Biological Safety Manual

U 2008 N I V Biological E R Safety S I Manual T Y

L O Finance and Administration

I D Division of Environmental Health & Safety A

University of Florida Finance and Administration Environmental Health and Safety

Box 112190 Gainesville, Florida 32611 (352) 392-1591 phone (352) 392-3647 fax Email: [email protected] Internet: http://www.ehs.ufl.edu/bio

November 2008

Forward By authority delegated from the University President, the Vice President for Finance and Administration is responsible for the safety of all University facilities. Under this authority, policies are developed to provide a safe teaching, research, service, housing and recreational environment.

The division of Environmental Health and Safety was established in 1974 and given the responsibility for the management of all safety practices and the administration of the program.

The mission of the division of Environmental Health and Safety (EH&S) 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, EH&S will adhere to standards or codes related to safety which have been adopted and promulgated by nationally recognized standards-setting organizations. The interpretation of safety codes and standards is the responsibility of the Division of Environmental Health and Safety.

In order to assure an effective Environmental Health & Safety program for the University of Florida, it is imperative that all individuals associated with the University comply fully with the policies and procedures set forth in the manual.

Cover photos are from Henry Wellcome Laboratory for Cell Imagery, University of East Anglia.

Dr. Michael Wormstone provided the one in the upper left corner. It’s a photo of FHL124 (immortalized human lens) cells labeled with an antibody to the transcription factor SMAD4 (a component of the TGF-beta signaling pathway) and an Alexa-Fluor-488-conjugated secondary antibody (green). The cells actin filaments were also labeled using Texas-Red-conjugated phalloidin

Mr. David Moss contributed the one in the lower right corner of A UE-1 (immortalized mouse inner ear epithelial) cell labeled with an antibody to alpha-tubulin (microtubules) and stained with an Alexa-Fluor-488-conjugated secondary antibody (green). The cells nucleus was counterstained with DAPI (blue).

Biosafety Manual November, 2008 i Policy Statement

It is the policy of the University of Florida to provide a safe working environment. The primary responsibility for insuring safe conduct and conditions in the laboratory resides with the principal investigator.

The UF 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, we provide this manual as a resource, to be used in conjunction with the CDC and NIH guidelines, the UF Select Agent Program, the Laboratory Safety Manual and other resource materials from Environmental Health & Safety.

ii November, 2008 Bio Safety Manual Introduction

The University of Florida 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 UF Laboratory Safety Manual, which provides more general safety information. These manuals describe policies and procedures that are required for the safe conduct of research at the University of Florida. Responsibilities

In the academic research/teaching setting, the principal investigator 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.

Employees who work with biological materials are responsible for reading this manual, carrying out the safety practices outlined here, and reporting any problems, accidents, and spills to the appropriate faculty member.

Environmental Health & Safety 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.

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Emergency Phone Numbers

General

University Police Department 392-1111

Gainesville Fire Department 911

Gas leak 911

Spills/Accidents

Asbestos 392-3392

Biological or Recombinant Materials 392-1591

Chemicals (laboratory) 846-2550

Mercury 392-8400

Pesticides 392-1904

Radioactive Materials Campus 392-7359

HSC 392-1589

Select Agents 392-1591 392-6369

Medical Emergency

Student Health Care Center 392-1161

Shands Hospital Emergency Center 395-0050

Shands Occupational Health Services 265-0250 (blood and body fluid exposures only)

Needle Stick Injury 1-866-477-6824 (OUCH)

iv November, 2008 Bio Safety Manual Environmental Health & Safety Phone Numbers

Biological Safety 392-1591 Bldg. 179 Box 112190 Business Office 392-1591 Bldg. 179 Box 112190 Chemical Waste 392-8400 Surge Area Box 112725 Director’s Office 392-1591 Bldg. 179 Box 112190 Diving Science & Safety 392-1661 Bldg. 21 Box 118205 Facility & Fire Safety 392-1904 Bldg. 179 Box 112200 Fire Equipment Services 392-2365 Bldg. 179 Box 112200 Hazardous Materials Management 392-8400 Surge Area Box 112725 Industrial Hygiene 392-3393 Bldg. 104 Box 112195 Laboratory Safety 392-1591 Bldg. 179 Box 112190 Occupational Safety and Health 392-1591 Bldg. 104 Box 112195 Occupational Medicine 392-1591 Bldg. 179 Box 112195 Pest Control Services 392-1904 Bldg. 175 Box 112205 Program Support 392-1591 Bldg. 179 Box 112190 Radioactive Waste 392-8400 Surge Area Box 112725 Radiation Control & Rad. Services 392-7359 Bldg. 175 Box 112205 Radiation Control Delivery Services 392-8700 Bldg. 175 Box 100252 Radiation Control Health Center 392-1589 HSC D8-17 Box 112205 Risk Management 392-1591 Bldg. 104 Box 112195 Select Agents 392-1591 Bldg. 179 Box 112190

Biosafety Manual November, 2008 v Table of Contents

1 -- Biological Safety ...... 1 Principles of Biological Safety ...... 2 Laboratory Practice and Technique ...... 2 Safety Equipment (Primary Barrier) ...... 3 Facility Design (Secondary Barrier) ...... 3 Biosafety Levels ...... 4 Animal Facilities ...... 6 Clinical Laboratories ...... 6 Importation and Interstate Shipment of Certain Biomedical Materials ...... 7 Biological Safety Levels ...... 7 Biosafety Level 1 ...... 8 Biosafety Level 2 ...... 9 Biosafety Level 3 ...... 13 Animal Biosafety Level 1 (ABSL-1) ...... 18 Animal Biosafety Level 2 (ABSL-2) ...... 20 Animal Biosafety Level 3 (ABSL-3) ...... 23 Agents List ...... 30 Biological Safety Level 1 (BSL-1) ...... 30 Biological Safety Level 2 (BSL-2) ...... 30 BSL-2 - Bacterial Agents Including Chlamydia ...... 31 BSL-2 - Fungal Agents ...... 32 BSL-2 - Parasitic Agents ...... 32 BSL-2 - Viruses ...... 33 Biological Safety Level 3 (BSL-3) ...... 35 BSL-3 - Bacterial Agents Including Rickettsia ...... 35 BSL-3 - Fungal Agents ...... 35 BSL-3 - Parasitic Agents ...... 35 BSL-3 - Viruses and Prions ...... 35 Biological Safety Level 4 (BSL-4) ...... 36 BSL-4 - Bacterial Agents ...... 36 BSL-4 - Fungal Agents ...... 36 BSL-4 - Parasitic Agents ...... 36 BSL-4 - Viral Agents ...... 36 2 -- Information for Researchers ...... 38 Project Registration ...... 39 Bio-Agent (BA) Registration ...... 39 Recombinant DNA (R-DNA) Registration ...... 39 Acute Toxins (AT) Registration ...... 40 Regulated Biological Materials ...... 40 Table 3: Toxin Table ...... 41 Select Agents ...... 43

vi November, 2008 Bio Safety Manual Select Agents List ...... 44 Minors in Research Laboratories or Animal Facilities ...... 46 Biological Waste Disposal Policy ...... 47 Categories ...... 47 1) Infectious/potentially infectious/R-DNA ...... 47 2) Non-infectious waste ...... 47 3) Mixed radioactive/biohazardous waste ...... 47 4) Mixed chemical/biohazardous waste ...... 48 5) Animal carcasses and materials ...... 48 6) Human remains ...... 48 Packaging ...... 48 1) Biohazard bags ...... 48 2) Sharps ...... 48 3) Corrugated cardboard boxes ...... 48 Labeling ...... 48 1) Date ...... 49 2) Name/Location/Phone Number ...... 49 3) Biohazard sign ...... 49 Transport ...... 49 Training ...... 49 Biological Waste Disposal Containers ...... 50 Biohazard Boxes ...... 50 Sharps Boxes ...... 50 Biohazard Bags ...... 51 Autoclave Use and Testing ...... 51 Autoclave Testing ...... 51 Record-Keeping ...... 52 Autoclave Operating Procedures ...... 52 Autoclave Operation and Safety Training ...... 53 Autoclave Guidelines ...... 54 Disinfectants ...... 55 Liquids ...... 55 Alcohols ...... 55 Chlorine compounds ...... 56 Formaldehyde ...... 56 Glutaraldehyde ...... 56 Hydrogen peroxide ...... 56 Iodine and Iodophors ...... 57 Phenol and phenolic compounds ...... 57 Quaternary ammonium compounds ...... 57 Gases ...... 57 Ethylene Oxide ...... 57 Vapor Phase Hydrogen Peroxide ...... 57 Chlorine Dioxide gas ...... 57

Biosafety Manual November, 2008 vii Ozone ...... 57 Formaldehyde Gas (from heating paraformaldehyde) ...... 58 Irradiation ...... 58 Ultraviolet, UV radiation ...... 58 Ionizing radiation ...... 58 Electron Beam ...... 58 Microwaves ...... 58 Disinfectants Bibliography ...... 58 Shipment of Biological Materials ...... 63 Biological materials subject to shipping & transport regulations: ...... 64 Transporting biological material within and around UF: ...... 64 Permits: ...... 65 Biological Safety Cabinets ...... 67 Biological Safety Cabinets (BSCs) ...... 67 The Class I BSC ...... 67 The Class II BSC ...... 67 The Class II, Type A BSC ...... 67 The Class II, Type B1 BSC...... 68 The Class II, Type B2 BSC...... 68 The Class II, Type B3 BSC...... 69 The Class III BSC ...... 69 Horizontal Laminar Flow “Clean Bench” ...... 69 Vertical Laminar Flow “Clean Bench” ...... 69 Operations within a Class II BSC ...... 69 Laboratory Hazards ...... 69 Decontamination ...... 71 Surface Decontamination ...... 71 Gas Decontamination ...... 71 Engineering Requirements ...... 72 Ultraviolet Lamps ...... 72 BSC Placement ...... 72 HEPA Filters ...... 72 Certification of BSCs ...... 72 Emergency Procedures/Telephone Numbers ...... 73 General Information ...... 74 Medical Emergency ...... 74 Accidental injection, cuts, skin exposures ...... 74 Splashes to face and eyes ...... 75 Accidental ingestion ...... 75 Animal bites and scratches ...... 76 Break in/Security Breach ...... 77 Handling Biological Spills ...... 77 Spill in the biosafety cabinet ...... 77 Spill in the centrifuge ...... 77 Spill inside the laboratory ...... 78 viii November, 2008 Bio Safety Manual Spill outside the laboratory ...... 79 Fire Safety ...... 80 Workplace Violence: ...... 81 Hurricane: ...... 81 Tornadoes and other natural disasters:...... 82 3 -- Programs ...... 83 Animal Contact Medical Monitoring Program ...... 84 UF Bloodborne Pathogen Program ...... 86 BBP Exposure Information ...... 86 4 -- Medical Surveillance ...... 87 HIV Research Laboratory Occupational Medicine Policy ...... 88 Pre-employment ...... 88 Continuing employment ...... 88 Post-exposure prophylaxis ...... 88 References ...... 89 Immunoprophylaxis ...... 90 Recommendations for Prophylactic Immunization of Laboratory Personnel Working with Infectious Agents ...... 91 Bacterial agents ...... 91 Rickettsial agents ...... 92 Viral agents ...... 92 Vaccinia Immunization Policy ...... 94 University of Florida ...... 95 Health Surveillance for Personnel Working with Infectious Agents ...... 96 Blood Serum Sampling ...... 96 Health Assessments ...... 96 Exposure to Mycobacterium Tuberculosis ...... 97 5 -- Appendix A – Forms ...... 98 R-DNA ...... 98 Bio-Agent ...... 98 Transgenic Animals ...... 98 Acute Toxin ...... 98 Project Addendum ...... 98

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List of Tables Table 1: Summary of Recommended Biosafety Levels for Infectious Agents ...... 28 Table 2: Summary of Recommended Biosafety Levels for Activities in Which Experimentally or Naturally Infected Vertebrate Animals Are Used ...... 29 Table 3: Toxin Table ...... 41 Table 4: Dilutions of Household Bleach ...... 56 Table 5: Summary and Comparison of Liquid Disinfectants (Page 1) ...... 59 Table 6: Summary of Practical Disinfectants ...... 61 Table 7: Reprocessing Methods for Equipment Used in the Health Care Setting ...... 62

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Biosafety Manual November, 2008 xi

1 -- Biological Safety

Principles of Biological Safety The following is from Biosafety in Microbiological and Biomedical Laboratories, 1999, HHS publication No. (CDC) 93-8395, 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

2 November, 2008 Biosafety Manual appropriate facility design and engineering features, safety equipment, and management practices.

