Biological Safety Manual

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1 Last Reviewed Date: 10/11/2016 Revised by: Tekechia Hester Effective Date: 02/25/2009 Applies To: Employees, Faculty, Students, Others For More Information Contact: Biosafety P a g e

2 Table of Contents Forward... 6 Incidents, Exposures and Emergencies... 7 Emergency Numbers... 7 On-Campus Medical Emergencies Requiring an Ambulance... 7 Rules, Regulations and Guidelines Governing the Use of Biological Agents... 8 National Institute of Health (NIH)... 8 Centers for Disease Control and Prevention (CDC) and the NIH... 8 Occupational Safety and Health Administration (OSHA)... 8 The Select Agent Rule... 8 United States Department of Agriculture (USDA)... 8 New York Department of Health... 8 Other Agencies and Entities... 9 Responsibilities and Administration Senior Biosafety Specialist Institutional Biosafety Committee (IBC) Academic Chairs, Department Heads Principal Investigator (PI) Laboratory Personnel Biosafety Requirements Biological Laboratory Survey Registration of Recombinant and Synthetic Nucleic Acid Molecule (r-sna) Experiments Human Blood, Body Fluids, Tissues and Other Potentially Infectious Materials Animal Use Biological Safety Cabinets (BSC), Laminar Flow Benches (LFB) and Chemical Fume Hoods Training Standard Operating Procedures Risk Assessment and Exposure Sources Risk Assessment Biological Agent Hazards Exposure Sources Risk Groups Routes of Exposure Percutaneous Exposure (through the skin) Inhalation of Aerosols Mucous Membrane Ingestion Biosafety Levels Signs and Labels Laboratory Practices Microbiological Standard Practices Hazard Controls P a g e

3 Engineering Controls Biological Safety Cabinets Centrifugation Vacuum Line Chemical Traps and Filters Syringes and Needles Safety Blenders, Sonicators, and Mixers Mechanical Pipetting Devices Lyophilizers Cryostats Miscellaneous Equipment (Waterbaths, Shakers, Cold Storage) Work Practice Controls Universal (Standard) Precautions Use of Engineering Controls General Work Practices Hand Washing Handling Disposable Needles and Syringes Reusable Sharps Footwear Slash and Aerosol Control Housekeeping for Laboratory Workers Chemical Sterilization and Disinfection Personal Protective Equipment (PPE) Laboratory Clothing Hand Protection Face and Eye Protection Respiratory Protection Disposable Booties and Shoe Covers Decontamination Liquid Disinfection Heat Vapors and Gases Radiation Regulated Medical Waste /Biological Waste Select Agents and Toxins Shipping, Receiving and Transportation of Biological Materials On Campus Transport of Biological Materials Federal Permits for Transport and Use Biosecurity Inventory Biological Exposure and Spill Response Procedures Steps to Consider with a Biohazard Exposure Basic Biological Spill Clean-up Procedures Reporting Incidents P a g e

4 Fire and Fire Related Emergencies In Laboratories Animal Areas Evacuation Appendices Appendix A: Disinfectants and Sterilization Methods Appendix B: Spill Response Cue Cards Appendix C: Supplemental Supportive Documents Appendix D: External Resources P a g e

5 Definitions Antiseptic is a chemical germicide formulated to be used on skin or tissue. Biological Agents: This Manual uses the Environmental Agency (EPA) originated definition of biological agents as preparations made from living organisms and their products. Biological agents include: all microorganisms and their toxins, viruses and sub-viral particles (including prions), recombinant products (plant, animal, microbial), parasites (microscopic, as well as, macroscopic), Cultured human and animal cells and the potentially infectious agents that these cells may contain, clinical specimens (human or animal blood, body fluids, cells, tissues, bone), whole animals and tissues from experimentally infected animals, allergens (such as molds, microbial spores, and animal allergens. Biohazards (Biological Hazards): Biohazardous materials will include any of the following requiring IBC oversight: recombinant or synthetic nucleic acid materials, biological agents and toxins, bacteria and their phages and plasmids, viruses, fungi, mycoplasmas, prions, and parasites; human and non-human primate tissues, body fluids, blood, blood byproducts, and cell lines, animal remains and insects that may harbor zoonotic pathogens. Biological Select Agent or Toxin: Specifically regulated pathogens and toxins as defined in Titles 42 CFR Part 73, 7 CFR Part 331, and 9 CFR Part 121, Possession, Use, and Transfer of Biological Agents and Toxins regulated by both DHHS and USDA (i.e., overlapping agents or toxins) and plant pathogens regulated by USDA alone. Decontamination refers to any procedure that eliminates or reduces microbial contamination to a safe or acceptable level with respect to the transmission of infection. Disinfection is the irreversibly inactivation of specific viruses, bacteria, or pathogenic fungi (but not necessarily spores) on inanimate surfaces. Exposure: Any eye, nose, other mucous membrane, skin, or parenteral contact with any biohazardous material. Laboratory Personnel: refers to anyone, including students of any kind, conducting research on or otherwise handling biohazard material. Recombinant and synthetic nucleic acid molecules: as defined by the NIH Guidelines, molecules that (a) are constructed by joining nucleic acid molecules and (b) that can replicate in a living cell, i.e., recombinant nucleic acids; nucleic acid molecules that are chemically or by other means synthesized or amplified, including those that are chemically or otherwise modified but can base pair with naturally occurring nucleic acid molecules, i.e., synthetic nucleic acids, or molecules that result from the replication of those described in (i) or (ii) above. Sterilization is the destruction of all microorganisms on inanimate surfaces. 5 P a g e

