructions on practices and procedures. An SOP manual is prepared to cover all procedures and activities. 8. Laboratory personnel receive appropriate training on the potential hazards associated with the work involved, the necessary precautions to prevent exposures, and the exposure evaluation procedures. Personnel receive annual updates, or additional training as necessary for procedural changes. 9. The laboratory director is responsible for insuring that all personnel demonstrate proficiency in standard microbiological practices and techniques, and in the practice and operations specific to the laboratory facility, before working with organisms at Biosafety Level 3. This might include prior experience in handling human pathogens or cell cultures, or a specific training program provided by the laboratory director or other competent scientist proficient in safe microbiological practices and techniques. 10. A high degree of precaution must always be taken with all contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels. Needles and syringes or other sharp instruments should be restricted in the laboratory for use only when there is no alternative, such as parenteral injection, phlebotomy, or aspiration of fluids from laboratory animal and diaphragm bottles. Plastic ware should be substituted for glassware whenever possible. a. Only needle-locking syringes or disposable syringe needle units (i.e., needle is integral to the syringe) are used for injection or aspiration of infectious materials. Used disposable needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal; rather, they must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal. Non-disposable sharps must be placed in a hard-walled container for transport to a processing area for decontamination, preferably by autoclaving. 22

23 b. Syringes that re-sheathe the needle, needle-less systems, and other safe devices should be used when appropriate. c. Broken glassware must not be handled directly by hand, but must be removed by mechanical means such as a brush and dustpan, tongs, or forceps. Containers of contaminated needles, sharp equipment, and broken glass should be decontaminated before disposal, in accordance with any local, state, or federal regulations. 11. All manipulations involving infectious materials are conducted in biological safety cabinets or other physical containment devices within the containment module. No work in open vessels is conducted on the open bench. 12. Laboratory equipment and work surfaces should be decontaminated with an appropriate disinfectant on a routine basis, after work with infectious materials is finished. It must be cleaned and decontaminated after overt spills, splashes, or other contamination with infectious materials. Contaminated equipment should also be decontaminated before it is sent for repair or maintenance. An Equipment Release Form, available on the EH&S website, should be used to assure that equipment has been properly decontaminated prior to servicing. In addition, it must be packaged for transport in accordance with applicable local, state, or federal regulations, before removal from the facility. Plastic-backed paper toweling can be used on non-perforated work surfaces within biological safety cabinets to facilitate clean up. 13. Cultures, tissues, or specimens of body fluids are placed in a container that prevents leakage during collection, handling, processing, storage, transport, or shipping. 14. All potentially contaminated materials (e.g., gloves, lab coats, etc.) from laboratories or animal rooms are decontaminated before disposal or reuse. 15. Spills of infectious materials are decontaminated, contained and cleaned up by appropriate professional staff, or others properly trained and equipped to work with concentrated infectious material. 16. Spills and accidents that result in overt or potential exposures to infectious materials are immediately reported to the laboratory director. Appropriate medical evaluation, surveillance, and treatment are provided and written records are maintained. 17. Animals and plants not related to the work being conducted are not permitted in the laboratory. C. Safety Equipment (Primary Barriers) 1. Properly maintained biological safety cabinets are used (Class II or III) for all manipulation of infectious materials. Biological Safety Cabinets (BSC) should be located away from doors, air supplies and other heavily traveled areas. Biosafety Cabinets must be certified at least yearly. 2. Outside of a BSC, appropriate combinations of personal protective equipment are used (e.g., special protective clothing, masks, gloves, face protection, or respirators) in combination with physical containment devices (e.g., centrifuge safety cups, sealed centrifuge rotors, or containment caging for animals). 23

24 3. Biological Safety Cabinets must be used for manipulations of cultures and clinical or environmental materials that may be a source of infectious aerosols. The aerosol challenge of experimental animals; harvesting of tissues or fluids from infected animals, and embryonated eggs, and necropsy of infected animals also require the use of BSCs. 4. Face protection (goggles and mask, or face shield) is worn for manipulations of infectious materials outside of a biological safety cabinet. 5. Respiratory protection is worn when aerosols cannot be safety contained (e.g., outside of a biological safety cabinet), and in rooms containing infected animals. 6. Protective laboratory clothing such as solid-front or wrap-around gowns, scrub suits, or coveralls must be worn in, and not worn outside, the laboratory. Reusable laboratory clothing is to be decontaminated before being laundered. Change protective clothing immediately if contaminated. 7. Gloves must be worn when handling infected animals and when hands may contact infectious materials and contaminated surfaces or equipment. Disposable gloves should be discarded when contaminated, and never washed for reuse. Always wash hands between glove changes. D. Laboratory Facilities (Secondary Barriers) 1. The laboratory is separated from areas that are open to unrestricted traffic flow within the building. Passage through two sets of self-closing doors is the basic requirement for entry into the laboratory from access corridors or other contiguous areas. A clothes change room (shower optional) may be included in the passageway. 2. Each laboratory contains a sink for hand washing. The sink is foot, elbow, or automatically operated and is located near the laboratory exit door. 3. The interior surfaces of walls, floors, and ceilings are smooth, hard, impervious, and water-resistant so that they can be easily cleaned and capable of disinfection. Penetrations around these surfaces are sealed or capable of being sealed to facilitate decontamination. Floors should be monolithic and coved to the walls. 4. Bench tops are smooth, hard and impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat. 5. Laboratory furniture is sturdy, and spaces between benches, cabinets, and equipment are accessible for cleaning. No fabric materials are allowed. 6. Windows in the laboratory are closed and sealed. 7. A method for decontaminating all laboratory wastes is available, preferably within the laboratory (i.e., autoclave, chemical disinfection, incineration, or other approved decontamination method). 8. A ducted exhaust air ventilation system is provided. This system creates directional airflow that draws air from clean areas into the laboratory toward contaminated areas. The exhaust air is not recirculated to any other area of the building, and, depending on risk assessment, discharged to the outside through a HEPA filtration system. The outside exhaust must be dispersed away from occupied areas and air intakes. Laboratory personnel must verify that the 24

25 direction of the airflow (into the laboratory) is proper. Visual monitoring devices, such as digital readout or maghihelic gauges are recommended. 9. The High Efficiency Particulate Air (HEPA)-filtered exhaust air from Class II or Class III biological safety cabinets is discharged directly to the outside or through the building exhaust system. If the HEPA-filtered exhaust air from Class II Type A biological safety cabinets is to be discharged to the outside through the building exhaust air system, it is connected to this system in a manner (e.g., thimble unit connection) that avoids any interference with the air balance of the cabinets or building exhaust system. Exhaust air from Class II type A biological safety cabinets may be exhausted into the laboratory if the cabinet is tested and certified at least every twelve months. Class III biosafety cabinets must be hard ducted to the building exhaust system. 10. Continuous flow centrifuges or other equipment that may produce aerosols are contained in devices that exhaust air through HEPA filters before discharge into the laboratory. 11. Vacuum lines are protected with liquid disinfectant traps or HEPA filters, or their equivalent, which are routinely maintained and replaced as needed. 12. An eyewash/safety shower is readily available within the BSL-3 suite. 13. Biosafety Level 3 facility design and operational procedures must be documented. The facility must be tested for verification that the design and operational parameters have been met prior to operation. Facilities should be reverified/certified at least annually (HEPAs and air systems). 14. Additional environmental protection (e.g. personnel showers, containment of other piped services and the provision for effluent decontamination) should be added when recommended by the agent summary statement, as determined by risk assessment, the site conditions or other applicable federal, state, or local regulations. 15. Level 3 laboratories and animal areas are audited by The EH&S Biosafety Office quarterly. 16. Openings such as around ducts, spaces between doors and frames and other penetrations must be capable of being sealed to facilitate decontamination. Animal Biosafety Level 1 (ABSL-1) Animal Biosafety Level 1 (ABSL-1) is suitable for work involving well characterized agents that are not known to cause disease in healthy adult humans, and that are of minimal potential hazard to laboratory personnel and the environment. 25

26 A. Standard Practices 1. The animal facility director establishes polices, procedures, and protocols for emergency situations. Each project is subject to pre-approval by the Institutional Animal Care and Use Committee (IACUC) and the Institutional Biosafety Committee (IBC). Any special practices are approved at this time. 2. Only those persons required for program or support purposes are authorized to enter the facility. Before entering, persons are advised of the potential biohazards and are instructed on the appropriate safeguards. 3. An appropriate medical surveillance program is in place. 4. A safety manual is prepared or adopted. Personnel are advised of special hazards, and are required to read and follow instructions on practices and procedures. 5. 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. 6. All procedures are carefully performed to minimize the creation of aerosols or splatters. 7. Work surfaces are decontaminated after use or after any spill of viable materials. 8. 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 disposal in compliance with applicable institutional or local requirements. As a means of incineration is not available on the NAU Campus, all animal wastes must be packaged and shrouded and delivered to the Vivarium Manager at the Biological Sciences Annex. 9. Policies for the safe handling of sharps are instituted. 10. Personnel wash their hands after handling cultures and animals, after removing gloves, and before leaving the animal facility. 11. A biohazard sign must be posted on the entrance to the animal room whenever infectious agents are present. The hazard warning sign identifies the infectious agent(s) in use, lists the name and telephone number of the responsible person(s), and indicates the special requirements for entering the animal room (e.g., the need for immunizations and respirators). 12. An insect and rodent control program is in effect. B. Special Practices: None C. Safety Equipment (Primary Barriers): 1. The wearing of laboratory coats, gowns, and/or uniforms in the facility is recommended. Laboratory coats remain in the animal room. Gowns and uniforms are not worn outside the facility. 2. Persons having contact with non-human primates should assess their risk of mucous membrane exposure and wear appropriate eye and face protection. D. Facilities (Secondary Barriers) 1. The animal facility is separated from areas that are open to unrestricted personnel traffic within the building. 2. External facility doors are self-closing and self-locking. Doors to animal rooms open inward, are self-closing, and are kept closed when experimental animals are 26

27 present. Cubicle room inner doors may open outward or be horizontal or vertical sliding. 3. The animal facility is designed, constructed, and maintained to facilitate cleaning and housekeeping. The interior surfaces (walls, floors, and ceilings) are water resistant. 4. Internal facility appurtenances, such as light fixtures, air ducts, and utility pipes, are arranged to minimize horizontal surface areas. 5. 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 are fitted with fly screens. 6. If floor drains are provided, the traps are always filled with water and/or an appropriate disinfectant. 7. 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 maintain negative pressure compared to adjoining hallways. 8. The facility has a hand washing sink. 9. Cages are washed manually or in a cage washer. The mechanical cage washer should have a final rinse temperature of at least 180F. 10. Illumination is adequate for all activities, avoiding reflections and glare that could impede vision. Animal Biosafety Level 2 (ABSL-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-1. A. Standard Practices 1. Aside from the standard policies, procedures, and protocols for emergency situations established by the facility director, appropriate special policies and procedures should be developed as needed and approved by the Institutional Animal Care and Use Committee (IACUC) and the Institutional Biosafety Committee (IBC). 2. Access to the animal room is limited to the fewest number of individuals possible. Personnel who must enter the room for program or service purposes when work is in progress are advised of the potential hazard. 27