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 biological safety cabinets (Class I, II, III) used in microbiological laboratories are described and illustrated in Appendix A. Open- fronted Class I and Class II biological safety cabinets 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 biological safety cabinet 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 biological safety cabinet 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 BSCs 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

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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 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 Air-conditioning (HVAC) published by the American Society of Heating, Refrigerating, and Air-conditioning Engineers (ASHRAE).

Biosafety Levels Four biosafety levels (BSLs) are described which consist of combinations of laboratory practices and techniques, safety equipment, and laboratory facilities used to control hazardous biological materials. 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 biosafety level(s) for the organisms represent those conditions under which the agent can ordinarily be safely handled. The laboratory director 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.

4 November, 2008 Biosafety Manual

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. Hepatitis B virus, Salmonellae, 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 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. 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

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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.

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

6 November, 2008 Biosafety Manual 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 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.

Please see the section entitled “Shipment of Biological Materials” for more information on permits.

Biological Safety Levels The following is from Biosafety in Microbiological and Biomedical Laboratories, 1999, HHS publication No. (CDC) 93-8395, Centers for Disease Control & Prevention/National Institutes of Health

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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:

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)

8 November, 2008 Biosafety Manual 1. Special containment devices or equipment such as a biological safety cabinet are generally not required for manipulations of agents assigned to Biosafety Level 1. 2. 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. 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.

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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 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 Biosafety manual is prepared or adopted. Personnel are advised of special hazards and are required to read and to follow instructions on practices and procedures.

10 November, 2008 Biosafety Manual 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 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)

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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 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 must be constructed of water impervious material that will stand up to harsh disinfectants. Carpeting is not allowed in laboratory facilities. 3. Bench tops are 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.

12 November, 2008 Biosafety Manual 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 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. It is strongly recommended that persons have a minimum of 120 hour work experience in a Level 2 laboratory to qualify to work at Level 3.

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

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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 off-site 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. 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.

14 November, 2008 Biosafety Manual 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 at-risk personnel. Additional serum specimens may be collected periodically, depending on the agents handled or the function of the laboratory. 7. A Biosafety Manual 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. 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, preferably 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 contaminated needles, sharp equipment, and broken glass should be decontaminated before disposal, in accordance with any local, state, or federal regulations.

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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. 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). 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.

16 November, 2008 Biosafety Manual 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 water-resistant so that they can be easily cleaned. Penetrations in 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 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 is 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 direction of the airflow (into the laboratory) is proper. Visual monitoring devices 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

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outside or through the building exhaust system. If the HEPA-filtered exhaust air from Class II or III 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 biological safety cabinets may be recirculated within the laboratory if the cabinet is tested and certified at least every twelve months. 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 and HEPA filters, or their equivalent, which are routinely maintained and replaced as needed. 12. An eyewash/safety shower is readily available. 13. A 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 re-verified/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 EH&S 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.

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

18 November, 2008 Biosafety Manual Institutional Biosafety Committee). 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. Incineration is recommended. 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

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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 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.

20 November, 2008 Biosafety Manual potentially present (e.g., hepatitis B vaccine, TB skin testing). When appropriate, a serum surveillance system should be implemented. 4. A biosafety 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. 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. Autoclaving of the contents prior to incineration is recommended. 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. Plasticware 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 prevent exposures, and the exposure evaluation procedures.

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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 self-closing 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 water resistant.

22 November, 2008 Biosafety Manual 4. Internal facility appurtenances, such as light fixtures, air ducts, and utility pipes, are arranged to minimize horizontal surface areas. 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

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the animal facility unless special procedures can eliminate the extra risk. Assessment should be made by the occupational health physician. 4. A biosafety 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 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. Incineration is recommended. The outer surface of the containers is disinfected prior to moving the material (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. Plasticware 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.

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B. Special Practices 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. All wastes from the animal room must be autoclaved prior to incineration or other appropriate terminal treatment. 4. 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.

Biosafety Manual November, 2008 25

These include necropsy of infected animals, harvesting of tissues or fluids from infected animals or eggs, or intranasal inoculation of animals. At BSL-3, all work should 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, 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 (air-lock) 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 water resistant. 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. 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

26 November, 2008 Biosafety Manual 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 biological safety cabinet can be recirculated into the animal room if the cabinet is tested and certified at least annually. When exhaust air from Class II safety cabinets is to be 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., an air gap between the cabinet exhaust and the exhaust duct). When Class III biological safety cabinets are used, they should be directly connected to the exhaust system. If the Class III cabinets are connected to the supply system, it is done in a manner that prevents positive cabinet pressurization. 10. Cages are washed in a cage washer. The mechanical cage washer has a final rinse temperature of at least 180°F. 11. An autoclave is available which is convenient to 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. 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 re-verified 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.

Biosafety Manual November, 2008 27

Table 1: Summary of Recommended Biosafety Levels for Infectious Agents

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

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Table 2: Summary of Recommended Biosafety Levels for Activities in Which Experimentally or Naturally Infected Vertebrate Animals Are Used

BSL Agents Practices Safety Equipment Facilities (Primary Barriers) (Secondary Barriers) 1 Not known to Standard animal care and management As required for normal care of each Standard animal facility consistently cause practices, including appropriate medical species • No recirculation of exhaust air disease in healthy surveillance programs • Directional air flow recommended adults • Hand washing Sink recommended 2 Associated with ABSL-1 practice plus: ABSL-1 equipment plus barriers: ABSL-1 facility plus: human disease, hazard • Limited access containment equipment appropriate for • Autoclave available = percutaneous injury, • Biohazard warning signs animal species; PPES; laboratory coats, • Hand washing sink available in ingestion, mucous • “Sharps” precautions gloves, face and respiratory protection animal room membrane exposure • Decontamination of all as needed. • Mechanical cage washer used infectious wastes and of animal cages prior to washing

3 Indigenous or exotic ABSL-2 practice plus: ABSL-2 equipment plus: ABSL-2 facility plus: agents with potential • Controlled access • Containment equipment for • Physical separation from access for aerosol • Decontamination of all waste housing animals and cage corridors transmission; disease • Cages decontaminated before dumping activities • Self-closing, double-door access may have serious or bedding removed • Class I or II BCSs available • Sealed penetrations lethal consequences • Disinfectant foot bath as for manipulative • Sealed windows needed procedures(inoculation, • Autoclave available in facility • Decontamination of all effluent necropsy) that may create infectious aerosols. PPEs: appropriate respiratory protection 4 Dangerous/exotic ABSL-3 practice plus: ABSL-3 equipment plus maximum ABSL-3 facility plus: agents which pose • Entrance through change room containment equipment (i.e. Class III • Separate building or isolated zone high risk of life- where personal clothing is BCS or partial containment equipment • Dedicated air supply and exhaust, threatening disease, removed and laboratory in combination with full body, air- vacuum and decontamination aerosol-transmitted clothing is put on; shower on supplied positive-pressure personnel systems lab infections; or exit suit) used for all procedures and • Other requirements outlined in the related agents with • All wastes are decontaminated activities text unknown risk of before removal from facility transmission

Biosafety Manual November, 2008 29

Agents List

The following agents have been listed according to the most appropriate Biological Safety Level to be used. The list presented below is based upon the risk groups given in Appendix B of the March 1996 Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines), the agent summary statements in the CDC/NIH publication, Biosafety in Microbiological and Biomedical Laboratories (BMBL), 4th edition (1999), guidance from state and local regulatory agencies, and recommendations of the CDC.

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 Biological Safety Office (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 Biological Safety Office 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 (<109 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.

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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 • Bacillus anthracis • 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, C. trachomatis, C. pneumoniae • Clostridium botulinum, Cl. chauvoei, Cl. 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 - all species except K. oxytoca (BSL-1) • 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. leprae, M. malmoense, M. marinum, M. paratuberculosis, M. scrofulaceum, M. simiae, M. szulgai, M. ulcerans, M. xenopi • Mycoplasma, except M. mycoides and M. agalactiae which are restricted animal pathogens • Neisseria gonorrhoeae, 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

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• Streptobacillus moniliformis • Streptococcus including S. pneumoniae, S. pyogenes • Treponema pallidum, T. carateum • Vibrio cholerae, V. parahemolyticus, V. vulnificus • Yersinia enterocolitica BSL-2 - Fungal Agents • Blastomyces dermatitidis • Cladosporium bantianum, C. (Xylohypha) trichoides • Cryptococcus neoformans • Dactylaria galopava (Ochroconis gallopavum) • Epidermophyton • Exophiala (Wangiella) dermatitidis • Fonsecaea pedrosoi • Microsporum • Paracoccidioides braziliensis • Penicillium marneffei • Sporothrix schenckii • Trichophyton BSL-2 - Parasitic Agents • Ancylostoma human hookworms including A. duodenal, 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) • Echinococcus including E. granulosis, E. multilocularis, E. vogeli • Entamoeba histolytica • Enterobius • Fasciola including F. gigantica, F. hepatica • Giardia including G. lamblia • Heterophyes • Hymenolepis including H. diminuta, H. nana • Isospora • Leishmania including L. braziliensis, L. donovani, L. ethiopia, L. major, L. mexicana, L. peruvania, L. tropica • Loa loa filaria worms • Microsporidium • Naegleria fowleri • 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

32 November, 2008 Biosafety Manual • Schistosoma including S. haematobium, S. intercalatum, S. japonicum, S. mansoni, S. mekongi • Strongyloides including S. stercoralis • Taenia solium • 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 BSL-2 - Viruses Adenoviruses, human - all types

Alphaviruses (Togaviruses) - Group A Arboviruses • Eastern equine encephalomyelitis virus • Venezuelan equine encephalomyelitis vaccine strain TC-83 • Western equine encephalomyelitis virus

Arenaviruses • Lymphocytic choriomeningitis virus (non-neurotropic strains) • Tacaribe virus complex • Other viruses as listed in the BMBL

Bunyaviruses Bunyamwera virus Rift Valley fever virus vaccine strain MP-12 Other viruses as listed in the BMBL

Calciviruses

Coronaviruses

Flaviviruses (Togaviruses) - Group B Arboviruses Dengue virus serotypes 1, 2, 3, and 4 Yellow fever virus vaccine strain 17D Other viruses as listed in the BMBL

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

Biosafety Manual November, 2008 33

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 in the BMBL

Papovaviruses • All human papilloma viruses • Bovine papilloma virus • Polyoma virus • Shope papilloma virus • Simian virus 40 (SV40)

Paramyxoviruses Newcastle disease virus Measles virus Mumps virus Parainfluenza viruses types 1, 2, 3, and 4 Respiratory syncytial virus

Parvoviruses Human parvovirus (B19)

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, Smallpox, and Whitepox (restricted to the CDC, Atlanta, GA)

Reoviruses - all types including Coltivirus, human Rotavirus, and Orbivirus (Colorado tick fever virus)

Retroviruses Avian leukosis virus Avian sarcoma virus Bovine leukemia virus Clinical samples from HIV-positive patients Feline immunodeficiency virus Feline leukemia virus Feline sarcoma virus

34 November, 2008 Biosafety Manual Gibbon leukemia virus Mason-Pfizer monkey virus Mouse mammary tumor virus Murine leukemia virus Murine sarcoma virus Rat leukemia virus

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 of detectable replication competent retrovirus can be maintained, handled, and administered, under BL1 containment.