6 Forward The NYU Biosafety Program is administered by the NYU Department of Environmental Health and Safety (EHS), which has primary responsibility for its content, implementation, and enforcement. Working with principal investigators, laboratory personnel, supervisors, and administrators, EHS, through this Program, seeks to minimize the health risks of laboratory personnel and the community at large and reduce environmental impacts while meet regulatory requirements. The purpose of the Manual is to provide general reference, guideline, policies and procedures to be followed in teaching, research, and clinical laboratories and for other activities that use biological agents. This Manual, in conjunction with NYU Safety Policies and training modules, forms the basis for the Biological Safety Program, which has the commitment to obtain the following goals: Protect laboratory personnel and visitors from exposure to infectious agents Prevent environmental contamination Provide an environment for research excellence while maintaining a safe work place Comply with applicable federal, state and local requirements In general, the handling and manipulation of biological agents and toxins, as well as recombinant or synthetic nucleic acid molecules, requires the use of a variety of precautionary measures depending on the material involved. The Manual provides assistance in the evaluation/assessment, containment and control of biological hazards. However, it is expected that all parties working with these materials will seek additional information and advice if necessary. All research personnel should be familiar with this manual. 6 P a g e

7 Incidents, Exposures and Emergencies Emergency Numbers NYU NYU Washington Square Public Safety NYU College of Dentistry Public Safety NYU Tandon School of Engineering DIAL 911 from a campus phone DIAL from a campus phone DIAL from a campus phone DIAL or for after hour emergencies Student Health Center DIAL On-Campus Medical Emergencies Requiring an Ambulance 1. Do not move a seriously injured person unless they are in further danger. 2. Dial 9-911(from campus phone) then X82222 or Tell the dispatcher the location and nature of the emergency. 3. Public Safety will coordinate the ambulance or transportation to either the Student Health Center or a local Hospital. 4. Employees and students must immediately notify their supervisor or instructor of all illnesses and injuries possibly related to an exposure of an infectious material. 5. The PI or Laboratory Supervisor is responsible for contacting EHS once you have received medical attention. 6. The PI or Laboratory Supervisors must complete an Incident Reporting form and submit it to Biosafety within 5 days of an exposure incident and within 7 days of an accidental spill. Accidents, injuries and exposures must also be reported on the Work Related Incident-Injury Form for anyone employed by the University. Non-employee students will complete an additional form at Student Health Services. 7. Spills resulting in an overt exposure must be reported immediately and the reporting form must be completed within 24 hours. 7 P a g e

8 Rules, Regulations and Guidelines Governing the Use of Biological Agents National Institute of Health (NIH) Guidelines for research involving Recombinant or Synthetic Nucleic Acid Molecules: The NIH Guidelines address the safe conduct of research and/or teaching activities that involve construction and handling of recombinant or synthetic nucleic acid molecules and organisms containing them. The Recombinant DNA Advisory Committee (RAC) was established in 1974 to determine appropriate biological and physical containment practices and procedures for experiments that potentially posed risks to human health and environment. The RAC published the initial version of the NIH Guidelines in The current NIH Guidelines include practices/procedures for research involving recombinant or synthetic nucleic acid molecules. Principal Investigators who are responsible for research and/or teaching activities that involve recombinant or synthetic nucleic acid molecules must include them on IBC registration forms. Centers for Disease Control and Prevention (CDC) and the NIH In 1984, the CDC and NIH published Biosafety in Microbiological and Biomedical Laboratories (BMBL). The BMBL describes combinations of standard and special microbiological practices, safety equipment and facilities that constitute four levels of biological containment (BSL 1-4) which are recommended for working with a variety of infectious (biological) agents in various laboratory settings. The BMBL also outlines requirements for four levels of animal biological safety containment (ABSL 1-4). The BMBL serves as the basis for NYU s biosafety manual. The CDC also issues permits for the importation of infectious agents, materials and host animals into the United States. Occupational Safety and Health Administration (OSHA) Bloodborne Pathogens Standard (BBP): The 1991 Bloodborne Pathogens regulations were originally created to address healthcare workers concerns regarding occupational health risks caused by exposure to human blood and other potentially infectious materials of human origin. While originally focused on healthcare workers, this standard applies to any personnel who may be exposed to human blood or other potentially infectious materials of human origin, including human cell lines. Refer to the BBP section of the Manual for specific information regarding the BBP standard and how it applies to occupationally exposed personnel. The Select Agent Rule Federal regulations regarding the use, storage and transfer of certain select biological agents and toxins were published starting in At the federal level, registration and approval of select agents is the responsibility of the USDA-APHIS/CDC National Select Agent Registry (NSAR). Additional information can be found in the Select Agent section of this Manual. United States Department of Agriculture (USDA) USDA issues permits for the possession, use and transport of biological agents and materials important to agriculture and at times, human health. Conditions of these permits vary for each type of agent or material. These conditions often include requirements for containment, handling and disposal of permitted materials. Additional information can be found in the Shipping, Receiving and Transportation section of this Manual. New York Department of Health Users of hypodermic syringes and needles must comply with the applicable New York State Department of Health regulations, and are responsible for appropriate certification, procurement, storage, and distribution. 8 P a g e

9 Non-medical and non-veterinary use of syringes and needles (e.g. teaching, research) require a Department of Health Certificate of Need. Other Agencies and Entities Shipping of regulated biological materials must comply with the US Department of Transportation (DOT) and the International Civil Aviation Organization and the International Air Transport Association (IATA) regulations. Personnel packaging and shipping regulated biological materials must be trained every two years. See Shipping, Receiving and Transportation of Biological Materials section of this Manual. Information Systems for Biotechnology s (Virginia Tech) A Practical Guide to Containment: Plant Biosafety in Research Greenhouses and The American Committee of Medical Entomology of the American Society of Tropical Medicine and Hygiene s Arthropod Containment Guidelines are examples of independent entities whose work has become Biosafety best practices. 9 P a g e