28 3. An appropriate medical surveillance program is in place. All personnel receive appropriate immunizations or tests for the agents handled or potentially present (e.g., hepatitis B vaccine, TB skin testing). When appropriate, a serum surveillance system should be implemented. 4. A is prepared or adopted. Personnel are advised of special hazards, and are required to read and follow instructions on practices and procedures. 5. 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. 6. All procedures are carefully performed to minimize the creation of aerosols or splatters. 7. Equipment and work surfaces in the room are routinely decontaminated with an effective disinfectant after work with the infectious agent, and especially after overt spills, splashes, or other contamination by infectious materials. 8. All infectious samples are collected, labeled, transported, and processed in a manner that contains and prevents transmission of the agent(s). All wastes from the animal room (including animal tissues, carcasses, contaminated bedding, unused feed, sharps, and other refuse) are transported from the animal room in leak-proof, covered containers for appropriate disposal in compliance with applicable institutional or local requirements. The outer surface of the containers is disinfected prior to moving the material. As a means of incineration is not available on the NAU Campus, all animal wastes must be packaged and shrouded and delivered to the Vivarium Manager at the Biological Sciences Annex. 9. Policies for the safe handling of sharps are instituted: a. Needles and syringes or other sharp instruments are restricted for use in the animal facility only when there is no alternative, such as for parenteral injection, blood collection, or aspiration of fluids from laboratory animals and diaphragm bottles. b. Syringes that re-sheathe the needle, needle-less systems, and other safe devices should be used when appropriate. c. Plastic-ware should be substituted for glassware whenever possible. 10. Personnel wash their hands after handling cultures and animals, after removing gloves, and before leaving the animal facility. 11. A biohazard sign must be posted on the entrance to the animal room whenever infectious agents are present. The hazard warning sign identifies the infectious agent(s) in use, lists the name and telephone number of the responsible person(s), and indicates the special requirements (e.g., the need for immunizations and respirators) for entering the animal room. 12. An insect and rodent control program is in effect. B. Special Practices 1. Animal care laboratory and support personnel receive appropriate training on the potential hazards associated with the work involved, the necessary precautions to 28

29 prevent exposures, and the exposure evaluation procedures. Personnel receive annual updates, or additional training as necessary for procedural or policy changes. Records of all training provided are maintained. In general, persons who may be at increased risk of acquiring infection, or for whom infection might be unusually hazardous, are not allowed in the animal facility unless special procedures can eliminate the extra risk. 2. Only animals used for the experiment(s) are allowed in the room. 3. All equipment must be appropriately decontaminated prior to removal from the room. 4. Spills and accidents which result in overt exposures to infectious materials must be immediately reported to the facility director. Medical evaluation, surveillance, and treatment are provided as appropriate and written records are maintained. C. Safety Equipment (Primary Barriers) 1. Gowns, uniforms, or laboratory coats are worn while in the animal room. The laboratory coat is removed and left in the animal room. Gowns, uniforms, and laboratory coats are removed before leaving the animal facility. Gloves are worn when handling infected animals and when skin contact with infectious materials is unavoidable. 2. Personal protective equipment is used based on risk assessment determinations. Appropriate face/eye and respiratory protection is worn by all personnel entering animal rooms that house nonhuman primates. 3. Biological safety cabinets, other physical containment devices, and/or personal protective equipment (e.g., respirators, face shields) are used whenever conducting procedures with a high potential for creating aerosols. These include necropsy of infected animals, harvesting of tissues or fluids from infected animals or eggs, or intranasal inoculation of animals. 4. When needed, animals are housed in primary biosafety containment equipment appropriate for the animal species. Filter top cages are always handled in properly designed and operating animal bio-containment cabinets recommended for rodents. D. Facilities (Secondary Barriers) 1. The animal facility is separated from areas that are open to unrestricted personnel traffic within the building. 2. Access to the facility is limited by secure locked doors. External doors are selfclosing and self-locking. Doors to animal rooms open inward, are self-closing, and are kept closed when experimental animals are present. Cubicle room inner doors may open outward or be horizontal or vertical sliding. 3. The animal facility is designed, constructed, and maintained to facilitate cleaning and housekeeping. The interior surfaces (walls, floors, and ceilings) are smooth, hard and impervious. 4. Internal facility appurtenances, such as light fixtures, air ducts, and utility pipes, are arranged to minimize horizontal surface areas. 29

30 5. Any windows must be resistant to breakage. Where possible, windows should be sealed. If the animal facility has windows that open, they are fitted with fly screens. 6. If floor drains are provided, the traps are always filled with an appropriate disinfectant. 7. Exhaust air is discharged to the outside without being recirculated to other rooms. Ventilation should be provided in accordance with criteria from Guide for Care and Use of Laboratory Animals, latest edition. The direction of airflow in the animal facility is inward; animal rooms should maintain negative pressure compared to adjoining hallways. 8. Cages are washed manually or in an appropriate cage washer. The mechanical cage washer should have a final rinse temperature of at least 180F. 9. An autoclave is available in the animal facility to decontaminate infectious waste. 10. A hand washing sink is in the animal room where infected animals are housed, as well as elsewhere in the facility. 11. Illumination is adequate for all activities, avoiding reflections and glare that could impede vision. Animal Biosafety Level 3 (ABSL-3) Animal Biosafety Level 3 involves practices suitable for work with animals infected with indigenous or exotic agents that present the potential of aerosol transmission and of causing serious or potentially lethal disease. ABSL-3 builds upon the standard practices, procedures, containment equipment, and facility requirements of ABSL-2. A. Standard Practices 1. Aside from the standard policies, procedures, and protocols for emergency situations established by the facility director, appropriate special policies and procedures should be developed as needed and approved by the Institutional Animal Care and Use Committee (IACUC) and the Institutional Biosafety Committee (IBC). 2. The laboratory or animal facility director limits access to the animal room to the fewest number of individuals possible. Personnel who must enter the room for program or service purposes when work is in progress are advised of the potential hazard. 3. An appropriate medical surveillance program is in place. All personnel receive appropriate immunizations or tests for the agents handled or potentially present (e.g., hepatitis B vaccine, TB skin testing). When appropriate, a serum surveillance system should be implemented. In general, persons who may be at increased risk of acquiring infection, or for whom infection might have serious consequences, are not allowed in the animal facility unless special procedures can eliminate the extra risk. Assessment should be made by the occupational health physician. 30

31 4. A is prepared or adopted. Personnel are advised of special hazards, and are required to read and follow instructions on practices and procedures. 5. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use should be done only in designated areas and are not permitted in animal or procedure rooms. 6. All procedures are carefully performed to minimize the creation of aerosols or splatters. 7. Equipment and work surfaces in the room are routinely decontaminated with an effective disinfectant after work with the infectious agent, and especially after overt spills, splashes, or other contamination by infectious materials. 8. All wastes from the animal room (including animal tissues, carcasses, contaminated bedding, unused feed, sharps, and other refuse animal tissues) are transported from the animal room in leak-proof, covered containers for appropriate disposal in compliance with applicable institutional or local requirements. The outer surface of the containers is disinfected prior to moving the material. As a means of incineration is not available on the NAU Campus, all animal wastes must be packaged and shrouded and delivered to the Vivarium Manager at the Biological Sciences Annex. (see Special Practices #3 below). 9. Policies for the safe handling of sharps are instituted: a. Needles and syringes or other sharp instruments are restricted in the animal facility for use only when there is no alternative, such as for parenteral injection, blood collection, or aspiration of fluids from laboratory animals and diaphragm bottles. b. Syringes that re-sheathe the needle, needle-less systems, and other safety devices should be used when appropriate. c. Plastic ware should be substituted for glassware whenever possible. 10. Personnel wash their hands after handling cultures and animals, after removing gloves, and before leaving the animal facility. 11. A biohazard sign must be posted on the entrance to the animal room whenever infectious agents are present. The hazard warning sign identifies the infectious agent(s) in use, lists the name and telephone number of the responsible person(s), and indicates the special requirements for entering the animal room (e.g., the need for immunizations and respirators). 12. All infectious samples are collected, labeled, transported, and processed in a manner that contains and prevents transmission of the agent(s). 13. Laboratory and support personnel receive appropriate training on the potential hazards associated with the work involved, the necessary precautions to prevent exposures, and the exposure evaluation procedures. As necessary, personnel receive updates and/or additional training on procedural or policy changes. Records of all training provided are maintained. 14. An insect and rodent control program is in effect. B. Special Practices 31

32 1. Cages are autoclaved or thoroughly decontaminated before bedding is removed and before they are cleaned and washed. Equipment must be decontaminated according to any local, state, or federal regulations before being packaged for transport or removal from the facility for repair or maintenance. 2. A spill procedure is developed and posted. Only personnel properly trained and equipped to work with infectious materials are to clean up spills. Spills and accidents that result in overt exposures to infectious materials must be immediately reported to the facility director. Medical evaluation, surveillance, and treatment are provided as appropriate and written records are maintained. 3. Materials not related to the experiment (e.g., plants, animals) are not permitted in the animal room. C. Safety Equipment (Primary Barriers) 1. Uniforms or scrub suits are worn by personnel entering the animal room. Wrap-around or solid-front gowns should be worn over this clothing. Front-button laboratory coats are unsuitable. The gown must be removed and left in the animal room. Before leaving the animal facility, scrub suits and uniforms are removed and appropriately contained and decontaminated prior to laundering or disposal. 2. Personal protective equipment used is based on risk assessment determinations. a. Personal protective equipment is used for all activities involving manipulations of infectious material or infected animals. b. Personnel wear gloves when handling infected animals. Gloves are removed aseptically and autoclaved with other animal room wastes before disposal. c. Appropriate face/eye and respiratory protection (e.g., respirators and face shields) is worn by all personnel entering animal rooms. d. Boots, shoe covers, or other protective footwear, and disinfectant foot baths are available and used where indicated. 3. The risk of infectious aerosols from infected animals or their bedding also can be reduced if animals are housed in containment caging systems, such as open cages placed in inward flow ventilated enclosures (e.g., laminar flow cabinets), solid wall and bottom cages covered with filter bonnets, or other equivalent primary containment systems. 4. Biological safety cabinets and other physical containment devices are used whenever conducting procedures with a potential for creating aerosols. These include necropsy of infected animals, harvesting of tissues or fluids from infected animals or eggs, or intranasal inoculation of animals. At ABSL-3, all work should 32

33 be done in a primary barrier; otherwise respirators should be worn by personnel in the room. D. Facilities (Secondary Barriers) 1. The animal facility is separated from areas that are open to unrestricted personnel traffic within the building. 2. Access to the facility is limited by a self-closing and self-locking door. This exterior entry door may be controlled by a key lock, card key, biometrics, or proximity reader. Entry into the animal room is via a double-door entry which includes a change room and shower(s). An additional double-door access (airlock) or double-doored autoclave may be provided for movement of supplies and wastes into and out of the facility, respectively. Doors to animal rooms open inward and are self-closing. Doors to cubicles inside an animal room may open outward or slide horizontally or vertically. 3. The animal facility is designed, constructed, and maintained to facilitate cleaning and housekeeping. The interior surfaces (walls, floors, and ceilings) are smooth, hard, impervious and capable of harsh disinfection. Penetrations in floors, walls and ceiling surfaces are sealed and openings around ducts and the spaces between doors and frames are capable of being sealed to facilitate decontamination. 4. A hands-free or automatically operated hand washing sink is provided in each animal room near the exit door. The sink trap is filled with an appropriate disinfectant after each use. 5. Internal facility appurtenances, such as light fixtures, air ducts, and utility pipes, are arranged to minimize horizontal surface areas. 6. Windows are not recommended. Any windows must be resistant to breakage and must be sealed. Direct viewing of animals is prohibited. 7. If floor drains are provided, they are always filled with an appropriate disinfectant. 8. Ventilation should be provided in accordance with criteria from the Guide for Care and Use of Laboratory Animals, latest edition. A ducted exhaust air ventilation system is provided. This system creates directional airflow which draws air into the laboratory from "clean" areas and toward "contaminated" areas. The exhaust air is not recirculated to any other area of the building. Filtration and other treatments of the exhaust air may not be required, but should be considered based on site requirements, and specific agent manipulations and use conditions. The exhaust must be dispersed away from occupied areas and air intakes, or the exhaust must be HEPA-filtered. Personnel must verify that the direction of the airflow (into the animal areas) is proper. It is recommended that a visual monitoring device that indicates and confirms directional inward airflow be provided at the animal room entry. Consideration should be given to installing an HVAC control system to prevent sustained positive pressurization of the animal spaces. Audible alarms should be considered to notify personnel of HVAC system failure. 9. HEPA-filtered exhaust air from a Class II type A biological safety cabinet can be exhausted into the animal room if the cabinet is tested and certified at least annually. When exhaust air from Class II type A safety cabinets is to be 33

