Biological Safety Guide
Biological Safety Office Environmental Health & Safety Division 1405 Goss Lane, CI 1001 Augusta, Georgia 30912
Revised: February 2014 STATEMENT OF AUTHORITY
Upon publication of these procedures, the Institutional Biosafety Committee (IBC) of the Georgia Regents University, is hereby authorized to act as agent for the Georgia Regents University in matters of review, control, and mediation arising from the use or proposed use of biological materials, including recombinant DNA, at the Georgia Regents University. A statement of composition of the Institutional Biosafety Committee and a delineation of authority is included in the following pages of this text.
Furthermore, it is hereby declared that the Biological Safety Office of the Georgia Regents University derives its authority directly from the Office of the President of the Georgia Regents University in all matters involving biological safety and/or violations of accepted rules of practice as described herein. The Biosafety Officer is hereby granted the authority to immediately suspend a project which is found to be a threat to health, property, or the environment.
______James J. Rush, Jr, Esq Date Chief Integrity Officer Georgia Regents University
Georgia Regents University Biosafety Guide-January 2012 Statement of Authority TABLE OF CONTENTS
List of Abbreviations ...... viii
Forward ...... x
1 Introduction ...... 1.1 1.1 Emergency Phone Numbers and Office of Environmental Health and Safety Contacts ...... 1.1
1.2 Responsibilities ...... 1-2 1.2.1 Department Chairperson and/or Center/Institute Director ...... 1-2 1.2.2 Principal Investigators, Clinical Directors and/or Instructional Course Directors ...... 1-2 1.2.3 Research and GRU Clinical Personnel ...... 1-5 1.2.4 Division of Environmental Health and Safety Biological Safety Office and Biosafety Officer ...... 1-5 1.2.5 GRU Institutional Biosafety Committee...... 1-7 1.2.6 The Georgia Regents University (GRU) ...... 1-8 1.2.6.1 Other Offices at GRU ...... 1-9 1.2.6.1.1 Facilities Operation & Maiantenance ...... 1-9 1.2.6.1.2 The Institutional Animal Care and Use Committee (IACUC) and Division of Laboratory Animal Services (DLAS) ...... 1-9 1.2.6.1.3 The Institutional Review Boards (IRBs) and the GRU Office of Human Research Compliance (OHRP)...... 1-10 1.2.6.1.4 Division of Sponsored Projects Administration (DSPA) ...... 1-11 1.2.6.1.5 Office of Institutional Audits and Compliance (OIAC) and the Compliance Oversight Council (COC) ...... 1-11 1.2.6.1.6 Office of Technology Transfer and Economic Development (OTTED)...... 1-12
2 Biosafety Requirements ...... 2-1
2.1 Registration for the Use of Biological Materials ...... 2-1 2.1.1 Annual Affirmation of Biosafety Protocol Information ...... 2-2 2.1.2 Ammendments to Biosafety Protocols ...... 2-3 2.1.2.1 Adding New Personnel to or Removal of Personnel from a Biosafety Protocol ...... 2-3 2.1.2.2 Amendments for New or Modified Biological Agents or Operations to a Biosafety Protocol ...... 2-4 2.1.2.3 Modifying the Locations or Equipment on a Biosafety Protocol ...... 2-4 2.1.2.4 Documenting New or Additional Project Titles Associated with a Biosafety Protocol (or Multiple Biosafety Protocols) ...... 2-5 2.2 Training Requirements ...... 2-6
2.3 Laboratory Establishment, Close-outs and Moves ...... 2-8 2.3.1 Incoming New Faculty to GRU ...... 2-8 2.3.2 Laboratory Close-outs ...... 2-9 2.3.3 Laboratory Moves ...... 2-10
2.4 Laboratory Record-keeping ...... 2-11
2.5 Paperwork Flow ...... 2-11 2.5.1 IBC Review/Approval Process for Biosafety Protocols (BSPs) and Amendments ..... 2-12 2.5.2 Verification that IBC has Received and approved Experiments, Agents, and Uses that were Proposed in a Particular Sponsored Project, Clinical Protocol and/or Animal Use Protocol to Other GRU Compliance Offices...... 2-14
Georgia Regents University Biosafety Guide January 2012 i 2.6 Biological Materials and Applications Which Must Be Documented on Biosafety Protocols ...... 2-17 2.6.1 Recombinant DNA Experiments ...... 2-17 2.6.1.1 Definition of Recombinant DNA ...... 2-17 2.6.1.2 Definition of Exempt vs. Non-Exempt Recombinant DNA ...... 2-18 2.6.1.3 Recombinant DNA Registration Requirements ...... 2-21 2.6.1.4 Human Gene Transfer/Therapy (HGT) ...... 2-21
2.6.2 Human, Animal and/or Agricultural Pathogens and/or Toxins (or Materials Potentially Contaminated with these Pathogens or Toxins) ...... 2-22 2.6.2.1 Toxins of Biological Origin ...... 2-23 2.6.2.2 Select Agents and Toxins ...... 2-23 2.6.2.2.1 Exclusions from Select Agents and Toxins Regulations ...... 2-26 2.6.2.2.2 Exemptions from Select Agents and Toxins Regulations for Clinical/Diagnostic Laboratories ...... 2-27 2.6.2.2.3 Criteria for Handling Select Agent or Toxins Contaminated Specimens for Diagnosis or Verification under the SAT Exemption Clause ...... 2-28 2.6.3 Human Blood, Body Fluids, Cells, Tissues and Other Potentially Infectious Material ...... 2-29 2.6.3.1 Cultures of Cell Lines of Human and/or Primate Origin ...... 2-31 2.6.3.1.1 Human Embryonic Stem (hES) Cells and Embryonic Germ Cell Lines ...... 2-32
2.6.4 Animals and the Use of Biological Materials in Animals ...... 2-33 2.6.5 Insects/Arthropods ...... 2-34
2.7 Experiments Prohibited at GRU ...... 2-34
3 Risk Assessment ...... 3-1
3.1 Risk Assessment and Management Table ...... 3-2
3.2 Considerations in a Risk Assessement ...... 3-3 3.2.1 Risk Groups: Hazardous Characteristics of a Biological Agent ...... 3-3 3.2.2 Routes of Exposure ...... 3-4 3.2.3 Operations with may be associated with additional risk of exposure ...... 3-7 3.2.3.1 Zoonotic Disease Risks/Animal Research Risks ...... 3-7 3.2.3.1.1 Non-Human Primates ...... 3-7 3.2.3.1.1.1 Cercopithecine Herpes Virus (CHV-1); Herpes B virus ...... 3-8 3.2.3.1.1.2 Other Viral Infections associated with NHPs ...... 3-9 3.2.3.1.1.3 Gastrointestinal Diseases associated with NHPs ... 3-9 3.2.3.1.1.4 Bacterial Respiratory Diseases associated with NHPs ...... 3-10 3.2.3.1.2 Birds...... 3-10 3.2.3.1.3 Dogs (Canines) ...... 3-11 3.2.3.1.4 Cats (Felines) ...... 3-11 3.2.3.1.5 Rabbits (Lagomorphs) ...... 3-12 3.2.3.1.6 Rodents (Mice, Rats, Hamsters, Gerbils, Guinea Pigs) ...... 3-12 3.2.3.1.7 Ferrets ...... 3-13 3.2.3.1.8 Pigs/Swine ...... 3-13 3.2.3.1.9 Large Hooved Mammals (Cows, Horses, Sheep, Goats) ...... 3-14 3.2.4 Amount of Material Present ...... 3-15 3.2.5 Experience of Staff ...... 3-15 3.2.6 Mode of Transmission ...... 3-15 3.2.7 Environmental Stability ...... 3-15 3.2.8 Institutional Public Image ...... 3-15
Georgia Regents University Biosafety Guide January 2012 ii 4 Risk Management: Biosafety Levels ...... 4-1
4.1 Laboratory Practices ...... 4.4 4.1.1 Human Factors and Attitudes in Relation to Laboratory Accidents ...... 4.4 4.1.2 Practices Associated with Biosafety Levels 1 through 2+ Laboratories ...... 4.4 4.1.2.1 Biosafety Level 1 (BSL-1/BL-1) ...... 4.4 4.1.2.2 Biosafety Level 2 (BSL-2/BL-2)...... 4-6 4.1.2.3 Biosafety Level 2+ (BSL2+) ...... 4-7 4.1.3 Practices Associated with Activities Involving Animals ...... 4-8 4.1.3.1 Animal Biosafety Level 1 (ABSL-1/BL-1N) ...... 4-9 4.1.3.2 Animal Biosafety Level 2 (ABSL-2/BL-2N) ...... 4-11 4.1.4 Tissue/Cell and Microbiological Culture Practices ...... 4-13 4.1.4.1 Tissue/Cell Culture Practices ...... 4-13 4.1.4.2 Microbiological Practices ...... 4-15 4.1.4.3 Glass Ampules ...... 4-16 4.1.4.4 Cryovials/Cryopreservation in Liquid Nitrogen ...... 4-17 4.1.4.4 Vacuum-packed tubes/vials ...... 4-17 4.1.4.5 Embryonated eggs ...... 4-17 4.1.5 Transport of Biohazards on Campus (between labs or buildings) ...... 4-18 4.1.6 Housekeeping ...... 4-18 4.1.6.1 Objectives of Housekeeping ...... 4-19 4.1.6.2 Scope ...... 4-19 4.1.6.3 Assignment of Responsibilities ...... 4-20 4.1.7 Standard Operating Procedure Development...... 4-20
4.2 Laboratory Equipment ...... 4-21 4.2.1. Personal Protective Equipment (PPE) ...... 4-21 4.2.1..1 Laboratory Clothing...... 4-22 4.2.1.1.1 Gloves ...... 4-23 4.2.1.1.2 Procedure for Removing Gloves ...... 4-24 4.2.1.1.3 Shoes ...... 4-24 4.2.1.1.4 Gowns, Lab Coats, Jumpsuits, Aprons and Other Protective Clothing ...... 4-24 4.2.1.1.5 Face and Eye Protection ...... 4-25 4.2.1.1.1.6 Respiratory Protection ...... 4-25 4.2.1.2 Selection of PPE ...... 4-27 4.2.1.3 PPE Requirements Table ...... 4-28 4.2.2 Biological Safety Cabinets ...... 4-30 4.2.3 Laminar Flow Hoods/Clean Benches ...... 4-32 4.2.4 Fume Hoods ...... 4-32 4.2.5 Centrifuges ...... 4-32 4.2.6 Vacuum Line Chemical Traps and Filters ...... 4-33 4.2.7 Syringes and Needles ...... 4-34 4.2.8 Pipettes ...... 4-35 4.2.9 Blenders, Mixers, Sonicators, and Cell Disruption Equipment ...... 4-36 4.2.10 Lyophilizers ...... 4-36 4.2.11 Microtome/Cryostat ...... 4-37 4.2.12 Fluorescence Activated Cell Sortiers (FACS) and FACS analyzers ...... 4-38 4.2.13 Miscellaneous Equipment (Waterbaths, Cold Storage, Shakers) ...... 4-39
4.3 Laboratory Facilities ...... 4-40 4.3.1 Physical Separation of Laboratory Spaces from Non-Laboratory Spaces ...... 4-41 4.3.2 Doors and Locks ...... 4-41 4.3.3 Laboratory and Biological Material Security ...... 4-42 4.3.4 Windows ...... 4-42 4.3.5 Floor Coverings, Walls and Ceilings ...... 4-42 4.3.6 Furnishings ...... 4-42 4.3.7 Lab Benches/Cabinetry ...... 4-43 4.3.8 Exhaust systems/Directional Airflow/HVAC ...... 4-43 4.3.9 Plumbing/Eyewashes/Showers ...... 4-43 4.3.10 Biosafety Cabinets ...... 4-44 4.3.11 Lighting, Air Ducts, Utility Pipes ...... 4-44
Georgia Regents University Biosafety Guide January 2012 iii 4.3.12 Additional Animal Facility Consdierations ...... 4-44
5 Medical Surveillance Program ...... 5-1
5.1 Tuberculosis (TB) Screening...... 5-2
5.2 Immunizations ...... 5-3 5.2.1 Vaccinia Virus and Immunization Recommendations ...... 5-4
5.3 Respiratory Protection Program ...... 5-7
5.4 Medical Restrictions ...... 5-7 5.4.1 Pregnancy ...... 5-8 5.4.1.1 Reproductive Biological Hazards ...... 5-8 5.4.2 Work with Animals ...... 5-9 5.4.3 Allergies ...... 5-9 5.4.3.1 Allergies to Laboratory Animals (ALAs) ...... 5-9 5.4.3.2 Latex Gloves and Related Allergies ...... 5-10 5.4.3.3 Antibiotic Allergies ...... 5-10 5.4.3.4 Mold Allergies ...... 5-11 5.4.4 Other Restrictions ...... 5-12
5.5 Minors in Laboratories, Clinics or Other Hazardous Areas...... 5-12 5.6 Employee Serum Storage ...... 5-13
6 Accidents, Exposures, Spill Response ...... 6-1
6.1 Emergency Procedures for Exposure Incidents ...... 6-1 6.1.1 Percutaneous Injury ...... 6-1 6.1.2 Splash to Face ...... 6-1 6.1.3 Aerosol Exposure ...... 6-1
6.2 Reporting Incident ...... 6-1
6.3 Medical Assistance...... 6-2
6.4 Spill Clean-up Procedures ...... 6-3 6.4.1 Composition of a Basic Biohazard Spill Kit ...... 6-3 6.4.2 Spills Occurring Outside of Laboratory Areas ...... 6-3 6.4.3 Spills Within Laboratory Areas ...... 6-4 6.4.3.1 Biosafety Level 1 (BSL-1) Spills ...... 6-4 6.4.3.2 Biosafety Level 2 (BSL-2) Spills ...... 6-4 6.4.3.3 Blood Spills ...... 6-5 6.4.3.4 Spill in a Biosafety Cabinet ...... 6-5 6.4.3.5 Centrifuge Spill...... 6-6 6.4.3.6 Spill of a Biohazardous Radioactive Material ...... 6-6
6.5 Investigation of Laboratory Accidents ...... 6-7
7 Decontamination of Biologicals ...... 7-1
7.1 Decontamination Methods ...... 7-1 7.1.1 Heat ...... 7-1 7.1.1.1 Autoclave Use and Maintenance ...... 7-1 7.1.2 Chemical Decontaminants ...... 7-2 7.1.2.1 Liquid Chemical Decontaminants...... 7-2
Georgia Regents University Biosafety Guide January 2012 iv 7.1.2.2 Vapors and Gases ...... 7-3 7.1.3 Physical Methods ...... 7-3
7.2 Characteristics of Chemical Decontaminants ...... 7-4 7.2.1 Properties of Some Common Decontaminants ...... 7-4 7.2.1.1 Chlorine/Hypochlorite ...... 7-4 7.2.1.2 Alcohols ...... 7-5 7.2.1.3 Aldehydes (Formaldehyde, Glutaraldehyde) ...... 7-5 7.2.1.4 Phenols ...... 7-5 7.2.1.5 Quaternary Ammonium Compounds (“Quats”) ...... 7-6 7.2.1.6 Iodine ...... 7-6 7.2.1.7 Peroxygens ...... 7-6 7.2.2 Selecting Chemical Disinfectants ...... 7-6 7.2.3 Characteristics of Some Liquid Disinfectants Table ...... 7-7
7.3 Deactivation of Biological Toxins...... 7-10
7.4 Deactivation of Prions ...... 7-13
8 Waste Management ...... 8-1
8.1 Purpose of Biological Waste Management Program ...... 8-1 8.2 Definition of Biomedical Waste ...... 8-1 8.3 Solid Biomedical Waste ...... 8-3 8.3.1 Proper Solid Biohazardous Waste Disposal Procedures ...... 8-4 8.3.2 Non-contaminated Glass ...... 8-6 8.3.3 Improper Solid Biohazardous Waste Disposal ...... 8-6 8.4 Pathological Wastes ...... 8-7 8.5 Liquid Biological Wastes ...... 8-7 8.6 Chemotherapy Wastes ...... 8-8 8.7 Mixed Waste ...... 8-8 8.7.1 Biological/Chemical Mixed Waste ...... 8-8 8.7.2 Biological/Radioactive Mixed Wasete ...... 8-9 8.8 Requisitions for New or Additional Biohazardous Waste Containers ...... 8-9 8.9 Requisition for Biohazardous Waste Pick-up ...... 8-9
9 Placard Signs and Labels ...... 9-1
9.1 Door Placards ...... 9-1
9.2 Labels and Color-Coding ...... 9-2
9.3 Labeling Equipment Sent Out for Repair or Disposal ...... 9-2
10 Shipping ...... 10-1
10.1 Training Requirements ...... 10-1
10.2 Shipping Overview ...... 10-4
10.3 Shipment Type ...... 10-4 10.3.1 Unregulated Biological Materials ...... 10-4 10.3.2 Infectious Substances ...... 10-7 10.3.2.1 Category A Infectious Substances ...... 10-7 10.3.2.1.1 Packaging ...... 10-7 10.3.2.1.2 Labeling ...... 10-10 10.3.2.2 Category B Infectious Substances...... 10-10
Georgia Regents University Biosafety Guide January 2012 v 10.3.2.2.1 Packaging ...... 10-10 10.3.2.2.2 Labeling ...... 10-10 10.3.3 Patient Specimens ...... 10-11 10.3.3.1 Packaging ...... 10-11 10.3.3.2 Labeling ...... 10-11 10.3.3.3 Dried Blood ...... 10-11 10.3.4 Biological Products ...... 10-11 10.3.5 Genetically Modified Organisms or Microorganisms ...... 10-12 10.3.5.1 Packaging ...... 10-12 10.3.5.2 Labeling ...... 10-12 10.3.6 Regulated Biomedical Waste ...... 10-12
10.4 Packaging Biological Material ...... 10-13 10.4.1 Triple Packaging ...... 10-13 10.4.2 Other Packaging Requirements ...... 10-14 10.4.2.1 Overpacks ...... 10-14 10.4.2.2 Dry Ice ...... 10-14 10.4.2.2.1 Hazard Identification ...... 10-14 10.4.2.2.2 Packaging Dry Ice ...... 10-15 10.4.2.3 Liquid Nitrogen ...... 10-17 10.4.2.4 Samples Preserved in Fixative ...... 10-18 10.4.2.4.1 In Aqueous Formaldehyde Solutions <25% (Formalin) ...... 10-18 10.4.2.4.2 In Aqueous Ethanol Solutions of 55-100% ...... 10-20
10.5 Shipper’s Declaration for Dangerous Goods ...... 10-21
10.6 Regulated Agents Which May Require Special Permits for Transfer ...... 10-25 10.6.1 .... CDC/USDA Select Agents & Toxins ...... 10-25 10.6.2 .... Agricultural Pests, Pathogens and Biological Agents ...... 10-25 10.6.3 .... Agents or Vectors of Human Disease ...... 10-26 10.6.4 .... Department of Commerce- Bureau of Industry and Security (BIS) ...... 10-26 10.6.5 .... FDA Import Permits ...... 10-28 10.6.6 .... Fish and Wildlife Service Permits ...... 10-28
10.7 International Shipments ...... 10-29 10.7.1 .... Exporting from the United States ...... 10-29 10.7.2 .... Importing into the United States ...... 10-29
10.8 Shipping Company Restrictions ...... 10-30 10.8.1 .... DHL ...... 10-30 10.8.2 .... FedEx ...... 10-30 10.8.3 .... United Parcel Service (UPS) ...... 10-30 10.8.4 .... United States Postal Service (USPS) ...... 10-30
10.9 United States Postal Service Mailings ...... 10-30 10.9.1 Mailing Category B Substances ...... 10-30 10.9.2 Mailing Exempt Human and Animal Specimens ...... 10-31 10.9.3 Mailing Non-regulated Materials...... 10-31 10.9.3.1 Non-Regulated Liquid Substance, Not Exceeding 50 ml ...... 10-31 10.9.3.2 Non-Regulated Liquid Substance, Exceeding 50 ml...... 10-31 10.9.3.3 Non-Regulated Dry Substance ...... 10-31
10.10 Transport as Checked Airline Baggage ...... 10-31
10.11 Transport in Ground Vehicles ...... 10-32 10.11.1 Passenger Automobiles...... 10-32 10.11.2 Public Modes of Transportation (e.g., Shuttle busses) ...... 10-32 10.11.3 Courier Services ...... 10-32
Georgia Regents University Biosafety Guide January 2012 vi Appendicies ...... A-1
Appendix A Biosafety Protocol Applications Forms ...... A-1 Appendix B Classification of Human Etiologic Agents on the Basis of Hazard ...... B-1 Appendix C Biosafety Levels ...... C-1 Appendix D BL2+ Work Practices ...... D-1 Appendix E SOP Development Template ...... E-1 Appendix F Human Gene Transfer Clinical Trials...... F-1 Appendix G Sources of Contamination ...... G-1 Appendix J Biosafety Notice for Decontamination of Equipment ...... J-1 Appendix K Shipping Labels ...... K-1 Appendix L Map of GRU Campus ...... L-1
Acknowledgements
Georgia Regents University Biosafety Guide January 2012 vii
LIST OF ABBREVIATIONS
AAV Adeno-associated Virus ABSA American Biological Safety Association AdV Adenovirus ALA Allergy to Laboratory Animals ANSI American National Standards Institute APHIS (U.S.D.A) Animal & Plant Health Inspection Sevice ARO Alternate Responsible Official BBP Bloodborne Pathogen BMBL Biosafety in Microbiological and Biomedical Laboratories (CDC/NIH Guidelines) BSC Biosafety Cabinet (a.k.a “Tissue Culture Hood”) BSO Biosafety Officer BSP Biosafety Protocol CCRI Chesapeake Research Review, Inc. (an GRU-authorized IRB) CDC Centers for Disease Control cDNA Complementary DNA CFR (U.S.) Code of Federal Regulations COC (GRU) Compliance Oversight Council DLAS Division of Laboratory Animal Services DOAS (Georgia State) Department of Administrative Services DOC (U.S.) Department of Commerce DOT (U.S.) Department of Transportation DSPA (GRU) Division of Sponsored Project Administration (formerly, Contracts & Grants) EHS Environmental Health and Safety EPA (U.S.) Environmental Protection Agency EPD (GA) Environmental Protection Division FACS Fluorescence Activated Cell Sorting FDA (U.S.) Food and Drug Administration GeMCRIS Genetic Modification Clinical Research Information System (NIH/FDA) HAC (GRU) Human Assurance Committee (GRU’s internal IRB) IACUC Institutional Animal Care and Use Committee IATA International Air Transport Association IBC Institutional Biosafety Committee ICAO International Civil Aviation Organization IND (FDA) Investigational New Drug IRB Institutional Review Board LAS Laboratory Animal Services (see also: DLAS)