10 Responsibilities and Administration Senior Biosafety Specialist The Senior Biosafety Specialist is responsible for the implementation and the administration of the biosafety program. The Senior Biosafety Specialist identifies potential areas of concern and suggests to the EHS Director safety objectives to be achieved. Ensures the department head, principal investigators, and managers are complying with the biosafety program, Coordinate the University biosafety program by enforcing applicable standards governing the safe use, storage, and disposal of biohazards, Conduct annual inspections of laboratories, Report to the IBC any problems, violations, and accidents or illnesses from activities that involve the use of biohazardous materials, Assist with the development of emergency plans for handling accidental spills and personnel contamination and investigate laboratory accidents involving biohazard materials, Review and approve the use of biohazard materials, Provide advice on lab security, Provide technical advice to PIs, lab personnel, and the IBC on biosafety procedures. Institutional Biosafety Committee (IBC) It is the IBC s responsibility to review and approve the use of recombinant and synthetic nucleic acid molecules (r-sna) and select agent materials as defined in this Manual. The IBC must assure that all University community members engaged in teaching and/or research activities follow the safety guidelines and requirements of the NIH Guidelines and the CDC/NIH publication Biosafety in Microbiological and Biomedical Laboratories. Teaching or research laboratory experiments that involve the use of r-sna molecule technology and/or biohazard material must be registered as described in this Manual research or teaching activities conducted at or sponsored by New York University (NYU) that use of the described materials through the review of registrations. Academic Chairs, Department Heads Academic departments have a responsibility to instill good scientific practices in their students and to assure that employees use and enforce safe work practices. Department Heads have overall responsibility for the teaching and research conduct of their faculty. This responsibility includes the maintenance of safe work practices/procedures in the handling of hazardous and potentially hazardous materials. Principal Investigator (PI) The role of Principal Investigator (PI) may be fulfilled by a Faculty member, Laboratory Director/Manager or course Instructor. The following are responsibilities of every PI: Complete the training required by EHS and other appropriate compliance committees, Assist in implementing the University s health and safety policy, use of the biosafety manual and all other university safety practices, Complete a biological laboratory survey for their laboratory, Register with the IBC for the use of r-sna or select agent materials, Prepare a risk assessment for all biological materials (see Risk Assessment), 10 P a g e

11 Implement the guidelines, policies and procedures outlined in this Manual, Institute appropriate safe work practices, Instruct laboratory personnel, (students and staff) on a continuing basis, of potential health hazards in the workplace, Assure all laboratory personnel receive required safety training, Provide appropriate training resources, Document and report accidents, injuries and exposures involving biological agents, Develop practices and procedures that serve to protect employees and students, Maintain workplaces and equipment under your direction in a safe, well maintained manner, Identify and meet the safety needs for personnel they relate to appropriate engineering controls, training, personal protective equipment and corrective measures for non-compliant issues, Conduct periodic self-audits to identify operational gaps in work practices and/or facilities. Laboratory Personnel It is the laboratory personnel's responsibility to: Complete the training required by EHS and other appropriate compliance committees, Know and adhere to all laboratory biosafety policies and emergency procedures, Receive an overview of the Laboratory Specific Biosafety Manual from the PI or Laboratory Supervisor and follow all laboratory practices established, Inform the PI of any personal health requirements that may require implementation of safety precautions, Report to the PI or the lab supervisor all problems, deviations in procedure, spills involving biohazardous materials, exposures, and/or safety/security concerns (e.g., suspicious persons or activities). 11 P a g e

12 Biosafety Requirements The following information describes the requirements for NYU researchers as defined by EHS. It is the responsibility of each PI to ensure the laboratory is in compliance. Biological Laboratory Survey Institutions with research and teaching activities are obligated by federal and/or state regulations to know which biological agents are used or stored in their buildings. The identification of the biohazard materials present in an institution is a crucial step in the development of an effective biological safety program. The Principal Investigators (PI) are required to complete a biological laboratory survey. It is used to identify laboratories and personnel who are handling biological materials. The information provided will be reviewed by the Senior Biosafety Specialist. The Biosafety review will: Determine appropriate risk groups and biological safety containment levels (BSL), Identify recombinant or synthetic nucleic acid molecule activities, Determine the adequacy of the laboratory for the research activities, Determine and establish appropriate biosecurity measures, Determine bloodborne pathogens exposure risk, Determine and develop necessary and appropriate training/training tools, Determine medical surveillance needs, Comply with all applicable regulations, Determine appropriate shipping and transport procedures, Identify related environmental issues (waste handling, facility ventilation), and Assist emergency response planning. The Survey must be updated for the following reasons: New agents and materials are brought in the laboratory Location of laboratory changes Removal or addition of personnel Registration of Recombinant and Synthetic Nucleic Acid Molecule (r-sna) Experiments PIs are required to register all r-sna activities with NYU Medical Center s Institutional Biosafety Committee. Committee approval is required prior to the initiation of all r-sna activities. The r-sna registration form is available on NYU School of Medicine s IBC webpage through the Kerberos Research Portal System. Human Blood, Body Fluids, Tissues and Other Potentially Infectious Materials The Occupational Safety and Health Administration (OSHA) created the Occupational Exposure to Bloodborne Pathogens Standard, 29 CFR Part (the Standard) to minimize or eliminate exposure to infectious agents that may be present in human blood, tissues or certain body fluids (bloodborne pathogens.) The Standard applies to all employers having employees that may be occupationally exposed to human blood or other potentially infectious materials. An employee is considered occupationally exposed if there is reasonably anticipated skin, eye, mucous membrane, or parenteral contact with human blood or other potentially infectious materials (OPIM) in the performance of an employee s duties. Other potentially infectious materials include: 12 P a g e