34 discharged to the outside through the building exhaust air system, the cabinets must be connected in a manner that avoids any interference with the air balance of the cabinets or the building exhaust system (e.g., a thimble unit, or an air gap between the cabinet exhaust and the exhaust duct). When Class III biological safety cabinets are used, they should be directly connected (hard ducted) to the exhaust system 10. Cages are washed in a cage washer. The mechanical cage washer has a final rinse temperature of at least 180F. 11. An autoclave is available which is convenient to (preferably within) the animal rooms where the biohazard is contained. The autoclave is utilized to decontaminate infectious waste before moving it to other areas of the facility. A Pass Through autoclave is recommended. 12. If vacuum service (i.e., central or local) is provided, each service connection should be fitted with liquid disinfectant traps and an in-line HEPA filter, placed as near as practicable to each use point or service cock. Filters are installed to permit in-place decontamination and replacement. 13. Illumination is adequate for all activities, avoiding reflections and glare that could impede vision. 14. The completed Biosafety Level 3 facility design and operational procedures must be documented. The facility must be tested for verification that the design and operational parameters have been met prior to operation. Facilities should be reverified at least annually against these procedures as modified by operational experience. 15. Additional environmental protection (e.g., personnel showers, HEPA filtration of exhaust air, containment of other piped services, and the provision of effluent decontamination) should be considered if recommended by the agent summary statement, as determined by risk assessment of the site conditions, or other applicable federal, state, or local regulations. If USDA oversight or funding is involved, HEPA filtered exhaust, personnel shower in/shower out and a Pass Through autoclave will be required. 34

35 Table 1: Summary of Recommended Biosafety Levels for Infectious Agents BSL Agents Practices Safety Equipment (Primary Barriers) 1 Not known to consistently cause disease in healthy adults 2 Associated with human disease, hazard = percutaneous injury, ingestion, mucous membrane exposure 3 Indigenous or exotic agents with potential for aerosol transmission; disease may have serious or lethal consequences 4 Dangerous/exotic agents which pose high risk of life-threatening disease, aerosol-transmitted lab infections; or related agents with unknown risk of transmission 35 Facilities (Secondary Barriers) Standard Microbiological Practices None required Open bench top Sink for hand washing BSL-1 practice plus: Limited access Biohazard warning signs Sharps precautions Biosafety manual defining any needed waste decontamination or medical surveillance policies BSL-2 practice plus: Controlled access Decontamination of all waste Decontamination of lab clothing before laundering Baseline serum Decontamination of all effluent BSL-3 practice plus: Clothing change before entering Shower on exit All material decontaminated on exit from facility Primary barriers = Class I or II BSCs or other physical containment devices used for all manipulations of agents that cause splashes or aerosols of infectious materials; PPE; laboratory coats; gloves; face protection as needed Primary barriers = Class I or II BSCs or other physical containment devices for all open manipulations of agents; PPE; protective lab clothing; gloves; respiratory protection as needed Primary barriers = All procedures conducted in Class III BSCs or Class I or II BSCs in combination with full-body air-supplied, positive pressure personnel suit BSL-1 plus: Autoclave available Single pass air with no recirculation Hard, smooth, impervious floor, walls, and countertops BSL-2 plus: Physical separation from access corridors Self-closing, doubledoor access Exhausted air not recirculated Negative airflow into laboratory (single pass) No floor drains Hard, smooth, impervious ceilings BSL-3 plus: Separate building or isolated zone Dedicated supply and exhaust, vacuum, and decontamination systems Other requirements outlined in the text

36 Table 2: Summary of Recommended Biosafety Levels for Activities in Which Experimentally or Naturally Infected Vertebrate Animals Are Used ABSL Agents Practices Safety Equipment (Primary Barriers) 1 Not known to consistently cause disease in healthy adults 2 Associated with human disease, hazard = percutaneous injury, ingestion, mucous membrane exposure Standard animal care and management practices, including appropriate medical surveillance programs ABSL-1 practice plus: Limited access Biohazard warning signs Sharps precautions Decontamination of all infectious wastes and of animal cages prior to washing As required for normal care of each species ABSL-1 equipment plus barriers: containment equipment appropriate for animal species; PPE; laboratory coats, gloves, face and respiratory protection as needed. Facilities (Secondary Barriers) Standard animal facility No recirculation of exhaust air Directional air flow recommended Hand washing sink recommended ABSL-1 facility plus: Autoclave available Hand washing sink available in animal room Mechanical cage washer used Hard, smooth, impervious walls, floors, ceilings and countertops 3 Indigenous or exotic agents with potential for aerosol transmission; disease may have serious or lethal consequences 4 Dangerous/exotic agents which pose high risk of lifethreatening disease, aerosol-transmitted lab infections; or related agents with unknown risk of transmission ABSL-2 practice plus: Controlled access Decontamination of all waste Cages decontaminated before bedding removed Disinfectant foot bath as needed Decontamination of all effluent ABSL-3 practice plus: Entrance through change room where personal clothing is removed and laboratory clothing is put on; shower on exit All wastes are decontaminated before removal from facility ABSL-2 equipment plus: Containment equipment for housing animals and cage dumping activities Class I or II BSCs available for manipulative procedures(inoculation, necropsy) that may create infectious aerosols. PPE: appropriate respiratory protection ABSL-3 equipment plus maximum containment equipment (i.e. Class III BSC or partial containment equipment in combination with full body, airsupplied positive-pressure personnel suit) used for all procedures and activities ABSL-2 facility plus: Physical separation from access corridors Self-closing, double-door access Sealed penetrations Sealed windows Autoclave available in facility ABSL-3 facility plus: Separate building or isolated zone Dedicated air supply and exhaust, vacuum and decontamination systems Other requirements outlined in the text 36

37 Agents List The following agents have been listed according to the most appropriate Biological Safety Level to be used. The lists presented on the following pages are based upon the most recent information available in the Risk Group Classifications for Infectious Agents section of the American Biological Safety Association (ABSA) Website. Every attempt to provide accurate classification for the agents listed has been made. As this is not a complete list of infectious agents, and these classifications are subject to change, please refer to the following website: for classification of other agents, and the most up to date information for those listed. Please note that Biological Safety Levels are not inherent to an agent but are performance recommendations and should be chosen after a risk assessment is completed. A proper risk assessment takes into account the characteristics of the agent involved, the activities to be performed, and the environment in which the work will be completed. Therefore, certain agents may be used at different Biological Safety Levels depending upon the circumstances. For instance, human clinical samples from HIV-positive patients may be safely handled at BSL-2. Growth of HIV in culture should be performed under BSL-3 containment. Biological Safety Levels may be higher or lower than what is given below for a particular agent depending upon the circumstances of its use. The EH&S Biological Safety Officer (BSO) reviews all projects involving recombinant DNA, infectious disease agents, and agents of concern to livestock and agriculture and will assist you in the risk assessment process. Once the Institutional Biosafety Committee (IBC) and/or the EH&S BSO assigns a Biological Safety Level, it must be adhered to unless new information to warrant a change, in most cases from peer-reviewed literature, is provided. The IBC and/or BSO will review the literature and make an adjustment, if warranted. Biological Safety Level 1 (BSL-1) Agents that are not associated with disease in healthy adult humans, are of minimal potential hazard to laboratory personnel, and of minimal potential hazard to the environment may be used at BSL-1. Agents that may be used at BSL-1 include Lactobacillus spp., asporogenic Bacillus subtilis or Bacillus licheniformis, Escherichia coli-k12 (cloning strains), Baculoviruses, and adeno-associated virus types 1 through 4 in low concentrations (<10 9 IP/ml) Those agents not listed under Biological Safety Levels 2, 3 and 4 are not automatically or implicitly classified as BSL-1; a risk assessment must be conducted based on the known and potential properties of the agents and their relationship to agents that are listed. Biological Safety Level 2 (BSL-2) Agents to be used at BSL-2 are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available. They are of moderate potential hazard to laboratory personnel and/or the environment. 37

38 BSL-2 - Bacterial Agents Including Chlamydia Acinetobacter baumannii (formerly Acinetobacter calcoaceticus) Actinobacillus Actinomyces pyogenes (formerly Corynebacterium pyogenes) Aeromonas hydrophila Amycolata autotrophica Archanobacterium haemolyticum (formerly Corynebacterium haemolyticum) Arizona hinshawii - all serotypes Bartonella henselae, B. quintana, B. vinsonii Bordetella including B. pertussis Borrelia recurrentis, B. burgdorferi Burkholderia (formerly Pseudomonas species) except those listed under BSL-3 Campylobacter coli, C. fetus, C. jejuni Chlamydia psittaci (non-avian strains), trachomatis, C. pneumoniae Clostridium haemolyticum, Cl. histolyticum, Cl. novyi, Cl. septicum, Cl. tetani Corynebacterium diphtheriae, C. pseudotuberculosis, C. renale Dermatophilus congolensis Edwardsiella tarda Erysipelothrix rhusiopathiae Escherichia coli - all enteropathogenic, enterotoxigenic, enteroinvasive and strains bearing K1 antigen, including E. coli O157:H7 Haemophilus ducreyi, H. influenzae Helicobacter pylori Klebsiella Legionella including L. pneumophila Leptospira interrogans - all serotypes Listeria Moraxella Mycobacterium (except those listed under BSL-3) including M. avium complex, M. asiaticum, M. bovis BCG vaccine strain, M. chelonei, M. fortuitum, M. kansasii, M. malmoense, M. marinum, M. paratuberculosis, M. scrofulaceum, M. simiae, M. szulgai, M. ulcerans, M. xenopi Neisseria gonorrhoea, N. meningitidis Nocardia asteroides, N. brasiliensis, N. otitidiscaviarum, N. transvalensis Rhodococcus equi Salmonella including S. arizonae, S. cholerasuis, S. enteritidis, S. gallinarum-pullorum, S. meleagridis, S. paratyphi, A, B, C, S. typhi, S. typhimurium Shigella including S. boydii, S. dysenteriae, type 1, S. flexneri, S. sonnei Sphaerophorus necrophorus Staphylococcus aureus Streptobacillus moniliformis Streptococcus including S. pneumoniae, S. pyogenes Treponema pallidum, T. carateum Vibrio cholerae, V. parahemolyticus, V. vulnificus Yersinia enterocolitica 38

39 BSL-2 - Fungal Agents Cryptococcus neoformans Dactylaria galopava (Ochroconis gallopavum) Epidermophyton Exophiala (Wangiella) dermatitidis Fonsecaea pedrosoi Microsporum Penicillium marneffei Sporothrix schenckii Trichophyton BSL-2 - Parasitic Agents Ancylostoma human hookworms including A. duodenale, A. ceylanicum Ascaris including Ascaris lumbricoides suum Babesia including B. divergens, B. microti Brugia filaria worms including B. malayi, B. timori Coccidia Cryptosporidium including C. parvum Cysticercus cellulosae (hydatid cyst, larva of T. solium) Entamoeba histolytica Enterobius Fasciola including F. gigantica, F. hepatica Giardia including G. lamblia Heterophyes Hymenolepis including H. diminuta, H. nana Isospora Leishmania ethiopia, L. major, L. mexicana, L. peruvania, L. tropica Loa loa filaria worms Microsporidium Necator human hookworms including N. americanus Onchoerca filaria worms including, O. volvulus Plasmodium including simian species, P. cynomologi, P. falciparum, P. malariae, P. ovale, P. vivax Sarcocystis including S. sui hominis Schistosoma including S. haematobium, S. intercalatum, S. japonicum, S. mansoni, S. mekongi Strongyloides including S. stercoralis Toxocara including T. canis Toxoplasma including T. gondii Trichinella spiralis Trypanosoma including T. brucei brucei, T. brucei gambiense, T. brucei rhodesiense, T. cruzi Wuchereria bancrofti filaria worms 39