LD50 Lethal Dose 50 (i.e. dose of agent required to kill 50% of recipient subjects) LFH Laminar Flow Hood GRU Georgia Regents University GRU HS Georgia Regents University Health Incorporated (the GRU hospital, a private entity, separate from GRU) GRURI Georgia Regents University Research Institute MLV Moloney Leukemia Virus (a mouse retrovirus) MSDS Material Safety Data Sheet NHP Non-human Primate NIH National Institutes of Health NIOSH National Institute for Occupational Safety and Health NSF National Sanitation Foundation, International
Georgia Regents University Biosafety Guide January 2012 viii
OBA (NIH) Office of Biotechnology Activities OCGA Official Code of Georgia OCIS (GRU) Office of Clinical Investigative Services OHRP (GRU) Office of Human Research Protection OIAC (GRU) Office of Internal Audits and Compliance OSHA (U.S.) Occupational Safety and Health Administration OTTED (GRU) Office of Technology Transfer and Economic Development (a.k.a. Intellectual Properties Office) PI Principal Investigator PPD Purified Protein Derivative (a.k.a. Mantoux) tuberculin sensitivity test (a common test for tuberculosis) RAC Recombinant DNA Advisory Committee (to NIH/OBA) RCL Replication Competent Lentivirus RCR Replication Competent Retrovirus rDNA Recombinant DNA RO Responsible Official SAE Serious Adverse Event SAT Select Agents and Toxins SOP Standard Operating Procedures UN United Nations USDA U.S. Department of Agriculture VPR Vice President for Research WHO World Health Organization
Georgia Regents University Biosafety Guide January 2012 ix
FORWARD
This Biosafety Manual has been developed by the Division of Environmental Health and Safety Biosafety Office and the Institutional Biosafety Committee (IBC) at the Georgia Regents University (GRU). This Guide is part of the GRU Biosafety Program instituted to accomplish the following goals:
To protect against exposures of personnel (GRU employees and students, community members and visitors) to biological agents To prevent environmental contamination To provide an environment for high quality research while maintaining a safe workplace To comply with applicable federal, state and local regulations and guidelines To comply with Guidelines implemented by federal funding agencies and accepted by GRU as a condition of funding eligibility To create a secure laboratory environment to prevent unauthorized utilization of a biological agent.
This Biosafety Manual provides university-wide safety guidelines, policies and procedures for the use, possession, manipulation and transport of biological materials. Although the implementation of these procedures is primarily the responsibility of the Principal Investigator (PI), its success depends largely on the cooperative efforts of laboratory supervisors, employees and students. Please read the section on responsibilities for additional information. Planning for and implementation of biological safety must be part of every laboratory activity in which potentially biohazardous material is used.
Recommendations in this Biosafety Manual define a “standard of practice” that laboratories should follow.
In general, the possession, handling, manipulation and transport or biological agents, including: Recombinant DNA molecules Infectious or potentially infectious agents, including human or non-human primate derived material, cultures and genetically modified cells Microbial agents (e.g. viruses, bacteria, mycobacteria, rickettsia, yeast, fungi, prions, parasites) or specimens which may be exposed to microbial agents Toxins of biological origin requires the use of various precautionary measures depending on the material(s), facilities, personnel and their experience, and procedures involved. This manual will provide assistance in the evaluation, containment and control of these biohazards. It is required that all parties involved and/or working with these material be familiar with the contents of this manual, complete the required training, and that they seek additional advice when necessary. The IBC Chairperson, IBC members, as well as the Biosafety Officer (BSO), are available to assist in this endeavor.
This manual focuses on Biosafety Levels 1 and 2, as all GRU laboratories fall within these designations as of May, 2008. A separate manual will be available for researchers working in Biosafety Level 3 research laboratories which will be developed as part of establishing these facilities. No work with Biosafety Level 4 agents may be conducted at GRU.
We urge you to use the manual as a road map to compliance within your laboratory. Consult the sections relevant to your research and apply the appropriate safety procedures. The Biosafety Office is available for consultation if you have any question or concern with any aspect of the Biosafety Program at GRU. The credo, “Think before you act,” and “If you do not know, ask,” are relevant to the use of this manual. If you are unsure of a requirement or biosafety practice, please contact the Biosafety Office at x1-2663 or [email protected] for assistance. We also would appreciate any feedback or comments that you may have with the use of this manual, and will incorporate any suggestions in future versions.
Georgia Regents University Biosafety Guide January 2012 x 1. INTRODUCTION
1.1 EMERGENCY PHONE NUMBERS AND SAFETY, COMPLIANCE AND OPERATIONS CONTACTS Emergency Telephone Numbers Ambulance/Fire/Police x1-2911 Occupational Health Office (FG-1174) x1-3418 Student Health Office (AF-1040) x1-3487 Biological/Chemical/Radiological Emergencies x1-2663 (Monday - Friday, 8:30 AM to 5:00 PM) (After at other times contact Campus Police at x1-2911) GRU Human Resources & Benefits (HS-1111) x1-3770 Georgia Department of Administrative Services 1-877-656-7475
Office of Environmental Health and Safety (EHS) Contacts (CI-1001) http://www.georgiahealth.edu/services/ehs/documents/phonenumbers.pdf
Office of Environmental Health and Safety Main Office (CI-1001) x1-2663 Dr. Danielle Daniely, Biosafety Officer x1-2663 Kelly Close, Environmental & Occupational Health Officer x1-2663 Kenneth Erondu, Chemical Safety Office x1-2663 Philip Maguire, Assistant Radiation Safety Officer x1-9826
Laboratory Animal Services (LAS) and Institutional Animal Care and Use Committee Contacts http://www.georgiahealth.edu/research/animal/contact.html
LAS Main Office (CB-1901) x1-3421 Dr. Victor Monterosso, LAS Director x1-0199 (Vacant), LAS Assoc. Director x1-8575 Rennie Wolfe, LAS Asst. Director x1-2685 Dr. Misty Williams-Fritze, LAS attending vet x1-0111 IACUC Office (CA-1094) x1-0198 Jenny Whitlock, IACUC compliance officer
Facility Management/Research Support & Compliance Office Telephone Numbers
Facilities Management x1-2434 Environmental Services x1-2434 (housekeeping services, biowaste disposal/containers) Laboratory Equipment Services (LES) (CB-0110) x1-6124 (lab equipment repair, biosafety cabinet certification) Information Technology x1-4000 Telecommunication x1-3985 Public Safety (non-emergency) x1-2914 Key/I.D. Badging Office x1-6287 GRUU HS Epidemiology x1-2224 Office of Technology Transfer and Economic Development (CA-2105) x1-9822 http://georgiahealth.edu/research/techtransfer/ Office of Human Research Protection (OHRP) (CJ-2103) x1-1478 http://www.georgiahealth.edu/research/ohrp/
Georgia Regents University Biosafety Guide- 2014 1-1 Office of Clinical Investigative Services (OCIS) (FF-100) x1-9680 http://www.georgiahealth.edu/OCIS/ Division of Sponsored Project Administration(DSPA) Contacts (CJ-3301) x1-2592 http://www.georgiahealth.edu/SPA/ Office of Institutional Audit and Compliance (HS-3135) x1-2661 http://www.georgiahealth.edu/audits/ 24-hour Compliance Hotline (for confidential reporting of compliance concerns) 1-800-576-6623 http://www.georgiahealth.edu/compliance/hotline.html Legal Affairs Office (AA-211) x1-4018 http://www.georgiahealth.edu/services/legal/
1.2 ROLES AND RESPONSIBILITIES 1.2.1 Department Chairperson and/or Center/Institute Director The Department Chairperson and/or Center/Institute Director bears overall responsibility for the implementation and maintenance of safe practices and procedures in the department. Department/Center/Institute Heads have the following responsibilities: To ensure that prior to initiation of work, each Principal Investigator of a Research laboratory, Clinical Director or each clinical laboratory, and/or Instructional Course Director within their department which may expose GRU personnel, students, animals, the environment or the public to biological material files a Biosafety Protocol (BSP) for review by the IBC through the Biological Safety Officer and that approval has been granted prior to the initiation of the research.
To ensure that students, staff and faculty within their department/center/institute have had instruction in safety procedures in research and teaching laboratories or field situations where biological agents are used or collected.
To ensure that resources are made available to researchers, health care providers, laboratory and/or clinic staff, instructors and/or students who require health screening and/or vaccination due to potential risk of exposure to particular biological materials.
To assume responsibility for maintaining the appropriate Biosafety standards and documentation of shared departmental facilities or delegate that responsibility to an appropriate faculty member within the department.
To provide leadership in laboratory or clinic safety at the management level in the department or institute.
1.2.2 Principal Investigators, Clinical Directors and/or Instructional Course Directors The Principal Investigator (PI), Clinical Director, and/or Instructional Course Director in which biological material is used bears the ultimate responsibility and authority for assessing risks, establishing policies and procedures, training personnel and maintaining the facility and equipment. The Principal Investigator, Clinical Director, and/or Instructional Course Director is responsible for: Performing appropriate risk assessment of research projects. The level of detail should be dependent on the hazard associated with the organism under study (e.g., an assessment of risk associated with research on Risk Group 1 agents might reasonably be less detailed than a risk assessment of a Risk Group 2 or unknown agents). Each evaluation should be completed before work is undertaken and the project should be reassessed periodically as new data is obtained. The
Georgia Regents University Biosafety Guide- 2014 1-2 assessment should include an analysis of the risks posed by the particular organism under investigation and of any specific research, clinical or teaching methods that may affect that risk (e.g., procedures requiring highly concentrated amounts of microorganisms or inoculation of laboratory animals). No human or animal pathogen should be studied without prior written approval of the GRU Institutional Biosafety Committee (IBC). The procedures for handling unclassified agents must also be reviewed by the GRU IBC, the Division of Environmental Health and Safety (EHS), as well as the GRU Division of Laboratory Animal Resources (DLAS) if work with animals is anticipated. The agents must be registered and information about these agents must be provided to EHS.
Registering research work involving biological materials, particularly non-exempt recombinant DNA and potential animal, human or plant pathogens with the Institutional Biosafety Committee (IBC). This Biosafety Protocol (BSP) application must detail the nature of the proposed experiments and an assessment of the levels of physical and biological containment required for them as established by the NIH and CDC guidelines. The research described in BSP applications must be accurate and complete description of the research projects in the laboratory and reflect the agents, locations, operations, experiments, manipulations, personnel and safety measures that will be employed in the laboratory. Changes in the protocols must be submitted as amendments to the BSPs to the IBC prior to its initiation. PIs, Clinical Directors and/or Course Directors are responsible for ensuring that no research is initiated by any laboratory personnel prior to review and approval by the IBC.
Developing, establishing and implementing appropriate safety practices and procedures within their laboratories prior to bringing new biological agents to campus and/or before initiating any new research project (independent of funding status) to ensure safe operation and instructing students and staff of potential hazards. This involves: • Being knowledgeable in good laboratory practices and maintaining current knowledge of new safety practices and/or equipment which may improve safety within the laboratory.
• Demonstrating a positive safety attitude.
• Making available to the laboratory staff copies of the written procedures that describe potential biohazards, precautions, and actions to be taken in response to spills and accidents to include decontamination procedures, and emergency procedures. These procedures and other information addressing Biological Safety-related issues will be produced in the form of a standard operating procedure (SOP) for the work. The SOP will reside within the laboratory and be easily accessible for reference and provided to the IBC for review as part of the Biosafety Protocol application.
• Maintaining up-to-date knowledge to changes in international federal, state, local regulations and guidelines pertaining to biological materials, in consultation with the Biosafety Office, and modifying the laboratory procedures to comply with these.
Approving research personnel to work in the laboratory and documenting that personnel are competent to conduct the work. PIs, Clinical Directors and/or Course Directors are responsible for the safety of personnel on their Biosafety Protocols (BSPs) and their actions. This includes: • Providing laboratory staff with documented formal and informal instruction and training in the practices and techniques required to ensure safety and in the procedures for dealing with accidental spills, personnel contamination, and other laboratory accidents or emergencies.
• Informing the laboratory staff of the risks involved with the biological agents in the laboratory and the reasons and provisions for any precautionary medical practices (e.g. physical examinations, serum collection, and vaccinations).
Georgia Regents University Biosafety Guide- 2014 1-3 • Making provisions for any precautionary medical practices, including occupational health physical examinations, vaccinations and/or other medical surveillance of personnel when required by the agents and nature of the experiments.
• Supervising and monitoring the performance of the staff to ensure that required safety practices and techniques are employed.
• Ensuring the authorized staff complete the appropriate IBC-required training modules and keeping these training records up-to-date.
Maintaining a liaison with the Biosafety Office. This includes: • Amending and modifying Biosafety Protocols (BSPs) to reflect changes in agents, personnel, locations, experimental application, operations and/or safety equipment.
• Reporting, in writing, any accident, potential exposure or exposure of personnel, suspected illness, and/or release from containment of any biohazardous agents. Any significant problems pertaining to the operation and implementation of containment practices, procedures or facilities should also be reported to the Biosafety Office.
• Providing accurate information for compliance verification processes when requested.
Maintaining compliance with all Federal, State and/or local regulations related to possession, use, transfer and/or disposal of Biohazardous materials. This would include the following regulations: • Federal Select Agent regulations (42 CFR §73.3, 7 CFR §331.3 and 9 CFR §121.4) for use, possession and transfer of Select Agents and Toxins.
• U.S. Department of Transportation Regulations (49 CFR), U.S. Public Health Service (42 CFR §72) and IATA guidelines for shipping and/or transport of hazardous or etiological materials.
• U.S. Departments of State, Commerce and Treasury Regulations related to Export control laws.
• U.S. Department of Agriculture Animal and Plant Health Inspection Service Regulations related to transportation, importation or exportation of animal or animal products, genetically engineered organisms, plants or plant products and/or soil samples.
• U.S. Public Health Service importation and/or exportation requirements for Etiologic Agents, any arthropod and/or other animal host or vector of human disease, including unsterilized specimens of human and animal tissues (such as blood, body discharges, fluids, excretions or similar material) containing an infectious or etiologic agent.
• O.C.G.A. §31-12-13 and OSHA (29 CFR §1910.1030) standards for blood-borne pathogen handling, medical surveillance, training and record-keeping.
• Georgia EPA Solid Waste Management Laws Chapter 391-3-4-.15 for Biomedical Waste.
Ensuring that the terms and conditions of NIH Grants Policy Statement are maintained within the laboratory for all research projects (independent of funding status or sponsor). This includes compliance with the NIH Guidelines for Research with Recombinant DNA Molecules, the Occupational Health and Safety Administration (OSHA) standards included in 29 CFR Part 1910, and other applicable safety guidelines, including those in the CDC/NIH publication Biosafety in Microbiological and Biomedical Laboratories.
Georgia Regents University Biosafety Guide- 2014 1-4
1.2.3 Research and GRU Clinical Personnel and GRU Students Research and GRU Clinical Personnel and GRU students are responsible for: Completing requirements for approval to work in the laboratory and ensure that all work is conducted in compliance with GRU, NIH, CDC, OSHA, state labor and waste management laws, DOT and other applicable guidelines and regulations and GRU IBC policies prior to initiation of any work with biological materials at GRU (See Section 2, Biosafety Requirements). Follow the GRU Biological Safety Guide except where superseded by the GRU BSL-3 Manual.
Learning the standard operating procedures (SOPs) for the laboratory, the potential hazards of the infectious agents in use and emergency procedures. Personnel are responsible for helping to maintain the facility in good working condition and maintaining compliance with the laboratory Biosafety Protocol and SOPs.