13 Human cell/tissue cultures, including embryonic stem cells, Organ cultures, Any unfixed tissue or organ, other than intact skin, from a human being (living or dead), HIV- or HBV-containing culture media or other solutions, Human body fluids, except urine, feces, saliva or tears unless visibly contaminated with blood, Blood, organs or other tissues from experimental animals infected with HIV or HBV or other bloodborne pathogens, An individual is also considered occupationally exposed if they do not have direct contact with blood or other potentially infectious material, but operate equipment that is used to process or store blood or OPIMs. The BMBL recommends that human and non-human primate (NHP) cells, tissues, and bodily fluids be handled using Biosafety Level 2 (BSL-2) practices and containment. Human and NHP materials, even in the absence of overt contamination, may contain adventitious viruses and/or other opportunistic pathogens or zoonotic agents. Since it is extremely difficult to screen for every pathogen, all human and NHP materials must be handled with standard precautions (e.g. treated as though they are contaminated with infectious agents) and utilize BSL-2 practices. Moreover, all cell lines and tissue cultures of human and NHP origin, including established cell lines and bodily fluids will be handled in accordance with the OSHA Bloodborne Pathogens Standard and under Biosafety Level 2 (BSL-2) containment. Other mammalian cell lines used to propagate viruses will also require BSL-2 practices. Laboratory personnel working with these cultures must maintain a written record of their annual training as required by OSHA Bloodborne Pathogens Standard. Contact EHS at for a training schedule. NYU s Exposure Control Program is located at the following website policy #110, Bloodborne Pathogens Exposure Control Program. Animal Use All research involving animals must be conducted in accordance with an approved University Animal Welfare Committee (UAWC) protocol. Additionally, animal research that contains biohazards must receive approval from the IBC prior to the conduct of research. Contact the UAWC for additional information. The UAWC is charged with responsibility for reviewing New York University s program for the humane care and use of animals in research and teaching labs. The Biosafety Officer works with the UAWC when protocols involve the use of biohazard materials and the IBC when materials are of an r-sna nature. Contact Biosafety if there are any questions or concerns regarding biohazards and animal use. Biological Safety Cabinets (BSC), Laminar Flow Benches (LFB) and Chemical Fume Hoods Clean air devices are equipment that use one or more HEPA filters to deliver clean, nearly particulate free, air to a work surface. BSC are designed to protect the user and the environment from contamination, most protect the product as well. Laminar flow benches (LFB) only protect the product from contamination. The efficacy of BSCs and LFBs depends upon the behavior of the operator, the unit s orientation in the facility, and the movement of personnel in the laboratory. Chemical fume hoods and other capture devices must be used for operations that might result in the release of toxic chemical vapors, fumes, or dusts. Benchtop use of chemicals that present an inhalation hazard is not permitted. Fume hoods must be used when conducting new experiments with unknown consequences from reactions or when the potential for a fire exists. 13 P a g e

14 EHS has implemented and Inspection Program, designated to ensure the health and safety of NYU s laboratory personnel and prevent environmental release of infectious materials or the inhalation of chemical vapors, fumes, or dust. This is done through annual certification of all BSCs, LFBs and fume hoods to ensure the unit s ability to perform the intended function. All clean air devices and fume hoods must be placed on the NYU service contract and be certified at least annually. Biosafety coordinates the certification with a contracted vendor. Units that are not certified annually will be rendered non-functional until such time as a certification is performed. The Office of Environmental Health and Safety will contact you to schedule the required annual certification. Notify Biosafety in advance when you plan to have BSCs or LFBs moved, placed in storage, transferred to a new owner, discarded, removed from NYU or obtained from another institution or manufacturer. Contact Biosafety if service or repairs (e.g., replacing fluorescent lamps) are needed for your unit. BSCs must be professionally decontaminated with formaldehyde, by a certified technician, before a unit is relocated, stored, serviced (interior) or discarded. Training Successful completion of certain EHS training may be required prior to the conduct of research or work assignment. The training program was developed and is offered by EHS via classroom and online (NYU Classes). A schedule of trainings is provided to the NYU community at the start of each semester. If a specific training is required for work in the laboratory, health center or clinic, please contact Environmental Health and Safety at x81450 or ehs@nyu.edu to schedule a training time. EHS maintains a list of individuals who have completed various training modules. Please contact EHS if you require proof of training. Principal investigators are responsible for providing laboratory personnel with information regarding specific hazards or situations not covered in EHS trainings. This may include information about health hazards, location and use of PPE, spill clean-up materials, and the presence of any particularly hazardous materials in the laboratory. Additional training, provided by the principal investigator, should be specific to the activities conducted in the laboratory. It should include: Health risks (biological, chemical and physical) posed by the experimental procedures conducted in their lab, Regulated Medical Waste (RMW) training and proper disposal methods for biological, chemical and hazardous materials, The existence and location of all designated areas in the laboratory, The selection and use of personal protective equipment appropriate for laboratory tasks. PI s can work with EHS to schedule laboratory or department specific trainings. For more information on training requirements visit the Training webpage. 14 P a g e