40 BSL-2 - Viruses Adenoviruses, human - all types except African Swine Fever virus Arenaviruses Lymphocytic choriomeningitis virus (non-neurotropic strains) Other viruses as listed by ABSA/CDC Bunyaviruses Bunyamwera virus Rift Valley fever virus vaccine strain MP-12 only Other viruses as listed by ABSA/CDC Calciviruses Norovirus Sapovirus Coronaviruses except SARS Flaviviruses (Togaviruses) - Group B Arboviruses Yellow fever virus vaccine strain 17D Other viruses as listed by ABSA/CDC Hepatitis A, B, C, D, and E viruses Herpesviruses - except Herpesvirus simiae (Monkey B virus), BSL-4 Cytomegalovirus Epstein Barr virus Herpesvirus ateles Herpesvirus saimiri Herpes simplex types 1 and 2 Herpes zoster Human herpesvirus types 6 and 7 Marek's disease virus Murine cytomegalovirus Pseudorabies virus Orthomyxoviruses Influenza viruses types A, B, and C Other tick-borne orthomyxoviruses as listed by ABSA/CDC Papovaviruses All human papilloma viruses Bovine papilloma virus Polyoma virus Shope papilloma virus Simian virus 40 (SV40) 40

41 Paramyxoviruses Newcastle disease virus (non-exotic, non-virulent strains) Measles virus Mumps virus Parainfluenza viruses types 1, 2, 3, and 4 Respiratory syncytial virus Parvoviruses Human parvovirus (B19) Canine Parvovirus (CPV) Picornaviruses Coxsackie viruses types A and B Echoviruses - all types Polioviruses - all types, wild and attenuated Rhinoviruses - all types Poxviruses Vaccinia - all types except Monkeypox virus (BSL-3) and restricted poxviruses including Alastrim, Camelpox, Goatpox, Smallpox, and Whitepox (restricted to the CDC, Atlanta, GA) Reoviruses - Coltivirus, human Rotavirus, and Orbivirus (Colorado tick fever virus) Retroviruses Bovine leukemia virus Clinical samples from HIV-positive patients Feline immunodeficiency virus Feline leukemia virus Feline sarcoma virus Gibbon leukemia virus Mason-Pfizer monkey virus Mouse mammary tumor virus Murine leukemia virus Murine sarcoma virus Rat leukemia virus NOTE: Containment requirements for research involving rdna Experiments involving recombinant DNA lend themselves the application of highly specific biological barriers. The containment levels required for research involving recombinant DNA associated with plants or animals is based on classification of experiments in Section III, of the NIH document Experiments Covered by the NIH Guidelines found at www4.od.nih.gov/oba/rac/guidelines_02/nih_guidelines_apr_02.htm#_section_iii._experi MENTS. Consult this document for containment requirements specific to your research. NOTE: Murine Retroviral Vectors Murine retroviral vectors to be used for human transfer experiments (less than 10 liters) that contain less than 50% of their respective parental viral genome and that have been demonstrated to be free 41

42 of detectable replication competent retrovirus can be maintained, handled, and administered, under BL1 containment. Rhabdoviruses Mokola Virus Vertebrate and Invertebrate lyssaviruses Togaviruses (see Alphaviruses and Flaviviruses) Rubivirus (rubella) Ross River Virus Biological Safety Level 3 (BSL-3) Agents to be used at BSL-3 are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available. BSL-3 - Bacterial Agents Including Rickettsia Bacillus anthracis Bartonella Brucella including B. abortus, B. canis, B. suis Burkholderia (Pseudomonas) mallei, B. pseudomallei Clostridium botulinum, Cl. chauvoei Coxiella burnetii Francisella tularensis (Types A and B) Mycobacterium bovis (except BCG strain, BSL-2), M. tuberculosis, M. leprae Mycoplasma, except M. mycoides and M. agalactiae which are restricted animal pathogens Pasteurella multocida type B - buffalo and other virulent strains Rickettsia akari, R. australis, R. canada, R. conorii, R. prowazekii, R. rickettsii, R, siberica, R. tsutsugamushi, R. typhi (R. mooseri) Yersinia pestis BSL-3 - Fungal Agents Blastomyces dermatitidis Cladosporium bantianum, C. (Xylohypha) trichoides Coccidioides immitis (sporulating cultures; contaminated soil) Histoplasma capsulatum, H. capsulatum var.. duboisii Paracoccidioides braziliensis BSL-3 - Parasitic Agents Echinococcus including E. granulosis, E. multilocularis, E. vogeli Leishmania including L. braziliensis, L. donovani Naegleria fowleri Plasmodium falciparum Taenia solium 42

43 BSL-3 - Viruses and Prions Alphaviruses (Togaviruses) - Group A Arboviruses Eastern equine encephalomyelitis virus Venezuelan equine encephalomyelitis vaccine strain TC-83 Western equine encephalomyelitis virus Semliki Forest virus Venezuelan equine encephalomyelitis virus (except the vaccine strain TC-83 is BSL-2) Other viruses as listed by ABSA/CDC Arenaviruses Lymphocytic choriomeningitis virus (LCM) (neurotropic strains) Flexal Bunyaviruses Hantaviruses including Hantaan virus Rift Valley fever virus Other viruses as listed by ABSA/CDC Flaviviruses (Togaviruses) - Group B Arboviruses Dengue virus serotypes 1, 2, 3, and 4 Japanese encephalitis virus St. Louis encephalitis virus Yellow fever virus wild type Other viruses as listed by ABSA/CDC Poxviruses Monkeypox virus Prions Transmissible spongioform encephalopathies (TME) agents, Creutzfeldt-Jacob disease and kuru agents (see BMBL for specific containment instruction) Retroviruses Avian leukosis virus Avian sarcoma virus Human immunodeficiency virus (HIV) types 1 and 2 Human T cell lymphotropic virus (HTLV) types 1 and 2 Simian immunodeficiency virus (SIV) Rhabdoviruses Rabies virus Vesicular stomatitis virus - laboratory adapted strains ONLY including VSV-Indiana, San Juan, and Glasgow 43

44 Biological Safety Level 4 (BSL-4) Agents to be used at BSL-4 are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available. BSL-4 - Bacterial Agents None BSL-4 - Fungal Agents None BSL-4 - Parasitic Agents None BSL-4 - Viral Agents Arenaviruses (Togaviruses) - Group A Arboviruses Guanarito virus Lassa virus Junin virus Machupo virus Sabia virus Bunyaviruses (Nairovirus) Crimean-Congo hemorrhagic fever virus Filoviruses Ebola virus Marburg virus Flaviruses (Togaviruses) - Group B Arboviruses Tick-borne encephalitis virus complex including Absetterov, Central European encephalitis, Hanzalova, Hypr, Kumlinge, Kyasanur Forest disease, Omsk hemorrhagic fever, and Russian spring-summer encephalitis viruses Herpesviruses (alpha) Herpesvirus simiae (Herpes B or Monkey B virus) Paramyxiviruses Equine morbillivirus Hemorrhagic fever agents and viruses as yet undefined. 44

45 2 - Information for Researchers Project Registration Northern Arizona University Insitutional Biosafety Committee Many research projects involve work with potentially hazardous biological agents, known infectious disease agents, or biological materials regulated by the federal or state government. Granting agencies require that the university monitor the use of biological hazards, infectious disease agents, and recombinant DNA in order for them to release funds to investigators. Therefore, we have developed a registration system to ensure that all biological materials are handled properly and disposed of appropriately. The EH&S Biological Safety Office administers four registration programs for research projects. The NAU Institutional Biosafety Committee (IBC) reviews all registrations. Approval by the NAU IBC, in writing, is required before ordering or working with any agents. Please contact the EH&S BSO for more information. Fillable registration forms are available on the EH&S website at: Biological Agent (BA) Registration Use of the following materials requires that the Principal Investigator completes and submits the bio-agent registration document for approval by the EH&S Biological Safety Officer. Agents characterized at Biosafety Level 1 require registration. These will be expedited and administratively approved barring any complications. Agents to be used at Biosafety Level 2 (BSL-2) or Biosafety Level 3 (BSL-3): 1. All human, animal, or plant pathogens that require BSL-2 or BSL-3 containment and handling (see previous section: Agents List ) must be registered. Please note that BSL-4 agents may not be stored or used at NAU. 2. Unknown human and animal pathogens must be registered. These are considered BSL-2 until identified. 3. Cell lines or cultures that 1) have been immortalized with a virus (such as EBV or a retrovirus), 2) are known to be tumorigenic in primates (including humans), or 3) are primary human tumor cells. These are considered BSL-2 (or higher in many cases). 4. Human blood or other tissues, when used in research, must be registered. 5. Human blood or other tissue known to be HIV positive or known to contain any human disease causing agent may require higher containment depending upon the IBC evaluation. Recombinant DNA (R-DNA) Registration All R-DNA projects that involve a recombinant organism (this excludes projects that involve DNA only, i.e. PCR) require registration with the EH&S Biological Safety Office. A subset of R-DNA 45

46 projects requires review and approval from the Institutional Biosafety Committee (IBC). The NAU IBC oversees all research projects and issues involving R-DNA at NAU. Use of the following requires that the principal investigator completes and submits an R-DNA registration document. 1. All R-DNA projects, including the growth of recombinant bacteria for probe isolation (plasmid or phage preparations) require registration. Projects must be registered regardless of where the material came from or who originally constructed it. 2. Projects that are exempt from the NIH Guidelines must also be registered. 3. The development of transgenic animals and plants requires registration. R-DNA projects are performed at BSL-1, BSL-2, BSL-3 or the corresponding levels for whole plant (BSL-1P, BSL-2P, BSL-3P) or whole animal (BSL-1N, BSL-2N, BSL-3N) work. The EH&S Biological Safety Officer, in conjunction with the IBC, will make the final determination. Acute Toxins (AT) Registration The use and storage of chemicals with a mammalian LD 50 of < 100 g/kg require registration. For a partial list, see the Toxins Table that follows. Acute toxins may only be ordered following written approval by the BSO or IBC. Regulated Biological Materials Agents, such as plant pathogens or exotic microorganisms, that are regulated by federal or state agencies (CDC, HHS, DOC, USDA/APHIS, EPA, FDA, DPI, etc.) shall be registered with the EH&S Biological Safety Office by submission of a biological agent registration form. All permits for transport, transfer, import or export of these regulated agents are the responsibility of the Principal Investigator. The NAU Responsible Official (RO) will be responsible for all Select Agent permits. Please allow a minimum of 6 weeks to obtain new federal permits. Project Amendment Changes to an existing registration can be done on an amendment unless said changes result in a dramatic change to the overall project or the containment level. Typical changes include: Addition or deletion of new personnel Addition or deletion of new agents (same containment level) Minor modifications to the protocol or procedures ( final decision to be made by the IBC) 46

47 Table 3: Toxin Table Toxins with a mammalian LD 50 of < 100 g/kg must be registered with the EH&S Biological Safety Office. Therefore, use of the following toxins requires registration. If a toxin is not on the list, it still may require registration, depending upon the LD 50. For more information, please contact the EH&S Biological Safety Office. Toxicity LD 50 ( g/kg)* Abrin 0.7 Aerolysin 7.0 Botulinin toxin A Botulinin toxin B Botulinin toxin C Botulinin toxin C Botulinin toxin D Botulinin toxin E Botulinin toxin F bungarotoxin 14.0 Caeruleotoxin 53 Cereolysin Cholera toxin 250 Clostridium difficile enterotoxin A 0.5 Clostridium difficile cytotoxin B 220 Clostridium perfringens lecithinase 3 Clostridium perfringens kappa toxin 1500 Clostridium perfringens perfringolysin O Clostridium perfringens enterotoxin 81 Clostridium perfringens beta toxin 400 Clostridium perfringens delta toxin 5 Clostridium perfringens epsilon toxin 0.1 Conotoxin Crotoxin 82 Diphtheria toxin 0.1 Listeriolysin 3-12 Leucocidin 50 Modeccin 1-10 Nematocyst toxins Notexin 25 Pertussis toxin 15 47