Maintaining their work areas neat and clean. All containers in which biological materials are placed should be appropriately labeled with biohazard stickers. Biohazardous waste must be disposed according to laboratory Standard Operating Procedures (SOPs).
Completing any medical surveillance requirements as required by the IBC on the Principal Investigator’s Biosafety Protocol Agreement with the IBC prior to initiation of work with biological materials at GRU.
Reporting to the Principal Investigator (PI), Clinical Directors and/or Course Directors any medical restrictions, reportable illnesses, and any event that may be an exposure or result in the creation of a potential hazard. Report all irregular conditions.
Bring to the attention of the PI, Clinical Director and/or Course Director any practice on the laboratory SOPs that is impossible or impractical to maintain operations within the laboratory so amendments to SOPs and Biosafety Protocols can be submitted which accurately reflect laboratory operations or provide suggestions of new SOPs which should be added to the laboratory SOP list to better facilitate communication of safety issues among the laboratory staff.
If inexperienced in handling human pathogens, tissue culture, recombinant DNA and/or microorganisms, receiving additional training from the Principal Investigator and demonstrating proficiency in these practices to the Principal Investigator prior to initiation.
Performing responsibilities assigned to them by the Principal Investigator, Clinical Director or Instructional Course Director. The operation of the facility is the responsibility of the users; therefore a number of tasks must be assigned. These tasks may include the following: • Training of other staff members • Autoclave maintenance and waste management • Freezer, refrigerator, equipment maintenance • Cleaning • Vacuum trap and filter maintenance • Maintenance of supplies, including personnel protective equipment • Security of infectious agents; i.e. store infectious agents in a locked freezer in a locked laboratory
1.2.4 Division of Environmental Health and Safety (EHS), Biological Safety Office and Biosafety Officer (BSO) GRU’s Biological Safety Office has responsibility for the daily administration of standards set by the GRU Institutional Biosafety Committee (IBC) and acts as the agent of the Committee in the implementation of
Georgia Regents University Biosafety Guide- 2014 1-5 their standards. In addition, the Biosafety Office serves as a resource to researchers, administration, compliance and maintenance departments, as resources allow. Responsibilities include: Providing information and consultation on operation of the laboratory to ensure compliance with CDC, NIH, USDA, OSHA, DOT, EPA, state and local requirements to researchers, administrators and other institutional compliance and maintenance offices.
Evaluation and inspection of laboratory facilities for work with infectious agents, recombinant DNA and other potentially hazardous biological agents. This includes advising on safety measures and equipment for new procedures that may be utilized to mitigate risks associated with working with potentially hazardous materials.
Administration and maintenance of records for the Institutional Biosafety Committee, including Biosafety Protocols, Personnel training records, meeting minutes and scheduling of IBC meetings.
Periodic review of rDNA experiments documented in the Biosafety Protocols to ensure compliance with the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines)
Providing general biosafety training programs related to proper handling of biological materials and maintenance of training records for compliance with federal, state and University requirements (laboratory-, agent- and operation-specific training is the responsibility of the Principal Investigator (see Section 1.2.2)
Providing advice and assistance in the event of large, high hazard or public biological material spills.
Investigation of laboratory incidents, accidents, exposures, potential exposures, illnesses which may have resulted from potential exposures to biological material in the laboratory, releases or possible releases from containment of biological materials to ensure appropriate emergency follow-up procedures have been followed. If alternate incident mitigation and/or management procedures are required to circumvent future similar incidents, the Division of Environmental Health and Safety will make recommendations to the appropriate safety committees to address these.
Coordinating/consulting with other institutional safety and compliance offices to maintain compliance and safety standards.
Serve as a point of contact and information for Facilities Maintenance, Security, IT, Public Safety, internal and external responders related to safety with biological materials and research activities within GRU facilities.
Identification and updating of areas of known and potential biohazards at GRU to the IBC on a regular basis.
Within the Biosafety Office, the Biological Safety Officer (BSO) is the Institutional Biological Safety Officer for recombinant DNA research, and required to maintain compliance with NIH Guidelines for Research with Recombinant DNA Molecules (NIH Guidelines). NIH assigns the BSO the following roles and responsibilities: Periodic inspections to ensure that laboratory standards are rigorously followed;
Reporting to the Institutional Biosafety Committee and the Institution any significant problems, violations of the NIH Guidelines, and any significant research-related accidents or illnesses of which the Biological Safety Officer becomes aware unless the Biological Safety Officer determines that a report has already been filed by the Principal Investigator;
Georgia Regents University Biosafety Guide- 2014 1-6 Developing emergency plans for handling accidental spills and personnel contamination and investigating laboratory accidents involving recombinant DNA research;
Providing advice on laboratory security;
Providing technical advice to Principal Investigators and the Institutional Biosafety Committee on research safety procedures.
The BSO also currently serves as the Institutional Responsible Official (RO) while the Associate Vice President of Environmental Health and Safety currently serves as the Alternate Responsible Official (ARO) for Compliance with the Federal Regulations on the Possession, Use, and Transfer of Select Agents and Toxins as described in 42 CFR 73, 7 CFR 331 and 9 CFR 121. As such, the RO and ARO have the authority and responsibility to act on behalf of the institution as dictated by these laws.
1.2.5 GRU Institutional Biological Safety Committee (IBC) The GRU Institutional Biosafety Committee (IBC) serves to maintain institutional compliance with the National Institutes of Health (NIH) Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines), the Centers for Disease Control (CDC) Biosafety in Microbiological and Biomedical Laboratories (BMBL), and International, Federal, state and local regulations pertaining to the handling of biological materials. The IBC shall also advise the President, Provost, Vice Presidents of Research and Administration and the Associate Vice President of the Division of Environmental Health and Safety (EHS) and/or other pertinent offices on policy matters concerned with the protection of personnel from biohazardous agents including both infectious organisms and allergens that may be present in either laboratory materials or the environment. The IBC shall also recommend guidelines relating to procedures and facilities used at the University, including such matters as safety training and health surveillance. The IBC shall offer its counsel to all University personnel regarding matters of biological safety. The President, Provost and/or Vice Presidents of Research or Administration may ask the Committee to inform the community about developments in the general area of biological safety. The IBC’s composition, roles and responsibilities adhere to those dictated in NIH Guidelines and the membership can be viewed at: : http://www.georgiahealth.edu/services/ehs/biosafe/IBCmembers.pdf. As such, the Committee is required to review applications for research involving recombinant DNA and biological materials to determine whether the facilities, procedures, and practices meet the standards required by the University and the NIH. It shall, in addition, have the responsibility to certify annually to the NIH that such facilities, procedures, and practices, and the training and expertise of personnel meet NIH standards. Meetings called for the purpose of such review and certification may be open to the public. Minutes of these meetings shall be kept and made available for public inspection. The IBC's responsibilities include: Review applications and perform comprehensive risk assessments to determine the appropriateness and adequacy of containment levels and safety measures proposed and/or used in research, clinical duties and teaching.
Assess the adequacy of containment facilities for biological agents and rDNA molecules as required by NIH or other funding or regulatory agencies. The IBC may down-grade or up-grade containment levels as appropriate to address the risks associated with the proposed activities.
Assess for adequacy the facilities, procedures, practices, training and expertise of personnel involved in the research, clinical duties and/or instructional activities.
When applications involve Select Agents, ensure that approval is granted only to those individuals who meet the access requirements stated in Federal Regulations on the Possession, Use, and Transfer of Select Agents and Toxins as described in 42 CFR 73, 7 CFR 331 and 9 CFR 121 and
Georgia Regents University Biosafety Guide- 2014 1-7 other applicable Federal regulations.
Develop with the Biological Safety Officer informational and training seminars and workshops on biohazards for the GRU community.
Periodically review biohazardous research, clinical duties, and/or instructional activities being conducted at GRU to ensure that the requirements of the University, funding sources, and regulatory agencies are being fulfilled.
Recommend to GRU Administration appropriate sanctions for noncompliance with Biological Safety standards, guidelines, or regulations.
Adopt emergency plans covering accidental spills and personnel contamination resulting from biohazardous research.
When experiments involve rDNA in humans, the IBC will grant no approval and ensure that no activities are conducted until NIH OBA Recombinant DNA Assurance Committee approval has been obtained and compliance with Appendix M of NIH Guidelines can be ensured.
In addition, the IBC shall oversee the activities of the Biosafety Office in the sense that it shall: review its objectives and performance goals;
monitor its progress in meeting those objectives and goals, and
recommend changes in the organization and activities of the Biosafety Office that the Committee may find desirable
The IBC shall meet regularly with the Associate Vice President of EHS and the Biological Safety Officer to receive progress reports and advise on specific safety issues as well as on general safety policy. On matters of oversight that involve the evaluation of performance by the Biosafety Office, the Committee may, at the discretion of the chair, meet in executive session. In such cases the Biological Safety Officer, the Associate Vice President EHS and any other EHS office representatives shall be excused from participation and voting.
1.2.6 The Georgia Regents University (GRU) The Georgia Regents University and its administrative officers are ultimately responsible for the following: Developing and maintaining appropriate policies regarding the conduct of potentially biohazardous research, education, and service activities.
Developing mechanisms for ensuring adherence to Biological Safety policies.
Providing the resources necessary for the construction of safe research, clinical and teaching facilities and for the implementation of the Biological Safety Program.
Providing adequate resources for IBC member training on biohazards and biological safety procedures, including training programs and workshops.
Providing resources for appropriate medical surveillance measures to protect the health and safety of employees.
Providing appropriate and sufficient legal protection for faculty and staff members who conduct
Georgia Regents University Biosafety Guide- 2014 1-8 activities in compliance with appropriate regulations and guidelines.
Coordinate efforts between institutional safety and compliance offices to ensure compliance with International, Federal, State and Local regulations and guidelines for research and clinical care.
1.2.6.1 Other Offices and Committees at the Georgia Regents University (GRU) 1.2.6.1.1 Facilities Operations & Maintenance The GRU Facility Operations and Maintenance Division is responsible for: Ensuring that the design and construction of all laboratory and clinical facilities meet the standards of containment to maintain compliance with Federal, State and Local Regulations, NIH Guidelines for Research with Recombinant DNA Molecules, the Centers for Disease Control Biosafety in Microbiological and Biomedical Laboratories and the GA Board of Regents among other standards and their intended purposes.
Making sure the mechanics and integrity of the facilities are maintained to ensure proper protection of workers within the facilities and proper protection of the environment from hazardous materials within the buildings.
Notifying the Biosafety Office, laboratory director and building manager any planned maintenance of the facilities which would require even short suspension of utility operations required to maintain safety in the laboratory. This may include tests or repairs of HVAC, electrical, plumbing or vacuum systems.
Immediately reporting any possible failures in facility containment that may have resulted in environmental release of biological materials or potential exposures of any personnel to biohazardous materials to the Biosafety Office (x1-2663 or [email protected]@georgiahealth.edu), as well as the building directors, departmental chairs and researchers.
Reporting any issues of non-compliance that are noticed while within laboratories, particularly if these may potentially place Facilities personnel at higher risk for exposure (e.g. inappropriate waste disposal for sharps or biological material).
Because proper design and function of laboratory facilities is one of the key components involved in management of risk within a laboratory, Facility Operations and Maintenance is encouraged to consult and coordinate with the Biosafety Office during design of new or renovated laboratory facilities and/or during construction or maintenance operations which may require untrained personnel to enter areas containing biohazardous materials.
1.2.6.1.2 The Institutional Animal Care and Use Committee (IACUC) and Division of Laboratory Animal Services (LAS) To comply with federal laws and institutional policies governing the humane care and treatment of laboratory animals, GRU requires that all use of invertebrate and vertebrate animals for research, education, or for any other purpose be documented in an Animal Use Protocol (AUP) which must be reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) prior to initiation of the work. The IACUC administrative office specifically supports the IACUC compliance mission, which works closely with the Division of Laboratory Animal Services (LAS), to address laboratory animal care and
Georgia Regents University Biosafety Guide- 2014 1-9 welfare issues The IACUC evaluates AUPs based on several criteria, most of which focus on the ethical and humane treatment and care of the animals, including: . Rationale and purpose of the proposed use of animals. . Justification of the species and number of animals requested. Whenever possible, the number of animals requested should be justified statistically. . Availability or appropriateness of the use of less-invasive procedures, other species, isolated organ preparation, cell or tissue culture, or computer simulation. . Adequacy of training and experience of personnel in the procedures used. . Unusual housing and husbandry requirements. . Appropriate sedation, analgesia, and anesthesia. . Unnecessary duplication of experiments. . Conduct of multiple major operative procedures. . Criteria and process for timely intervention, removal of animals from a study, or euthanasia if painful or stressful outcomes are anticipated. . Postprocedure care. . Method of euthanasia or disposition of animal. . Safety of working environment for personnel.
Although the IBC’s oversight of the health and safety of the (human) researchers, the community and the environment (including control of infection within the animal care areas) may overlap with the IACUC’s concerns for animal welfare and caretaker safety, each Committee reviews research protocols from different perspectives. Therefore, PIs may need to submit both AUP and BSP applications to the IACUC and IBC, respectively, in order to receive both Committees’ approvals to maintain compliance. To address common concerns, the IACUC and IBC (as does LAS and Biosafety Office) work in close collaboration with each other. IBC approval may be required by IACUC before initiation of projects involving biohazardous materials or recombinant DNA in conjunction with live animals or prior to completion of processing IACUC approvals.
1.2.6.1.3 The Institutional Review Boards (IRBs) and the GRU Office of Human Research Compliance (OHRP) The purpose of the GRU-approved Institutional Review Boards (IRBs) (i.e., the Human Assurance Committee (HAC) or Chesapeake Research Review, Inc.) is to ensure the principles outlined in the Department of Health and Human Services (DHHS) policies, the Belmont Report, the Nuremberg Code and the Declaration of Helsinki are maintained in all GRU investigations involving human subjects. These principles were established to safeguard the rights and welfare of human subjects of research investigations, and to fulfill the moral and legal obligations and commitments of the institution. The IRB administrative office and compliance officers are located within the GRU Office of Human Research Compliance (OHRP). The IRBs evaluate Human Use/Clinical Protocols (i.e., HAC or CCRI applications) based on several criteria, most of which focus on the ethical and legal obligations of GRU toward human research subjects including: . The rights and welfare of the human research subjects involved. . The appropriateness of the methods used to obtain informed consent from research subjects. . The risks to the human subjects and the potential benefits of the investigations to the patients and mankind.
Georgia Regents University Biosafety Guide- 2014 1-10 With the exception of protocols which may involve the delivery of recombinant DNA or other biological materials (e.g., cells, tissues, toxins of biological origin), IBC reviews focus more on the health and safety of the researchers, the community and the environment; therefore, IRB and Biosafety Protocol (BSP) applications and the information required of each differs. Therefore, PIs may need to complete two separate applications and receive both Committees’ approvals to maintain compliance with both the human use and biosafety requirements. Although the IRB and IBC reviews are independent of one another, some of the issues (e.g. delivery/collection of biohazardous material or recombinant DNA into/from human research subjects) overlap; therefore, the IRB and IBC (and consequently the Office of Human Research Protection and Biosafety Office) work in close collaboration. IBC approval may be required by OHRP before completion of IRB approvals for projects involving biohazardous materials or recombinant DNA in conjunction with human research subjects.
1.2.6.1.4 Division of Sponsored Projects Administration (DSPA) The Division of Sponsored Program Administration (DSPA), formerly the Office of Grants and Contracts, serves as the principal interface between the Georgia Regents University and external agencies providing sponsored program support, including the Georgia Regents University Research Institute (GRURI). As part of each application for sponsored funding, DSPA acts on behalf of the Senior Vice President of Research to ensure that each research project or contract complies with Federal, State, funding agency and institutional standards. The routing form that accompanies each application for funding serves as a way for DSPA to document that the Principal Investigator verifies that all compliance requirements, including those related to Biological Materials, have been completed. Any use of biological material needs to be fully disclosed on the routing sheet(s), and any Biosafety protocol (BSP) Authorization number(s) listed on the routing form must refer to the Biosafety Protocol(s) (BSPs) in which the biological materials, operations, facilities, personnel and equipment which will be used to accomplish the experiments described in the project application, have been fully described, reviewed and approved by the IBC. As indicated on the routing form, you “may be subject to criminal, civil or administrative penalties should any of the information contained on this form or in the submitted application… be false, fictitious, or include fraudulent statements.” Prior to establishment of an account associated with a sponsored project or contract, DSPA contacts the relevant compliance offices (OHRP, IACUC, Biosafety, Chemical Safety, Radiation Safety) to request verification from each office regarding the compliance status of the specific project. If compliance cannot be verified by any one of these offices for the project in question, DSPA will not establish the account associated with the sponsored project.
1.2.6.1.5 Office of Institutional Audits and Compliance (OIAC) and the Compliance Oversight Council (COC) The Office of Institutional Audit and Compliance (OIAC), formerly the Division of Internal Audits, is responsible for the institution’s internal audit function and compliance oversight activities. The OIAC maintain a Compliance Hotline to which any complaints or concerns related to any compliance issues can be reported (confidentially, if desired). Compliance hotline information can be accessed at the following URL: http://www.georgiahealth.edu/compliance/hotline.html
Georgia Regents University Biosafety Guide- 2014 1-11 In addition to their financial audit responsibilities, OIAC administers training and addresses questions regarding compliance with the Health Insurance Portability and Accountability Act of 1996 (HIPAA), Family Educational Rights and Privacy Act of 1974 (FERPA), and other research and business ethical standards. The OIAC is also currently serving as a central point of contact for compliance with the Departments of Commerce, State and Treasury Export Control Regulations, which may limit access and shipping of materials with potential commercial, military or economic value, including some biological materials. Further information and contacts can be accessed at their web page: http://www.georgiahealth.edu/compliance/coc.html The Compliance Oversight Council (COC) is made up of member’s from GRU’s executive management and is chaired by the Director of the Office of Institutional Audits and Compliance. Their purpose is to coordinate and monitor, as necessary, other GRU campus compliance efforts, collect annual compliance information and assemble an institutional compliance report, coordinate and track the GRU/MCGHI/PPG compliance hotline issues. Information on the COC can be found at: http://www.georgiahealth.edu/compliance/matrix.html
1.2.6.1.6 Office of Technology Transfer and Economic Development (OTTED) The Office of Technology Transfer and Economic Development (OTTED) (often referred to as the Intellectual Properties Office) is designed to help faculty, staff and student researchers with their inventions by serving as a resource on matters related to intellectual property. By identifying, evaluating and protecting novel ideas, the OTTED serves the inventor but also GRU and the GRU Research Institute. Because of its potential value, sharing of Intellectual Property among collaborators must be documented in writing using a Material Transfer Agreement (MTA). The OTTED will assist researchers in setting up MTAs to document shipment or receipt of Intellectual properties. MTAs are not typically viewed by the Biosafety Office or the IBC. Receipt of new biological materials on campus must be documented on the Principal Investigator’s Biosafety Protocol (BSP) and approved by the Institutional Biosafety Committee (IBC) prior to receipt on campus to ensure appropriate containment is available for the risks that the agent(s) may pose. Therefore, receipt and documentation of any new biological material on an MTA does not, by any means, substitute for documentation and approval by the IBC for receipt of new biological materials.