15 Work Involves Required Training Delivery Method Working in a lab Working with chemicals Human or NHP blood, other potentially infectious materials including human, NHP cell lines (e.g. HeLa, HEK 293, Vero), infectious (capable of causing disease in healthy adults) microorganisms; or viral vectors Laboratory Safety Hazardous Waste (initial) Annual Refresher Bloodborne Pathogens (initial) Training Annual Refresher In- person In- person On-line In-person In-person or On-line Animal subjects Laboratory Animal Care and Use Contact UAWC Shipping biological materials IATA/DOT Shipping Training 2 year refresher In-person In-person Biohazard work that may generate aerosols Respirator Training In-person Working with recombinant-synthetic r-sna training On-line PPT slides nucleic acid materials 3 year Refresher On-line PPT slides Table 1: EHS Laboratory Science Trainings Standard Operating Procedures Standard operating procedures (SOPs) are intended to provide individuals with general guidance on how to safely work with a specific class of biological agent or hazard. While SOPs provide only general guidance, observance of all the safety practices listed in them is mandatory. If compliance with all the requirements of a specific standard operating procedure is not possible, the principal investigator must develop a written procedure that will be used in its place. This alternate procedure must provide the same level of protection as the SOP it replaces. Biosafety is available to provide guidance during the development of alternate procedures. SOPs, at minimum, should include step-by-step instructions of the tasks emphasizing safety controls, (i.e. Personal Protective equipment, use of fume hoods). They should also state the type of personnel authorized to perform the task. In order to provide flexibility, standard operating procedures are generic in nature. They address the use and handling of substances by hazard class only. In some instances, multiple SOPs may be applicable for a specific biological agent. All SOPs should be incorporated into a Laboratory Specific Biosafety Manual and be accessible to all laboratory personnel. If you have questions concerning the applicability of any item listed in an SOP, contact the Principal Investigator of your laboratory or Biosafety at P a g e

16 Risk Assessment and Exposure Sources Risk Assessment Risk is the probability that harm, injury, or, in the context of this document, an occupationally acquired infection will occur. The foundation of any safety program is the use of control measures appropriate for the risk posed by the activities and the agents in use. To characterize the risk and facilitate control measures, microorganisms and clinical materials are assigned to one of four Biosafety Levels (BSL). For each BSL there is a unique set of safety equipment, facility design features, and practices that will reduce the risk of laboratory-acquired infections. Biosafety Level classifications are appropriate for typical laboratory operations. The PI or laboratory supervisor is responsible for implementing more (or less) stringent practices based on laboratory specific conditions. Such a decision is ultimately the result of a risk assessment process that accounts for the following: Biological Agent Hazards Virulence - the severity of disease Capacity to infect and cause disease in a susceptible host Infectious dose - the dose required causing infection in humans or animals Stability in the environment Host range Endemic nature Agent origin Reports of laboratory acquired infections Availability of treatment and prophylaxis Probable routes of transmission in laboratory All of the above factors are inherent to a particular microbe. The use of r-sna materials may alter any of the risk factors and investigators should take these modifications into consideration when working with r-sna microorganisms. Exposure Sources Exposure sources in the laboratory are hazards that could result in the infection of researchers or the public through work with biological agents. Some of the more common hazard considerations include the following: Aerosol generation (e.g. pipetting, mixing, sonicating, centrifuging, vortexing) Animal handling (e.g. bites and scratches) Manipulation with sharps Contact with blood, bodily fluids, or other potential infectious materials Spills and splashes into the mucous membranes When performing a risk assessment of laboratory procedures, all potential routes of exposure should be addressed. Most laboratory-acquired infections have resulted from inhalation of aerosols, splashes or sprays, and needlesticks. It is good practice to look for potential exposures via ingestion, inoculation, inhalation, and contamination of skin and mucous membranes. It is up to the Principal Investigator (PI) to determine which controls will be implemented. During periodic biosafety audit, Biosafety works with the PI/facility personnel to provide improvements to procedures, as necessary. It is recommended that the PI or his designee perform regular self-audits of their laboratory areas and related facilities. Biosafety safety checklists can be found on the Biosafety website for this purpose. 16 P a g e

17 Risk Groups The NIH Risk Group and World Health Organization (WHO) classifications should be used when a risk assessment for a microorganism is being conducted. The classification system assigns organisms to one of four categories of risk based on the agent hazard criteria listed above. Risk Group 1 (RG1) Risk Group 2 (RG2) Risk Group 3 (RG3) Risk Group 4 (RG4) Agents that are not associated with disease in healthy adult humans Agents that are associated with human disease which is rarely serious and for which preventative or therapeutic interventions are often available Agents that are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available (high individual risk but low community risk) Agents that are likely to cause serious or lethal human disease for which preventative or therapeutic interventions are not usually available (high individual risk and high community risk) Visit the Risk Group webpage for a comparison of international risk group classifications. A complete description of work practices, safety equipment, and facility design features for BSL-1 through BSL-4 is available in the CDC/NIH publication Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th Edition, specifically, Section IV. The NIH s Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines) also provides additional guidance for risk assessment of microorganisms and materials containing r-sna molecules, which may alter the risk of the activities. 17 P a g e