Rhabdoviruses Rabies virus - all strains Vesicular stomatitis virus - laboratory adapted strains ONLY including VSV-Indiana, San Juan, and Glasgow Togaviruses (see Alphaviruses and Flaviviruses) Rubivirus (rubella)

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 • Bartonella • Brucella including B. abortus, B. canis, B. suis • Burkholderia (Pseudomonas) mallei, B. pseudomallei • Coxiella burnetii • Francisella tularensis • Mycobacterium bovis (except BCG strain, BSL-2), M. tuberculosis • 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 • Coccidioides immitis (sporulating cultures; contaminated soil) • Histoplasma capsulatum, H. capsulatum var.. duboisii BSL-3 - Parasitic Agents None BSL-3 - Viruses and Prions Alphaviruses (Togaviruses) - Group A Arboviruses • Semliki Forest virus • St. Louis encephalitis virus

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• Venezuelan equine encephalomyelitis virus (except the vaccine strain TC-83 is BSL- 2) • Other viruses as listed in the BMBL

Arenaviruses • Lymphocytic choriomeningitis virus (LCM) (neurotropic strains) • Flexal

Bunyaviruses • Hantaviruses including Hantaan virus • Rift Valley fever virus

Flaviviruses (Togaviruses) - Group B Arboviruses • Japanese encephalitis virus • Yellow fever virus • Other viruses as listed in the BMBL

Poxviruses • Monkeypox virus

Prions • Transmissible spongiform encephalopathies (TME) agents, Creutzfeldt-Jacob disease and kuru agents (see BMBL for specific containment instruction)

Retroviruses • Human immunodeficiency virus (HIV) types 1 and 2 • Human T cell lymphotropic virus (HTLV) types 1 and 2 • Simian immunodeficiency virus (SIV)

Rhabdoviruses • Vesicular stomatitis virus

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

36 November, 2008 Biosafety Manual • 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.

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2 -- Information for Researchers

Project Registration Some research projects involve work with potentially hazardous biological agents, known infectious disease agents, or biological materials regulated by the federal or state government. Many 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 Biological Safety Office administers four registration programs for research projects.

Bio-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 Biological Safety Office.

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 used at UF. 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 that are known to be HIV positive (or positive for any human disease-causing virus or other agent), when used in research, must be registered.

Recombinant DNA (R-DNA) Registration All R-DNA projects that involve a living recombinant organism (this excludes projects that involve DNA only, i.e. PCR) require registration with the Biological Safety Office. A subset of R-DNA projects requires review and approval from the Institutional Biosafety Committee (IBC). The UF IBC oversees all research projects and issues involving R-DNA at UF. 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 Biological Safety Office, in conjunction with the IBC, will make the final determination. Biosafety Manual November, 2008 39

Acute Toxins (AT) Registration

The use and storage of chemicals with a mammalian LD50 of < 100 μg/kg. For a partial list, see the Toxins Table that follows.

Regulated Biological Materials Agents, such as plant pathogens or exotic microorganisms, that are regulated by federal or state agencies (USDA/APHIS, EPA, FDA, DPI, etc.) shall be registered with the Biological Safety Office by submission of a biological agent registration form and a photocopy of the permit and permit conditions that have been granted by that agency. No special form is required unless the agent fits into one of the first three categories.

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Table 3: Toxin Table

Toxins with a mammalian LD50 of < 100 μg/kg must be registered with the Biological Safety Office. Therefore, use of the following toxins may require registration. If a toxin is not on the list, it still may require registration, depending upon the LD50. For more information, please contact the Biological Safety Office at 392-1591.

Toxicity

LD50 (μg/kg)*

Abrin 0.7 Aerolysin 7.0 Botulinin toxin A 0.0012 Botulinin toxin B 0.0012 Botulinin toxin C1 0.0011 Botulinin toxin C2 0.0012 Botulinin toxin D 0.0004 Botulinin toxin E 0.0011 Botulinin toxin F 0.0025 β-bungarotoxin 14.0 Caeruleotoxin 53 Cereolysin 40-80 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 13-16 Clostridium perfringens enterotoxin 81 Clostridium perfringens beta toxin 400 Clostridium perfringens delta toxin 5 Clostridium perfringens epsilon toxin 0.1 Conotoxin 12-30 Crotoxin 82 Diphtheria toxin 0.1 Listeriolysin 3-12 Leucocidin 50 Modeccin 1-10 Nematocyst toxins 33-70 Notexin 25 Pertussis toxin 15

Biosafety Manual November, 2008 41

Toxicity

LD50 (μg/kg)*

Pneumolysin 1.5 Pseudomonas aeruginosa toxin A 3 Ricin 2.7 Saxitoxin 8 Shiga toxin 0.250 Shigella dysenteriae neurotoxin 1.3 Streptolysin O 8 Staphylococcus enterotoxin B 25 Staphylococcus enterotoxin F 2-10 Streptolysin S 25 Taipoxin 2 Tetanus toxin 0.001 Tetrodotoxin 8 Viscumin 2.4-80 Volkensin 1.4 Yersinia pestis murine toxin 10

*Please note that the LD50 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: 86-94 2. 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: 405-412 3. Registry of toxic effects of chemical substances (RTECS): comprehensive guide to the RTECS. 1997. 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

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Select Agents

The following lists of agents, toxins, and pathogens are classified by the Federal government as Select Agents. Any possession, use, transfer or shipment of these materials is strictly controlled by regulation.

See the EH&S web site at www.ehs.ufl.edu/bio for additional information and the UF Select Agent Policy currently in place.

Researchers considering work with any of these materials must first contact the UF Responsible Official at 392-1591 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.

Biosafety Manual November, 2008 43

Select Agents List

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Maximum Toxin Amounts Excluded from Regulation

HHS Toxins Amount Abrin 100mg Conotoxin 100mg Diacetoxyscirpenol (DAS) 1000mg Ricin 100mg Saxitoxin 100mg Shiga-like ribosome inactivating proteins 100mg Tetrodotoxin 100mg

Overlap toxins Amount Botulinum neurotoxins 0.5mg Staphylococcal enterotoxins 5.0mg Clostridium perfringens epsilon toxin 100mg Shigatoxin 100mg T-2 toxin 1000mg

Biosafety Manual November, 2008 45

Minors in Research Laboratories or Animal Facilities

Unless enrolled as a University of Florida student, minors are not allowed to work or conduct research in University of Florida 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.

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. See ACS Policy # ACS-PY-012. (Note that this does not apply to individual Principal Investigator animal research laboratories).

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 above) if the following requirements are met in full:

a. The University of Florida EH&S Policy titled; Minors In Research Laboratories Or Animal Facilities (www.ehs.ufl.edu/bio/minors.htm) has been read and understood. b. A minor’s research proposal registration form (www.ehs.ufl.edu/bio/MinorReg.pdf) is submitted to and approved by the University of Florida, Division of Environmental Health and Safety, or the Institutional Biosafety Committee. Included in this form is The Potential Hazards information sheet which requires a parental/guardian signature indicating he/she has read of potential risks c. Hazard specific safety training is completed by the Principal Investigator/Sponsor with the minor as approved by EH&S. 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 570.35. g. The laboratory is in full compliance with all applicable University of Florida safety programs and regulations.

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Biological Waste Disposal Policy This policy is intended to provide guidance and insure compliance with the NIH/CDC guidelines, the State of Florida Administrative Code 64E-6, and restrictions of the Alachua 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. 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 wastes 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.

IV packs test tubes petri dishes needles* razor blades* tissue culture flasks syringes culture dishes scalpels* flasks broken glass and plasticware** 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 many radioactive materials can be autoclaved safely, please check with the Radiation Safety

Biosafety Manual November, 2008 47

Office (392-7359) 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 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 Biological Safety Office (392-1591) for guidance regarding particular chemicals. 5) Animal carcasses and materials The disposal of animal carcasses and other animal materials shall be through the Animal Care Services incinerators or the Veterinary Medicine tissue digesters only. These incinerators and digesters are for animal materials only. Please contact Animal Care Services (392-2977) for further information. 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 (392-3588) 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. These bags must be placed in cardboard boxes (see #3, below) prior to disposal. 2) Sharps Needles, scalpels and razor blades are required to be containerized in red plastic sharps containers. These are provided by Building Services (392-4414) at the Health Center. All other sharps (broken glass and plasticware, pipettes, etc.) shall be containerized in puncture-resistant cardboard boxes (see #3, below). These are also available from Building Services. 3) Corrugated cardboard boxes All biological waste must be containerized in rigid, leak proof, puncture resistant boxes as the terminal receptacle. The appropriate boxes are available from Building Services at the Health Center, 392-4414.

Labeling All packages containing biological waste shall be labeled with indelible ink marker (i.e., Sharpie®) as follows:

48 November, 2008 Biosafety Manual 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. Corrugated boxes (biomedical waste boxes) shall be labeled with the date the biohazardous waste was treated. Boxes used for non-biohazardous waste collection shall be dated when the box is sealed. 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. 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 or a biomedical waste box with the universal biohazard symbol. Only corrugated biomedical boxes 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 must be avoided. Any leakage/spills from these containers must be immediately reported to the Biosafety Office at 392-1591. 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 UF 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 Biological Safety Office at (392-1591).

According to Florida 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.

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Biological Waste Disposal Containers The following waste disposal containers for biohazardous or biomedical waste are to be used for teaching and research purposes at the University of Florida:

ALL BIOHAZARDOUS WASTE MUST BE TREATED BEFORE DISPOSAL

Biohazard Boxes Biohazard boxes lined with red bags shall be used to dispose of treated (autoclaved or bleached) biomedical waste. These boxes are available from HSC Building Services (392-4414). Boxes shall be labeled with principal investigator’s name, phone number, date of treatment, and lab location (room number). The ¾ full boxes shall be labeled with indelible ink markers (e.g. Sharpie®). The ¾ full boxes shall be sealed with a leak- resistant, clear, strapping tape in an H pattern. All edges where leakage could occur shall be taped. Trained research or custodial staff only shall handle the full boxes.

H-Pattern Taping Sequence

1st 2nd 3rd

Disposal of biomedical waste in the regular trash or dumpster is prohibited. The Alachua 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 Red plastic sharps boxes are used for disposal of needles, razor blades, scalpels, and small Pasteur pipettes. These are available from HSC Building Services (2-4414). 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). Trained research or custodial staff only shall handle the full sharps boxes.

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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 biohazard boxes. They may be transported to the biomedical waste receptacle by trained laboratory personnel or by trained custodial 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 and fluid. Full, untreated biohazard bags shall be stored only in the lab where accumulation occurred. Full biohazard bags must be treated within 24 hours.

Fold box flaps down. Seal bag by twisting Double upper section Insert labeled liner and fold and taping. over and tape to over edges. double seal the bag. Autoclave Use and Testing Rules governing the use and testing of autoclaves are based on Rule 64E-16 of the Florida Administrative Code.

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) Every 6 months Required for autoclaves that are used to inactivate other material. 2) Method A commercially available test indicator kit that uses bacterial spores (Geobacillus stearothermophilus) is the approved method of testing autoclave efficiency. Most spore vial test kits require 56°C to 60°C incubation of the autoclaved test vial Biosafety Manual November, 2008 51

along with a non-autoclaved 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 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

52 November, 2008 Biosafety Manual 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.

Autoclave Operation and Safety Training Autoclave training is available from the EH&S Biological Safety Office both quarterly and 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.

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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 --- 100 MINUTES OR LONGER

SINGLE BAG (FULL) --- 90 MINUTES

PARTIAL BAG --- 60 MINUTES

DRY GOODS

GLASSWARE, EMPTY, INVERTED --- 15 MINUTES

INSTRUMENTS, WRAPPED --- 30 MINUTES

UTENSILS, WRAPPED --- 30 MINUTES

LIQUIDS (Bottles with vented caps ½ FULL)

75mL --- 25 MINUTES 250mL --- 30 MINUTES 500mL --- 40 MINUTES 1000mL --- 45 MINUTES 1500mL --- 50 MINUTES 2000mL --- 55 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.

Questions? Please call Gary Smith at 392-1591

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Disinfectants “Disinfectant” refers to an agent that is applied to treat (usually) inanimate objects to render them free of pathogenic or infectious non-sporeforming microorganisms. In contrast, the term “sterilant” refers to an agent that renders items free of all microorganisms, including spores. 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 (http://www.epa.gov/spdpublc/snap/sterilants/index.html).