Georgia Regents University Biosafety Guide- 2014 1-12
2. BIOSAFETY REQUIREMENTS
The following information describes the requirements for GRU researchers as defined by the GRU Institutional Biosafety Committee (IBC) and the GRU Biosafety Office. It is the responsibility of each Principal Investigator, Clinical Director and/or Instructional Course Director to ensure their workplaces are in compliance.
2.1 REGISTRATION FOR THE USE OF BIOLOGICAL MATERIALS
All Principal Investigators working with biological materials are required to complete and submit a Biosafety Protocol (BSP) Application prior to bringing new biological materials to campus or initiation of the research independent of its funding status. Incoming Principal Investigators establishing a new laboratory at GRU must submit the application prior to bringing biological materials to campus (see Section 2.3.1, Incoming New Faculty to GRU). The BSP must be kept current to accurately reflect the biological materials and their manipulation, personnel handling the material and the locations in which the material may be handled or stored (see Section 2.1.2, Amendments to Biosafety Protocols).
A copy of the Biosafety Protocol Application form can be found in Appendix A and forms can be accessed online at: http://www.georgiahealth.edu/research/ibc/apps.html. The risk assessment and mitigation methods associated with the PI’s research project(s) must be performed and documented in this application (See Section 3, Risk Assessment and Section 4, Risk Management for further information) to enable the Institutional Biosafety Committee (IBC) to evaluate and perform a mandated independent comprehensive risk assessment. In particular, BSPs require documentation of the following materials and operations: Non-exempt recombinant DNA (as defined by the NIH Guidelines for Research with Recombinant DNA), mammalian cells, tissues, organs or fluids requiring ≥BSL-2 containment Risk Group ≥2 microbial agents (as described in the Centers of Disease Control publication Biosafety in Microbiological and Biomedical Laboratories and the American Biosafety Association) Large scale (≥10 liters) cultures of biological materials Biological materials delivered into animal or human subjects Toxins of biological origin with LD50 ≤100µg/kg body weight Shipping of biological materials, toxins of biological origin, materials on dry ice or liquid nitrogen, or genetically modified organisms
The BSP application is a modular form and consists of several parts:
1. The Primary Application Form for Research Involving Biological Materials and/or Recombinant DNA. This form is required for all applications and is intended to describe: • the overall project aims • the general nature of biological materials/recombinant DNA and manipulations of these • the locations in which the biological materials/recombinant DNA may be located at any moment in time • personnel working on the project, and their experience levels (note: completion of all current required training modules by all personnel and PIs on the BSP is required by the IBC prior to release of IBC approvals)
2. Biosafety Schedule(s), as required to document and address the risks associated with each BSP’s agents and research applications. The Biohazardous Material Compliance Worksheet on p. iii of the Primary Application Form (see Appendix A) is intended as a guide to help assess which, if
Georgia Regents University Biosafety Guide- June 2008 2-1 any, additional supplemental schedules may be required for completion of the BSP. These schedules are as follows:
A: Non-exempt recombinant DNA B: Handling and/or culture of mammalian cells, tissues, and/or organs requiring ≥ BSL-2 containment (including human or primate blood, tissues or fluids; cell, tissue or organ cultures of human or non-human primate origin, or animal cells, tissues or organs which have been potentially infected with Risk Group ≥2 agents C: Research involving large-scale (≥10 liter) cultures D: Research involving potentially infectious microbial agents (Risk Group ≥2) or specimens exposed to these infectious microbial agents (excluding any material previously described in either Schedule A or B) E: The use of biological materials in association with live animals F: Human gene transfer/therapy G: HHS/USDA regulated Select Agents and Toxins O: Use of biological agents and/or recombinant DNA in applications for sponsored funding or IRB approval
3. A copy of the laboratory’s Standard Operating Procedures (SOPs). The risks associated with the experiments involving the agents, operations, locations, personnel and environment described in the forms above should be addressed by the mitigating measures in these SOPs. (Laboratory SOPs should also be provided to incoming laboratory staff members and reviewed with the PI as a training measure within the laboratory).
4. A copy of the laboratory’s Self-Audit form.
Once completed the above forms should be submitted to the Biosafety Office. Hard-copies can be submitted via campus mail to: Biosafety Office, EHS, CI-1001 or electronic copies can be sent to [email protected].
Please note that all Biosafety Protocol applications and amendments are reviewed for compliance with the annual training requirements as described in Section 2.2, Training Requirements, below, to comply with specifications with the Georgia Board of Reagents (BOR), NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines), the Centers of Disease Control and the Georgia State Department of Labor. The Biosafety Office will also assist in updating information during periodic the biological lab inspections.
2.1.1 Annual Affirmation of Biosafety Protocol Information
The Division of Environmental Health and Safety must maintain accurate information regarding the use of biological materials (e.g., microorganisms, cell lines, human materials, animals, and toxins) by GRU personnel. Approvals for BSPs typically issued by the IBC are for an indefinite period of time, as long as the research, personnel, practices and locations have not changed. Therefore, the IBC requires all Principal Investigators, Clinical Directors and/or Instructional Course Directors to annually review their BSPs to ensure that the information provided continues to accurately reflect the current situation in their laboratories, and submit a written affirmation to the IBC that their BSP(s) are up-to-date and accurate. Protocol Renewal forms can be found at: http://www.mcg.edu/research/ibc/apps.htm and can be submitted via campus mail to the Biosafety Office, Division of Environmental Health and Safety, CI-1001.
Georgia Regents University Biosafety Guide- June 2008 2-2 2.1.2 Amendments to Biosafety Protocols
Amendments must be submitted to the Biosafety Office by the Principal Investigator, Clinical Director and/or Instructional Course Director and approved prior to implementing any change(s) to IBC-approved Biosafety Protocol. Documentation should be provided for changes in any of the following: Addition or deletion of biological materials and/or non-exempt recombinant DNA. Changing the design, operations, scope or location of existing work, including alterations in: • Any non-exempt recombinant DNA, including new vectors, inserts or operations • Administration or exposures of new target cells, organs, organisms and/or animals • Employing new techniques which may increase risk of exposure of personnel (see Risk Assessment and Risk Management Sections 3 & 4 of this Biosafety Guide) • Creation of a new transgenic strain of animal • Embarking upon large-scale culture (≥10 liters) • Changes in locations • Changes in major equipment used (e.g., Biosafety cabinets, centrifuges) Addition or deletion of any personnel to your list of authorized users who may be handling your biological materials. Providing infectious agents to another investigator on or off campus. Arranging for visiting researchers and/or volunteers to work in your laboratory.
Amendments must comprehensively describe the new proposed changes, and document any new risk assessment(s) and proposed risk management methods, as appropriate. Amendments can be submitted by altering the original Biosafety Protocol Application forms to include the new information and re-submitting them to the Biosafety Office, or via a fully detailed email to the Biosafety Office. Any amendment which involves an alteration in risk relative to the previously-approved BSP will be submitted to the IBC for review and approval; simple amendments involving no alteration in risk (i.e., personnel changes, additions of grant/contract/clinical protocol titles to previously-approved BSPs) will be handled administratively by the Biosafety Office. All questions can be addressed to the Biosafety Office (x1-2663 or [email protected]).
2.1.2.1 Adding New Personnel to or Removal of Personnel from a Biosafety Protocol Before a new employee, student, volunteer, or visitor may handle the biological material documented on a Biosafety Protocol, their name must be added to the list of authorized personnel on the BSP. PIs should first submit the name(s) of new personnel to the Biosafety Office ([email protected]). In doing so, the PI takes responsibility for ensuring that all training and laboratory entrance requirements listed in the laboratory SOPs, BSP document and Institutional Biosafety Committee Authorization document have been completed by this new person before authorizing them to work in the laboratory.
The Biosafety Office will verify that the new person has completed the IBC-required training modules (as described in Section 2.2, Training, in addition to any special training requirements as required by the IBC for the BSP and documented in the PI’s Institutional Biosafety Committee Authorization document) before administratively adding their names to the list of authorized personnel of the Biosafety Protocol.
As with all personnel listed in the BSP, the PI, Clinical Director and/or Instructional Course Director is responsible for communicating the risks associated with the biological material and procedures in the laboratory, as well as the laboratory-specific mitigation methods. Therefore, the PI should review the laboratory standard operating procedures (SOPs), biosafety protocol(s) and GRU Biosafety Guide with any new personnel, including emergency response procedures and entrance requirements before allowing them to start work within the laboratory. The PI is also responsible for ensuring proper supervision and training is provided to any new personnel until proficiency in handling the materials and mastery of the techniques can be demonstrated.
Georgia Regents University Biosafety Guide- June 2008 2-3
New personnel should also be offered and provided employee health counseling and/or vaccinations as documented in the Biosafety Protocol prior to initiation of work within the laboratory.
Written notification should be provided to the Biosafety Office by the PI, Clinical Director or Instructional Course Director if someone should be removed from their list of authorized personnel upon their departure from the laboratory.
2.1.2.2 Amendments for New or Modified Biological Agents or Operations to a Biosafety Protocol
Any changes in the Biological Agents to be utilized in the laboratory that are not covered in the previously-approved BSP, must be documented to enable the comprehensive risk assessment by the IBC and evaluation whether the documented mitigation methods are appropriate for the new materials or operations.
In particular, any changes in non-exempt recombinant DNA materials or applications must be documented and submitted to the full IBC for review to maintain strict compliance with NIH Guidelines for Research with Recombinant DNA Molecules. This includes changes in inserts, vectors or target cells/tissues/organisms or operations. This is a condition of NIH funding to the entire institution and applies to all research at the institution, independent of its funding source.
Any change which might alter the risk of a BSP must also be documented. For instance, if the infectious potential status of biological material handled differs from that previously documented and approved in a BSP, an amendment must be submitted. For instance, if a previous BSP included approval for handling blood samples obtained from non-infectious patients and the PI wishes to initiate a study involving taking blood specimens from infectious patients (e.g., HIV+ patients), this change must be submitted and approved by the IBC prior to initiation.
Similarly, if new operation will be employed with the biological agent(s) which may involve additional risk, this must also be documented. For instance, if a previous BSP included approval for handling the biological agents in vitro, and the PI wishes to initiate a study involving in vivo administration of the biological agent into animals or humans, this change must be submitted and approved by the IBC prior to initiation. Another example of operational changes that may alter the risk of a BSP includes utilizing new procedures with the biological which may involve additional splash, spray or aerosol exposure risks (e.g., sonication, homogenization, FACS sorting, etc.). See Section 3, Risk Assessment for further details.
BSP Amendments involving changes in agents or operations are reviewed and approved by the IBC via the same procedures as the original BSP (see Section 2.5.1 for further details).
2.1.2.3 Modifying the Locations or Equipment on a Biosafety Protocol
If a PI’s biological agents will be handled or stored in locations other than those documented on their IBC-approved BSPs, the new location(s) must be documented and approved by the IBC in order to ensure that the containment is appropriate to handle the risks presented by the agents. A laboratory assessment by the Biosafety Office will likely be required prior to approval of the amendment.
Some common locations (such as core laboratories or common storage rooms) may have previously been approved by the IBC for other BSPs involving similar agents/operations;
Georgia Regents University Biosafety Guide- June 2008 2-4 amendments to add these locations to a BSP are often approved administratively through the Biosafety Office. However, any area proposed for handling or storage of biological materials which has not been previously reviewed and approved by the IBC will need to be authorized by the IBC before its use to ensure appropriate containment.
Because major equipment used to contain or handle biological agents, such as Biosafety Cabinets or centrifuges, may not only impact the risk of the protocol, but may also require certification documentation prior to use, such amendments must also be submitted to the Biosafety Office.
2.1.2.4 Documenting New or Additional Project Titles Associated with a Biosafety Protocol (or Multiple Biosafety Protocols)
Biosafety Protocols (BSPs) are intended to document the risks and mitigations associated with working with biological agents, including which agents, operations, personnel, locations and major equipment to be utilized, independent of the funding status of the project. If a PI, Clinical Director or Instructional Course Director wishes to initiate a new project which involves identical agents, operations, personnel, locations, etc. as previously described on an IBC-approved BSP, the IBC considers the new project “covered” by the terms of the previously-approved BSP.
This is often the case with many clinical research laboratories. If the study agents administered to the patients are not biological in nature (i.e., they do not involve administration of rDNA, cells, biological fluids, or toxins of biological origin), the infectious status of the patients are identical, the operations involved are the same (e.g., serum preparation, aliquotting and shipping specimens to sponsors), and the locations and personnel handling the materials are identical for multiple clinical protocols, a single BSP may “cover” these multiple clinical protocols.
However, information may be required to “link” each of these new projects to an existing BSP to enable the Biosafety Office to provide IBC verification information to DSPA, OHRP or IACUC, as required (see Section 2.5.2 for more information about requirements for documentation of IBC compliance to DSPA, OHRP or IACUC). To accomplish this “linkage”, the PI listed on the BSP must provide verification that the information provided in his/her existing BSP fully “covers” the new project. Also, by submitting this verification, the PI on the BSP signifies that he/she takes responsibility in ensuring that the terms of the IBC Agreement document will be followed in the accomplishment of the new project or protocol.
Verification can be accomplished via completion and submission of a Schedule O form (via email or submission of a hard-copy, signed by the PI) or by providing the following information via email to the Biosafety Office ([email protected]):
a. The PI name on the project/protocol if other than the PI name on the BSP (this may be the case for student or post-doctoral fellowships accomplished under the supervision of a laboratory director) b. The BSP # which covers the new project/protocol c. The BSP title (if available) d. The BSP approval date e. The project/protocol title f. The project/protocol sponsor or funding agency g. The anticipated funding period (until the next competitive renewal application) h. The HAC or CCRI # (if verification to OHRP is required for IRB approval processing)
The Biosafety Office also recognizes that some projects or protocols may be collaborative in nature, and may involve handling of biological material under the oversight of two or more different collaborating laboratory directors. As long as the BSPs dovetail to seamlessly document
Georgia Regents University Biosafety Guide- June 2008 2-5 the agents, their handling and operations and transport between the laboratories, multiple BSPs may be required to “cover” a single project or protocol. Again, the PIs on each of the BSPs required to “cover” the project/protocol must provide documentation of verification of “coverage”.
Random audits will be performed to compare the experiments and agents described in the grants, contracts, or protocol descriptions verified by the PIs with the contents of their IBC-approved BSPs to ensure compliance. During these audits, the PIs will be requested to provide the requested grant, contract or protocol information to the Biosafety Office for review.
2.2 TRAINING REQUIREMENTS
Successful completion of a range of biosafety training programs may be required prior to the initiation of your work at GRU.
Please review the following table for information on the IBC-required training modules. The Biosafety Office must be able to document completion of the required training modules listed in this table prior to completion of any IBC-approval paperwork:
Intended for all You must satisfactorily Training Options personnel who complete the following work on GRU training: projects or areas, who: Work at GRU or on Basic Awareness Right-to- http://georgiahealth.edu/services/ehs/chemsafe/RTKTraining.html GRU projects Know Training
Required once upon initial hire Work with chemicals Chemical Specific Right-to- http://www.usg.edu/ehs/training/chemical/ Know Training
Required once prior to initiation of work and annually thereafter Work with or may Hazardous Waste http://www.usg.edu/ehs/training/hazwaste/ produce hazardous Awareness Training waste (including chemicals, and blood Required once prior to and other biological initiation of work and materials) annually thereafter Work with human or Bloodborne pathogens http://www.usg.edu/ehs/training/pathogens/
GA Board of Regents Requirements Regents of Board GA primate blood, tissues, training fluids other potentially infectious materials Required once prior to and bloodborne initiation of work and pathogens annually thereafter
Georgia Regents University Biosafety Guide- June 2008 2-6
Are listed on the Biosafety for Basic Didactic initial training offered monthly. Contact EHS for personnel lists of BSPs Research* next scheduled date/time. and work with biological materials in a basic An Initial Training session is Annual refresher training sessions may be offered in a laboratory setting. didactic training format per request of the department required prior to initiation of Includes Recombinant (typically in conjunction with all EHS sections) DNA, microbial agents work. OR ≤ RG2, cell culture & Online refresher training module may be accessed at: biological materials with Annual Refresher required http://georgiahealth.edu/services/ehs/biosafe/biotraining.html animals thereafter.
Are listed on BSPs that Biosafety for Clinical Didactic initial training offered monthly. Contact EHS for pertain to simple clinical Research* next scheduled date/time. protocols only. Involves taking human blood, Annual refresher training sessions may be offered in a tissues or fluid didactic training format per request of the department An Initial Training session is specimens from patients (typically in conjunction with all EHS sections) who are not considered required prior to initiation of OR infectious with any work. Online refresher training module may be accessed at: agent ≥RG3, preparation http://georgiahealth.edu/services/ehs/biosafe/biotraining.html of serum or plasma Annual Refresher required samples, aliquotting of thereafter. specimens and shipping to off-site sponsors.
Does NOT cover the delivery of any biological materials (recombinant DNA, IBC Training Requirements for all personnel listed on BSPs on listed personnel all for Requirements Training IBC biotoxins, cells/tissues) into patients or on-site GRU analysis or culture of specimens. Are responsible for Shipping of Hazardous Currently, a CD Training module developed by Saf-T-Pak packaging, shipping, Materials can be obtained on loan from the Biosafety Office. Because transporting, or of limited numbers available, please contact the office (x1- receiving Biohazards 2663 or [email protected]) to determine if Required biennially (i.e. once (infectious agents, one is available or to be placed on a waiting list. biological toxins, human every 2 years) clinical specimens, animal diagnostic
IATA/DOT specimens, dry ice, Requirements liquid nitrogen, genetically modified organisms)
Work with agents or IBC-required need-specific Any special training requirements will be listed as a Special applications which, training Condition of the IBC Authorization document. Assistance in based on the risk, the locating or obtaining the training can be obtained from the IBC assesses a need for Biosafety Office (x1-2663 or Special
Training a specific training. [email protected]). *Personnel listed on BSPs are required to take only one of these basic Biosafety classes, not both. The “Biosafety for Clinical Research” is a slightly abbreviated, more clinically-oriented version of the more comprehensive “Biosafety for Basic Research” class. The “Biosafety for Clinical Research” class is intended as an option only for those whose work is not anticipated to ever involve recombinant DNA, culture of biological materials or higher risk agents or operations.
Georgia Regents University Biosafety Guide- June 2008 2-7 In addition to the above training modules, the IBC expects that the following training will be provided to any person who is listed on a Biosafety Protocol prior to initiation of work within the laboratory. Documentation of completion of the following training should be maintained by the Principal Investigator, Clinical Director and/or Instructional Course Director:
May be in contact with Infection Control Training Currently, this training should be provided by the Clinical or patients or clinical Bloodborne Pathogens and Laboratory Director and involve review of the specific material in an GRU Tuberculosis Training Exposure Control Plan for the particular clinical area. clinical setting Control Control Infection Infection Standards
May be in contact with Laboratory-specific Training The Principal Investigator, Clinical director or Instructional Biological materials Course Director is responsible for educating all personnel in
- (includes review of SOPs, BSPs, and hands-on training) the laboratory related to the laboratory-specific risk issues, and mitigation methods. The PI or director is also Lab specific specific
Training responsible for providing supervision training for all personnel in the laboratory-specific operations.