18 Routes of Exposure There are four exposure routes that must be addressed when working with biological materials and many work practices can be viewed in the context of eliminating a particular exposure pathway. Percutaneous Exposure (through the skin) Percutaneous injuries can result from needle sticks and cuts or abrasions from contaminated items. These exposures are particularly serious because of the potential for immediate entry of the agent into the circulation. Sharps must be handled and disposed as noted in the Regulated Medical Waste Disposal section. Inhalation of Aerosols Many laboratory procedures can cause the aerosolization of infectious agents. Some of these procedures include the use of vortexes, blenders and sonicators. Proper work practices must be implemented to minimize the aerosolization of all materials, especially whose main route of exposure is through inhalation (e.g., Mycobacterium tuberculosis, SARS.). Please see the Laboratory Practice and Technique section for more information on how to minimize and contain aerosols. Mucous Membrane Compared to intact skin, infectious agents can more readily enter the body through contact with the eyes, nose, mouth, or by inadvertent inoculation of these areas via contaminated hands. Eye and face protection should always be used if there is a potential of splash or splatter. Ingestion Accidental ingestion of biohazardous materials can result from improper personal hygiene in the laboratory. Food and drink are prohibited in all areas of the laboratory. Hands must be washed immediately if visible contamination occurs, after removing gloves and always before leaving the laboratory. 18 P a g e

19 Biosafety Levels Although the most important aspect of biohazard control is the awareness and care taken by personnel in handling infectious materials, certain features of laboratory design, ventilation, and safety equipment can prevent dissemination of pathogens should accidental release occur. The CDC/NIH publication, Biosafety in Microbiological and Biomedical Laboratories (BMBL) defines four levels of containment based on the risks assigned to the agents and procedures. Each biosafety levels describes the standard microbiological and safety practices, safety equipment (primary barriers and personal protective equipment), and laboratory facilities recommended to protect personnel and the environment from exposure and contamination. Special microbiological practices enhance worker safety, environmental protection, and address the risk of handling agents requiring increasing levels of containment. During your initial or annual laboratory inspection a biosafety level will be assigned depending upon the risks associated in each lab space. Biosafety Level 1 Biosafety Level 1 is appropriate for laboratories, and for other facilities in which work is done with wellcharacterized agents not known to cause disease in healthy adult humans. Work in a BSL 1 laboratory is typically conducted on an open bench top using standard microbial practices and universal precautions. Agents: defined and characterized strains of microorganisms not known to consistently cause disease in healthy adults (e.g., B. subtilis, S. cerevesiae, non-pathogenic E. coli.) Includes recombinant DNA activities using such non-pathogenic organisms as hosts for the expression of genes incorporated into bacterial plasmids or low risk viral vectors such as baculovirus or Adeno Associated Virus. Work practices: standard microbiological practices/aseptic technique. Safety equipment: none required-gloves, lab coats and eye protection recommended. Facilities: bench top sink available for hand washing. Biosafety Level 2 Biosafety Level 2 is applicable to work done with a broad spectrum of indigenous agents present in the community and associated with moderate-risk to humans. Agents: associated with human diseases of varying severity (e.g., Hepatitis B and C, HIV, S. typhi, human retroviruses, S. aureus. Includes r-sna activities using viral vector systems such as Adenoviruses and some Retroviral vectors, particularly Lentiviral vectors, and expression of recombinant DNA in BSL-2 organisms.) Transmission: inoculation and other percutaneous injuries, ingestion, mucous membrane exposure Work practices: BSL-1 practices, with the addition of: limited access, Biohazard signs, sharps precautions, defined procedures for Regulated Medical Waste (RMW) disposal and medical surveillance (as needed). Safety equipment: Class II Biological Safety Cabinet (BSC) or equivalent containment for manipulations with potential for aerosolization or splashing; lab coats, gloves, eye/face protection. Facilities: BSL-1 facilities, with the addition of: available autoclave, directional airflow, no air recirculation, disinfection/decontamination procedures are in place. Please note: Biosafety level 3 and 4 work requires specially engineered laboratory facilities. 19 P a g e

20 These facilities are not available at New York University. No work may be conducted with agents requiring BSL-3 or BSL-4 containment at NYU Washington Square, Tandon School of Engineering, or the College of Dentistry. Biosafety Level 3 Biosafety Level 3 is applicable to work done using indigenous or exotic agents with a potential for respiratory transmission and which may cause serious and potentially lethal infection. Agents: serious or lethal diseases transmissible via aerosols (e.g., M. tuberculosis, SARS. Recombinant DNA activities using genetic material from BSL-3 organisms or such organisms as host cells.) Transmission: aerosol inhalation, inoculation and other percutaneous injuries, ingestion, mucous membrane exposure Work practices: BSL-2 practices, with the addition of: respiratory protection training and policy; individualized proof of competency, controlled access, on-site decontamination of all waste and lab clothing, medical surveillance. Safety equipment: Class II Biological Safety Cabinet (BSC) or equivalent containment for all open manipulations of agents; lab coats, gloves, eye/face, and respiratory protection (as needed). Facilities: BSL-2 facilities, with the addition of: physical separation from access corridors, double-door entry, directional air flow into lab, no recirculation of exhaust air, back-up ventilation and filtration systems, in-lab autoclave. No work is conducted on BSL-3 organisms at New York University, and no facilities currently exist here for BSL-4 work. Biosafety Level 4 Biosafety Level 4 is for work using dangerous and exotic agents, which pose a high individual risk of lifethreatening disease, which may be transmitted via the inhalation route and for which there is no available vaccine or therapy. No work is conducted on BSL-4 organisms at New York University, and no facilities currently exist here for BSL-4 work. Biosafety Level 2 Plus There are no standard criteria for this intermediate biosafety level. The enhancements or additions to the standard BSL-2 are dependent on the risk assessment. It is preferred that the BSL2+ laboratory be self-contained with the equipment required for the experiment located in the laboratory. Additions also include mandatory use of personal protective equipment such as a surgical mask, safety glasses, a class II biological safety cabinet, and secondary containment is utilized for centrifugation and other potential aerosol generating procedures. Respiratory protection may be recommended especially in animal facilities. Please follow the link to Section IV of the BMBL Laboratory Biosafety Level Criteria for more complete details. A table summarizing the laboratory recommendation is provided on the next page. The BMBL also provides guidance for the use of experimentally infected animals housed in indoor research facilities (e.g., vivaria), and also in the maintenance of laboratory animals that may naturally harbor zoonotic infectious agents. The four animal biosafety levels provide increasing levels of protection to personnel and to the environment, and are recommended as minimal standards for activities involving infected laboratory animals. The four ABSLs describe animal facilities and practices applicable to work with animals infected with agents assigned to Biosafety Levels 1-4, respectively. Information on Vertebrate Animal Biosafety Level Criteria can be found in Section V of the BMBL. 20 P a g e