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% (v/v) in water. Both higher and lower concentrations are less effective. Alcohols are active against vegetative bacteria, fungi, and lipid containing viruses but not against spores. Their action on lipid-containing viruses is variable. 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

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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. 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. Clorox) 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 1:1 5.25 50,000 1 9 1:10 0.5 5,000 1 99 1:100 0.05 500 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 kills all microorganisms and spores but not prions. It is used for space decontamination and to decontaminate biological safety cabinets, a dangerous process requiring specifically trained personnel. Formaldehyde dissolved in water is active at 1-8% solutions and can be used to decontaminate hard surfaces. However, because formaldehyde is an irritant at low concentrations (0.1 to 5 ppm) and a probable 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 2% 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 all microorganisms, but is toxic, an irritant, 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 to decontaminate work surfaces of laboratory benches and biosafety cabinets. It is active against a wide array of microorganisms. However, it is an oxidizing

56 November, 2008 Biosafety Manual

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, 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 non-lipid viruses and, when properly formulated, show anti-mycobacterial activity. There are many common disinfectants based on phenol and they should be used according to the manufacturer’s recommendations. Quaternary 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 Inactivates micro-organisms, cellular disruption; used only as surface sterilizer Temp 50°C-60°C, Humidity 30-40% Conc. 400-800 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°C-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 25°C-30°C, pre-humidification required Limited to surfaces, no penetration Corrosive to some materials Mucous membrane irritant Ozone Biosafety Manual November, 2008 57

Oxidizing properties, inhibits bacterial growth, reacts with amino acids, RNA/DNA Temp 25°C Conc. 2-5 mg/liter Systems convert oxygen to ozone Limited penetration Formaldehyde Gas (from heating paraformaldehyde) Temp 20°C-22°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, 250-260 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 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, most common technique used for commercial sterilization of single- use items, 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. 1991. Seymour S. Block ed., Lea & Fibiger, Philadelphia Laboratory Biosafety Manual. Third edition. 2004. World Health Organization, Geneva. Biological Safety: Principles and practices. Third edition. 2000. Diane O. Fleming et al. eds. ASM Press, Washington DC Manual of Clinical Microbiology. Eighth edition. 2003. Patrick Murray ed., ASM press, Washington DC Prudent Practices in the Laboratory: Handling and disposal of chemicals. 1995 National Research Council. National Academy Press, Washington

58 November, 2008 Biosafety Manual 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 70% Isopropyl • Cleaning some • Changes protein • Fairly inexpensive • <50% solution not very •Flammable alcohol instruments structure of effective •Eye Irritant solution • Cleaning skin microorganism • Not active when organic •Toxic • Presence of water matter present assists with killing action • Not active against certain types of viruses • Evaporates quickly - contact time not sufficient for killing Chlorine • Spills of human body • Free available chlorine • Kill hardy viruses • Corrode metals such as • Follow spill procedure • Bleach compounds fluids combines with contents (e.g., hepatitis) stainless, aluminum and dilution instructions solutions • Bactericidal - Good within microorganism - • Kill a wide range of • Organics may reduce • Make fresh solutions (sodium • Fungicidal - Good reaction byproducts cause organisms activity before use hypochlorite) • Sporicidal - Good at its death • Inexpensive • Increase in alkalinity • Eye, skin, and • Clorox >1000 ppm Sodium • Need 500 to 5000 ppm • Penetrates well decreases bactericidal property respiratory irritant • Cryosan Hypochlorite • Produce chemical • Relatively quick • Unpleasant taste and odor •Corrosive • Purex combination with cell microbial kill • Tuberculocidal - with •Toxic substances • May be used on food extended contact time • Depend upon release of prep surfaces hypochlorous acid Gluteraldehyde • Bactericidal - Good • Coagulates cellular • Non-staining, • Not stable in solution (shelf •Eye, skin and • Calgocide • Fungicidal - Good proteins relatively non- life 14 days) respiratory irritant. 14 • Tuberculocidal – corrosive • Has to be in alkaline •Sensitizer • Cidex slow acting • Useable as a sterilant solution •Toxic • Vespore • Virucidal - Good on plastics, rubber, • Inactivated by organic • Sporicidal - Good lenses, stainless steel, material and other items that can’t be autoclaved

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

Barbara Fox Nellis (8-23-96)

60 November, 2008 Biosafety Manual Table 6: Summary of Practical Disinfectants

Quaternary Ammonium Phenolic Chlorine Isopropyl Compounds Compounds Compounds Iodophor Ethyl Alcohol Alcohol Formaldehyde Glutaraldehyde Inactivates Vegetative Bacteria + + + + + + + + Lipoviruses + +a + + +a + + + Nonlipid Viruses - - + + - - + + Bacterial Spores - - + + - - + + Treatment Requirements Use Dilution 0.1-2.0% 1.0-5.0% 500ppmb 25-1600ppmb 70-85% 70-85% 0.2-0.8% 2% Contact Time-minutes Lipovirus 10 10 10 10 10 10 10 10 Broad Spectrum NE NE 30 30 NE NE 30 30 Important Characteristics Effective Shelf Life + + - + + + + + > 1 weekc Corrosive - + + + - - - - Flammable - - - - + + - - Explosion Potential ------Inactivated by organic + - + + - - - - matter Skin Irritant + + + + - - + + Eye Irritant + + + + + + + + Respiratory Irritant - - + - - - + + Toxicd + + + + + + + + Applicability Waste Liquids - - + - - - - - Dirty Glassware + + + + + + + + Equipment, Surface + + + + + + + + Decontamination Proprietary Productse A-33, CDQ, End-Bac, Hll-Phene, Chloramine Hy-Sine, Ioprep, Sterac Cidex Hl-Tor, Mikro-Quat Metar, Mikro- T, Clorox, Mikroklene, Bac, O-Syl Purex 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: 104-105 + Yes a Variable results depending on virus d By skin or mouth or both b e - No Available Halogen 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.

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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.

Steam under pressure (autoclave), gas (ethylene oxide), dry heat or immersion in an approved chemical “sterilant” (e.g., US Environmental Methods: 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 Use: 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.

High-Level Destroys: All forms of microbial life except high numbers of bacterial spores. Disinfection: Hot water pasteurization (80-100°C, 30 minutes) or exposure to an approved (e.g., US EPA-approved) “sterilant” chemical as above, except for a Methods: short exposure time (10-45 minutes or as directed by the manufacturer).

Use: For reusable instruments or devices that come into contact with mucous membranes (e.g., laryngoscope blades, endotracheal tubes, etc.).

Intermediate- Destroys: Mycobacterium tuberculosis, vegetative bacteria, most viruses and most fungi, but does not kill bacterial spores. Level Disinfection: Approved (e.g. US EPA-approved) “hospital disinfectant” chemical germicides that have a label claim for tuberculocidal activity; commercially Methods: available hard-surface germicides or solutions containing at least 500 ppm free available chlorine (a 1:10 dilution of common household bleach- about 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 Use: contaminated with blood or bloody body fluids. Surfaces must be pre-cleaned of visible material before the germicidal chemical is applied for disinfection.

Low-Level Destroys: Most bacteria, some viruses, some fungi, but no Mycobacterium tuberculosis or bacterial spores. Disinfection: Methods: Approved (e.g., US EPA-approved) “hospital disinfectants” (no label claim for tuberculocidal activity).

Use: Use these excellent cleaning agents for routine housekeeping or removal of soiling in the absence of visible blood contamination.

Environmental Environmental surfaces that have become soiled should be cleaned and disinfected using any cleaner or disinfectant that is intended for

Disinfection: 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.

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Shipment of Biological Materials The following regulations apply to the packaging and shipment of biological materials:

U.S. Department of Transportation, 49 CFR Parts 171-180 and amendments U.S. Public Health Service, 42 CFR Part 72, Interstate Shipment of Etiologic Agents U.S. Department of Labor, Occupational Safety and Health Administration, 29 CFR Part 1910.1030, 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.

Many biological materials fall into the category of “dangerous goods” for shipping purposes. All individuals involved in the transport of dangerous goods or the preparation of dangerous goods for transport must be trained to do so properly and safely. In addition, we require safe transport of items within facilities and around campus. These topics are covered in the “Shipping and Transport of Biological Materials” training given by the Biosafety Office.

You must have a training certificate from UF EH&S to ship biological materials. Call us or see our list of scheduled classes at http://www.ehs.ufl.edu/Bio/default.asp.

Training is valid for 2 years and is a federal requirement designed to protect yourself, your co-workers, the public - drivers, airline staff, crew, pilots, passengers, and package recipients. Training also ensures successful shipments; carriers or Federal regulators may open, delay, or reject your shipment if it’s not correct. Training also prevents penalties for violations; civil penalties range from $250 to $27,500 per violation per day, and criminal penalties for willful violations range up to $500K and 5 years in jail.

Classes cover the following:

1. Classifying the material – Is it regulated? Is it forbidden for transport? 2. Identifying the material – select the proper shipping name 3. Choosing the right packaging 4. Packaging it correctly

5. Marking & Labeling the shipment correctly 6. Supplying additional required documentation – dangerous goods declaration forms 7. Making shipping arrangements – i.e. permits, customs documents for overseas shipments 8. Transporting things safely around UF – hand carrying & vehicle transport

Biological materials subject to shipping & transport regulations:

In the context of shipping regulations, “Dangerous Goods” are “Articles or substances which are capable of posing a risk to health, safety, property or the environment & which are shown in the list of dangerous goods in the Regulations or which are classified according to these Regulations.” (49 CFR Parts 100-185 & IATA 1.0).

Biological materials that fall under this definition include:

Biological toxins Infectious substances Diagnostic specimens Biomedical waste Cultures Genetically Modified Organisms

Other biological material that may be regulated via state or federal permits:

Plants, Plant pests Insects Cell cultures Live animals

Items that frequently accompany shipments of biological material are also regulated:

Dry ice Environmental pollutants (e.g. formalin) Alcohol Fixative solutions

Transporting biological material within and around UF:

The following general guidelines apply:

64 November, 2008 Biosafety Manual Double contain the items plastic leak-proof containers within sturdy outer packaging. Include absorbent material within the containers as well as padding to minimize movement of the container(s) within the outer packaging. Wipe the outer container with an appropriate disinfectant before removing it from the laboratory and apply a biohazard sticker if applicable. Put your name and contact information on the package. Individuals transporting biohazardous agents should be knowledgeable about handling spills.

UF policy states that dangerous goods are not to be transported in your personal vehicle. This is both a safety and liability issue. Use a state vehicle.

Shipping radioactives? Call 392-7359 Shipping chemicals? Call 392-8400

Permits: Federal or state permits may be required for some biological materials. See below or contact us at 392-1591 for more information. Permits are issued in the name of the PI who is required to keep them updated and current as necessary; the Biosafety Office does not hold any “centralized” permits.

1. CDC Import Permit http://www.cdc.gov/od/eaipp/ for import of etiological agents causing disease in humans, non-sterilized human or animal tissues/fluids known or suspected to contain disease agents, hosts/vectors known or suspected to contain disease agents

2. USDA/APHIS Veterinary Permit http://www.aphis.usda.gov/vs/ncie/ needed for import of materials derived from (livestock/poultry) animals or exposed to (livestock/poultry) animal-source materials, including: animal tissues, blood, cells or cell lines of livestock or poultry origin, RNA/DNA extracts, hormones, enzymes, monoclonal antibodies for IN VIVO use in non-human species, certain polyclonal antibodies and antisera, bulk shipments of test kit reagents, arthropod vectors of livestock diseases, and microorganisms infectious to livestock including bacteria, viruses, protozoa, and fungi. 3. Interstate movement of microorganisms infectious to livestock/poultry including bacteria, viruses, protozoa, and fungi, arthropod vectors of livestock/poultry diseases, and tissues, blood, serum, or cells from known infected livestock/poultry.

Note: A courtesy letter to the Florida Department of Agriculture and Consumer Services Division of Animal Industry http://www.doacs.state.fl.us/ai/contact.shtml is required for possession or use of any of the State of Florida reportable animal diseases http://www.doacs.state.fl.us/ai/main/ani_diseases_main.shtml.

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4. USDA/APHIS Plant Protection & Quarantine Permit http://www.aphis.usda.gov/ppq/permits/ is needed for import or interstate movement of plant pests, plant pathogens, biological control agents, bees, plant pest diagnostic laboratories, soil microbe isolation laboratories, federal noxious weeds and parasitic plants

5. USDA Biotechnology Regulatory Services Notification or Permit http://www.aphis.usda.gov/brs/fieldtesting_importation.html for the import, interstate movement, or field release of genetically engineered plants, arthropods, and plant-associated microorganisms.

6. Florida Department of Agriculture and Consumer Services Division of Plant Industry Permit http://www.doacs.state.fl.us/onestop/plt/entnempath.html for the import into Florida of: arthropods, plant pathogens, nematodes, noxious weeds, genetically altered (insects, nematodes, plants, plant pests) organisms, biological control agents. 7. The transfer of Select Infectious Agents and Toxins is also regulated by the USDA/CDC. Each shipment of listed agents must be registered with the USDA/CDC through a responsible facility official at both the shipping and receiving entities. Please contact EH&S Biosafety before sending or requesting any Select Agents. The current list of Select Agents and Toxins can be found on in this manual or on our website at http://www.ehs.ufl.edu/Bio/select.htm. 8. Export of Etiologic Agents of Humans, Animals, Plants and Related Materials is regulated by the US Department of Commerce, Dept. of State, and Dept. of the Treasury. A wide variety of etiologic agents of human, plant and animal diseases, including genetic material, and products which might be used for culture or production biological agents, will require an export license. Information may be obtained by calling the Biosafety Office at 352-392-1591. Export to certain countries is prohibited.