2.3 LABORATORY ESTABLISHMENT, CLOSE-OUTS, AND MOVES
To ensure appropriate containment is maintained, safety measures are implemented and compliance with all Federal, State, local regulations and guidelines for all biological materials at the Georgia Regents University have been met, documentation must be provided, reviewed and approved by the IBC prior to moving any biological agents into any new facilities (which includes any materials which may be brought to campus by incoming new faculty or materials which may be moved to new locations not previously authorized as part of the Principal Investigator’s, Clinical Director’s or Instructional Course Director’s IBC-approved Biosafety Protocol). In addition, to ensure that the biological materials are properly removed and facilities are decontaminated prior to closing of a laboratory, PIs, Clinical Directors or Instructional Course Directors must document compliance with appropriate close-out procedures listed below.
2.3.1 Incoming New Faculty to GRU
New GRU researchers must receive authorization from the IBC prior to transfer of any biological material to campus prior to shipment of this material. Initial applications to transfer biological agents to GRU campus must, at minimum, document:
The biological agents to be transferred to GRU The method of transfer (which must be in compliance with IATA/DOT standards, and any required USDA or CDC permits for infectious materials must be obtained by the PI prior to shipment. See Section 10, Shipping for further information) The location(s) which these materials will be stored prior to the new faculty member’s laboratory establishment The GRU personnel responsible for the materials prior to arrival of the new faculty member (if applicable)
The IBC will review the above to ensure containment issues have been addressed and compliance with regulations, guidelines and policies are met, and will typically issue authorization only for the transfer of the material. Prior to initiation of research in GRU facilities, the incoming faculty member is expected to:
Complete and submit a complete Biosafety Protocol Application to the Biosafety Office for IBC review and approval (including documentation of all of the locations in which their biological materials may be handled or stored and submission of the laboratory-specific Standard Operating Procedures)
Georgia Regents University Biosafety Guide- June 2008 2-8 Ensure all laboratory personnel listed on the BSP have completed the IBC training requirements (see Section 2.2, Training for further details) Have all laboratory facilities assessed by the Biosafety Office. Once the facilities have been equipped for research, the PI should contact the Biosafety Office to make an appointment for a full laboratory assessment (x1-2663 or [email protected]).
Incoming faculty members are encouraged to contact the Biosafety Office (706-721-2663 or [email protected]) for assistance with these BSP submission processes as early as possible. The Biosafety Office is more than happy to help “walk” new faculty members through the process and assist them in obtaining any permits or authorizations required to transfer materials to campus.
New faculty members are advised that review and approval of BSPs involving non-exempt recombinant DNA or any materials which may pose a higher risks than those previously approved at GRU may take several (3-7) weeks due to requirements for full IBC review (the IBC meets once per month, and the deadline for submission for Biosafety Protocols for review within that month is the first day of the month). Because of these time constraints, the IBC recommends that Department chairs, Institute Directors, and/or Departmental Managers notify the Biosafety Office of any incoming faculty members in their programs as soon as possible after recruitment to initiate these application processes to enable the faculty member to begin research as promptly as possible after arrival at GRU.
2.3.2 Laboratory Close-outs
Any faculty member who may be vacating a laboratory needs to ensure that all biological materials have been removed from the laboratory and the laboratory has been decontaminated prior to departure. This is one of the terms of the IBC Authorization agreement between the IBC and the PI.
Biological materials should be removed from the laboratory by one of the following methods: Decontamination/deactivation and disposal of the biological material (as per the PI’s SOPs) Disposal in the authorized Biohazard waste containers (as per the PI’s SOPs) Transfer of the biological materials to another authorized user (or to another authorized location) • If the material is to be transferred to another GRU laboratory, written documentation of this transfer should be provided to the Biosafety Office (an email to [email protected] is sufficient). If the materials are being transferred to a different PI, the receiving PI should have authorization to possess this material in the new locations prior to transfer. The receiving PI must also agree to take responsibility for the new materials. • If the material is to be transferred outside of GRU, this must be done in accordance with IATA/DOT standards by personnel with documented training (since many biological materials and dry ice are considered hazardous materials by the federal government). Any transport, import and/or export permits must also be obtained by the PI prior to transfer for shipping (see Section 10 for more details). Contact the Biosafety Office for assistance (x1-2663 or [email protected]).
After removal of the biological materials from the laboratory, all surfaces and equipment must be decontaminated using the appropriate disinfection procedures as documented in the laboratory standard operating procedures prior to departure from the laboratory. Some equipment, such as biosafety cabinets, may require additional decontamination methods, such as gaseous fumigation with paraformaldehyde or vapor hydrogen peroxide (VHP), prior to removal from the laboratory. Contact the Laboratory Equipment Services (LES) office (x1-6124) to arrange for decontamination of biosafety cabinets prior to moving.
After removal of all biological materials and decontamination of the laboratory and equipment, the Biosafety Office must be notified to complete the “clearance” process.
Georgia Regents University Biosafety Guide- June 2008 2-9 2.3.3 Laboratory Moves
If biological materials or equipment are to be transferred from one GRU laboratory to a new GRU laboratory, the PI, Clinical Director and/or Instructional Course Director must: Submit a BSP amendment request to the Biosafety office for IBC authorization of the new locations for the biological materials prior to moving the materials (see Section 2.1.2, Amendments to Biosafety Protocols for more details). Transport the biological materials in a method which complies with their laboratory standard operating procedures. Contact the Biosafety Office (x1-2663 or [email protected]) for assistance and see below for special notes about transport considerations. Arrange for a full laboratory assessment with the Biosafety Office (x1-2663 or [email protected]) to ensure appropriate containment measures are in place in the new laboratory prior to initiation of work in the new laboratory (this is usually a stipulation in the IBC approval of any new location) Complete the laboratory close-out procedures (as described above in 2.3.2, Laboratory Close-outs) for the former laboratory.
Standard Operating Procedures for biological materials typically require the biological material to be transported outside of the laboratory: By authorized personnel (i.e., those personnel listed on the BSP who are familiar with the risks associated with the biological material, the emergency spill and exposure/release procedures). Contained in a sealed, leakproof primary container inside a well-labeled sealed, leakproof, durable secondary container.
However, transport of large amounts of biological materials in laboratory moves poses unique challenges. First, laboratory moves are often accomplished with the help of non-authorized personnel (e.g., professional movers or personnel from the GRU’s Materials Management Office). PIs are reminded that these personnel are not authorized to handle the biological materials because they have not been fully educated in the risks, received appropriate medical evaluation/vaccinations and are not prepared for any emergency procedures should spills or releases occur en route. Therefore, whenever possible, equipment containing biological materials should be transported separately by authorized personnel. However, if this is not feasible (due to the size/weight constraints of the equipment), the equipment containing the biological materials should be securely sealed to prevent moving personnel from exposure (e.g., refrigerators and freezers should be locked and/or securely taped shut), the exterior of the equipment should be decontaminated prior to the movers handling them and during the move, the movers and equipment should be accompanied by at least one member of the authorized laboratory staff equipped with the appropriate spill clean-up materials to ensure proper emergency procedures are followed should a spill/release occur en route.
Secondly, biological materials are often transported in refrigerators, freezers and cryotanks during laboratory moves to maintain the integrity of the samples. In preparation of moving, any glass, breakable items or other hazardous materials (e.g., chemicals or radiological materials) should be removed from any refrigerators or freezers. Biological materials should be contained in one or more forms for sealed, durable containment within the refrigerator or freezer and secured to prevent spillage or scattering of biological materials or samples within the freezer. Keep in mind: freezers and refrigerators require tipping to transport on moving dollies; a good deal of tipping is often required to get large equipment into tight spaces, such as elevators. For instance, sealed microcentrifuge tubes should be contained within boxes with lids (not in open racks); plastic tubes should have tightly secured lids and should be in fitted containers which would prevent movement during transport (or contained within ziplock bags). Filling any open spaces within the freezers/refrigerators with packing materials will also prevent shifting of materials during transport (foam rubber works well for this purpose). The refrigerator or freezer should be sealed shut (locked or securely taped shut) and the exterior decontaminated prior to moving.
Because cryotanks cannot be completely sealed during transport to prevent leakage of liquid nitrogen if tipped, and any vehicle transporting these materials on public roads may require special placarding for
Georgia Regents University Biosafety Guide- June 2008 2-10 DOT regulatory compliance, these items should not be transported via moving truck. Alternate methods of moving this equipment should be discussed with the Biosafety Office prior to moving.
The need for gaseous decontamination of a Biosafety cabinet (BSC) must be evaluated by the Biosafety office prior to moving. Should a BSC require decontamination, arrangements must be made in advance with Laboratory Equipment Services (LES) (x1-6124) to perform this service before the BSC can be moving the BSC.
2.4 LABORATORY RECORD-KEEPING
At least one copy of the Laboratory Biosafety Manual should be maintained within each laboratory for training and reference of all laboratory personnel. This manual should include the following material: • A copy of the GRU Biosafety Guide. • The Laboratory Biosafety Protocol(s), amendments and renewal documentation. • The Laboratory-specific Standard Operating Procedures (SOPs). • Copies of documentation of training of all laboratory staff. This should include: o Copies of certificates of completion of any BOR or IBC-required training modules o Documentation of completion of IATA/DOT shipping training, o Documentation that all authorized personnel listed on the BSP have received laboratory- specific education and training from the PI or his/her designated representative. Signed/dated statements should affirm that training included: . Laboratory-specific risk communication and training. This includes providing references and a discussion of the risks associated with the materials within the laboratory, any possible signs/symptoms which may suggest occupational exposure has occurred and any incident/emergency procedures that may be required. . Each staff member should verify that they have reviewed and understand the materials documented within the Biosafety Guide, Laboratory Biosafety Protocols and the Laboratory SOPs. • Copies of signed documentation that each person authorized to work within the laboratory has been offered Employee Health screening/counseling and vaccinations by the PI as appropriate for the agents within the laboratory and as described in the laboratory SOPs and IBC Approval documents. • Records of any incident, exposure, possible exposure, spill, release from primary containment or environmental exposure of any ≥Risk Group 2 biological agent (see Section 3, Risk Assessment for further information on Risk Groups). This should include the date of the incident, the agents, locations and personnel involved, a narrative description of the circumstances and outcome, the dates when the Biosafety Office was informed and any follow-up measures taken (e.g., was health care sought for those potentially exposed? are subsequent tests/health care follow-up measures required and performed and when? were any SOPs altered to prevent similar incidents in the future?)
2.5 PAPERWORK FLOW
There are two critical routine administrative processes that occur within the Biosafety Office: 1. Receipt and administration of new Biosafety Protocols (BSPs) or amendments, presentation of these to the IBC for review and processing of IBC approvals. 2. Verification that IBC approval has been received for a particular project, clinical protocol or AUP by a Principal Investigator to other campus compliance offices (DSPA, OHRP or IACUC).
Georgia Regents University Biosafety Guide- June 2008 2-11 2.5.1 IBC Review/Approval Process for Biosafety Protocols and Amendments
Biosafety Protocols and amendments are first reviewed by the Biosafety Office to determine the most appropriate review/approval track for the protocol. See Figure 2.5.1 for a diagram of the paperwork flow for Biosafety protocol reviews, which describe the three review tracks available for BSPs: 1. All BSPs or amendments involving non-exempt recombinant DNA agents, or those involving higher risk organisms (some RG2 and all RG3 organisms, and/or Select Agents) are required to be reviewed by the full IBC during a convened meeting in which minutes are recorded as per NIH and CDC Guidelines. o The IBC meets monthly; typically on the third Wednesday of the month o All BSPs and amendments received on or before the first day of the month will be submitted to the IBC for review within that month. o Delays on the order of approximately 3-7 weeks from the date of submission of a complete BSP application and training documentation until IBC review approval should therefore be anticipated 2. BSPs or amendments which do not involve recombinant DNA or involve only exempt recombinant DNA are eligible for an expedited review and conditional approval by an IBC subcommittee. Responses are requested from subcommittee members within 3 days of receipt of the completed application or amendment. 3. Simple amendments of previously-IBC approved Biosafety protocols (BSPs) which do not involve additional risk (e.g. an addition of a grant title/clinical protocol title to the protocol; most personnel changes) are handled administratively within the Biosafety Office.
During the IBC meeting, the Committee may vote to: approve the BSP/amendment without stipulations, approve the BSP/amendment with stipulations, disapprove or table the proposed BSP/ amendment. The PI will be notified of the IBC decision subsequent to the meeting.
If the BSP/amendment is approved with stipulations, the PI will be notified and will be given 2 months to address these stipulations. The BSP/amendment is not considered finally approved until all stipulations have been met. If the stipulations are not met within the 2 month time window after receipt of the stipulations, the BSP/amendment application will be considered withdrawn and must be re-submitted for further IBC reconsideration.
Georgia Regents University Biosafety Guide- June 2008 2-12
[INSERT FIGURE 2.5.1 HERE]
Georgia Regents University Biosafety Guide- June 2008 2-13 2.5.2 Verification that the IBC Has Reviewed and Approved Experiments, Agents and Uses that Were Proposed in a Particular Sponsored Project, Clinical Protocol or Animal Use Protocol to Other Compliance Offices
Part II of the NIH Grants Policy Statement, describes the terms and conditions of NIH Grant Awards http://grants.nih.gov/grants/policy/nihgps_2003/NIHGPS_Part4.htm#_Toc54600062 . Among these are specific requirements for institutions receiving NIH funds to meet Federal, State and local health and safety standards and guidelines for all research at their institution. Grantee institutions are responsible for meeting Federal, State, and local health and safety standards and for establishing and implementing necessary measures to minimize their employees’ risk of injury or illness in activities related to NIH grants.
Among these are the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines) (May 2011 or latest revision), which apply to all research projects that involve recombinant DNA and are conducted at or sponsored by an organization that receives NIH support for recombinant DNA research. The IBC is required to review each proposed project for recombinant DNA experiments and certify that the procedures, project, personnel, and facilities are adequate and in compliance with the NIH Guidelines.
In addition to applicable Federal, State, and local laws and regulations, the following regulations must be followed when developing and implementing health and safety operating procedures and practices for both personnel and facilities: 29 CFR 1910.1030, Bloodborne pathogens; 29 CFR 1910.1450, Occupational exposure to hazardous chemicals in laboratories; and other applicable occupational health and safety standards issued by the Occupational Health and Safety Administration (OSHA) and included in 29 CFR Part 1910. These regulations are available at http://www.osha.gov/comp-links.html.
The NIH recommends following these guidelines for use in developing and implementing health and safety operating procedures and practices for both personnel and facilities. Therefore, these have been adopted as part of GRU IBC Policies.:
• Biosafety in Microbiological and Biomedical Laboratories, CDC and NIH, HHS. This publication is available at http://www.cdc.gov/biosafety/publications/bmbl5/index.htm
• Prudent Practices for Safety in Laboratories (1995), National Research Council, National Academy Press, 500 Fifth Street, NW, Lockbox 285, Washington, DC 20055 (ISBN 0-309- 05229-7). This publication can be obtained by telephoning 800-624-8373. It also is available at http://www.nap.edu/catalog/4911.html .
Although grantee organizations are not typically required to submit documented assurance of their compliance with or implementation of the health and safety regulations and guidelines, if requested by the awarding office, the grantee institutions should be able to provide evidence that applicable Federal, State, and local health and safety standards have been considered and have been put into practice. NIH expects the grantee institutions to provide safe working conditions for their employees and foster work environments conducive to high-quality research.
Therefore to maintain these standards, and to ensure compliance with all Federal, State and Local laws, and maintain appropriate documentation of compliance GRU compliance offices have a verification process for all research involving hazardous materials, including biological hazards. To maintain compliance the Biosafety Office is asked to verify that the specific agents, experiments, operations, locations, personnel and equipment used in each sponsored project proposal, clinical protocol submission for IRB review, and/or animal use protocol, have been reviewed and approved by the IBC prior to account establishment, IRB or IACUC approval processing, respectively. See Figure 2.5.2 for guidance on when IBC approvals need to be completed relative to verification to DSPA, OHRP or IACUC.
In order to facilitate communication between these offices, the Biosafety Office asks that you provide
Georgia Regents University Biosafety Guide- June 2008 2-14 verification and information required to “link” a particular biosafety protocol to a particular sponsored project or clinical protocol. As previously described in Section 2.1.2.4, Documenting New or Additional Project Titles Associated with a Biosafety Protocol (or Multiple Biosafety Protocols), this type of amendment is handled administratively by the Biosafety Office and can be accomplished by submitting a Biosafety Protocol Schedule O form form http://www.mcg.edu/research/ibc/pdf/New/ScheduleO-DSPA.pdf or providing written documentation (e-mail to [email protected] is fine) for the Biosafety Office records from the PI on the BSP in which he/she verifies that the project/protocol information should be “linked” to the Biosafety Protocol(s) (BSP) indicated: i. The PI name on the project/protocol if other than the PI name on the BSP (this may be the case for student or post-doctoral fellowships accomplished under the supervision of a laboratory director) j. The BSP # (or #s) which covers the new project/protocol k. The BSP title(s) (if available) l. The BSP approval date m. The project/protocol title n. The project/protocol sponsor or funding agency o. The anticipated funding period (until the next competitive renewal application) p. The HAC or CCRI # (if verification to OHRP is required for IRB approval processing) Please see Section 2.1.2.4, Documenting New or Additional Project Titles Associated with a Biosafety Protocol (or Multiple Biosafety Protocols) for further information.
Georgia Regents University Biosafety Guide- June 2008 2-15
[INSERT FIGURE 2.5.2 HERE]
Georgia Regents University Biosafety Guide- June 2008 2-16 2.6 BIOLOGICAL MATERIALS AND APPLICATIONS WHICH MUST BE DOCUMENTED ON BIOSAFETY PROTOCOLS
2.6.1 Recombinant DNA Experiments
As a condition for receipt of NIH funding, GRU must ensure that all such research conducted at or sponsored by the institution, irrespective of the source of funding, shall comply with the NIH Guidelines for Research Involving Recombinant DNA Molecules. The NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines) is available via the NIH Office of Biotechnology Activities (NIH OBA) web page: http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.htm.
On behalf of the institution, the GRU IBC is required by NIH to review all recombinant DNA research conducted at or sponsored by the institution and perform a comprehensive risk assessment of containment levels, facilities, procedures, practices, training and expertise of personnel for proposed research to ensure compliance with the NIH Guidelines.
In addition, the IBC is expected to periodically review recombinant DNA research conducted at the Institution and report any significant problems with or violations of the NIH Guidelines and any significant research-related accidents or illnesses to the appropriate institutional official and NIH/OBA within 30 calendar days, unless the Institutional Biosafety Committee determines that a report has already been filed by the Principal Investigator (Section IV, NIH Guidelines: http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.htm). Reporting of adverse events specifically associated with Human Gene Transfer protocols must be accomplished even faster than 30 days (see Appendix M, NIH Guidelines for details: http://oba.od.nih.gov/oba/rac/Guidelines/APPENDIX_M.htm). The GRU Institutional Biosafety Committee (IBC) therefore requires registration and/or approval is required prior to the initiation of all recombinant DNA experiments.