21 Table 2: Summary of Recommended Biosafety Levels for Infectious Agents 21 P a g e

22 Signs and Labels All persons entering a laboratory must be advised of the actual or potential hazards present and must be informed of any specific entry/exit requirements. This may be accomplished by the use of signs posted at the entrances to the laboratory. Biosafety will post a biosafety level 2 sign incorporating the biohazard symbol and the words biohazard authorized personnel only. The sign can also be downloaded by accessing the Biosafety website. The lab must also post the NYU Emergency Contact Card. Warning labels must be affixed to containers of regulated waste, refrigerators, freezers, incubators and other devices used to store, transport or ship infectious or potentially infectious materials. Biosafety will provide adhesive labels for equipment. The labels shall be fluorescent orange or orange-red with lettering and symbols in a contrasting color (black). 22 P a g e

23 Laboratory Practices Microbiological Standard Practices The most important element of containment is strict adherence to standard microbiological practices and techniques. Persons working with biological materials must be aware of potential hazards, and must be trained and proficient in the practices and techniques required for handling them safely. Standard microbiological practices are the basis for biosafety practices. This list highlights some of these practices. Following sections of the manual will provide more details. Never mouth pipette. Avoid hand to mouth or hand to eye contact in the laboratory. Never smoke, eat, drink, take medication or apply cosmetics in the laboratory. Use aseptic technique. Thorough hand washing is essential after handling microorganisms, cells, mammalian tissues or body fluids and prior to exiting the laboratory. Wear laboratory garments, gloves and other recommended personnel protective equipment that protect street clothing from contamination in Biosafety Level-2 laboratories. It is strongly recommended that similar precautions be taken in Biosafety Level-1 labs as well. Discard laboratory gloves and remove lab coats upon leaving the laboratory area. Autoclave lab coats if working with cultured human pathogens before sending them to the laundry. Use great care and caution when handling syringes and needles, sharps or glassware in the microbiological laboratory. Never attempt to recap a used needle prior to disposal. Dispose of syringe needle assemblies in sharp-proof, autoclavable containers. Handle human pathogens or infectious materials containing pathogens in certified biosafety cabinets. Use the cabinets wisely, free of clutter and materials that block airflow and vent rids. Never leave pathogenic materials or contaminated labware open to the environment outside the biosafety cabinet. Store all biohazardous materials securely in clearly labeled, sealed containers. Storage units, incubators, freezers or refrigerators should be labeled with the Universal Biohazard sign when they house infectious material. Biosafety Level-2 laboratories must be posted with the Universal Biohazard symbol and must list infectious agent(s) in use. Avoid aerosol-generating procedures when working with pathogenic materials. Needle clipping, pipette mixing, sonication, centrifugation and other seemingly innocuous procedures can produce substantial aerosols. Use safety equipment when aerosol generation is unavoidable. Always close the laboratory doors when working with pathogenic materials. Know the laboratory biosafety plan for handling and the most suitable disinfectant for decontaminating the pathogens you use. Be familiar with the laboratory plan for managing an accidental spill of pathogenic materials. Always keep an appropriate spill kit available in the lab. Clean up the laboratory work surfaces thoroughly with a proven disinfectant after using pathogenic materials. The containment laboratory must not be cluttered in order to permit proper floor and work area cleaning. Never allow contaminated, infectious waste materials to leave the laboratory non-sterilized as normal trash or to be put in the sanitary sewer without being decontaminated. Use incineration or autoclaving at adequate temperature (121 ), pressure (18 psi) and time (based on the size of the waste load) and always use a sterile indicator tag to denote sterilization. Expose all materials being sterilized to the flowing steam principle. When shipping or moving pathogenic materials to another laboratory, always use DOT approved, leakproof, sealed and properly packed containers (primary and secondary containers). 23 P a g e

24 Report all accidents, occurrences and unexplained illnesses to your work supervisor. Understand the pathogenesis of the biohazards with which you work. Protect your fellow workers and the public from the pathogens you use. Think safety at all times during laboratory operations. The greater the hazard, the greater care that must be exercised. If you do not understand the proper safety procedures or the use of the appropriate safety equipment, do not work with the biohazard until you receive proper instruction. Seek the advice of the appropriate individuals. Hazard Controls Hazard controls are the steps necessary to protect workers from exposure to a substance. Steps to control a hazard include: Elimination (including substitution) - removing the hazard from the workplace, or substituting hazardous materials with less hazardous ones. Administrative controls- controls that alter the way the work is done, including policies, timing of work, training and work practices such as standard operating procedures. Engineering controls- methods that are built into the design of a lab, equipment or process to minimize the hazard. Personnel Protective Equipment The most effective hazard control is elimination, although this may not be practical when it comes to biological agents. This is because to eliminate the hazard the agent must be dead. An alternative to elimination is substitution. Substitution occurs when a substance (in this case biological agent) that is less hazardous is used instead of another. When conducting activities with biohazardous materials, this may be possible if a less hazardous species or strain of a biological agent exists or has been engineered in such a way that it does not cause any harmful effects. If the hazards cannot be eliminated or diminished then a way to contain the hazard and shield the worker from exposure must be explored in the form of engineering controls. Administrative Controls should be in place before addressing Engineering Controls, Work Practices and Personal Protection. These controls include the elimination and/or substitution of hazardous agents and procedures, creation of standard operating procedures (SOP), laboratory and agent specific training and appropriate supervision of workers during hazardous or potentially hazardous procedures. 24 P a g e