48 Toxicity LD 50 ( g/kg)* Pneumolysin 1.5 Pseudomonas aeruginosa toxin A 3 Ricin 2.7 Saxitoxin 8 Shiga toxin Shigella dysenteriae neurotoxin 1.3 Streptolysin O 8 Staphylococcus enterotoxin B 25 Staphylococcus enterotoxin F 2-10 Streptolysin S 25 Taipoxin 2 Tetanus toxin Tetrodotoxin 8 Viscumin Volkensin 1.4 Yersinia pestis murine toxin 10 *Please note that the LD 50 values are from a number of sources (see below). For specifics on route of application (i.v., i.p., s.c.), animal used, and variations on the listed toxins, please go to the references listed below. Reference: 1. Gill, D. Michael; 1982; Bacterial toxins: a table of lethal amounts; Microbiological Reviews; 46: Stirpe, F.; Luigi Barbieri; Maria Giulia Battelli, Marco Soria and Douglas A. Lappi; 1992; Ribosome-inactivating proteins from plants: present status and future prospects; Biotechnology; 10: Registry of toxic effects of chemical substances (RTECS): comprehensive guide to the RTECS Doris V. Sweet, ed., U.S. Dept of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health; Cincinnati, Ohio Select Agents The following lists of agents and toxins are classified by the Federal government as Select Agents. Any possession, use, transfer or shipment of these materials is strictly controlled by regulation. Researchers considering work with any of these materials must first contact the NAU Responsible Official for the approvals, permits, clearances and other necessary paperwork. Be aware that government clearance can take as much as 6 months to complete in advance of any project. Failure to comply with these Federal Regulations is punishable by both fines and imprisonment. 48

49 HHS and USDA Select Agents and Toxins 7CFR Part 331, 9 CFR Part 121, and 42 CFR Part 73 HHS SELECT AGENTS AND TOXINS Abrin Botulinum neurotoxins* Botulinum neurotoxin producing species of Clostridium* Conotoxins (Short, paralytic alpha conotoxins containing the following amino acid sequence X 1 CCX 2 PACGX 3 X 4 X 5 X 6 CX 7 ) 1 Coxiella burnetii Crimean-Congo haemorrhagic fever virus Diacetoxyscirpenol Eastern Equine Encephalitis virus 3 Ebola virus* Francisella tularensis* Lassa fever virus Lujo virus Marburg virus* Monkeypox virus 3 Reconstructed replication competent forms of the 1918 pandemic influenza virus containing any portion of the coding regions of all eight gene segments (Reconstructed 1918 Influenza virus) Ricin Rickettsia prowazekii SARS-associated coronavirus (SARS-CoV) Saxitoxin South American Haemorrhagic Fever viruses: Chapare Guanarito Junin Machupo Sabia Staphylococcal enterotoxins A,B,C,D,E subtypes T-2 toxin Tetrodotoxin Tick-borne encephalitis complex (flavi) viruses: Far Eastern subtype Siberian subtype Kyasanur Forest disease virus Omsk hemorrhagic fever virus Variola major virus (Smallpox virus)* Variola minor virus (Alastrim)* Yersinia pestis* OVERLAP SELECT AGENTS AND TOXINS Bacillus anthracis* Bacillus anthracis Pasteur strain Brucella abortus Brucella melitensis Brucella suis Burkholderia mallei* Burkholderia pseudomallei* Hendra virus Nipah virus Rift Valley fever virus Venezuelan equine encephalitis virus 3 USDA SELECT AGENTS AND TOXINS African horse sickness virus African swine fever virus Avian influenza virus 3 Classical swine fever virus Foot-and-mouth disease virus* Goat pox virus Lumpy skin disease virus Mycoplasma capricolum 3 Mycoplasma mycoides 3 Newcastle disease virus 2,3 Peste des petits ruminants virus Rinderpest virus* Sheep pox virus Swine vesicular disease virus USDA PLANT PROTECTION AND QUARANTINE (PPQ) SELECT AGENTS AND TOXINS Peronosclerospora philippinensis (Peronosclerospora sacchari) Phoma glycinicola (formerly Pyrenochaeta glycines) Ralstonia solanacearum Rathayibacter toxicus Sclerophthora rayssiae Synchytrium endobioticum Xanthomonas oryzae *Denotes Tier 1 Agent 49

50 1 C = Cysteine residues are all present as disulfides, with the 1st and 3rd Cysteine, and the 2nd and 4th Cysteine forming specific disulfide bridges; The consensus sequence includes known toxins α-mi and α-gi (shown above) as well as α-gia, Ac1.1a, α-cnia, α-cnib; X1 = any amino acid(s) or Des-X; X2 = Asparagine or Histidine; P = Proline; A = Alanine; G = Glycine; X3 = Arginine or Lysine; X4 = Asparagine, Histidine, Lysine, Arginine, Tyrosine, Phenylalanine or Tryptophan; X5 = Tyrosine, Phenylalanine, or Tryptophan; X6 = Serine, Threonine, Glutamate, Aspartate, Glutamine, or Asparagine; X7 = Any amino acid(s) or Des X and; Des X = an amino acid does not have to be present at this position. For example if a peptide sequence were XCCHPA then the related peptide CCHPA would be designated as Des-X. 2 A virulent Newcastle disease virus (avian paramyxovirus serotype 1) has an intracerebral pathogenicity index in day-old chicks (Gallus gallus) of 0.7 or greater or has an amino acid sequence at the fusion (F) protein cleavage site that is consistent with virulent strains of Newcastle disease virus. A failure to detect a cleavage site that is consistent with virulent strains does not confirm the absence of a virulent virus. 3 Select agents that meet any of the following criteria are excluded from the requirements of this part: Any low pathogenic strains of avian influenza virus, South American genotype of eastern equine encephalitis virus, west African clade of Monkeypox viruses, any strain of Newcastle disease virus which does not meet the criteria for virulent Newcastle disease virus, all subspecies Mycoplasma capricolum except subspecies capripneumoniae (contagious caprine pleuropneumonia), all subspecies Mycoplasma mycoides except subspecies mycoides small colony (Mmm SC) (contagious bovine pleuropneumonia), and any subtypes of Venezuelan equine encephalitis virus except for Subtypes IAB or IC, provided that the individual or entity can verify that the agent is within the exclusion category. 9/10/13 Minors in Research Laboratories or Animal Facilities Unless enrolled as a Northern Arizona University student, minors are not allowed to work or conduct research in Northern Arizona University research laboratories, greenhouses or animal facilities except as identified specifically below. In addition, minors are prohibited from operating farm machinery or state vehicles and from working in machine shops. All applicable forms can be found on the EH&S Forms webpage at 1. All Minors are prohibited from working or conducting research in the following: a. Any laboratory or facility designated as BSL-3, ABSL-3 or higher for recombinant or infectious organisms. b. Any laboratory where select agents or explosives are used or stored. c. Any Animal Care Services (ACS) housing or procedure area/lab/facility. 2. Minors are prohibited from working with any of the following materials. a. Radioactive materials or radiation (X-rays) b. Acute Toxins 3. Minors are allowed to work or conduct research in laboratories (not listed in #1 or #2 above) if the following requirements are met in full: a. Northern Arizona University EH&S Biosafety Office Policy titled; Minors In Research Laboratories Or Animal Facilities (as been read and understood. b. A Minor s Research Proposal Registration Form is submitted to and approved by Northern Arizona University Institutional Biosafety Committee which includes: i. A description of the project and all materials ii. A parental consent form 50

51 iii. A sponsor consent form c. Hazard specific safety training is completed by the Principal Investigator/Sponsor with the minor as approved by EH&S BSO. d. Personal protective equipment, specific to the hazard, is provided to the minor with instructions for use and disposal. e. The minor is supervised at all times while in the laboratory and never left alone. f. Hours of work comply with Federal Regulation 29 CFR The laboratory is in full compliance with all applicable Northern Arizona University safety programs and regulations. Responsibilities of the Principle Investigator during the Conduct of the Research The Principal Investigator shall: 1. Supervise the safety performance of the laboratory staff to ensure that the required safety practices and techniques are employed. 2. Report any significant incident, violation of the NIH Guidelines, or any significant, research-related accidents and illnesses immediately by contacting the Biosafety Officer. Examples of incidents and violations include: a. Overt exposures (exposures that result in direct personnel exposure to biohazards such as injection, spills, splashes or aerosol inhalation) b. Potential exposures (exposures that have a high risk of exposing personnel to biohazards such as spills, containment failure while working with the agent or equipment failure that may produce aerosols) c. Any exposure (overt or potential) in a BSL-3 laboratory d. Overt exposure in BSL1 or BSL-2 laboratories e. Any illness that may be caused by the agents used in the laboratory f. Incidents involving the improper disposal of biohazards. 3. Ensure the integrity of the physical containment (e.g., biological safety cabinets) and the biological containment (e.g., purity and genotypic and phenotypic characteristics) and correct procedures or conditions that might result in release of or exposure to biohazards. 4. Limit or restrict access to the laboratory when work with biohazards is in progress; this includes making a determination of who may be at increased risk. 5. Establish policies and procedures to limit access exclusively to those individuals who have been advised of the potential hazards and meet specific entry requirements. 6. Ensure that laboratory personnel are offered, at no cost, appropriate immunizations or tests for the infectious agents handled or potentially present in the laboratory (e.g., hepatitis B vaccine, tuberculosis skin testing). Reportable Incidents and Violations Incidents/problems involving biohazards must be immediately reported to the Biosafety Officer. Examples of reportable significant incidents include but are not limited to: 1. Any overt exposure, such as a needle stick, splash, and contamination due to equipment failure. 51

52 2. Any potential exposure in a BSL-3 facility. A significant event may also occur from a containment breach, which may be subsequently determined to pose either an overt or potential exposure to individuals or the environment. It should be noted that waste from recombinant or synthetic nucleic acid research is also considered biohazardous and incidents involving improper disposal of recombinant or synthetic nucleic acids must also be reported. Questions regarding reportable incidents should be directed to the Biosafety Officer. Failure by research personnel to follow federal and institutional regulations, guidelines, policies and/or procedures may also require reporting to the appropriate institutional, local, state and/or federal agencies. Violations may include but are not limited to conduct of new or ongoing research without appropriate federal or institutional registration, review, approval or oversight. Institutional Reporting Responsibilities The Institutional Biosafety Committee is required, by the NIH Guidelines, to report to the appropriate University official and to the NIH/OBA within thirty days any significant incidents, violations of the NIH Guidelines, or any significant findings of research-related accidents and illnesses. The IBC will be responsible to determine what actions, if any, are necessary. For example, the IBC may determine the need to make changes to the frequency of laboratory inspections or biosafety containment level of the research, based on results of the incident. Other IBC reporting requirements (to OBA and other agencies) include but are not limited to: 1. Research involving biohazards conducted without prior IBC approval 2. Lax security, unsafe procedures used in a laboratory setting, improper disposal of recombinant or synthetic nucleic acid waste 3. Changes to research risk that have been initiated without prior approval by IBC Some incidents must be reported to OBA on an expedited basis. Spills or accidents in BSL-2 laboratories involving recombinant or synthetic nucleic acids that result in an overt exposure must be immediately reported to OBA. In addition, spills or accidents involving recombinant or synthetic nucleic acids occurring in high containment (BSL-3 or higher) laboratories resulting in an overt or potential exposure must be immediately reported to OBA. The IBC working through the IBC Chair and the BSO will report to the Institutional Official, who, in turn will oversee the report to OBA, any of the above-described incidents. Institutional violations that will also be reported to the appropriate College or department head may include but are not limited to: 1. Lapses in disclosure approval 2. Failure to comply with institutional and federal regulations, guidelines, and policies 3. Unsafe work practices 52