2.6.1.1 Definition of Recombinant DNA
The NIH Guidelines (Section 1-B) defines recombinant DNA molecules as either: i) molecules that are constructed outside living cells by joining natural or synthetic DNA segments to DNA molecules that can replicate in a living cell, or ii) molecules that result from the replication of those described in (i) above.
NIH Guidelines also specifies that synthetic DNA segments which are likely to yield a potentially harmful polynucleotide or polypeptide (e.g., a toxin or a pharmacologically active agent) are considered as equivalent to their natural DNA counterpart. If the synthetic DNA segment is not expressed in vivo as a biologically active polynucleotide or polypeptide product, it is exempt from the NIH Guidelines.
Therefore, according to NIH Guidelines, the following are technically not considered “recombinant DNA”: Isolation of genomic DNA or RNA from natural sources. PCR amplification of genomic DNA or cDNA from non-cloned templates (e.g. for genotyping purposes), as long as the resultant amplicons are not subsequently cloned. Chemically synthesized oligonuleotides such as those used in some siRNA/shRNA or DNA sequencing applications. Please note, methods of producing siRNA or shRNA other than chemical synthesis of oligonucleotides typically involve recombinant DNA templates and/or cloning techniques and are therefore not exempt from NIH Guidelines (e.g. production via in vitro transcription, expression of siRNA from an expression plasmid or viral vector, or expression of a PCR-derived siRNA expression cassette).
Georgia Regents University Biosafety Guide- June 2008 2-17
2.6.1.2 Definition of Exempt vs. Non-Exempt Recombinant DNA
NIH categorizes recombinant DNA experiments into six categories, as described in Sections IIIA through F of NIH Guidelines for Research with Recombinant DNA Molecules. Five of these categories (described in Sections IIIA through E of NIH Guidelines) refer to research that the NIH considers “non-exempt” from the requirements for notification and/or approval of Internal Institutional Review Committee(s) (i.e. the IBC and/or IRB) and/or external agencies (NIH Office of Biotechnology Activities (OBA), the Recombinant DNA Advisory Committee (RAC) and/or the NIH Director). Although the NIH does not explicitly require notification or approval for recombinant DNA experiments which fall into a sixth (“exempt rDNA”) category (as described in Section III-F of NIH Guidelines) prior to initiation of the experiment, the GRU IBC asks researchers register and/or discuss their non-exempt rDNA experiments with the Biosafety Office in an effort to avoid accidental misclassification of rDNA experiments.
The factors that distinguish non-exempt rDNA research vs. exempt rDNA research involve one or a combination of the following, any one of which may alter the risk of the overall research proposal:
Vectors Typically, vectors that are contain large regions or are derived from genomes or episomes of Risk Group ≥ 2 agents (i.e. infectious material or material capable of infection of human cells) are considered “non-exempt” unless/until proven incapable of infection or transmitting harmful effects upon target cells (e.g. viral vectors—including those that are designed to be replication-incompetent and/or are commercially available. Please note that viral vectors are still infectious, even if they are designed to be replication-incompetent!)
Inserts Some inserts, by themselves, will relegate a recombinant DNA protocol to the non-exempt category, particularly those which: o confer drug resistance trait to microorganisms that are not known to acquire the trait naturally if such acquisition could compromise the use of the drug to control disease agents in humans, veterinary medicine, or agriculture (See Section III-A of NIH Guidelines for further information: http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.htm#_Toc72 61560). o are derived from genomes or episomes of RG ≥ 2 organisms (i.e. pathogens) or Select Agents (http://www.cdc.gov/od/sap/docs/salist.pdf) which may transmit a harmful effect upon an intended or unintended target cell. o are biological toxins with LD50 < 100 µg/kg body weight. Those toxins with LD50 < 100 ng/kg body weight (e.g. botulinum toxins, tetanus toxin, diphtheria toxin, and Shigella dysenteriae neurotoxin) require NIH/OBA approval; those with LD50s ≥100 ng/kg but < 100 µg/kg may require NIH/OBA registration prior to initiation (e.g. Staphylococcus aureus alpha toxin, Staphylococcus aureus beta toxin, ricin, Pseudomonas aeruginosa exotoxin A, Bordetella pertussis toxin, the lethal factor of Bacillus anthracis, the Pasteurella pestis murine toxins, the oxygen-labile hemolysins such as streptolysin O, certain neurotoxins present in snake venoms and other venoms, cholera toxin, the heat labile toxins of Escherichia coli, Klebsiella, and other related proteins that may be identified by neutralization with an antiserum monospecific for cholera toxin, and the heat stable toxins of
Georgia Regents University Biosafety Guide- June 2008 2-18 Escherichia coli and of Yersinia enterocolitica) See Appendix F NIH Guidelines and the CDC/USDA Select Agent Program (http://www.cdc.gov/od/sap/final_rule.htm) for further information. In addition, expression of some inserts in non-exempt vectors may alter the risk associated with the research proposal. For instance, the biological effect of over- or under-expression of DNA inserts in non-exempt vectors which modulate any of the following biologically active molecules may require special additional consideration by the IBC during the comprehensive risk assessment process: o Oncogenes or proto-oncogenes o Anti-apoptotic molecules o Immunomodulatory molecules o Toxins
Volumes Large-scale culture volumes ≥10 liters require special operations as per NIH Guidelines (see Appendix K, NIH Guidelines).
Applications/ Targets Delivery of rDNA into the following targets may relegate a recombinant DNA protocol to non-exempt status o Risk Group ≥ 2 microorganisms (i.e. potential pathogens) o Human subjects (i.e. Human Gene Transfer/Therapy—see Section 2.6.1.4, below for additional information) o Live Animals . Introduction of recombinant DNA into animals (e.g. injection of rDNA or transplantation of genetically engineered cells) may fall into the non-exempt rDNA categories as per NIH Guidelines. . Creation of a new strain of Transgenic (Tg) and/or Knock-out (KO) animals via: • Standard Tg/KO techniques (rDNA delivery into ES cells or blastocysts) • Cross-breeding 1+ strain of Tg/KO animals with another strain of different genetic background, thus creating a new strain. However, the purchase or transfer of existing transgenic or KO rodent strains (e.g. from commercial suppliers, such as Jackson, Harlan or Charles River Laboratories or from other IBC-authorized investigators) is exempt from the NIH Guidelines under Section III-F (see Appendix C-VI, NIH Guidelines) if these animals are appropriately contained at BSL-1.
The following guideline, reproduced in the Biosafety Protocol Application Form is intended to further assist researchers in properly classifying their recombinant DNA experiments (see Table 2.6.1.2). Please note that experiments 1-3 listed in Table 2.6.1.2 under “Experiments which must be registered with the IBC and approved prior to initiation” require registrations/approvals of external agencies (the NIH Director, RAC, and/or NIH/OBA), so IBC processing of these protocols will require the most time to complete. Please contact the Biosafety Office x1-2663 or [email protected] for any additional assistance:
Georgia Regents University Biosafety Guide- June 2008 2-19
“Non-exempt” Recombinant DNA “Exempt” Experiments which must be registered with the Experiments that require registration with the Recombinant DNA experiments that do not require IBC and approved prior to initiation: IBC simultaneous with initiation: IBC approval: 1. Deliberate transfer of a drug trait to a 1. Experiments using as vectors < 2/3 of the genome 1. rDNA containing less than 1/2 of an eukaryotic microorganism not known to acquire it naturally of a eukaryotic virus, demonstrated to be free of viral genome propagated in cell culture (with the (if it could compromise the use of the drug to helper virus or complementing helper virus exception of expression of DNA from Risk Group4 control disease agents in humans, animals or components. 2, 3, 4 or restricted agents); agriculture)2. (Note: this would likely exclude most subcloning procedures using antibiotic 2. rDNA work involving E. coli K12 derivatives, S. selectable markers in E. coli K12 derivatives) 2. Transgenic or knockout rodent experiments for cerevisiae, and B. subtilis host-vector systems (with 4 which BSL-1 containment is appropriate (NOTE: the exception of expression of DNA from Risk 2. Cloning of DNA encoding toxic molecules lethal the purchase of transgenic rodents for BSL-1 Group 2, 3, 44 or restricted agents). to vertebrates at an LD50 of <100 µg/kg body experiments falls into the “exempt” category; weight2,3. however, breeding of these animals with different strains of mice is considered non-exempt rDNA 3. Human gene transfer/therapy experiments2; experiments).
4. Cloning using human or animal pathogens as host-vector systems.
5. Cloning of DNA from all Risk Group4 2, 3, 4 or restricted human or animal pathogens (including HIV and related viruses and viruses capable of infecting human cells).
6. Experiments using more than 2/3 of the genome of infectious animal or plant viruses or defective viruses grown in the presence of helper virus or complementing helper virus components.
7. Recombinant DNA experiments involving whole animals, including transgenic or knockout rodent experiments requiring BSL24 containment; or transplantation of genetically engineered cells into animals.
8. Large scale DNA projects (≥10 liter cultures at any moment in time).
1For further explanations/details, please see: : http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.htm
2Please note that these experiments may require approval of NIH Director, NIH’s Office of Biotechnology Activities (OBA) approvals or registration and/or the Recombinant DNA Advisory Committee (RAC) review. Please contact the Biosafety Office (x1-2663) as soon as possible if your experiments fall into these categories for assistance.
3 LD50 for many toxins can be viewed on commercially available Material Safety Data Sheets (MSDSs), e.g. http://www.sigmaaldrich.com/Area_of_Interest/The_Americas/United_States.html or contact the Biosafety Office x1-2663 if you need assistance.
4See the following links for assistance in Risk Group classification and recommended Biosafety Level usage: • American Biosafety Association Risk Group Guide: http://www.absa.org/riskgroups/index.html • Public Health Agency of Canada MSDSs: http://www.phac-aspc.gc.ca/msds-ftss/ • NIH Guidelines for Recombinant DNA Research: http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.htm • CDC’s Biosafety in Microbiological and Biomedical Laboratories (BMBL): http://www.cdc.gov/biosafety/publications/bmbl5/index.htm
Table 2.6.1.2 Simplified Guideline for Classifying Recombinant DNA Experiments according to NIH Guidelines for Research with Recombinant DNA Molecules. GRU IBC requests that all recombinant DNA experiments be registered with the Biosafety Office prior to initiation to avoid potential mis-classification errors. (Table based on that from Yale University’s Biosafety Manual)
Georgia Regents University Biosafety Guide- June 2008 2-20 2.6.1.3 Recombinant DNA Registration Requirements
Principal Investigators and/or Lab Supervisors must submit or amend Biosafety Protocols: before bringing new non-exempt recombinant DNA materials to campus which had not already been approved by the IBC. before initiating any new non-exempt recombinant DNA work.
New submissions of Biosafety Protocol describing new work must be submitted by the PI using the Biosafety Protocol Application forms, which are available in Appendix A of the Biosafety Guide and are also available online on the GRU Biosafety Office web site (http://georgiahealth.edu/research/ibc/apps.html).
Amendments must be submitted by the PI fully describing the new work if the scope of the work has changed from that approved by the IBC. Changing the scope of the work would include changes in: vectors or inserts, administration or exposures of new target materials or animals, creation of a new transgenic strain or species, or embarking upon large-scale culture (≥10 liters), changes in locations, personnel or major equipment (see Section 2.1.2 regarding BSP amendment procedures). Amendments can be submitted by altering the original Biosafety Protocol Application forms to include the new information and re-submission to the Biosafety Office, or via a fully detailed email. All questions can be addressed to the Biosafety Office (x1-2663 or [email protected]).
2.6.1.4 Human Gene Transfer/Therapy (HGT)
Proposed clinical trials involving the deliberate transfer of recombinant DNA, or DNA or RNA derived from recombinant DNA, into human research participants are defined as human gene transfer experiments. Human gene transfer for therapeutic purposes is referred to as human gene therapy.
Human gene transfer experiments require registration and approval from at least two Campus Safety Committees (the IBC and the IRB) as well as review by federal agencies (the NIH Office of Biotechnology’s (NIH/OBA’s) Recombinant DNA Advisory Committee (RAC) and the Federal Drug Administration (FDA)) before enrollment of the first subject. These requirements are described in Section III-C and Appendix M of NIH Guidelines (http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.htm#_Toc7261564).
In addition to Appendix M of NIH Guidelines, the NIH has provided reference information for researchers involved in Human Gene Transfer Research on their web page. Principal Investigators should review these materials and implement the guidance in preparation of protocol applications. The NIH Guidance for the development of Informed Consent documents specifically related to Gene Transfer Research can be found at: http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.htm#_Toc7261564. NIH has also provided answers to Frequently Asked Question (FAQ) about the NIH Review Process for Human Gene Transfer Trials at: http://oba.od.nih.gov/rdna/rdna_faq_list.html .
The NIH/FDA Genetic Modification Clinical Research Information System (GeMCRIS) maintains a comprehensive information resource and analytical tool for scientists, research participants, sponsors, institutional oversight committees, federal officials, and others with an interest in human gene transfer research. GeMCRIS allows public users to access basic reports about human gene transfer trials registered with the NIH and to develop specific queries based on their own information needs. GeMCRIS information can be accessed via ttp://oba.od.nih.gov/rdna/adverse_event_oba.html .
Georgia Regents University Biosafety Guide- June 2008 2-21 The IBC is required by the terms of NIH Guidelines to ensure compliance with all aspects of Appendix M, which includes review of materials which are more typically considered the domain of the Institutional Review Board (IRB). Therefore, in addition to the standard BSP Application forms and protocol-specific SOPs, PIs of human gene transfer protocols must include the Clinical Protocol materials, Investigator’s Brochure, the Informed Consent Document, Recombinant DNA Advisory Committee (RAC) submission information and documentation of review/responses from the RAC, and any pertinent IND information, including preliminary animal or human studies and SAE report information in their application for IBC review and approval.
After IBC review and approval, a record of the IBC review and comments will be provided to the Institutional Review Board (IRB) for consideration during their review and a copy will be provided to the Office of Human Research Protection (OHRP) for their files.
Copies of any adverse event reports must be provided to the sponsor, the IRB, the NIH/OBA and IBC within the time frames described in Appendix M of NIH Guidelines. Human gene transfer protocols must be reviewed by the IBC at least annually. Therefore, any updated information related to the protocol must be provided to the Biosafety Office no later than 11 months after the IBC approval anniversary.
2.6.2 Human, Animal and/or Agricultural Pathogens or Toxins (or Materials Potentially Contaminated with these Pathogens or Toxins)
Special considerations may be required prior to possession and/or transfer of any known human, animal and/or agricultural pathogens or toxins or materials which may be potentially contaminated with these pathogens or toxins to GRU campus to comply with CDC and/or USDA regulations. This may include laboratory inspections for compliance with Biosafety standards and permits for transfer of these agents. The Biosafety Office can assist researchers in determining whether special inspections and/or permits may be required.
The GRU Institutional Biosafety Committee (IBC), the Institutional Animal Use and Care Committee (IACUC) and the Division of Laboratory Animal Services (LAS) must approve all experiments involving the introduction of infectious agents or potentially hazardous biological materials into animals prior to initiation.
All researchers working with etiologic agents (Risk Group 2 or higher) must receive training in both biosafety and the microbiological procedures that will be utilized for the experiment. Biosafety training sessions for new staff and faculty are provided through the Division of Environmental Health and Safety on a monthly basis (call x1-2663, [email protected] or check EHS web site for the next scheduled training date), The Principal Investigator is responsible for ensuring that all researchers are trained in the appropriate procedures and techniques used in the laboratory.
Consultation in Risk Group classification of biological agents can be sought through the Biosafety Office (x1-2663 or [email protected]). Information can also be found at the following URLs: CDC BMBL: http://www.cdc.gov/biosafety/publications/bmbl5/index.htm NIH Guidelines: http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.htm ABSA Risk Group Classification Database: http://www.absa.org/riskgroups/index.html Public Health of Canada Biological MSDSs: http://www.phac-aspc.gc.ca/msds-ftss/
Work with Risk Group 3 agents (or those requiring Biosafety Level 3 containment) and/or Select Agents or Toxins will require additional registration and training requirements by the PI and laboratory staff as specified in the BSL-3 Laboratory Manual. Contact the Biosafety Office for additional information. Work with Risk Group 4 agents or experiments requiring Biosafety Level 4 containment are not permitted at GRU.
Georgia Regents University Biosafety Guide- June 2008 2-22
2.6.2.1 Toxins of Biological Origin
Because of the risks posed by the acute toxicity of some biological toxins, the IBC will require documentation of Standard Operating Procedures (SOPs) to ensure safety handling and disposal procedures for any toxin of biological origin with an LD50 ≤ 100 µg/kg body weight as part of the Principal Investigator’s Biosafety Protocol (BSP). These should incorporate the risk assessment and management considerations as described in Appendix I of the CDC/NIH publication Biosafety in Microbiological and Biomedical Laboratories (BMBL) http://www.cdc.gov/biosafety/publications/bmbl5/BMBL5_appendixI.pdf .
These SOP considerations should include: • Special training should be provided to all personnel related to the unique risk characteristics of toxins and particular emergency spill and/or exposure procedures should be provided. • Special procedures and protective equipment required to prevent exposure to any dry/freeze-dried toxins, since the potential for electrostatic dispersal of concentrated forms of toxin pose uniquely high risks. Respiratory protective measures must be considered. • The containment practices and equipment required to reduce the risks of accidental exposure by direct contamination of mouth, eyes or other mucous membranes by inadvertent aerosol generation; and by needle-sticks or other accidents that may compromise the normal barrier of the skin while working with solutions of biotoxins. When working with toxins that pose direct percutaneous hazards, special care must be taken to select gloves that are impervious to the toxin and the diluents or solvents employed. • Restricted access to designated isolated rooms while toxins are in use. The room should be clearly posted: “Toxins in Use—Authorized Personnel Only” while toxin work is on- going and unrelated and nonessential work should be restricted from areas where stock solutions of toxin or organisms producing toxin are used. • Special deactivation/decontamination procedures may be required for surfaces and liquids contaminated with the toxins. Toxins may not be deactivated by typical disinfectants used against pathogens, and therefore, special procedures should be documented and followed for deactivation and disposal (see Section 7.3 for further information and guidance on deactivation of biological toxins). • Additional security measures which may be required to limit access to the stored biological toxins.
2.6.2.2 Select Agents and Toxins (SATs)
A subset among human, animal and/or agricultural pathogens and toxins are the Select Agents and Toxins (SATs). Select agents/toxins are agents that the U.S. Department of Health and Human Services (HHS) and/or the Department of Agriculture/Animal & Plant Health Inspection Service (USDA/APHIS) considers to have the potential to pose a severe threat to human health, animal or plant health, or to animal or plant products (See Table 2.6.2.2 for a list of these Select Agents of Toxins or http://www.selectagents.gov/select%20agents%20and%20Toxins%20list.html).