66 November, 2008 Biosafety Manual 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). The Class II, Type A BSC An unducted Class II Type A BSC is not to be used for work involving volatile or toxic chemicals. The buildup of chemical vapors in the cabinet (by recirculated air) and in the

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laboratory (from exhaust air) could create health and safety hazards. Generally, BSCs are not for use with chemicals. Small quantities of chemicals and chemotherapeutic agents may be used in ducted BSCs.

It is possible to duct the exhaust from a Type A cabinet out of the building. However, it must be done in a manner that does not alter the balance of the cabinet exhaust system, thereby disturbing the internal cabinet air flow. The typical method of ducting a Type A cabinet is to use a “thimble,” or canopy unit, which maintains a small opening (usually 1 inch) around the cabinet exhaust filter housing. The volume of the exhaust must be sufficient to maintain the flow of room air into the space between the thimble unit and the filter housing (contact manufacturers for any additional specifications). The thimble must be removable or be designed to allow for operational testing of the cabinet. The performance of a cabinet with this exhaust configuration is unaffected by fluctuations in the building exhaust system.

“Hard-ducting” (i.e., direct connection) of Class II Type A cabinets to the building exhaust system is not recommended. The building exhaust system must be precisely matched to the airflow from the cabinet in both volume and static pressure. However, fluctuations in air volume and pressure that are common to all building exhaust systems make it difficult, if not impossible, to match the airflow requirements of the cabinet. The Class II, Type B1 BSC Some biomedical research requires the use of small quantities of certain hazardous chemicals, such as carcinogens. The powdered form of these carcinogens should be weighed or manipulated in a chemical fume hood or a static-air glove box. Carcinogens used in cell culture or microbial systems require both biological and chemical containment.

Type B1 cabinets must be hard-ducted to their own dedicated exhaust system. As indicated earlier, blowers on laboratory exhaust systems should be located at the terminal end of the ductwork. A failure in the building exhaust system may not be apparent to the user, as the supply blowers in the cabinet will continue to operate. A pressure- independent monitor should be installed to sound an alarm and shut off the BSC supply fan, should failure in exhaust airflow occur. Since all cabinet manufacturers do not supply this feature, it is prudent to install a sensor in the exhaust system as necessary. To maintain critical operations, laboratories using Type B BSCs should connect the exhaust blower to the emergency power supply. The Class II, Type B2 BSC This BSC is a total-exhaust cabinet; no air is recirculated within it. This cabinet provides simultaneous primary biological and chemical containment.

Should the building or cabinet exhaust fail, the cabinet will be pressurized, resulting in a flow of air from the work area back into the laboratory. Cabinets built since the early 1980's usually have an interlock system installed by the manufacturer to prevent the supply blower from operating whenever the exhaust flow is insufficient. Presence of

68 November, 2008 Biosafety Manual such an interlock system should be verified; systems can be retrofitted if necessary. A pressure-independent device should monitor exhaust air movement. The Class II, Type B3 BSC This BSC is a ducted Type A cabinet. All positive pressure contaminated plenums within the cabinet are surrounded by a negative air pressure plenum. Thus, leakage in a contaminated plenum will be into the cabinet and not into the environment. The Class III BSC The Class III BSC was designed for work with biosafety level 4 microbiological agents, and provides maximum protection to the environment and the worker. It is a gas-tight enclosure with a non-opening view window.

Long, heavy-duty rubber gloves are attached in a gas-tight manner to ports in the cabinet and allow for manipulation of the materials isolated inside. Although these gloves restrict movement, they prevent the user's direct contact with the hazardous materials. The trade-off is clearly on the side of maximizing personal safety. Depending on the design of the cabinet, the supply HEPA filter provides particulate-free, albeit somewhat turbulent, airflow within the work environment.

Horizontal Laminar Flow “Clean Bench” Horizontal laminar flow clean air benches are NOT BSCs. They discharge HEPA-filtered air across the work surface and toward the user. These devices only provide product protection. They can be used for certain clean activities, such as the dust-free assembly of sterile equipment or electronic devices. These benches should never be used when handling cell culture materials or drug formulations, or when manipulating potentially infectious materials. The worker can be exposed to materials (including proteinaceous antigens) being manipulated on the clean bench, which may cause hypersensitivity. Horizontal clean air benches should never be used as a substitute for a biological safety cabinet in research, biomedical or veterinary laboratories and/or applications.

Vertical Laminar Flow “Clean Bench” Vertical laminar flow clean benches also are NOT BSCs. They may be useful, for example, in hospital pharmacies when a clean area is needed for preparation of intravenous drugs. While these units generally have a sash, the air is usually discharged into the room under the sash, resulting in the same potential problems as the horizontal laminar flow clean benches.

Operations within a Class II BSC

Laboratory Hazards Many common procedures conducted in BSCs may create splatter or aerosols. Good microbiological techniques should always be used when working in a biological safety cabinet. For example, techniques to reduce splatter and aerosol generation will minimize the potential for exposure to personnel from infectious materials manipulated within the

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cabinet. Class II cabinets are designed so that horizontally nebulized spores will be captured by the downward flowing cabinet air within fourteen inches of travel. Therefore, as a general rule of thumb, keeping clean materials at least one foot away from aerosol- generating activities will minimize the potential for cross-contamination.

The general workflow should be from “clean to contaminated (dirty).” Materials and supplies should be placed in such a way as to limit the movement of “dirty” items over “clean” ones.

Several measures can be taken to reduce the chance for cross-contamination when working in a BSC. Work at least 6” back from the front edge and never cover the front grill. Opened tubes or bottles should not be held in a vertical position. Investigators working with Petri dishes and tissue culture plates should hold the lid above the open sterile surface to minimize direct impact of downward air. Bottle or tube caps should not be placed on the toweling. Items should be recapped or covered as soon as possible.

Open flames are not permitted in the near microbe-free environment of a biological safety cabinet. On an open bench, flaming the neck of a culture vessel will create an upward air current that prevents microorganisms from falling into the tube or flask. An open flame in a BSC, however, creates turbulence that disrupts the pattern of air supplied to the work surface. When deemed absolutely necessary, touch-plate microburners equipped with a pilot light to provide a flame on demand may be used. Internal cabinet air disturbance and heat buildup will be minimized. The burner must be turned off when work is completed. Small electric “furnaces” are available for decontaminating bacteriological loops and needles and are preferable to an open flame inside the BSC. Disposable sterile loops should be used to eliminate the need for heat or flame.

Aspirator bottles or suction flasks should be connected to an overflow collection flask containing appropriate disinfectant, and to an in-line HEPA or equivalent filter. This combination will provide protection to the central building vacuum system or vacuum pump, as well as to the personnel who service this equipment. Inactivation of aspirated materials can be accomplished by placing sufficient chemical decontamination solution into the flask to kill the microorganisms as they are collected. Once inactivation occurs, liquid materials can be disposed of appropriately as noninfectious waste.

Investigators must determine the appropriate method of decontaminating materials that will be removed from the BSC at the conclusion of the work. When chemical means are appropriate, suitable liquid disinfectant should be placed into the discard pan before work begins. Items should be introduced into the pan with minimum splatter, and allowed appropriate contact time as per manufacturer's instructions. Alternatively, liquids can be autoclaved prior to disposal. Contaminated items should be placed into a biohazard bag or discard tray inside the BSC. Water should be added to the bag or tray prior to autoclaving.

When a steam autoclave is to be used, contaminated materials should be placed into a biohazard bag or discard pan containing enough water to ensure steam generation during

70 November, 2008 Biosafety Manual the autoclave cycle. The bag should be taped shut or the discard pan should be covered in the BSC prior to removal to the autoclave. The bag should be transported and autoclaved in a leak-proof tray or pan.

Decontamination

Surface Decontamination All containers and equipment should be surface decontaminated and removed from the cabinet when work is completed. At the end of the workday, the final surface decontamination of the cabinet should include a wipe-down of the work surface, the cabinet's sides and back, and the interior of the glass. If necessary, the cabinet should also be monitored for radioactivity and decontaminated when necessary. Investigators should remove their gloves and gowns and wash their hands as the final step in safe microbiological practices.

Small spills within the BSC can be handled immediately by removing the contaminated absorbent paper toweling and placing it into the biohazard bag. Any splatter onto items within the cabinet, as well as the cabinet interior, should be immediately wiped with a towel dampened with decontaminating solution. Gloves should be changed after the work surface is decontaminated and before placing clean absorbent toweling in the cabinet. Hands should be washed whenever gloves are changed or removed.

Spills large enough to result in liquids flowing through the front or rear grilles require more extensive decontamination. All items within the cabinet should be surface decontaminated and removed. After ensuring that the drain valve is closed, decontaminating solution can be poured onto the work surface and through the grille(s) into the drain pan.

Thirty minutes is generally considered an appropriate contact time for decontamination, but this varies with the disinfectant and the microbiological agent. Manufacturer's directions should be followed. The spilled fluid and disinfectant solution on the work surface should be absorbed with paper towels and discarded into a biohazard bag. The drain pan should be emptied into a collection vessel containing disinfectant. A flexible tube should be attached to the drain valve and be of sufficient length to allow the open end to be submerged in the disinfectant within the collection vessel. This procedure serves to minimize aerosol generation. The drain pan should be flushed with water and the drain tube removed.

Should the spilled liquid contain radioactive material, a similar procedure can be followed. Radiation safety personnel should be contacted for specific instructions (392- 7359). Gas Decontamination BSCs that have been used for work involving infectious materials must be decontaminated before HEPA filters are changed, internal repair work is done or before a BSC is relocated. The most common decontamination method uses formaldehyde gas,

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although more recently, hydrogen peroxide vapor has been used successfully. This environmentally benign vapor is useful in decontaminating HEPA filters, isolation chambers, and centrifuge enclosures. Call the Biosafety Office (392-1591) for the current BSC vendor who does the gas decontaminations and certifications. All BSCs must be re- certified following any gas decontamination, maintenance or relocation.

Engineering Requirements

Ultraviolet Lamps Ultraviolet (UV) lamps are not required in BSCs. If installed, UV lamps must be cleaned weekly to remove any dust and dirt that may block the germicidal effectiveness of the ultraviolet light. The lamps should be checked periodically with a meter to ensure that the appropriate intensity of UV light is being emitted. UV lamps must be turned off when the room is occupied to protect eyes and skin from UV exposure, which can burn the cornea and cause skin cancer. Do not depend on UV lamps to disinfect the area. BSC Placement The ideal location for the biological safety cabinet is remote from the entry (e.g., the rear of the laboratory away from traffic), since people walking parallel to the face of a BSC can disrupt the air curtain. The air curtain created at the front of the cabinet is quite fragile, amounting to a nominal inward and downward velocity of 1 mph. Open windows, air supply registers, or laboratory equipment that creates air movement (e.g., centrifuges, and vacuum pumps) should not be located near the BSC. Similarly, chemical fume hoods must not be located close to BSCs. HEPA Filters HEPA filters, whether part of a building exhaust system or part of a cabinet, will require replacement when they become so loaded that sufficient air flow can no longer be maintained. Filters must be decontaminated before removal. Certification of BSCs All BSCs must be certified (according to a National Sanitation Foundation standard) annually according to UF policy. Please contact the EH&S Biological Safety Office for the name and phone number of the current contractor performing this service. Prices and quality vary widely, so only BSO-approved contractors may be used.

72 November, 2008 Biosafety Manual Emergency Procedures/Telephone Numbers

In the event of a major disaster affecting the campus, the UF Homepage http://www.ufl.edu/ is the official source of UF emergency related information.

Emergency Phone Numbers Normal Business Evenings & Weekends Hours Fire/Police/Medical Emergency Note: callers should be able to accurately state 911 their location to emergency responders. Write the Note that you 911 may need to location/address of the lab here: Note that you may need to dial 9 first to get dial 9 first to an outside line. get an outside line.