53 Biological Waste Disposal Policy This policy is intended to provide guidance and insure compliance with the NIH/CDC guidelines, the State of Arizona Administrative Code, and restrictions of the Coconino County Landfill. Categories 1) Infectious/potentially infectious/r-dna a) human pathogens b) animal pathogens c) plant pathogens d) recombinant DNA e) human and primate blood, blood products and other body fluids f) human and primate tissue g) any material containing or contaminated with any of the above (test tubes, needles*, syringes, tubing, culture dishes, flasks, etc.) This waste must be inactivated prior to disposal. The preferred method is steam sterilization (autoclaving), although chemical inactivation or incineration may be appropriate in some cases. Per NAU policy, even autoclaved or otherwise inactivated wastes must be disposed of as biohazardous waste. Storage of non-inactivated waste is restricted to within the generating laboratory. The material may not be stored longer than 24 hours prior to inactivation. 2) Non-infectious waste This category includes waste that is not contaminated with any of the biological materials listed in category 1. It includes solid waste and sharps generated in clinical or laboratory settings. Sterile or unopened biomedical materials that require disposal are also considered biological waste, and must be treated as such. IV packs test tubes Petri dishes needles* razor blades* tissue culture flasks syringes culture dishes scalpels* flasks broken glass and plastic ware** pipettes This material does not require sterilization prior to disposal. *must be packaged in plastic sharps boxes. **must be within a box or other puncture proof container before adding to waste. 3) Mixed radioactive/biohazardous waste The biohazardous component of mixed radioactive/biohazardous waste shall be inactivated prior to its release to Radiation Safety for disposal as radioactive waste. Steam-sterilization or chemical inactivation shall be employed as above. Although some radioactive materials can be autoclaved safely, please check with the EH&S Radiation Safety Officer regarding the best method to employ with any given radionuclide. 4) Mixed chemical/biohazardous waste The biohazardous component of mixed chemical/biohazardous waste shall be inactivated prior to its release for chemical disposal. Precautions should be taken to prevent the generation and release of 53

54 toxic chemicals during the inactivation process. In general, autoclaving is not recommended because flammable or reactive compounds should not be autoclaved due to the explosion hazard. Please check with the NAU Chemical Hygiene Officer for guidance regarding particular chemicals. 5) Animal carcasses and materials As there are no tissue digesters or incinerators present on the NAU Campus, all animal materials must be packaged, shrouded and delivered to the Vivarium Manager. No animal bodies or material shall be disposed of as regular trash or through the biomedical waste receptacle. 6) Human remains Please contact the State Anatomical Review Board for information regarding the final disposition of human remains and body parts. Packaging 1) Biohazard bags These are used for the initial collection of certain biological wastes. All biohazard bags must meet impact resistance (165 grams), tearing resistance (480 grams), and heavy metal concentration (<100 PPM total of lead, mercury, chromium and cadmium) requirements. Written documentation (a test report) from the manufacturer regarding these requirements must be on file. Individual departments are responsible for obtaining the appropriate biohazard bags. These bags must be placed in bulk biohazard containers as described within this section. 2) Sharps Needles, scalpels and razor blades must be containerized in red plastic sharps containers. NAU departments are responsible for obtaining their own sharps containers. All other sharps (broken glass and plastic ware, pipettes, etc.) must be containerized in puncture-resistant cardboard boxes (see #3, below). 3) Containers All biological waste must be containerized in rigid, leak proof, puncture resistant boxes or plastic tubs/barrels as the terminal receptacle. NAU Departments are responsible for obtaining the appropriate containers from the EH&S Hazardous Waste Coordinator. Labeling All packages containing biological waste shall be labeled with indelible ink marker (i.e., Sharpie ) as follows: 1) Date Biohazard bags shall be labeled with the date they were put into use. Please note that biohazard bags must be labeled even though they will be placed inside a secondary container for final disposal. Sharps containers shall be labeled with the date the container is full. 2) Name/Location/Phone Number Generator s (principal investigator s) name, lab location (room number) and phone number will be clearly printed on each container. 54

55 3) Biohazard sign Only manufactured containers with the preprinted universal biohazard symbol and the words biomedical, biohazardous, or infectious shall be used. Transport The transport of biohazardous waste outside of the laboratory (i.e., to an autoclave or incinerator) must be in a closed, leak-proof container that is labeled biohazard. Only trained personnel may transport biomedical waste. Labeling may be accomplished by use of a red biohazard bag with the universal biohazard symbol. Only biomedical bags and red plastic sharps containers may be used to transport biological waste to the biomedical waste receptacle. Waste receptacle personnel are instructed not to accept any other type of containers. Transportation of red-bagged waste must be in closed, leak-proof containers, properly labeled as Biohazards. Movement of regulated/biological waste through public corridors, along carpeted hallways, and on public elevators should be avoided whenever possible. Any leakage/spills from these containers must be immediately reported to the NAU Biosafety Office. Signs must be displayed to prevent tracking of the spills to other areas. Training All employees who handle biological waste shall be trained annually regarding its proper handling. All new employees shall be trained before they are allowed to handle biological waste. Training may be accomplished through the NAU Bloodborne Pathogen Training Program, informally in the lab setting, or through formal training programs set up by individual departments or divisions. For assistance, please call the NAU Biological Safety Officer or Hazardous Waste Coordinator. According to Arizona Statute (Ch. 64E-16 F.A.C.), records of the training session shall be maintained for each employee, along with an outline of the training program. Training records shall be retained for a period of three (3) years. Biological Waste Disposal Containers The following waste disposal containers for biohazardous or biomedical waste are to be used for teaching and research purposes at Northern Arizona University: ALL BIOHAZARDOUS WASTE MUST BE TREATED BEFORE DISPOSAL. Bulk Biohazard Receptacles The EH&S currently provides departments who generate biohazardous and biomedical waste with large receptacles for the disposal of full biohazard bags and sharps containers. Individual departments can contact the EH&S Hazardous Waste Coordinator for pickup when those containers are full. 55

56 PLEASE NOTE: PER NAU POLICY, EVEN AUTOCLAVED WASTES MUST BE TREATED AS BIOHAZARDOUS WASTES. Disposal of biomedical waste in the regular trash or dumpster is prohibited. The Coconino County Landfill does not accept any items that resemble biomedical devices and will reject the entire load. In some cases, the individual principal investigator has been made responsible for the costs incurred for sorting and disposal of improperly disposed waste. Sharps Boxes NAU is subject to the requirements of Arizona Administrative Code Rule , Medical Sharps. This regulation requires that a generator who treats bio-hazardous medical waste on site shall place medical sharps in a sharps container after rendering them incapable of creating a stick hazard by using an encapsulation agent or any other process that prevents a stick hazard. Red plastic sharps boxes are used for disposal of needles, razor blades, scalpels, and small Pasteur pipettes. Sharps boxes that contain infectious materials must be inactivated by autoclaving the closed box. These boxes shall be labeled (with indelible ink marker as above) with the principal investigator s name, phone number, date the sharps box is full, and lab location (room number). Only trained staff/research staff shall handle the full sharps boxes. Full sharps containers can be disposed of in the bulk biohazard receptacles described above. Biohazard Bags Red plastic biohazard bags shall be used for collection of biohazardous tissue culture items, petri dishes, and other non-sharp items. These biohazard bags shall be immediately labeled with an indelible ink marker (e.g. Sharpie ) when the bag is first put into use. Labels shall include the principal investigator s name, phone number, the date the first biohazard item is placed into the bag, and lab location (room number). The biohazardous waste items must be inactivated by autoclaving in biohazard autoclave bags or treatment with bleach within 30 days of accumulation (Ch. 64E-16 F.A.C.). After treatment, the bags shall be tightly sealed and placed in the bulk containers described above. They may be transported to the biomedical waste receptacle by trained laboratory personnel or by trained staff. Biohazard bags should never be handled by non-research staff or placed in the hallway. Double bagging may be required to prevent leakage, however, absorbent material must be added to prevent the presence of any liquid or fluid. Full, untreated biohazard bags shall be stored only in the lab where accumulation occurred. Full biohazard bags must be treated within 24 hours. As previously noted, even autoclaved wastes must be disposed of as biohazardous waste. Autoclave Use and Testing Rules governing the use and testing of autoclaves are based on Arizona Administrative Code, Rule , Biohazardous Medical Waste Treated on Site. The following summarize the requirements set by this administrative rule. A generator who autoclaves bio-hazardous medical waste on site shall: 56 1) Further process by grinding, shredding, or any other process, any recognizable animals and human tissue, organs, or body parts, to render such wasted nonrecognizable and ensure effective treatment.

57 2) Operate the autoclave at the manufacturer's specifications appropriate for the quantity and density of the load. 3.) Keep records of operational performance levels for six months after each treatment cycle. Operational performance level recordkeeping includes all of the following: a. Duration of time for each treatment cycle. b. The temperature and pressure maintained in the treatment unit during each cycle. c. The method used to determine treatment parameters in the manufacturer's specifications. d. The method in manufacturer's specifications used to confirm microbial inactivation and the test results. e. Any other operating parameters in the manufacturer's specifications for each treatment cycle. 4) Keep records of equipment maintenance for the duration of equipment use that include the date and result of all equipment calibration and maintenance. A generator shall also do the following: 1) Package the treated medical waste according to the waste collection agency's requirements; 2) Attach to the package or container a label, placard, or tag with the following words: "This medical waste has been treated as required by the Arizona Department of Environmental Quality standards" before placing the treated medical waste out for collection as a general solid waste. The generator shall ensure that the treated medical waste meets the standards of R ) Upon request of the solid waste collection agency or municipal solid waste landfill, provide a certification that the treated medical waste meets the standards of R ) Make treatment records available for Departmental inspection upon request. A generator of medical sharps shall handle medical sharps as prescribed in R A generator of chemotherapy waste, cultures and stocks, or animal waste shall handle that waste as prescribed in R Autoclave Testing Autoclaves shall be tested before being placed into service and then periodically for effectiveness. 1) Periodicity a) Every 40 hours of use Required for autoclaves that are used to inactivate human or non-human primate blood, tissues, clinical samples, or human pathogens. b) Annually Required for autoclaves that are used to inactivate other material. 2) Method A commercially available test indicator kit that uses bacterial spores (Bacillus stearothermophilus) is the approved method of testing autoclave efficiency. Most spore vial test kits require 56 to 60 C incubation of the autoclaved test vial along with a nonautoclaved control vial. Incubation causes surviving spores to grow. a) New autoclaves Before placing an autoclave into service, a test load approximating the weight and density of the type of waste generated shall be autoclaved with test spore vials. The 57

58 spore vials should be placed at the bottom, top, front, rear, and center of the autoclave chamber. This can be achieved by either: -placing vials at those positions within one large test load, OR -making several smaller test packs with 1 vial at the center of each and placing the packs at those locations within the chamber. The appropriate parameters for sterilization including temperature, pressure, and treatment time shall be determined in this way. b) Autoclaves already in use For periodic testing, place a spore vial in the very center of a test load prior to autoclaving. 3) Storage Information Please read the product information sheet for appropriate storage information. Spore vials should not be frozen. Each batch of vials has an expiration date. Vials should not be used after their expiration date. Record-Keeping The following records regarding autoclave use must be kept: 1) On-site maintenance records 2) Autoclave use log must be available near the autoclave. Each load of material inactivated shall be logged as follows: a) Date, time, and operator's name b) Contact Information: Lab, room number, phone number c) Is this biohazardous material? d) Confirmation of sterilization: i) Record the temperature, pressure, and length of time the load is sterilized. Please note that temperature sensitive autoclave tape is not sufficient to indicate that the load reached sterilization conditions because the tape will change color at lower temperatures. ii) Save the autoclave printout, if the autoclave has a working printer. Autoclave Operating Procedures A written sterilization procedure shall be in place for each workplace. This shall include the following: 1) Parameters Appropriate parameters for sterilization shall be determined from the testing with spore vials. The time it takes to sterilize a load will change depending upon the load density and the sterilization cycle one chooses. Tests have been performed which imitate these various situations. Please follow the established guidelines. 2) Protocol Identification of standard treatment containers and proper load placement shall be made. 3) Cleaning The autoclave and work areas shall be cleaned after every use and the work area shall be disinfected, as needed. 58

59 Autoclave Operation and Safety Training Autoclave training is available from the EH&S Biological Safety Office upon request. The training is geared toward the research staff and goes over proper use of autoclaves and how they may be maintained and used properly. Safety training is also given. 59