The SAT regulations pertain not only to the intact agents or toxins, but also several genetic elements or recombinant nucleic acids from these agents and recombinant Select Agent organisms. The HHS and USDA originally enacted interim regulations governing the possession, use, and/or transfer of these agents on December 13, 2002, and subsequently published their final rules on March 18th, 2005 and these can be viewed here:
Georgia Regents University Biosafety Guide- June 2008 2-23 42 CFR Parts 72 and 73 Office of Inspector General 42 CFR Part 1003: Possession, Use, and Transfer of Select Agents and Toxins; Final Rule: http://www.selectagent.gov/resources/42_cfr_73_final_rule.pdf
7 CFR Part 331 and 9 CFR Part 121: Agricultural Bioterrorism Protection Act of 2002; Possession, Use, and Transfer of Biological Agents and Toxins; Final Rule: http://www.selectagent.gov/resources/APHISFinalRule.pdf
The National Select Agent Registry has established a URL that serves as an informational center, as well as a source of all registration materials, and forms. http://www.selectagent.gov/
Georgia Regents University Biosafety Guide- June 2008 2-24 Georgia Regents University Biosafety Guide- June 2008 2-25 Among these requirements of these regulations is that any entity (defined by HHS/CDC and USDA/APHIS as “any government agency (Federal, State, or local), university, corporation, company, partnership, society, association, firm, sole proprietorship, or other legal entity”) wishing to use, possess, or transfer SATs must register with either the Centers of Disease Control (CDC) or USDA/APHIS. This registration process requires the following:
Designation of a Responsible Official (RO) for the entity, which is the person “designated by an entity to act on its behalf…” and that “…this individual must have the authority and control to ensure compliance with the regulations in this part”. Currently, the GRU RO is the Biosafety Officer. An Alternate Responsible Official (ARO) may also be designated by the entity and who is similarly required to ensure compliance with the regulations. Currently, the GRU ARO is the Associate Vice President of Environmental Health and Safety. Approval of the HHS or USDA Secretary or Administrator based on a Security Risk Assessment (SRA) by the U.S. Attorney General for any individual or entity who may potentially have access or control over any Select Agent or Toxin. Note: an application for an individual may be denied or a certificate of registration revoked or suspended if an individual is reasonably suspected by any Federal law enforcement or intelligence agency of: o Committing a crime specified in 18 U.S.C. 2332b(g)(5), o Knowing involvement with an organization that engages in domestic or international terrorism (as defined in 18 U.S.C. 2331) or with any other organization that engages in intentional crimes of violence, or o Being an agent of a foreign power (as defined in 50 U.S.C. 1801) Identification of the particular physical location in which these SATs may be present, and the associated comprehensive documentation must be provided and approved by the CDC and/or USDA for the specific locations, agents and experiments proposed for use in these facilities: o Safety plans o Security plans o Incident/Emergency response plans o Precise Inventories and all records of transfer and use Agent- and Use-specific training of all who may have access must be documented and approved, and must include drills and exercises. Notifications of any theft, loss, or unaccounted samples, releases from containment and possible exposures must be made to the CDC and/or USDA, and may require further Federal investigation and cooperation.
Any individual who intends to possess, use or transfer any Select Agent or Toxin to GRU campus which does not qualify for any of the exclusions and exemptions listed below in Sections 2.6.2.2.1 and 2.6.2.2.2, must contact the Biosafety Office (x1-2663 or [email protected]) immediately to discuss registration procedures and the adequacy of the facilities to secure the agents or toxins in question, and the measures that will need to be taken to qualify for registration. They must refer to the BSL-3/Select Agent and Toxin Manual and receive additional training as required by Federal law.
2.6.2.2.1 Exclusions from Select Agents and Toxins Regulations
HHS/USDA select agents or toxins that meet any of the following specific criteria are excluded from the Select Agent of Toxin Regulations as described in 42 CFR §73.3 (d) & (e), 7 CFR §331.3 (d) & (e) and 9 CFR §121.4 (d) & (e):
1. Any HHS/USDA select agent or toxin that is in its naturally occurring environment provided the select agent or toxin has not been intentionally introduced, cultivated, collected, or otherwise extracted from its natural source.
Georgia Regents University Biosafety Guide- June 2008 2-26 2. Non-viable HHS/USDA select agents or nonfunctional HHS/USDA toxins. 3. HHS/USDA toxins under the control of a principal investigator, treating physician or veterinarian, or commercial manufacturer or distributor, if the aggregate amount does not, at any time, exceed the following amounts:
Abrin 100 mg Botulinum neurotoxins 0.5 mg Clostridium perfringens epsilon 100 mg toxin Conotoxins 100 mg Diacetoxyscirpenol 1,000 mg Ricin 100 mg Saxitoxin 100 mg Shiga-like ribosome inactivating 100 mg proteins (i.e. verotoxins) Shigatoxin 100 mg Staphylococcal enterotoxins 5.0 mg Tetrodotoxin 100 mg T–2 toxin 1,000 mg
The regulations in 42 CFR Part 73 and 9 CFR Part 121 also include a procedure by which an attenuated strain of a select biological agent or toxin that does not pose a severe threat to public health and safety, animal health, or animal products may be excluded from the list of select biological agents and toxins upon application and approval of the HHS or USDA (See 42 CFR §73.3 (e), 7 CFR § 331.3 (e) and 9 CFR §121.4 (e).
Based upon consultations with subject matter experts and a review of relevant published studies and information provided by the entities requesting the exclusions, the HHS and USDA have determined that some attenuated strains are not subject to the requirements of 42 CFR §73, 7 CFR §331, and 9 CFR§121 if used in basic or applied research, as positive controls, for diagnostic assay development, or for the development of vaccines and therapeutics. A list of excluded attenuated strains of SATs can be viewed at: http://www.selectagents.gov/Select%20Agents%20and%20Toxins%20Exclusions.html . However, please note that an individual or entity that possesses, uses, or transfers an excluded attenuated strain will be subject to the regulations if there is any reintroduction of factor(s) associated with virulence or other manipulations that modify the attenuation such that virulence is restored or enhanced.
All Select Agents Toxins and their uses must still be documented on the associated Biosafety Protocol even if exclusion levels are not met or exceeded. Because of the very specific requirements to maintain exclusion criteria, the IBC will likely require additional documentation or standard operating procedures (SOPs) for possession use and/or transfer of any excluded Select Agent or Toxin. This may include testing to prove attenuated/non-viable agents/toxins are, indeed, attenuated/non-viable upon receipt and maintaining additional security on any room or refrigerator or freezer which may contain the SAT, and/or maintaining an accurate log book to document the amount of Select Agent Toxin received/used in the laboratory to document the aggregate amounts in the laboratory at any moment in time.
2.6.2.2.2 Exemptions from Select Agents and Toxins Regulations for Clinical/Diagnostic Laboratories
Georgia Regents University Biosafety Guide- June 2008 2-27 As described in 42 CFR §73.5, 7 CFR §331.5 and 9 CFR §121.5, provisions have been made specifically for clinical and/or diagnostic laboratories or other entities that possess, use, or transfer an HHS/USDA select agent or toxin under certain specified conditions when the SAT is: Contained in a specimen presented for diagnosis or verification (see below for conditions) Contained in a specimen presented for proficiency testing A product that is, bears or contains SATs that is cleared, approved, licensed, or registered under any of the following laws, insofar as its use meets the requirements of such laws: • The Federal Food, Drug, and Cosmetic Act (21 U.S.C. 301 et seq.), • Section 351 of the Public Health Service Act pertaining to biological products (42 U.S.C. 262) • The Act commonly known as the Virus-Serum-Toxin Act (21 U.S.C. 151–159) • The Federal Insecticide, Fungicide, and Rodenticide Act (7 U.S.C. 136 et seq.) An agent or product for which the HHS Secretary or USDA Administrator has granted a special exemption as per their application/approval process (CDC/USDA Form 5). These special circumstances may include an investigational/experimental product that is, bears, or contains a select agent or toxin • when such product is being used in an investigation authorized under any Federal Act and additional regulation under this part is not necessary to protect public, animal or plant health and safety or protect animal or plant product safety. • being used, possessed or transferred to allow for timely participation of the individual or entity in response to a domestic or foreign public health emergency, agricultural emergencies or outbreaks, or in endemic areas. Is a diagnostic reagent or vaccine that is, bears, or contains USDA Veterinary Service Select agents or Toxins that are produced at USDA diagnostic facilities.
The Biosafety Officer/Responsible Official must be contacted immediately (x1-2663) when any material containing a Select Agent or Toxin is identified or is likely to be transferred to campus (even if it may qualify for an exemption or exclusion) to ensure that the strict compliance standards with these Federal laws are maintained.
2.6.2.2.3 Criteria for Handling Select Agents or Toxins Contaminated Specimens in Clinical/Diagnostic Laboratories under the Exemption Clause
Please note, if a SAT is contained in a specimen presented for diagnosis or verification, please be aware that the following conditions must be met: 1. The SAT needs to be transferred or destroyed on-site within 7 calendar days in accordance with specific guidance in the Federal laws. 2. The SAT must be secured against theft, loss or release prior to transfer or destruction. 3. With the exception of certain agents which have requirements for immediate reporting to the CDC/USDA (see below), once the diagnosis/identification of SATs in any specimens must be reported to the CDC and/or APHIS with seven (7) calendar days after identification and other appropriate authorities when required by law.
Georgia Regents University Biosafety Guide- June 2008 2-28 The following agents must be reported to the CDC immediately by telephone, facsimile, or e-mail, followed by submission of CDC/USDA Form 4: • Ebola viruses • Lassa fever virus • Marburg virus • Variola major virus (Smallpox virus) • Variola minor (Alastrim) • Yersinia pestis • South American Haemorrhagic Fever viruses (Junin, Machupo, Sabia, Flexal, Guanarito)
The following agents must be reported to the USDA immediately by telephone, facsimile, or e-mail, followed by submission of CDC/USDA Form 4: • African horse sickness virus • African swine fever virus • Avian influenza virus (highly pathogenic) • Bovine spongiform encephalopathy agent • Classical swine fever virus • Foot-and-mouth disease virus • Newcastle disease virus (velogenic) • Rinderpest virus • Swine vesicular disease virus
2.6.3 Human Blood, Body Fluids, Cells, Tissues and Other Potentially Infectious Materials
The Occupational Safety and Health Administration (OSHA) created the Occupational Exposure to Bloodborne Pathogens (BBP) Standard, 29 CFR Part 1910.1030 (Bloodborne Pathogens Standard) to minimize or eliminate exposure to infectious agents that may be present in any human blood, tissues or certain body fluids (bloodborne pathogens). Georgia does not have an OSHA-approved state plan for occupational safety and health. Consequently, while federal OSHA requirements do apply to most private employers in the state, they do not extend to public employers such as government agencies. However, Georgia Code §31-12-13 requires public employers to adopt a bloodborne pathogen standard governing occupational exposure of public employees to blood and other potentially infectious materials. This standard must be "at least as prescriptive" as the OSHA standard. Lastly, as described in Section 2.6.1, one of the terms and conditions for acceptance of NIH Grant awards that any grantee institution adheres to the OSHA Bloodborne Pathogen Standard 29 CFR 1910.1030. http://grants.nih.gov/grants/policy/nihgps_2003/NIHGPS_Part4.htm#_Toc54600062. For these reasons, it is GRU’s IBC policy to adhere to the OSHA standards for Bloodborne Pathogens.
The Bloodborne Pathogens Standard applies to all employers having employees that are “occupationally exposed” to human blood, materials which may have been 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 [via injection, infusion, cut exposure, or transdermal methods] contact with human blood or other potentially infectious materials in the performance of an employee’s duties.” An individual is also considered occupationally exposed even if they do not have direct contact with blood or other potentially infectious material, if the employee uses equipment that is used to process or store blood, other potentially infectious materials or bloodborne pathogens.
Georgia Regents University Biosafety Guide- June 2008 2-29 Potentially infectious materials other than human blood may include: Human cell, tissue or organ cultures Any unfixed human tissue or organ, other than intact skin, from a human being (living or dead) Human body fluids, except urine, feces, saliva or tears unless contaminated with blood. This includes: • semen • vaginal secretions • cerebrospinal fluid • synovial fluid • pleural fluid • pericardial fluid • peritoneal fluid • amniotic fluid • saliva in dental procedures All body fluids in situations where it is difficult or impossible to differentiate between body fluids or is contaminated with blood HIV-containing cell, tissue or organ cultures HIV- or HBV-containing culture medium or other solutions Blood, organs, or other tissues from experimental animals infected with HIV or HBV or other bloodborne pathogens
OSHA has determined that occupational exposure to human blood, tissues and body fluids poses a significant health risk because these may contain bloodborne pathogens such as: • Human Immunodeficiency Virus (HIV) • Hepatitis B virus (HBV) • Hepatitis C virus • Hepatitis D virus • Plasmodium species • Treponema species • Babesia species • Borrelia species • Brucella species • Leptospira species • Francisella species • Streptobacillus moniliformis • Colorado Tick Fever viruses • Arboviruses • Spirillum minus • Creutzfeldt-Jakob virus • Human T-lymphotropic Virus Type I • Hemorrhagic Fever viruses
All occupationally exposed employees are required by OSHA to attend a Bloodborne Pathogens training session prior to beginning work and annually thereafter. Employees must receive training when they are first assigned to tasks where occupational exposure to bloodborne pathogens may occur. The training must be repeated annually, and must be provided at no cost to the employees and during work hours.
Employees must receive additional training when changes occur that affect the employees' occupational exposure, such as task or procedural modifications or the institution of new tasks or procedures. This additional training only needs to address new exposures created.
Training must include: • making accessible a copy of the regulatory text of the standard and explanation of its contents • general discussion on bloodborne diseases and their transmission
Georgia Regents University Biosafety Guide- June 2008 2-30 • an explanation of the exposure control plan and how the employee can obtain a copy of the written plan • how to recognize tasks and other activities that may involve exposure to blood and other potentially infectious materials • use and limitations of methods for preventing or reducing exposure, including engineering and work practice controls and personal protective equipment • information on the hepatitis B vaccine • response to emergencies involving blood or other potentially infectious materials • how to handle exposure incidents • the post-exposure evaluation and follow-up program • information about signs/labels/color-coding related to bloodborne pathogens
Due to the importance of information regarding occupational exposure to potentially life-threatening bloodborne pathogens, employees must have the opportunity to ask questions and receive answers from a knowledgeable trainer. Laboratory and production facility workers must receive additional specialized initial training.
Consult the Bloodborne Pathogen Training Manual for Clinical and Laboratory Personnel for additional information on the exposure control plan, training requirements, work practices, housekeeping, engineering controls, personal protective equipment, signs/label requirements, Hepatitis B vaccination, emergency actions, exposure incident procedures, post-exposure evaluation and follow-up, and recordkeeping. Contact the Biosafety Office at 706-721-2663 or [email protected] for assistance with exposure determination and for training information.
2.6.3.1 Cultures of Cell lines of Human and/or Primate Origin
Human cell lines are commonly used in biomedical research, yet appropriate biosafety requirements for handling human cell lines are often subject to debate within the scientific community. While human blood, most body fluids, unfixed human tissues and organs were clearly included within the scope and application of the OSHA Bloodborne pathogen standard (29 CFR Part 1910.1030), the inclusion of human cell lines was ambiguous.
In 1994, OSHA issued a letter of interpretation related to the applicability of the BBP Standard towards human cell lines: http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_table=INTERPRETATIONS&p_id =21519.
According to the interpretation, human cell lines are considered to be potentially infectious and within the scope of the BBP Standard unless the specific cell line has been characterized to be free of hepatitis viruses, HIV, Epstein-Barr virus, papilloma viruses and other recognized bloodborne pathogens. In alignment with this interpretation, the American Type Culture Collection (ATCC) recommends that all human cell lines be accorded the same level of biosafety consideration as a line known to carry HIV: http://www.atcc.org/TechnicalInfo/faqCellBiology.cfm#Q53 Comment [L1]: Couldn’t find the correct link
Moreover, Appendix H of the Fifth Edition of the CDC publication, Biosafety in Microbiological and Biomedical Laboratories, recommends that human and other primate cells should be handled using Biosafety Level 2 (BSL2) practices and containment http://www.cdc.gov/od/ohs/biosfty/bmbl5/APPENDIX%20H.pdf .
Tumorigenic human cell lines also present a potential hazard as the result of self-inoculation. In fact, there has been at least one reported case of development of a tumor resulting from an accidental needlestick (Gugel EA and Sanders ME. (1986) Needle-stick transmission of human
Georgia Regents University Biosafety Guide- June 2008 2-31 colonic adenocarcinoma. N Engl J Med. 315(23):1487; PMID: 3785302). Because of the extra protection that the laboratory worker’s functioning immune system affords him/her in case of accidental needlestick or exposure, laboratory workers should never handle autologous cells or tissues.
In consideration of the aforementioned regulatory interpretation and consensus guidelines and other factors, the GRU Institutional Biosafety Committee considers that all cell and organ cultures of human or primate origin, including well-established cell lines should be handled in accordance with the OSHA Bloodborne Pathogens Standard and under Biosafety Level 2 (BSL-2) containment.
2.6.3.1.1 Human Embryonic Stem (hES) Cell and Embryonic Germ Cell Lines
On August 9, 2001, at 9:00 p.m. EDT, President George W. Bush announced his decision to allow Federal funds to be used for research on existing human embryonic stem cell lines as long as prior to his announcement 1. the derivation process (which commences with the removal of the inner cell mass from the blastocyst) had already been initiated and 2. the embryo from which the stem cell line was derived no longer had the possibility of development as a human being
In addition, the President established the following criteria that must be met: 1. The stem cells must have been derived from an embryo that was created for reproductive purposes; 2. The embryo was no longer needed for these purposes; 3. Informed consent must have been obtained for the donation of the embryo; 4. No financial inducements were provided for donation of the embryo.
In order to facilitate research using human embryonic stem cells, the NIH created the Human Embryonic Stem Cell Registry, which lists the human embryonic stem cell lines—at varying stages of development—that meet the eligibility criteria. A list of the embryonic stem cell lines which meet the President’s criteria and are therefore eligible for federal funding can be viewed at: http://stemcells.nih.gov/research/registry/ . No federal funds may be used, either directly or indirectly, to support research on human embryonic stem cell lines that do not meet the criteria established by President Bush on August 9, 2001. Thus, research on lines (or their derivatives) not listed on the NIH Human Embryonic Stem Cell Registry may not be supported by federal funds. This includes considerations to strictly prevent any indirect costs, or F&A costs, to be utilized to support any research with any unallowable human embryonic stem cell lines. Strict compliance with cost allocation methodologies described in the circular, including the Cost Accounting Standards, must prevent the shifting of unallowable stem cell research costs to federally sponsored programs. A properly documented F&A proposal utilized in the establishment of F&A rates should demonstrate that none of the costs of unallowable stem cell research or other unallowable activities have been shifted to federally sponsored activities. Generally, this means that separate facilities, supplies and equipment must be maintained for research with any unallowable hES lines to prevent inadvertent use of Federal indirect funds for the support of unallowable hES line research. See: http://stemcells.nih.gov/info/faqs.asp for more information.