1-800-222- Florida Poison Information Center 1-800-222-1222 1222 Suspected Gas Leak 392-1121 392-1121 Needlestick/Sharps Injury 1-866-477-6824 Chemical Spill 392-8400 or 392-1111 http://www.ehs.ufl.edu/RiskMgmt/emergncy/chemspill.htm 1591 Radiation Spill 392-7359 or 392-1111 http://www.ehs.ufl.edu/Rad/RCGuide/rcg2.htm#2IX 1589 Biological Spill 392-1591 392-1111 http://www.ehs.ufl.edu/RiskMgmt/emergncy/biospill.htm Physical Plant Trouble Desk* 392-1121 392-1121 HSC Physical Plant Trouble Desk 392-4411 392-4411 IFAS Facilities Operations/Maintenance 392-1984 392-1121 * 24-hour building/maintenance repair hotline. Other Contacts PI or lab Supervisor home number and/or cell number(s): ______(fill in)

Karen Gillis, UF BSO, 392-1591

Mark Yanchisin, UF lab safety Coordinator, 392-1591

Phil Collis, Assoc. Director of EH&S, 392-1591

Student Health Care Center 352-392-1161

Alachua County Emergency Management 352-264-6500

Red Cross, Alachua County Headquarters 352-376-4669

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General Information It is important that persons handling biological agents have the proper training and experience to work with these materials safely. This includes how to handle an accidental release, exposure, injury, or evidence of agent theft or loss. • Personnel should know what constitutes a potential exposure or release and report this to the PI or lab supervisor immediately. The PI or lab supervisor must then report this to the biosafety office and, as appropriate, to medical care providers. • All personnel must also be familiar with symptoms of disease or illness associated with the materials they handle so that a previously undetected exposure can be discovered and measures taken to prevent further exposures. • Time and situation permitting, contain and secure all biological agents during any and all emergencies. Agent storage areas shall remain locked. • Know ahead of time the location and operation of emergency equipment such as the eyewash and safety shower, first aid kit, chemical and biological spill kits, emergency numbers, fire extinguishers, emergency exits. • PIs or lab supervisors should periodically review this information with lab staff/ students and display this information in the laboratory. • When possible, lab staff, PI/lab supervisor should meet and escort emergency personnel on site.

Medical Emergency 1) Remain calm. 2) Initiate lifesaving measures, as required. 3) Call for EMERGENCY RESPONSE - MEDICAL EMERGENCY 9-1-1. 4) Calling 911 automatically alerts the UF campus Police. 5) Do not remove injured person unless there is a danger of further harm. 6) Keep injured person warm. 7) Notify the PI/ supervisor.

Accidental injection, cuts, skin exposures 1) Remove protective clothing or PPE; place it in biowaste or red bag if contaminated. 2) Wash hands. 3) Wash the effected part. Use soap if available, but avoid strong chemical disinfectants that can damage skin, e.g. bleach. 4) Apply an appropriate disinfectant from the first aid kit (e.g. antibiotic ointment). 5) Notify the PI or lab supervisor and inform them of the circumstances of the injury, including what was being handled at the time. 6) If medical treatment is needed: a) During working hours go to the UF Student Health Center (SHCC):

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i) Main Campus (Infirmary) location: Fletcher Drive next to the Florida Gym and the Racquet Club, 392-1161 ii) UF Health Science Center Satellite location: Second floor of the Dental Towers of the J. Hillis Miller Health Science Center. Take a Dental Tower elevator to the second floor, turn left, and straight ahead is SHCC@Shands in Room D2-49, phone 392-0627

b) After hours or on weekends, seek treatment at the nearest emergency room. c) The PI or supervisor must notify the Biosafety office of the incident.

7) If exposed to a Blood Borne Pathogen: a) Wash the area thoroughly with soap & water b) In Gainesville, call 1-866-477-6824, the Needle Stick Hotline. c) In Jacksonville, 7:00 – 4:00, Go to Employee Health Suite 505 in Tower 1. Other hours, go to the ER d) Other areas: Go to nearest medical facility e) Get immediate medical attention (1-2 hr max) f) Notify supervisor g) Allow Medical to follow up with the appropriate testing & the required written opinion.

Splashes to face and eyes 1) Go to the eyewash station and activate it 2) Rinse face/mouth/nose/eyes 3) Eyes should be flushed for at least 15 minutes. 4) Forcibly hold eye open to ensure effective rinsing behind eyelids. 5) Have injured worker move eye side-to-side and up-down during rinsing. 6) Remove contact lenses. 7) Always obtain medical attention for a hazardous material splash to the eye. 8) Place contaminated clothing in a red biohazard bag for decontamination. 9) Watch for symptoms of exposure or delayed onset effects. 10) Report incident to PI/supervisor and Biological Safety Office (392-1591).

Accidental ingestion 1) Seek medical treatment: a) During working hours go to the UF Student Health Center (SHCC): i) Main Campus (Infirmary) location: Fletcher Drive next to the Florida Gym and the Racquet Club, 392-1161 ii) UF Health Science Center Satellite location: Second floor of the Dental Towers of the J. Hillis Miller Health Science Center. Take a Dental Tower elevator to the second floor, turn left, and straight ahead is SHCC@Shands in Room D2-49, phone 392-0627

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b) After hours or on weekends, seek treatment at the nearest emergency room. 2) The PI or supervisor must notify the Biosafety office of the incident.

Animal bites and scratches 1) For small wounds – allow to bleed freely. If necessary, control bleeding by applying direct pressure with a sterile gauze or bandage. 2) Immediately wash with copious quantities of soap and water. Wash for at least 15 minutes. A chlorhexidine soap such as Nolvason is recommended. Povidone – iodine or Betadine surgical soap may be used but is more likely to cause skin irritation and cellular damage. If eyes or mucous membranes are exposed, irrigate the area for at least 15 minutes with water. 3) Seek medical treatment: a) During working hours go to the UF Student Health Center (SHCC): i) Main Campus (Infirmary) location: Fletcher Drive next to the Florida Gym and the Racquet Club, 392-1161 ii) UF Health Science Center Satellite location: Second floor of the Dental Towers of the J. Hillis Miller Health Science Center. Take a Dental Tower elevator to the second floor, turn left, and straight ahead is SHCC@Shands in Room D2-49, phone 392-0627 b) After hours or on weekends, seek treatment at the nearest emergency room. 4) The PI or supervisor must notify the Biosafety office of the incident. 5) If the bite or scratch is from a non-human primate, contact the following physician/specialists regarding Monkey B virus (Herpesvirus Simiae, CHV-1) exposure: a) Dr. Reuben Ramphal, Work phone: 352 392-4058, Pager: 352-413-7837 b) Dr. Kenneth Rand, Work phone: 352 392-5621, Pager: 888-553-2503 c) The physicians will evaluate the injury and may decide to culture the wound for B-virus (Herpesvirus simiae) or collect blood for a baseline titer against B-virus, or use prescription drugs for preventative therapy. The physician directing the care of the patient will contact the Director of Animal Care Services for instructions regarding the need for cultures or serology from the monkey inflicting the injury upon the patient. d) Symptoms suggestive of B virus infection should be reported immediately to the medical consultant. When the possibility of B virus illness is seriously entertained, appropriate diagnostic studies should be performed and specific antiviral therapy should be instituted. The physician may wish to consult the Viral Exanthems and Herpesvirus Branch, Division of Viral and Rickettsial Diseases, CDC (Dr. Scott Schmitt, (404) 639-0066 or cell 404-725-5652 or Terri Hyde (404) 639-2696, for laboratory assistance, the National B Virus Resource Center at GSU, (Dr. Julia Hilliard, (404) 651-0808). e) The above procedures also apply to the employees of private contractors in non- human primate facilities.

76 November, 2008 Biosafety Manual Break in/Security Breach 1. Call 352-392-1111 for University police department. 2. Notify PI/lab supervisor and the Biosafety Office of the break in. 3. Escort University Police personnel at the scene. 4. The PI and Biosafety Office will conduct an inventory check and report results to appropriate authorities as needed.

Handling Biological Spills Advance preparation for management of a spill is essential. A “bio spill kit” should be available and contain the following: • Forceps/scoop for broken glass/sharps • Paper towels or absorbent material • Disinfectant • Respirators, if necessary • Latex or nitrile gloves and safety glasses • Red bag

Spill in the biosafety cabinet 1) Leave the cabinet on/running to prevent escape of contaminants from the cabinet. 2) Cover the area with paper towels or other absorbent material. 3) Pour appropriate disinfectant (e.g. a fresh 1:10 dilution of household bleach, 0.5% sodium hypochlorite) over the spill as a disinfectant solution to inactivate biohazardous material. If necessary, sufficient disinfectant solution shall be used to ensure that the drain pans and catch basins below the work surface contain disinfectant. Disinfect under the front exhaust grill if needed. Walls and equipment in the biological safety cabinet that may have been splashed shall be wiped with disinfectant. 4) Let disinfectant solution sit for 30 minutes. 5) Pick up absorbent materials and wipe up excess disinfectant solution. 6) Place material in biohazard bag. 7) Use tweezers, forceps, or tongs to pick up broken sharps and place in a sharps container. 8) Rinse all disinfected areas with water and allow to dry. 9) Follow with 70% ethanol.

Spill in the centrifuge

1) Allow aerosols to settle for 30 minutes before attempting to clean up the spill. Keep the centrifuge closed during this time. Post a sign on the centrifuge so others don’t try to open it. 2) Gently open the centrifuge to prevent re-aerosolization. 3) Place absorbent materials in the chamber and pour a fresh 1:10 dilution of household bleach (0.5% sodium hypochlorite) over them. Let sit 30 minutes.

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4) Carefully remove carriers to a tub containing a fresh 1:10 dilution of household bleach (0.5% sodium hypochlorite). Soak 30 minutes. 5) Wipe the interior and lid of the centrifuge with a fresh 1:10 dilution of household bleach (0.5% sodium hypochlorite). 6) Use tweezers, forceps, or tongs to pick up broken sharps – place in a sharps container. 7) Wipe all areas with plenty of water to prevent corrosion. Dry and follow with 70% ethanol.

Spill inside the laboratory

1) Notify room occupants of the spill. 2) All persons should leave the laboratory immediately. 3) Close the door. 4) If clothing is known (or suspected) to be contaminated, remove the clothing with care, folding the contaminated area inward. Place the clothing into a red bag for autoclaving. 5) Wash all potentially contaminated body areas as well as the arms, face and hands. Shower if necessary. 6) Any exposed persons should seek medical advice or treatment: a) During working hours go to the UF Student Health Center (SHCC): i) Main Campus (Infirmary) location: Fletcher Drive next to the Florida Gym and the Racquet Club, 392-1161 ii) UF Health Science Center Satellite location: Second floor of the Dental Towers of the J. Hillis Miller Health Science Center. Take a Dental Tower elevator to the second floor, turn left, and straight ahead is SHCC@Shands in Room D2-49, phone 392-0627 b) After hours or on weekends, seek treatment at the nearest emergency room. 7) Post a sign on the door to keep people out. 8) No one should enter for approximately 30 minutes so that aerosols can be cleared by the ventilation system or allowed to settle. 9) During this time, notify the PI or lab supervisor. Notify the biosafety office for large spills, those you are uncomfortable handling, those involving BSL-3 agents, or those involving select agents. 10) Protective clothing should be worn when entering the laboratory to clean the spill area. Latex or nitrile gloves, autoclavable, or disposable footwear, safety glasses, and an outer garment. If you have been issued an N95 respirator to work with this agent, put that on. 11) Take the “bio spill kit” into the laboratory room, and place paper towels, spill pillows, or other absorbent materials around and on the spill. If the spill was on the floor, do not use a surgical gown that may trail on the floor when bending down. 12) Carefully pour a fresh 1:10 dilution of household bleach (0.5% sodium hypochlorite) over the spill as a disinfectant solution; avoid splashing and work from the outside towards the center. 13) Let disinfectant solution sit for 30 minutes.

78 November, 2008 Biosafety Manual 14) Use paper or cloth towels to wipe up the disinfectant and spill, working toward the center of the spill. Discard all towels and other clean up materials into a discard container/red bag as they are used. 15) Wipe the outside of the discard containers, especially the bottom, with a towel soaked in a disinfectant. 16) Place the discard container and other materials in an autoclave and sterilize. 17) Remove shoes or shoe covers, outer clothing, respirator, and gloves and sterilize by autoclaving. 18) Wash hands, arms and face; shower if necessary. 19) If gaseous decontamination of the room is required, contact the Biosafety Office.