60 Autoclave Guidelines AUTOCLAVE GUIDELINES STERILIZATION TIMES (Drying Time Not Included) 121 C / 250 F and 15 p.s.i. BIO WASTE IN AUTOCLAVE BAGS, LOOSELY TIED MULTIPLE BAGS MINUTES OR LONGER SINGLE BAG (FULL) MINUTES PARTIAL BAG MINUTES DRY GOODS GLASSWARE, EMPTY, INVERTED INSTRUMENTS, WRAPPED UTENSILS, WRAPPED MINUTES MINUTES MINUTES LIQUIDS (Bottles with vented caps ½ FULL) 75ml MINUTES 250ml MINUTES 500ml MINUTES 1000ml MINUTES 1500ml MINUTES 2000ml MINUTES The above times should be used as a guide in determining the length of time items should be autoclaved in order to achieve sterilization. 60

61 Disinfectants Disinfectant refers to an agent that is applied to treat (usually) inanimate objects to render them free of pathogenic or infectious microorganisms. In contrast, the term, sterilant, refers to an agent that renders items free of all microorganisms. The two are not the same and should not be confused. Disinfectants are used in laboratory and chemical settings to 1) treat a surface or an item before or after routine use, or 2) to treat a surface or an item after a spill or contaminating event. Because disinfectants are antimicrobial, they may, by their nature, also have a toxic effect to the user. Therefore, Material Safety Data Sheets and other manufacturer s product information should be available and thoroughly reviewed before using these products. There are many different types and formulations of disinfectants. The researcher or clinician should ensure that the proper product, one that is effective against the specific microorganism being studied, is used. The FDA regulates those products that are marketed as sterilants or sanitizing agents on medical devices. They have published a list of products currently on the market that are labeled as sterilants. ( The EPA regulates moderate and low level disinfectants as chemical germicides under their pesticide regulations. They have published a list of 1) those products that are effective against Mycobacterium spp. (tubercle bacilli), and 2) those products that are effective against HIV-1 (human immunodeficiency virus). Please contact the Biological Safety Office for information about any of these lists or for a list of manufacturers. Be aware that most disinfectants assume pre-cleaning to remove gross organic/protein prior to use. Whenever a disinfectant or sterilant is used, proper safety precautions must be followed. Appropriate clothing (gloves, safety goggles, aprons) must be worn. In addition, these compounds must be used in well-ventilated areas. Following is a discussion of general categories of disinfectants. Please note that there are several different products and different formulations in each category. Liquids Alcohols The most commonly used alcohols, ethanol and isopropanol, are most effective at concentrations of 70% in water. Both higher and lower concentrations are less effective. Alcohols are active against vegetative bacteria, fungi, and lipid viruses but not against spores. They are only moderately effective against nonlipid viruses. Alcohols are difficult to use as contact disinfectants because they evaporate rapidly and do not penetrate organic matter well. When using alcohols, it is best to clean an object, then submerge it in alcohol for the appropriate time. Alcohols are often used in concert with other disinfectants such as formaldehyde (but see toxicity warning below) or chlorine (2000 ppm chlorine in alcohol). Alcohol is NOT a registered tuberculocidal or HIV listed disinfectant. 61

62 Chlorine compounds The most commonly used and generally effective disinfectant is sodium hypochlorite (common household bleach). It is a strong oxidizing agent and therefore can be corrosive to metal. A 1:50 dilution, supplying 1000 ppm available chlorine, of the common household product (e.g. chlorox) is very effective as a general laboratory disinfectant and a 1:10 dilution supplying 5000 ppm available chlorine is effective against spills involving blood or other organic material. Please note that the presence of high concentrations of protein can inactivate the action of chlorine products. Dilute hypochlorite solution must be prepared daily to be maximally effective. There are more concentrated sodium hypochlorite solutions available for industrial use, so please read the product information carefully to determine the proper dilution. Table 4: Dilutions of Household Bleach Volume Volume Dilution Sodium Available Of Bleach of Water Ratio Hypochlorite % Chlorine (mg/l) Undiluted 0 1: , : , : Formaldehyde Formaldehyde is a gas that is available either dissolved in water and methanol as a 37% formaldehyde solution, known as formalin, or as a solid, paraformaldehyde, that may be melted to release the gas. Formaldehyde gas is very active against a variety of microorganisms and is used for space decontamination and to decontaminate biological safety cabinets. Formaldehyde dissolved in water is active at 1-8% solutions and can be used to decontaminate hard surfaces. However, because formaldehyde is very irritating at low concentrations (0.1 to 5 ppm) and a known carcinogen, its use as a hard surface disinfectant is limited to situations in which it is particularly needed. Due to its toxic effects, there are no EPA-registered disinfectants that contain formaldehyde. Glutaraldehyde Glutaraldehyde is usually supplied as a 20% solution and requires activation by the addition of an alkaline agent prior to use. The activated product may be kept for about two weeks and should be discarded when turbid. Glutaraldehyde is active against almost all microorganisms, but is toxic, irritating, and mutagenic and should be used only when necessary. Please follow the manufacturer s guidance when using glutaraldehyde-based products because there are many different formulations that have been designed for specific uses. Hydrogen peroxide Hydrogen peroxide is usually available as a 30% solution. It may be diluted 1:5 for use as a disinfectant. It is active against a wide array of microorganisms. However, it is an oxidizing agent and should not be used on aluminum, copper, zinc, or brass. Hydrogen peroxide is unstable at high temperatures and in light. Iodine and Iodophors Iodine and iodophors, compounds in which the iodine is combined with a solubilizing or carrier agent, are general, all-purpose disinfectants with an action similar to that of chlorine products. The appropriate concentration for iodine-containing products is 75 ppm available iodine for disinfecting work surfaces. Concentrations may be much higher for other purposes. Like chlorine compounds, 62

63 the effectiveness of iodine compounds may be diminished in the presence of protein/organic material. Iodophor compounds that are used for antisepsis (germicide applied to tissue or skin) are not appropriate for use as hard surface disinfectants and vice versa. Please read the product material for appropriate dilutions and applications. Phenol and phenolic compounds Phenolic compounds are active at 0.2 to 3% concentrations against all forms of vegetative microorganisms but not against spores. They have only limited effectiveness against nonlipid viruses. There are many common disinfectants based on phenol and they should be used according to the manufacturer s recommendations. Phenol is a hazardous chemical and requires hazard assessment as well as proper PPE selection and use. Quatenary ammonium compounds Compounds in this class are active at concentrations of 0.1-2%. They are active against vegetative bacteria, lipid viruses, but not against bacterial spores, non-lipid viruses, or tubercle bacilli. These compounds should be used only when a low-level disinfectant is required. Gases Ethylene Oxide (Not currently allowed at NAU) Inactivates micro-organisms, cellular disruption Temp C, Humidity 30-40% Conc mg/liter Time 2 hrs at 60 C or 24 hrs at 20 C Aeration required after cycle for 8 hrs Biological indicators needed to confirm kill Suspected carcinogen with explosive properties Vapor Phase Hydrogen Peroxide Temp 4-60 C,> temp results in > activity Conc 30%, less than 10 mg/liter Non-toxic end products of water and oxygen Limited to surfaces, no penetration Corrosive to some materials Degrades natural rubber and nylon Chlorine Dioxide gas Dilute chlorine gas and sodium chlorite, less than 25 mg/liter Temp C, prehumidification required Limited to surfaces, no penetration Corrosive to some materials Mucous membrane irritant Ozone Oxidizing properties, inhibits bacterial growth, reacts with amino acids, RNA/DNA Temp 25 C Conc 2-5 mg/liter Systems convert oxygen to ozone 63

64 Limited penetration Formaldehyde Gas (from heating paraformaldehyde) Temp C, humidity 60-85% Conc 0.3 gm/cu ft of volume Time 6-8 hours Toxic irritant and suspected carcinogen Limited penetration, primarily surface action Requires aeration and time for formaldehyde to off-gas, usually 8 hours Irradiation Ultraviolet, UV radiation Virucidal action correlates with shorter wavelengths of the UV spectrum, nm. Mechanism of UV radiation injury attributed to absorption by and resultant damage to nucleic acids Due to low energy, the power of penetration is poor. Dust and thin layers of proteins on surfaces reduce the virucidal activity Do not rely on Biosafety Cabinet UV lights to disinfect surfaces. Ionizing radiation Penetrates products and micro-organisms Releases high energy, disrupts cells, DNA & RNA and results in kill Damage done to cell membrane and other cellular structures Cobalt 60 and Cesium 137 Electron Beam Limited penetration Accelerator generates high energy electrons Microwaves Thermal and non-thermal activity Still under study for the most part Disinfectants Bibliography The following materials were consulted or used in this chapter: Disinfection, Sterilization, and Preservation. Fourth edition Seymour S. Block ed., Lea & Febiger, Philidelphia Laboratory. Second edition World Health Organization, Geneva. Laboratory Safety: Principles and practices. Second edition Diane O. Fleming et al. eds. ASM Press, Washington DC Manual of Clinical Microbiology. Fifth edition Albert Balows ed., ASM, Washington DC Prudent Practices in the Laboratory: Handling and disposal of chemicals National Research Council. National Academy Press, Washington 64

65 Table 5: Summary and Comparison of Liquid Disinfectants (Page 1) Commonly used disinfectants, recommended when appropriate. Class Recommended Use How They Work Advantages Disadvantages Comments/Hazards Examples Cleaning some Fairly inexpensive Flammable instruments Eye Irritant Cleaning skin Toxic 70% Isopropyl alcohol solution Chlorine compounds Gluteraldehyde Spills of human body fluids Bactericidal - Good Fungicidal - Good Sporicidal - Good at >1000 ppm Sodium Hypochlorite Bactericidal - Good Fungicidal - Good Tuberculocidal - Excellent Virucidal - Good Sporicidal - Good Changes protein structure of microorganism Presence of water assists with killing action Free available chlorine combines with contents within microorganism - reaction byproducts cause its death Need 500 to 5000 ppm Produce chemical combination with cell substances Depend upon release of hypochlorous acid Coagulates cellular proteins Kill hardy viruses (e.g., hepatitis) Kill a wide range of organisms Inexpensive Penetrates well Relatively quick microbial kill May be used on food prep surfaces Non-staining, relatively non-corrosive Useable as a sterilant on plastics, rubber, lenses, stainless steel, and other items that can t be autoclaved <50% solution not very effective Not active when organic matter present Not active against certain types of viruses Evaporates quickly - contact time not sufficient for killing Corrode metals such as stainless, aluminum Organics may reduce activity Increase in alkalinity decreases bactericidal property Unpleasant taste and odor Tuberculocidal - with extended contact time Not stable in solution Has to be in alkaline solution Inactivated by organic material Follow spill procedure and dilution instructions Make fresh solutions before use Eye, skin, and respiratory irritant Corrosive Toxic Eye, skin and respiratory irritant. Sensitizer Toxic Bleach solutions (sodium hypochlorite) Clorox Cryosan Purex Calgocide 14 Cidex Vespore 65

66 Table 5: Summary and Comparison of Liquid Disinfectants (Page 2) Class Recommended Use How They Work Advantages Disadvantages Comments/Hazards Examples Iodophors (iodine with carrier) Phenolic Compounds Quaternary ammonium compounds (QUATS) Disinfecting some semi-critical medical equipment Bactericidal - Very Good Fungicidal - Excellent Virucidal - Excellent Bactericidal - Excellent Fungicidal - Excellent Tuberculocidal - Excellent Virucidal - Excellent Ordinary housekeeping (e.g., floors, furniture, walls) Bactericidal - Excellent Fungicidal - Good Virucidal - Good (not as effective as phenols) Free iodine enters microorganism and binds with its cellular components Carrier helps penetrate soil/fat Need 30 to 50 ppm Probably by disorder of protein synthesis due to hindrance and/or blocking of hydrogen bonding Gross protoplasmic poison Disrupts cell walls Precipitates cell proteins Low concentrations inactivate essential enzyme systems Affect proteins and cell membrane of microorganism Release nitrogen and phosphorous from cells Kill broad range of organisms Highly reactive Low tissue toxicity Kill immediately rather than by prolonged period of stasis Not affected by hard water May be used on food prep surfaces Nonspecific concerning bactericidal and fungicidal action When boiling water would cause rusting, the presence of phenolic substances produces an anti-rusting effect Contain a detergent to help loosen soil Rapid action Colorless, odorless Non-toxic, less corrosive Highly stable May be used on food prep surfaces May stain plastics or corrode metal May stain skin/laundry Stains most materials Odor Some organic and inorganic substances neutralize effect Tuberculocidal - with extended contact time Sporicidal - some Unpleasant odor Some areas have disposal restrictions Effectiveness reduced by alkaline ph, natural soap, or organic material Sporicidal - NO Do not eliminate spores, TB bacteria, some viruses Effectiveness influenced by hard water Layer of soap interferes with action Dilution critical - follow directions! Use only EPAregistered hard surface iodophor disinfectants Don t confuse skin antiseptic iodophors for disinfectants Skin and eye irritant Corrosive Toxic Skin and eye irritant Sensitizer Corrosive Toxic Select from EPA list of hospital disinfectants Skin and eye irritant Toxic Barbara Fox Nellis ( ) Bactergent Hy-Sine Ioprep Providoneiodine; betadine Wescodyne Hil-Phene Lph Metar Vesphene Coverage 258 End-Bac Hi Tor 66