25 Engineering Controls Engineering Controls include equipment, devices or supplies that reduce the risk of occupational exposure by removing the hazard or isolating the worker from the exposure. Biological safety cabinets, autoclaves, safety centrifuges, medical sharps containers, splashguards, mechanical pipetting devices, and self-sheathing needles/needle devices are examples of engineering controls. Supervisors are responsible for providing the necessary engineering controls in the workplace. Utilize appropriate engineering controls whenever possible. Engineering controls are most effective when they are combined with good work practices. Equipment will require preventive maintenance or periodic replacement to provide worker protection. It is the direct responsibility of the supervisor to insure these controls operate properly. Equipment such as biological safety cabinets, safety centrifuges and mechanical pipetting devices are to be decontaminated immediately (or as soon as feasible) when overtly contaminated, or after a spill of biological agents, blood or other potentially infectious materials. Engineering controls should also be routinely decontaminated at the end of the work shift. Biological Safety Cabinets Biological Safety Cabinets (BSCs) offer personal, product and environmental protection. They prevent biohazards from contaminating the worker by confining contaminants within the operating cabinet and removing them through High Efficiency Particulate Air (HEPA) or Ultra High Efficiency Particulate Air (ULPA) filters. The intake air must pass through the filter before flowing into the BSC work area. The exhaust air is also filtered by a HEPA or ULPA filter. Any aerosols generated within the cabinetwork area are contained within the BSC. Proper airflow is essential to the containment nature of BSCs and other CADs. BSCs must be certified by a qualified and reputable certifier after installation, and before use, after being relocated and at least annually thereafter. BSCs in use or with uncertain or undetermined usage are to be professionally decontaminated before relocation, storage or service to the interior of the unit. Contact EHS Biosafety to coordinate a BSC certification and/or repair services. Additional considerations for BSC use include the following: Small table-top centrifuges, sonicators, stirrers and shakers may be operated within a biological safety cabinet to protect workers from any aerosols generated during their operation and when opening pressurized containers. Biological safety cabinets with microscope modifications are available when the use of a microscope in a clean environment is necessary. Centrifugation Centrifuge safety cups or buckets are designed to retain the contents of the centrifuge tube in the event of breakage or leakage. When centrifuging biological agents, use sealed tubes and safety buckets that seal with O- rings. Open these inside a BSC to contain the release of any potential aerosols. The contents may be under pressure. 25 P a g e

26 Centrifuge Safety Cup Safety Centrifuges (Centrifuges with automatic locking mechanisms or solid lids) prevent the centrifuge lid from being opened while the rotor is still in motion or prevent aerosol release while the centrifuge is in operation. The locking device releases after the centrifuge head has stopped revolving. Vacuum Line Chemical Traps and Filters Vacuum line chemical traps and filters prevent suction of human blood and other potentially infectious materials into the vacuum lines. The trap systems also prevent vacuum lines from clogging with non-infectious material. Laboratory workers protect maintenance personnel and vacuum line by installing and maintaining vacuum line traps and filter systems. PLEASE NOTE: Filters are to be placed between the chemical traps and the vacuum line. Do not allow Erlenmeyer flasks to overfill. Filters are to be examined and replaced if clogged or if liquid makes contact with the filter. Insure all connections or seals are tight to assure the vacuum is adequate. Spent filters are to be discarded in the regulated medical waste stream. There are now a variety of inexpensive HEPA in-line filter traps on the market. Contact the EHS Biosafety for information regarding vacuum line filters. Syringes and Needles The left suction flask (A) is used to collect contaminated fluids into a suitable decontamination solution; the right flask (B), serves as a fluid overflow collection vessel. An in-line HEPA filter (C) is used to protect the vacuum system (D) from aerosolized microorganisms. Use flask(s) large enough to collect a day s worth of aspirate, nothing larger. Keep flasks in the BSC, not on the floor, to avoid accidental breakage or spilling. Filters may be obtained through VWR, Fisher, and other lab supply companies. Empty flasks daily. Needle-sticks may occur in situations where needles require manipulation or disassembly. Devices or systems such as Catheter Safety Systems, I.V. Access Safety Systems, Needle Protection Devices, Air Bubble Removal Devices, Syringe and Needle Shields reduce the need to use needles and lessens the danger of accidental needle-sticks. There are also safety sharps devices such as retractable lancets and disposable scalpels with safety features. Contact EHS for information about safer sharps devices. Sharps contaminated with biological agents, human blood, or other potentially infectious material and capable of puncturing the skin must be disposed of in sharps containers. Waste items that can puncture or tear plastic bags are considered sharps and should also be disposed of in sharps containers. Sharps disposal containers are to be used to contain and discard used and unused sharps waste. Containers for sharps disposal should be easily accessible and located as close as possible to the immediate area where sharps are used or found. Sharps containers should be puncture-resistant, closeable and leak-proof on the sides and 26 P a g e

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