Spill outside the laboratory

Safe transport of biohazardous material outside the laboratory is essential. Materials should be packaged securely (triple contained in unbreakable containers) to avoid such spills. In addition, the person transporting the material should be knowledgeable about the hazards of the material and how to respond to a spill. In the event of a spill outside the lab:

1) Clear area of all personnel and keep them out of the spill 2) Have someone call the University of Florida Police Dept., 392-1111 and the Biosafety Office 392-1591, for help. 3) If clothing is known (or suspected) to be contaminated, remove the clothing with care, folding the contaminated area inward. Place the clothing into a bag for autoclaving. 4) Wash all potentially contaminated body areas as well as the arms, face and hands. Shower if necessary. 5) Any exposed persons should seek medical advice or treatment: a) During working hours go to the UF Student Health Center (SHCC): i) Main Campus (Infirmary) location: Fletcher Drive next to the Florida Gym and the Racquet Club, 392-1161 ii) UF Health Science Center Satellite location: Second floor of the Dental Towers of the J. Hillis Miller Health Science Center. Take a Dental Tower elevator to the second floor, turn left, and straight ahead is SHCC@Shands in Room D2-49, phone 392-0627 b) After hours or on weekends, seek treatment at the nearest emergency room. 6) Spill will be cleaned up by lab staff (if a spill kit is available) or by EH&S staff. 7) Protective clothing should be worn to clean the spill area. Latex or nitrile gloves, autoclavable, or disposable footwear, safety glasses, and an outer garment. If you have been issued an N95 respirator to work with this agent, put that on. 8) Take the “bio spill kit” to the area, and place paper towels, spill pillows, or other absorbent materials around and on the spill. If the spill was on the floor, do not use a surgical gown that may trail on the floor when bending down.

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9) Carefully pour a fresh 1:10 dilution of household bleach (0.5% sodium hypochlorite) over the spill as a disinfectant solution; avoid splashing and work from the outside towards the center. 10) Let disinfectant solution sit for 30 minutes. 11) Use paper or cloth towels to wipe up the disinfectant and spill, working toward the center of the spill. Discard all towels and other clean up materials into a discard container/red bag as they are used. 12) Wipe the outside of the discard containers, especially the bottom, with a towel soaked in a disinfectant. 13) Place the discard container and other materials in an autoclave and sterilize. 14) Remove shoes or shoe covers, outer clothing, respirator, and gloves and sterilize by autoclaving. 15) Wash hands, arms and face; shower if necessary.

Fire Safety

Predetermine two means of egress from your normal workplace. Learn the location of the nearest fire alarm pull station and portable fire extinguisher.

If you discover a fire in a University of Florida building, do the following: 1) Pull the fire alarm and call 911. 2) Fire alarm pull stations are normally located near each exit. If the building is not equipped with a fire alarm system, notify other occupants as you exit the building. 3) Do not attempt to fight the fire with portable fire extinguishers or fire hoses unless the fire is small and you have been trained in their proper use. DO NOT PUT YOUR LIFE IN DANGER WHILE ATTEMPTING TO CONTROL A FIRE. When in doubt, evacuate. 4) Remain calm while talking to the operator. Be prepared to answer several questions as to location, size of fire, your name, number of persons in building (if known) and any injuries. Remain on the line until the operator is finished. 5) Meet fire or police personnel when they arrive at the building. Stand by to answer any questions they may have concerning the fire. 6) Once out of the building DO NOT RE-ENTER THE BUILDING FOR ANY REASON, unless emergency personnel have given the "ALL CLEAR" signal.

If the fire is INSIDE your room: Leave your room and close the door. Pull the fire alarm and call 911. Or, if the fire is small and you have been trained to use a fire extinguisher, attempt to put it out. (Again, DO NOT PUT YOUR LIFE IN DANGER WHILE ATTEMPTING TO CONTROL A FIRE. When in doubt, evacuate.) Remember the acronym “PASS” PASS = P Pull the pin. A Aim at the base of the fire.

80 November, 2008 Biosafety Manual S Squeeze the trigger. S Sweep the nozzle from side to side.

If the fire is NOT in your room, but in a room you must pass through to get out or exit: 1) With your hands, test the door for heat before opening. 2) If the door is HOT: a) Stay in your room or lab. b) Phone for help. c) Stay calm. d) Seal cracks with wet towels. e) Wait for help. 3) If the door is COOL: a) Take your room key. b) Open the door slowly. c) WALK to the nearest exit and leave the building. d) If the exit is unsafe, return to the room and remain there. e) If the hall is smoky, stay low or crawl out on your hands and knees.

DO NOT USE THE ELEVATOR !!!

Workplace Violence:

All threats and other inappropriate behavior that create an immediate concern for safety should be reported immediately to the University Police Department (UPD) at (352) 392- 1111or local law enforcement if off campus. You may also dial 911, but remember, you must first dial 9 to get an outside line. Examples of behavior requiring a call to authorities include: Direct or veiled threats Written sexual or violent notes – intimidation verbally or physically Carries a weapon (Florida Statutes and University Policy prohibit firearms and certain other articles that could be weapons on state property) Makes suicidal comments or threats Involved in fights or assaults Stalks co-workers or their family See the UF Workplace violence policy at http://www.hr.ufl.edu/policies/workviolencepolicy.pdf

Hurricane:

1) All biological agents shall be secured and contained in preparation for a hurricane.

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2) See the checklist to prepare lab areas for an impending hurricane: http://www.ehs.ufl.edu/disasterplan/LabPrep.rtf 3) The UF homepage is the official source of UF Information. Official emergency information for Alachua County is broadcast on i) Radio: WUFT-FM / WJUF-FM / WLUF-LP / WRUF / AM850 / Rock 104 ii) Television: WUFT-TV iii) Additional information regarding safety and preparedness may be found at: www.ufl.edu/emerg.html

Tornadoes and other natural disasters:

Tornado Watch - conditions are favorable for the formation of tornados. Tornado Warning - indicates that a tornado has been sighted or is indicated on radar. There is generally little or no warning given at the approach of a tornado. In the event of a tornado: 1) IMMEDIATELY seek shelter, preferably in the hallway of a main office building, an interior bathroom or an interior closet. 2) AVOID windows; flying debris can kill. Protect yourself by getting under a heavy desk or table. Also, remember to protect your head. 3) If time permits, open at least two windows to provide complete ventilation for each building.

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3 -- Programs

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Animal Contact Medical Monitoring Program The University of Florida Animal Contact Medical Monitoring Program grew out of the recommendations of the American Association for the Accreditation of Laboratory Animal Care (AAALAC) reviewers. According to the US Public Health Service, an occupational health program is required for institutions that employ personnel who have animal contact.

The UF program is designed to protect our employees, students, and volunteers having animal contact from occupational exposure to conditions that may result in animal related illnesses. The requirements of the program are based upon those outlined in the Public Health Services document, Guide for the Care and Use of Laboratory Animals, published by the National Research Council. The current UF program requirements were revised and approved, and put into place May 2006.

Individuals who will be working with animals or who will be working in proximity to animals are required to participate in the medical monitoring program. They are provided with animal contact medical monitoring information and immunizations relating to their animal contact as part of their pre-placement health assessment. In addition, the Institutional Animal Care and Use Committee (IACUC) verifies that all personnel listed on new and continuing projects are registered with the UF Animal Contact Medical Monitoring Program. The IACUC committee notifies the PI of any personnel who are not thus cleared for animal contact. Principal Investigators are responsible for ensuring that all personnel (including employees, students, colleagues, collaborators, and volunteers) involved with their IACUC-approved project are given program information. Investigators who do not respond to requests for registration may have their approval rescinded by the IACUC. Individuals that have animal contact must participate in a risk assessment that includes contact information, health questionnaire and health assessment (physical examination, medical history, blood serum banking) based upon the type of animal contact, and immunizations as needed. This risk assessment is provided at no cost to the employee. Short-term visitors to UF from other institutions must provide documentation of participation in their home institution’s animal contact program or they must register in the UF Animal Contact Program. Individuals involved in isolated one-time, non-recurrent exposures shall be informed of potential dangers and medical precautions, but are not required to participate in the program. The primary responsible party (principal investigator, research director, student research coordinator, etc.) shall be responsible for assuring compliance with the notification requirements for these individuals.

84 November, 2008 Biosafety Manual Elements of the Program 1. Risk Assessment

• A Risk Assessment form is required to be completed by everyone working with animals at UF or entering UF animal facilities. This form includes contact information and a health questionnaire that will be evaluated by UF’s Occupational Medicine Physician or Licensed Health Care Professionals to assess the risk of exposure and determine whether additional information and/or interaction is necessary.

• A Renewal Risk Assessment form is required every three years or when any new species is contacted. This update will allow UF’s Occupational Medicine Physician or Licensed Health Care Professionals to evaluate and, if necessary, address potential health risks to you resulting from a change to your health status or changes to the type of animal exposure. 2. Specific requirements

• Tetanus Immunization within 10 years - All participants

• Rabies Immunization Series/Booster or Positive Titer within 2 years – All individuals handling unvaccinated carnivores or their tissue

• Respirator Clearance and Fit Test – All individuals required by the Q-Fever Policy or as medically necessary to prevent allergic reactions

• Serum Banking – All individuals who work with non-human-primates, all who handle blood from alligators or birds housed outdoors, all who are required by the Q-Fever Policy and all pre-menopausal females who handle cats or cat waste.

• TB Screening within 12 months – All individuals who enter any room with non- human primates.

• Medical consultation – As determined by the Occupational Medicine Physician. Examples are individuals with chronic disease, work-related injuries or illness, environmental or animal allergies. 3. Exemptions from the program Individuals working on projects that involve observation of birds or other animals outdoors/in their natural habitat are exempt from the program. See http://www.ehs.ufl.edu/Bio/Animal/acweb.htm for more information.

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UF Bloodborne Pathogen Program In December 1991, OSHA published the final rule covering occupational exposure to bloodborne pathogens. This was adopted by the state of Florida and written into the Florida Administrative Code in January 1993. UF instituted its program in the spring of 1993.

The rule requires that those who handle human blood or other potentially infectious (human) materials as part of their job duties participate in an employer-generated program. This program shall include development and annual review of a site specific Exposure Control Plan, annual training regarding exposures, offer of hepatitis B vaccinations free-of-charge, and post-exposure health care services.

Environmental Health and Safety (EH&S) manages this program through the Biological Safety Office. Each January, EH&S sends program materials and instructions to department chairs and directors of groups identified as having employees with occupational exposure. These materials provide instructions and information necessary to achieve annual training compliance. The BBP program materials and information is available on our website at http://www.ehs.ufl.edu/bio/BBP/default.htm .

In addition to the program materials, EH&S provides “train-the-trainer” sessions annually.

Hepatitis B vaccinations are given by the Student Health Care Center (SHCC) or, in some cases, Shands Occupational Health Services, or other providers. Official program medical records are kept by SHCC. There is a requirement that Shands or other providers send vaccination and post-exposure records to SHCC for record-keeping purposes. Declination statements from those that decline the vaccination series are kept by EH&S.

EH&S monitors for UF compliance by requiring training and vaccination documentation and by confirming BBP participation during the annual laboratory safety survey which is conducted in each laboratory each year.

For further information, please call the Biological Safety Office at 392-1591.

BBP Exposure Information In Gainesville, call the Needlestick Hotline 24 hours a

day following any exposure.

In Jacksonville, go to the Employee Health Office in Suite 505, Tower 1, 5th and Jefferson from 7 – 4:30. Go to the ER after hours. Phone: 904 244-9576

For exposure incidents more than 1 hour away from Gainesville, go to the nearest medical facility.

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4 -- Medical Surveillance

HIV Research Laboratory Occupational Medicine Policy

This policy is designed to protect employees who conduct research with HIV. Researchers who handle, manipulate, or assay live HIV cultures are covered under this policy.

Pre-employment Prior to beginning work in an HIV research lab, an employee shall be given, at no cost to the employee, the following:

1. A baseline serum sample shall be stored with the UF Student Health Care Center. This is a requirement to work in the HIV lab. 2. A confidential HIV test shall be offered to the employee. The HIV test is available at the UF Student Health Care Center. The test must be offered by the employer, but may be declined by the employee. This test shall include HIV counseling. Results of the test shall be given to the employee in person in a face- to-face meeting with a health care professional. No exceptions. All results of HIV testing shall remain completely confidential and shall be stored in a separate section in the employee’s medical record. At no time, will the employer or any administrator or official of UF have access to the employee’s confidential record concerning HIV testing. 3. If the HIV researcher also works with human blood or other potentially infected material, all aspects of the UF Bloodborne Pathogen Program under the OSHA Bloodborne Pathogen standard shall be implemented for that employee. The employer shall offer free HBV immunization, annual training, and have a written exposure control plan in the workplace.