67 Table 6: Summary of Practical Disinfectants Quatenary Ammonium Compounds Phenolic Compounds Chlorine Compounds Iodophor Ethyl Alcohol Isopropyl Alcohol Formaldehyde Glutaraldehyde Inactivates Vegetative Bacteria Lipoviruses + + a a Nonlipid Viruses Bacterial Spores Treatment Requirements Use Dilution % % 500ppm b ppm b 70-85% 70-85% % 2% Contact Time-minutes Lipovirus Broad Spectrum NE NE NE NE Important Characteristics Effective Shelf Life > 1 week c Corrosive Flammable Explosion Potential Inactivated by organic matter Skin Irritant Eye Irritant Respiratory Irritant Toxic d Applicability Waste Liquids Dirty Glassware Equipment, Surface Decontamination Proprietary Products e A-33, CDQ, End-Bac, Hl-Tor, Mikro-Quat Hil-Phene, Metar, Mikro- Bac, O-Syl Chloramine T, Clorox, Purex Hy-Sine, Ioprep, Mikroklene, Wescodyne Source: Adapted from Laboratory Safety Monograph, A Supplement to the NIH Guidelines for Recombinant DNA Research. National Institutes of Health, Office of Research Safety, National Cancer Institute, and the Special Committee of Safety and Health Experts, Bethesda, Maryland. January 1989: Yes a Variable results depending on virus d By skin or mouth or both - No b Available Halogen e Space limitations preclude listing all products available. Individual listings (or omissions) NE Not Effective c Protected from light and air do not imply endorsement (or rejection) of any product by the National Institutes of Health. Sterac Cidex 67

68 Table 7: Reprocessing Methods for Equipment Used in the Health Care Setting Sterilization: Destroys: All forms of microbial life including high numbers of bacterial spores. High-Level Disinfection: Intermediate- Level Disinfection: Low-Level Disinfection: Environmental Disinfection: Methods: Use: Destroys: Methods: Use: Destroys: Methods: Use: Destroys: Methods: Use: Steam under pressure (autoclave), gas (ethylene oxide), dry heat or immersion in an approved chemical sterilant (e.g., US Environmental Protection Agency-approved) for prolonged period of time, e.g., 6-10 hours or according to manufacturer s instructions. Note: Liquid chemical sterilants should be used only on those instruments that are impossible to sterilize or disinfect with heat. For those instruments or devices that penetrate skin or contact normally sterile areas of the body, e.g., scalpels, needles, etc. Disposable invasive equipment eliminates the need to reprocess these types of items. When indicated, however, arrangements should be made with a health-care facility for reprocessing of reusable invasive instruments. All forms of microbial life except high numbers of bacterial spores. Hot water pasteurization ( C, 30 minutes) or exposure to an approved (e.g., US EPA-approved) sterilant chemical as above, except for a short exposure time (10-45 minutes or as directed by the manufacturer). For reusable instruments or devices that come into contact with mucous membranes (e.g., laryngoscope blades, endotracheal tubes, etc.). Mycobacterium tuberculosis, vegetative bacteria, most viruses and most fungi, but does not kill bacterial spores. Approved (e.g. US EPA-approved) hospital disinfectant chemical germicides that have a label claim for tuberculocidal activity; commercially available hard-surface germicides or solutions containing at least 500 ppm free available chlorine (a 1:10 dilution of common household bleachabout ¼ cup bleach per gallon of tap water). For those surfaces that come into contact only with intact skin, e.g., stethoscopes, blood pressure cuffs, splints, etc., and have been visibly contaminated with blood or bloody body fluids. Surfaces must be pre-cleaned of visible material before the germicidal chemical is applied for disinfection. Most bacteria, some viruses, some fungi, but no Mycobacterium tuberculosis or bacterial spores. Approved (e.g., US EPA-approved) hospital disinfectants (no label claim for tuberculocidal activity). Use these excellent cleaning agents for routine housekeeping or removal of soiling in the absence of visible blood contamination. Environmental surfaces that have become soiled should be cleaned and disinfected using any cleaner or disinfectant that is intended for environmental use. Such surfaces include floors, woodwork, ambulance seats, countertops, etc. Important: To assure effectiveness of any sterilization or disinfection process, equipment and instruments must first be thoroughly cleaned of all visible soil. 68

69 Shipment of Biological Materials The following regulations apply to the packaging and shipment of biological materials: U.S. Department of Transportation, 49 CFR Parts and amendments U.S. Public Health Service, 42 CFR Part 72, Interstate Shipment of Etiolgic Agents U.S. Department of Labor, Occupational Safety and Health Administration, 29 CFR Part , Bloodborne Pathogens International Air Transport Association (IATA), Dangerous Goods Regulations U.S. Postal Service, 39 CFR Part 111, Mailability of Etiologic Agents, Mailability of Sharps and Other Medical Devices, and Publication 52, Acceptance of Hazardous, Restricted or Perishable Matter International Civil Aviation Organization, Technical Instructions for the Safe Transport of Dangerous Goods by Air United Nations, Recommendations of the Committee of Experts on the Transportation of Dangerous Goods All North American airlines and FedEx, the largest shipper of infectious materials, use the IATA regulation (also referred to as the Dangerous Goods Regulation or DGR) as their standard. Meeting the conditions of this standard will ensure meeting the provisions of the other US regulations. General information is provided below, but please contact the EH&S Biological Safety Office for specific information. Note that for any biological materials for which a state or federal permit or license is required, registration with the EH&S Biological Safety Office is also required. Any NAU faculty/staff, regardless of job or task, must be trained to package, ship or receive packages of materials/samples classified by regulation as dangerous goods. This training is available from EH&S. Regulation requires retraining every other year OR whenever the regulations change. 69

70 Laboratory Spills A spill of biological materials that takes place in the open laboratory may create a serious problem. Every effort should be taken to prevent spilling materials. A spill poses less of a problem if it happens inside a biological safety cabinet, provided splattering to the outside of the cabinet does not occur. Direct application of concentrated liquid disinfectant and a thorough wipe down of the internal surfaces of the cabinet will usually be effective for decontaminating the work zone, but gaseous sterilants will be required to disinfect the interior sections of the cabinet and HEPA filter(s). Spill in the Open Laboratory Advance preparation for management of a spill is essential. A spill kit may include the following: Leak-proof containers Forceps Paper towels Disinfectant Respirators, if necessary Rubber gloves and other personal protective equipment Absorbent powder or other material Red bag If potentially hazardous biological material is spilled in the laboratory, avoid inhaling any airborne material by holding your breath and leaving the laboratory. Warn others in the area by posting signs and go directly to wash or change room area. If clothing is known (or suspected) to be contaminated, remove the clothing with care, folding the contaminated area inward. Discard the clothing into a bag or place the clothing directly in an autoclave. Wash all potentially contaminated body areas as well as the arms, face and hands. Shower if facilities are available. Re-entry into the laboratory should be delayed for a period of at least thirty minutes to allow reduction of the aerosol generated by the spill. Protective clothing should be worn when entering the laboratory to clean the spill area. Rubber gloves, autoclavable or disposable footwear, an outer garment, and a respirator equipped with a HEPA filter should be worn. If the spill was on the floor, do not use a surgical gown that may trail on the floor when bending down. Take the spill kit into the laboratory room, place a discard container near the spill, and transfer large fragments of material into it; replace the cover. Using a freshly prepared hypochlorite containing 5000 ppm (10% by volume) available chlorine, iodophor solution containing 1600 ppm iodine, or other appropriate EPA registered disinfectant, carefully pour the disinfectant around and into the visible spill. Avoid splashing and work from the outside toward the center. Allow 30 minutes' contact time. Use paper or cloth towels to wipe up the disinfectant and spill, working toward the center of the spill. Discard all towels and other 70

71 clean up materials into a discard container as they are used. Wipe the outside of the discard containers, especially the bottom, with a towel soaked in a disinfectant. Place the discard container and other materials in an autoclave and sterilize. Alternately, place all materials in the appropriate biomedical waste system for incarceration. Remove shoes or shoe covers, outer clothing, respirator, and gloves and sterilize by autoclaving. Wash hands, arms and face, or if possible, shower. If gaseous decontamination of the room is required, contact the Biosafety Office. Spill in a Biological Safety Cabinet A spill that is confined to the interior of the biological safety cabinet should present little or no hazard to personnel in the area. However, chemical disinfection procedures should be initiated at once while the cabinet ventilation system continues to operate to prevent escape of contaminants from the cabinet. Spray or wipe walls, work surfaces, and equipment with a disinfectant. A disinfectant with a detergent has the advantage of detergent activity that will help clean the surfaces by removing both dirt and microorganisms. A suitable disinfectant is a 3% solution of an iodophor such as Wescondyne or a 1:100 dilution of household bleach (e.g. Chlorox) with 0.7% nonionic detergent. The operator should wear gloves during this procedure. Use sufficient disinfectant solution to ensure that the drain pans and catch basins below the work surface contain the disinfectant. Lift the front exhaust grill and tray and wipe all surfaces. Wipe the catch basin and drain the disinfectant into a container. The disinfectant, gloves, wiping cloth and sponges should be discarded into an autoclave pan and autoclaved. The above procedure will not disinfect the filters, blower, air ducts, or other interior parts of the cabinet. If the entire interior of the cabinet needs to be sterilized, contact the EH&S Biosafety Office for the name of the current contractor. 71

72 Biological Safety Cabinets The following is excerpted from Primary Containment for Biohazards: Selection, Installation and Use of Biological Safety Cabinets, 1995, Centers for Disease Control & Prevention/National Institutes of Health This chapter presents information on the selection, function, and use of biological safety cabinets (BSCs), which are the primary means of containment developed for working safely with infectious microorganisms. BSCs are designed to provide personnel, environment, and product protection when appropriate practices and procedures are followed. Three kinds of biological safety cabinets, designated as Class I, II, and III, have been developed to meet varying research and clinical needs. Biological safety cabinets use high efficiency particulate air (HEPA) filters in their exhaust and/or supply systems. Biological Safety Cabinets (BSCs) The similarities and differences in protection offered by the various classes of biosafety cabinets are reflected in Table 1. The Class I BSC This type of cabinet is not for aseptic or sterile technique. The Class I BSC provides personnel and environmental protection, but no product protection. It is similar in air movement to a chemical fume hood, but has a HEPA filter in the exhaust system to protect the environment. The Class II BSC The Class II (Types A and B) biological safety cabinets provide personnel, environmental, and product protection. Air flow is drawn around the operator into the front grille of the cabinet, which provides personnel protection. In addition, the downward laminar flow of HEPA-filtered air provides product protection by minimizing the chance of cross-contamination along the work surface of the cabinet. Because cabinet air has passed through the exhaust HEPA filter, it is contaminant-free (environmental protection), and may be recirculated back into the laboratory (Type A BSC) or ducted out of the building (Type B BSC). 72