Continuing employment HIV researchers shall be given the following at no cost to the employee: 1. Annual serum banking shall be offered to the employee, but may be declined. 2. Annual HIV tests shall be offered to the employee, but may be declined. 3. Annual training regarding HIV and post-exposure prophylaxis, including the risks of chemoprophylaxis, shall be given to the employee. This is a required, not optional, component.

Post-exposure prophylaxis An exposure to an HIV culture in a research laboratory is considered a “highest risk exposure” according to Public Health Service CDC guidelines. The following shall be implemented:

88 November, 2008 Biosafety Manual 1. If the exposure is to intact or broken skin, or in the event of a puncture wound, immediately wash the affected area with water for five minutes. Soap may be used, if immediately available. If the exposure is to the eyes, they shall be rinsed for five minutes in an eyewash station. Other exposed mucous membranes (nose and mouth) shall be rinsed for five minutes with water. 2. The employee shall proceed immediately to the nearest health care facility. It is important for the employee to seek treatment within the first two hours of exposure. 3. The employee may be seen at the Student Health Care Center between the hours of 8:00 a.m. and 4:00 p.m. After hours, or on weekends, employees should proceed to Shands Emergency Department, where they will be seen immediately, free-of-charge. 4. The current Public Health Service (CDC) chemoprophylactic treatment/ post- exposure prophylaxis (PEP) shall be recommended and offered to an employee with the highest risk exposure, within the first two hours of exposure. However, medical use of chemoprophylaxis is a decision that shall be made by a health care professional in conjunction with the employee, based upon the most current CDC recommendations, the nature of the exposure event, and other medical factors. Shands pharmacy is well stocked with the appropriate medication. 5. Baseline tests and informed consent statements shall be required for PEP. 6. Counseling concerning the risks and benefits of the chemoprophylactic treatment shall be given to the employee at the time of the exposure event. 7. The employee may contact an infectious disease specialist, if desired. 8. All HIV exposures that occur in the workplace are covered under Worker’s Compensation. Shands OHS at UF personnel are familiar with the paperwork to process this type of Worker’s Compensation claim for UF employees. 9. Post-exposure follow-up, including the offer of HIV testing and counseling, shall be given in accordance with the CDC recommendations and the OSHA Bloodborne Pathogen rule. The Shands protocol on HIV exposures will be followed.

References UF Bloodborne Pathogen Program Compliance Materials; http://www.ehs.ufl.edu/Bio/BBP/bio_bbp.htm OSHA. 29 CFR Part 1910.1030. Occupational Exposure to Bloodborne Pathogens, Final Rule, Dec. 6, 1991 CDC. Update: Provisional public health service recommendations for chemoprophylaxis after occupational exposure to HIV; MMWR 1996; 45(22):468-472 Human immunodeficiency virus (HIV) and acquired immune deficiency syndrome (AIDS) exposure (includes chemoprophylaxis treatment protocol); Shands OHS at UF, rev. July 17, 1996.

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Immunoprophylaxis The University of Florida follows the recommendations of the Centers for Disease Control and Prevention (CDC) and the Public Health Service Advisory Committee for Immunization Practices (ACIP) for vaccination of at-risk personnel.

Currently, UF has written policies regarding the need for hepatitis B, tetanus, rabies, and vaccinia vaccination for certain at-risk personnel. Other vaccinations may be required or recommended, as needed. Particular attention is given to individuals who are or may become immunocompromised, as recommendations for vaccine administration may be different than for immunologically competent adults. Various other factors may be taken into account such as pregnancy, history of allergy, or HIV status.

When considering the need for immunization, a risk assessment will be conducted by the health care provider in conjunction with information regarding the experimental agent provided by the UF EH&S Biological Safety Office.

90 November, 2008 Biosafety Manual Recommendations for Prophylactic Immunization of Laboratory Personnel Working with Infectious Agents Please note, the following is derived from the CDC publication, Biosafety in Microbiological and Biomedical Laboratories, Fourth Edition, 1999 and the CDC publication, “Immunization of Health-Care Workers: Recommendations of the Advisory Committee on Immunization Practices (ACIP) and the Hospital Infection Control Practices Advisory Committee (HICPAC)”, 1997, MMWR vol. 46. Bacterial agents 1. Bacillus anthracis A licensed vaccine is available through the CDC. However, immunization of laboratory personnel is not recommended unless frequent work with clinical specimens or diagnostic cultures is anticipated (e.g., animal disease diagnostic laboratory) and in settings in which production quantities, high concentrations, or procedures with a high potential for aerosol (all BSL-3 work) are encountered. In these settings, vaccination is recommended for all persons working with the agent, all persons working in the laboratory room where the cultures are handled, and all persons working with infected animals. 2. Clostridium botulinum A pentavalent (ABCDE) botulism toxoid is available through the CDC as an investigational new drug (IND). This toxoid is recommended for personnel working with cultures of C. botulinum or its toxins. 3. Clostridium tetani The adult diphtheria-tetanus toxoid is highly recommended for laboratory personnel who manipulate cultures or toxins and is required by the UF Animal Contact Program for all UF personnel who work with animals. This immunization should be given every 10 years. 4. Corynebacterium diphtheriae The adult diphtheria-tetanus toxoid every 10 years is recommended for laboratory personnel who work with this agent. 5. Francisella tularensis A live attenuated vaccine is available. It is recommended for persons working with infectious materials or infected rodents, and for persons working in or entering laboratories or animal rooms where cultures or animals are maintained. 6. Neisseria meningitidis The use of licensed polysaccharide vaccines should be considered for personnel regularly working with large volumes or high concentrations of infectious materials. 7. Salmonella typhi Licensed vaccines, which have been shown to protect 70-90% of recipients, may be a valuable adjunct to good safety practices in personnel regularly working with cultures or clinical materials which may contain S. typhi. 8. Yersinia pestis Although field trials have not been conducted to determine the efficacy of a licensed inactivate vaccine, experience with this product has been favorable. Immunization is recommended for personnel working regularly with cultures of Y. pestis or infected rodents.

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Rickettsial agents 1. Coxiella burnetii An investigational new Phase I Q fever vaccine (IND) is available from the Special Immunizations Program, U.S. Army Medical Research Institute for Infectious Diseases (USAMRIID), Fort Detrick, MD. The use of this vaccine should be limited to those at high risk of exposure (e.g., individuals engaged in research with pregnant sheep, or live C. burnetii organisms) and who have no demonstrated sensitivity to Q fever antigen. Viral agents 1. Hepatitis A virus A vaccine is available and recommended for those who work with hepatitis A virus. 2. Hepatitis B virus Hepatitis B virus infection is a major infectious hazard for health care personnel. A vaccine is available and highly recommended for workers performing tasks involving exposure to blood or blood contaminated body fluids, according to the OSHA rule and the UF Bloodborne Pathogen Program. 3. Influenza: Recommended during influenza season for health care workers involved in patient care, those with chronic medical conditions, to prevent staff illness and reduce spread of the disease. 4. Poliovirus All laboratory personnel who work directly with the agent are required to have documented polio vaccination or demonstrated serologic evidence of immunity to all three poliovirus types. 5. Poxviruses: vaccinia, cow pox, monkey pox Vaccination is required for those who work with or around orthopox viruses. 6. Rabies virus Immunization is recommended for all individuals prior to work with rabies virus or infected animals, or engaging in diagnostic, production, or research activities with rabies virus. Immunization is also recommended for all individuals entering or working in the same room where rabies virus or infected animals are used. The UF Animal Contact Program requires immunization for all employees (e.g. veterinarians and animal control personnel) who come into contact with unvaccinated carnivores. 7. Arboviruses For the following arthropod-borne viruses there is a vaccine available and it is recommended for those who work with the virus. Eastern equine encephalitis* Junin Cabassue Western equine encephalitis* Mucambo Rift Valley Fever Venezuelan equine encephalitis* Chikungunya Everglades Yellow Fever (*No commercial vaccine available for human infections)

92 November, 2008 Biosafety Manual 8. Measles, Mumps, and German measles virus (Rubeola, paramyxovirus, and rubellavirus) Immunizations are usually given in childhood. Vaccines are highly recommended for all unvaccinated or serologically negative adults.

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Vaccinia Immunization Policy

All personnel who work in an area where vaccinia, orthopoxviruses, or animals vaccinated with those agents are used, handled or housed must have received a vaccinia vaccination within the last ten years. Individuals must declare their vaccination status prior to engaging in such work. This may be accomplished by using the UF Pox Virus Vaccine Form.

To fulfill this requirement:

1. Request a Vaccinia Vaccination Information Packet from Environmental Health & Safety (EH&S) at 392-1591.

2. Following review of the material, questions should be directed as follows:

a. Medical questions – Student Health Care Center’s Occupational Medicine Program (391-1161 X4259) b. Virology questions – EH&S and/or principal investigator.

3. All projects involving “pox” viruses require vaccination.

4. Any medical condition precluding vaccination will also preclude participation in a project involving infectious orthopoxvirus.

It shall be the responsibility of each principal investigator to insure that:

1. Each project is evaluated to determine whether vaccination is required. These evaluations are conducted by EH&S and its various committees.

2. Each participant complies with the vaccine policy requirements prior to participation in any covered research project.

3. EH&S is notified of vaccination dates for all researchers working with vaccinia.

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University of Florida Pox Virus Vaccine Form

Name______Department ______UF ID ______Title ______Date ______Position # ______Phone ______

I have read the informational package concerning biosafety considerations for working with vaccinia virus and related orthopoxviruses and I have had an opportunity to ask questions about this information of both a subject expert and a health care professional. I understand that I may obtain a smallpox vaccination administered by university medical personnel at no cost to me.

I understand that failure to consent to vaccine will prohibit me from working with any “pox” viruses.

Previously Vaccinated

Date: ______Location: ______Administered by: ______Employee Signature: ______

Consent to Vaccination

I authorize and request the University of Florida and its designated employees to administer the vaccine to me.

Employee Signature: ______Date Given: ______Lot Number: ______Administered By: ______

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Health Surveillance for Personnel Working with Infectious Agents A health surveillance program is available for laboratory personnel who use agents that require it. Laboratory personnel should receive immunizations (see chapter entitled “Immunoprophylaxis”), such as hepatitis B vaccination, and medical tests, such as tuberculosis skin testing, when appropriate. The Animal Contact and Bloodborne Pathogen Programs (see appropriate chapters) provide for health assessments, risk assesments, medical tests, and immunizations for certain at-risk personnel.

Blood Serum Sampling For those laboratory personnel working with certain BSL-2 or BSL-3 agents, baseline serum samples may be appropriate (see chapter entitled “Biological Safety Levels”). The collection and maintenance of baseline serum samples provide a tool for monitoring serological changes that may result from the employee’s work. For example, staff who work with non-human primates may be required to bank a blood sample.

The Biological Safety Office, with advice from the appropriate health care professional, will provide information regarding the need for, and the frequency of, blood serum sampling on a case-by-case basis. The need for serum sampling will be dependent upon the agent used and the type of research in which an individual is engaged. For example, researchers who work with large quantities of HIV are required to have serum sampling monitored annually and upon accidental exposure.

Health Assessments The UF Occupational Medicine Program provides for pre-placement and other health assessments depending upon the type of work in which an employee will be engaged.

Laboratory personnel who work with agents that are transmitted by aerosol or with certain chemicals or acute toxins may require a respirator at some stage of the research project. The EH&S Occupational Medicine Respiratory Protection Program provides for health assessments that will determine an employee’s fitness for respirator use.

The EH&S Occupational Medicine Program also includes the Pesticide Medical Monitoring Program, the Hearing Conservation Program, the Diving Science and Safety Program, and the Asbestos Monitoring Program, as well as the Animal Contact Program, discussed in a separate section. Occasionally, researchers who work with infectious agents will require a health assessment based on the requirements of one of these other programs.

96 November, 2008 Biosafety Manual Exposure to Mycobacterium Tuberculosis

All personnel who can be occupationally exposed to either TB research or patients with active TB should have a yearly TB test for health surveillance.

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5 -- Appendix A – Forms

R-DNA http://www.ehs.ufl.edu/bio/Files/rdna.doc

Bio-Agent http://www.ehs.ufl.edu/bio/Files/bio.doc

Transgenic Animals http://www.ehs.ufl.edu/bio/Files/rdnata.doc

Acute Toxin http://www.ehs.ufl.edu/bio/Files/toxin.doc

Project Addendum http://www.ehs.ufl.edu/bio/Files/addendum.doc

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