GRU hES research should also adhere to Guidelines for Human Embryonic Stem Cell Research developed by the National Research Council of the National Academies of Sciences. http://www.nap.edu/catalog.php?record_id=11278 . While GRU does not currently support an Institutional Embryonic Stem Cell Research Oversight Committee (ESCRO),
Georgia Regents University Biosafety Guide- June 2008 2-32 as recommended by the Guidelines, the ethics and compliance oversight will still be monitored by the IRB, IBC and IACUC. Much of the recommendations in these Guidelines relate to ensuring the source and consent of the donors and/or recipients of these cells (i.e. issues generally under review by the Institutional Review Board); however, please note the Guidelines specifically recommends against the following applications for hES research: • Research involving in vitro culture of any intact human embryo, regardless of derivation method, for longer than 14 days or until formation of the primitive streak begins, whichever occurs first. • Research in which hES cells are introduced into nonhuman primate blastocysts or in which any ES cells are introduced into human blastocysts. • No animal into which hES cells have been introduced at any stage of development should be allowed to breed.
Research use of human embryonic germ cells derived from fetal tissue with Federal funds requires review of compliance with the NIH Guidelines for Research Involving Human Pluripotent Stem Cells. The review process is described at: http://grants.nih.gov/grants/guide/notice-files/NOT-OD-02-049.html .
2.6.4 Animals and the use of Biological Materials in Animals
All research experiments involving animals must be conducted in accordance with the associated GRU Institutional Animal Care and Use Committee (IACUC) approved animal use protocol (AUP). If the animals are infected, infectious or pose a higher risk for human infection such that the animals or their tissues require Biosafety Level-2 containment or higher (see Section 4, Risk Management for further information), research with these animals will require IBC authorization, as well. This would include working with non-human primates (NHP), their tissues and cells.
Because of the evolutionary similarities between humans and non-human primates, NHPs are at higher risk of carrying bloodborne pathogens infectious to humans. In addition, most species of macaque monkeys are at high risk of being carriers of Cercopithecine Herpes Virus 1 (CHV-1, Herpes B virus) (see Section 3.2.3.1, Zoonotic Disease Risks/Animal Research Risks for further information). For these reasons, a minimum of BSL-2 containment is recommended for handling of NHP species, their tissues and cells by the CDC BMBL, Appendix H (http://www.cdc.gov/biosafety/publications/bmbl5/BMBL5_appendixH.pdf). Materials and practices must be documented on a BSP Application, and all personnel who may be at risk of exposure to NHP materials should receive BBP training and preventative measures (see Section 2.6.3 for further information).
Animal research that involves the introduction of biological hazards (e.g., agents which require ≥BSL-2 containment or recombinant DNA) must have an approved Biosafety Protocol (BSP) to reflect these uses and fully describe and disclose the use of these biological agents/recombinant DNA in their AUP and BSP applications. The Biosafety Office must approve work with pathogens or recombinant DNA in animals (including transgenic animals) prior to initiation. Biosafety Protocol Application forms can be found at: http://georgiahealth.edu/research/ibc/apps.html or please contact the Biosafety Office (x1-2663 or [email protected]) for assistance.
Once approved by the IBC, the GRU Division of Laboratory Animal Resources Center (LAS) will be contacted prior to initiation to ensure that a safety protocol has been established and appropriate facilities are obtained for these experiments. Contact LAS at 706-721-3421 for additional information.
Georgia Regents University Biosafety Guide- June 2008 2-33
2.6.5 Insects/Arthropods
Insects present unique challenges to containment which are not encountered with either microbial pathogens or biological toxins. In addition, because some arthropods may serve as vectors for disease transmission, special containment considerations, including facilities (insectaries) and practices specifically designed to prevent accidental escape of arthropods, are crucial for health and environmental safety. With this in mind, the American Committee of Medical Entomology (ACME), a subcommittee of the American Society of Tropical Medicine and Hygiene (ASTMH) have drafted Arthropod Containment Guidelines (ACG) and related materials which should be considered during the risk assessment process: http://www.astmh.org/SIC/acme.cfm.
Currently, GRU has no areas appropriately configured as insectaries to handle any insects which could serve as disease vectors. This limits research projects involving live insects only to those involving insects which do generally not serve as disease vectors (e.g., Drosophila melanogaster (i.e., fruit flies)), which may or may not carry exempt (RG-1) recombinant DNA. Even with these experiments, special precautions should be taken to prevent accidental escape/release of laboratory insects, particularly those which have been genetically engineered, to prevent environmental contamination.
2.7 EXPERIMENTS PROHIBITED AT GRU
The following experiments are prohibited at GRU:
Experiments using agents or organism that require containment facilities and/or equipment which are not available at the Georgia Regents University. Experiments using any organism or agent that is prohibited by any federal or state agency from importation into Georgia or for which the required permits/authorizations have not been received. Experiments involving recombinant DNA manipulation of plants or plant pathogens Research with venomous insects or animals Experiments involving purposeful environmental release of any biological agent and/or recombinant DNA materials. Experiments with agents or organisms which are not permitted as a term of maintaining our federal fundability status (e.g., human embryonic stem cells which are not eligible for federal funding) Experiments which violate the ethical standards laid out by the National Institutes of Health and/or the National Research Council of the National Academies of Sciences (e.g., see Section 2.6.3.1.1).
Georgia Regents University Biosafety Guide- June 2008 2-34 3. RISK ASSESSMENT
Responsibility for biosafety exists at all levels and is shared throughout the University. The President, Provost and Senior Vice President of Research acknowledge the institution’s role in providing a safe workplace and have given the Institutional Biosafety Committee (IBC) and Biosafety Office the authority to administer the campus biosafety program. The Institutional Biosafety Committee (IBC) establishes policies for the safe use of biohazards and for compliance with all applicable regulations and guidelines to secure the safety of the university, personnel, research assets, the environment and the surrounding community. The Biosafety Office acts as the agent of the IBC in ensuring compliance with these policies, disseminating pertinent information; consulting with faculty, staff, students and visitors, and monitoring for non-compliance.
The researchers, clinicians, students and technicians who perform work with biohazards are perhaps the most important component of the biosafety program, as they must incorporate the biosafety requirements and safety precautions into all facets of their work.
The Principal Investigator is ultimately responsible for safety within the laboratory (See Section 1.2.2, Responsibilities, Principal Investigator). An integral part of this responsibility is to conduct a review of proposed work to identify potential hazards (risk assessment) and to adopt appropriate safety procedures before initiation of the experiments (risk management). The PI must also monitor the work to ensure that the safety procedures are being utilized by the staff and assess whether any improvements should be made based on logistics of the experiments or additional safety concerns.
Certain experiments require advanced registration and Biological Safety Committee approval prior to initiation (See Section 2).
A risk assessment/risk management matrix has been prepared to illustrate key elements of the process (see below). Relevant sections providing additional details are indicated within the matrix. Information on the routes of exposure is included at the end of this section.
The five P’s of risk assessment and risk management are: Pathogen – hazardous biological agent. Procedures – proposed experimental manipulations and safe work practices. Personnel – appropriate training and skills. Protective equipment – protective clothing and safety equipment. Place – laboratory design.
Consider the five P’s in each facet of laboratory work. Properly conducted, risk assessment can help prevent exposure to biohazards and minimize the potential for laboratory acquired infection. Remember that prior planning prevents poor performance from both a biosafety and research integrity standpoints.
After reading this section and relevant sections of the Biological Safety Manual contact the Biosafety Office at x1-2663 or [email protected]@georgiahealth.edu for help applying the principles of risk assessment and risk management to experimental procedures.
Georgia Regents University Biosafety Guide- June 2008 3-1 3.1 RISK ASSESSMENT AND MANAGEMENT TABLE
Risk Assessment Risk Management Pathogen • Agent classification (See Appendix B) • Registration – See Section 2 . • Routes of infection/exposure . Biosafety Office • Infectious disease process . Biological Safety Committee • Virulence, pathogenicity, quantity, . State of Georgia- certain infectious agents concentration, incidence in community, . USDA – restricted agents or Select Agents/Toxins presence of vectors . CDC – Select Agents/Toxins . FDA/NIH - human gene therapy • Ensure strain identity, attenuation, replication competency (safety tests & documentation) Procedures • Aerosol risk: sonicating, centrifuging, • Written set of standard operating procedures (SOPs) with homogenizing, blending, shaking, etc. safety practices incorporated • Percutaneous risk: needles, syringes, • Adherence to basic biosafety principles glass Pasteur pipettes, scalpels, cryostat • Label labs, areas, and equipment housing BL2 or higher blade/knife, etc. agents • Splash/splatter risk: pipetting, microbial • Conduct lab inspections to review practices and containment loop, etc. equipment • Transport/shipping of agents outside of • Use trial experiments with non-infectious material to test new laboratory procedures/equipment
Personnel • Host immunity • Safety training . Neoplastic disease • Prior work experience with biohazards . Infection • Demonstrated proficiency with techniques . Immunosuppressive therapy • Prompt reporting of all exposure incidents, near misses, as . Age, race, sex, pregnancy well as signs and symptoms of related disease to PI and . Surgery (splenectomy, Employee Health & Biosafety Office gastrectomy) • Investigation/review of incidents/spills, etc. to prevent future . Diabetes, Lupus occurrence • Immunization • Post-exposure prophylaxis • Serum banking • Attitude toward safety • Comfort • Open wounds, non-intact skin, eczema, dermatitis
Protective Protection (containment) for: • Personal protective equipment (PPE): Equipment . Respirators – HEPA, N-99, N-95, etc. • Aerosols – respirable size particles . Surgical masks (for droplets) <5µm . Face (eye, nose, mouth) protection – mask and safety • Droplets/splatter glasses, or chin length face shield • Sharps . Solid front gown or lab coat . Gloves • Biological safety cabinets • Centrifuge safety buckets/rotors • Plexiglass shielding, glove box
Place – • Risk group/biosafety level requirements • Basic lab – door, sink, surfaces easily cleaned, eyewash, Laboratory • Aerosol risk screens on windows that open facility • Restricted access • Labels • Containment laboratory with directional airflow
Georgia Regents University Biosafety Guide- June 2008 3-2
3.2 CONSIDERATIONS IN A RISK ASSESSMENT
In general, a risk assessment includes considerations of the following three items for all of the following factors listed below:
a. Identification of the potential hazards in an experiment.
b. Characterization of the relative potential for the hazards to become an issue.
c. Characterization of the potential severity of the consequences if the hazard exposure did occur (to personnel, community, environment, institutional image)
3.2.1 Risk Groups: Hazardous Characteristics of a Biological Agent
The principal hazardous characteristics of an agent are:
Its capability to infect and cause disease or pernicious response in a susceptible human or animal host, which includes the following considerations about the agent: • Infective dose • Attenuation • Allergenicity • Physiological activity • Oncogenicity Is virulence as measured by the severity of disease that results from infection, which includes the following considerations: • Pathogenicity • Attenuation • Genetic modifications (toxic effects, oncogenecity, allergenicity, physiological activity) The availability of preventive measures and effective treatments for the disease, which includes the following considerations about the agents: • Are there vaccinations or treatment modalities available against the agent? • Is this a drug resistant strain?
The World Health Organization (WHO), NIH Guidelines for Research with Recombinant DNA Molecules, and CDC/NIH publication Biosafety in Microbiological and Biomedical Laboratories (BMBL) established a risk-group (RG) classification system based on the risks that the agent, alone, presents to the health of healthy human adults based on an assessment of the characteristics described above. See Table 3.2.1 for a general description of these risk groups. The risk group of an agent should be only one factor to be considered in association with mode of transmission, procedural protocols, amount of material present, experience of staff, and other factors in determining the appropriate biosafety containment level (BSLs) which the work will be conducted.
Keep in mind that since these guidelines are based on the effects on healthy human adults, and do not account for individual health considerations, such as allergies, pregnancy, breast feeding, medication effects, a compromised immune system (due to illnesses or medical treatments such as steroids or chemotherapy) or other illnesses which may make individuals more susceptible to agents. In addition, the potential for differential effects of these agents in the immature systems of minors are also not considered in these guidelines. Therefore, the guidelines represent only a starting point for an biological agent’s risk assessments. Other known health considerations should also be factored in when performing a comprehensive risk assessment. Also, for this reason, for their own safety, any individual with special health concerns is strongly encouraged to discuss these with the Principal Investigator, Clinical Director or Instructional Course Director prior to initiation of work within the laboratory. In turn, PIs, Clinical
Georgia Regents University Biosafety Guide- June 2008 3-3 Directors or Instructions Course Directors should offer the opportunity for the individual to seek Occupational Health Counseling through the Employee Health Office to discuss their potential special risks (See Section 5, Medical Surveillance Program, for more information).
Additional guidance in determining the appropriate Risk Group for microbial agents can be found at: NIH Guidelines, Appendix B (http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.htm) American Biological Safety Association Risk Group Database: http://www.absa.org/riskgroups/index.html CDC/NIH Biosafety in Microbiological and Biomedical Laboratories (BMBL), particularly Section VIII: http://www.cdc.gov/biosafety/publications/bmbl5/BMBL5_sect_VIII.pdf Public Health of Canada MSDSs for biological agents: http://www.phac-aspc.gc.ca/msds-ftss/
Table 3.2.1 NIH Guidelines (2002) Definitions of Risk Groups
Risk Group Description of Risk of Agents (RG) Classification RG 1 Agents that are not associated with disease in health human adults
RG 2 Agents that are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available.
RG 3 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).
RG 4 Agents that are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available (high individual risk and high community risk).
3.2.2 Routes of Exposure
In order for microbiological agents to cause disease, they must:
1. Enter or invade the body in sufficient numbers. Routes of entry include oral, respiratory, and parenteral, via mucous membrane and/or via animal contacts (bites, scratches). See the table below for additional information on routes of exposure or contact the Biosafety Office at x1-2663 or [email protected].
Any risk assessment process should consider the possible unique routes of entry presented during the course of the proposed experiment in addition to the risks associated with potential spread of the infectious organism outside of the laboratory should a researcher be inadvertently infected. Please note: these infectious routes of entry may differ. For instance, although a parasitic infection may normally require a specific arthropod vector to transmit the infectious agents, and the risk of having the arthropod vector within the laboratory may be extremely low, the researcher must also consider that a parasitic infection may arise in the laboratory after accidental parenteral introduction of the organism (via a needlestick, for instance).
It is difficult to determine a minimum infectious dose when discussing biohazards. The same dose of a pathogen may produce no disease symptoms in one individual but may cause serious or even fatal disease in another. There are microorganisms for which it is thought one organism entering the body is sufficient to invade and promote the disease process; Mycobacterium tuberculosis, the bacteria that causes tuberculosis or Coxiella burnetii, the causative agent of Q
Georgia Regents University Biosafety Guide- June 2008 3-4 fever are examples. For many pathogens, 10 to 100 or more organisms must enter the body to cause infection leading to disease.
2. Once inside the body, microbial agents must colonize in a hospitable area and establish an infection in the host body cells, tissues and/or organs
3. Lastly, the microorganisms must overcome the host body’s natural defense mechanisms and/or mutate or adapt to body changes to persevere within the host organism.
Other factors contribute to an individual’s susceptibility to the disease process. These include age, immunological state, occupation, physical and geographic environment and predisposing conditions (such as alcoholism and other drug abuse, pregnancy and diseases such as diabetes).
Therefore all factors must be considered in a comprehensive risk assessment process. When performing risk assessments using recombinant DNA materials, all of the above factors must also be considered, in addition to issues more specific to recombinant DNA, including the recombinant agents’ ability to replicate once established inside the host, its ability to spread horizontally (to others or to unintended regions of the host’s body), and/or the ability to transfer recombinant DNA vertically via germ cells into future generations.
Once the risks are assessed, appropriate mitigation/management measures can be developed which specifically address the risks posed by the agents and how these will be used (See Section 4, Risk Management, for further information).
Georgia Regents University Biosafety Guide- June 2008 3-5 Routes of Transmission for Infectious Agents
Georgia Regents University Biosafety Guide- June 2008 3-6
3.2.3 Operations which may be associated with additional risk of exposure
The following laboratory operations are associated with additional risk for aerosol, splash and/or spray production. Therefore, additional risk considerations must be factored in during the risk assessment, and these activities must be documented on all Biosafety Protocols for the IBC to review.
• Blowing out pipettes • Homogenizing • Cell sorters (FACS) • Blending • Shaking , vortexing or stirring tubes • Grinding • Opening lyophilized cultures • Cell disruption with French press • Opening snap top tubes • Intranasal inoculation of animals • Breakage of culture containers • Cage cleaning, changing animal • Flaming loops or slides bedding • Pulling needles out of septums • Harvesting infected material from • Filling a syringe animals, eggs, and other virology • Placing liquids under pressure procedures • Pouring liquids • Necropsies of infected animal • Centrifugation • Sonication
3.2.3.1 Zoonotic Disease Risks/Animal Research Risks
Use of animals and animal tissues may pose additional inherent risks due to the somewhat uncontrollable nature of animals as well as the potential risk of exposure to zoonotic diseases, which are diseases which are communicable from animals to humans and in many cases, visa versa, under natural conditions. Some animals are natural carriers of infections which may be associated with mild symptoms in animals, and therefore difficult to diagnose; however, some of these diseases can be quite serious or lethal in humans. Several of these illnesses may pose a particular hazard to pregnant women or their developing fetuses (Q-fever, toxoplasmosis); therefore pregnant workers should consider discussing their special risks and protective measures with a physician. Familiarization of the potential symptoms of the zoonotic diseases which may be carried by the research animals used in the laboratory should be part of the laboratory-specific training/education measures provided to all personnel working with the materials and should be communicated to any health care provider, should any exposures or symptoms present. Protective measures should always be addressed in each laboratory’s standard operating procedures (SOPs) to mitigate the risk of these diseases. A recommended initial reference for information on zoonotic diseases and their associated pathogens is the interactive tables developed by the Air Force Public Health Services: http://phsource.us/PH/ZD/DiseasesTable.htm.
Additional risks associated with working with animals may include those related simply with the logistics for working in animal facilities. Heavy and large equipment is often used in these facilities, such as cage racks or changing stations, and the metal caging can often become bent in the day-to-day activities in the animal laboratory. These additional risks, including risks for crush injuries or lifting hazards must also be considered by the Facility Director.
3.2.3.1.1 Non-human primates Because of primates evolutionary similarities to humans, many of the diseases that affect one species can affect the other; albeit, often with different symptoms. Care and consideration must not only be given to the protection of the workers while working with NHP, but also to protecting the NHP colonies to infections brought in my human
Georgia Regents University Biosafety Guide- June 2008 3-7 caregivers and researchers, which can quickly devastate a primate colony. A minimal BSL-2 containment facilities and safety measures should be followed when working with NHP and materials derived from NHP.
3.2.3.1.1.1 Cercopithecine Herpes Virus (CHV-1); Herpes B virus Most species of macaque monkeys (rhesus, cynomolgus, pigtail) can carry a virus known as B virus, Herpesvirus simiae or Cercopithecine Herpes Virus I (CHV-1). CHV-1 is to primates what human herpes simplex virus-1 (i.e. the cold sore virus) or -2 (i.e. genital herpes) are to humans. In monkeys, it is a relatively mild disease; most of the time, one cannot tell whether a monkey is infected by sight. The virus tends to "lie dormant" in the body, but it can cause "breakouts" with occasional mucous membrane or genital lesions, especially during periods of high stress. It's during the "breakouts" that monkeys can shed the virus and potentially expose humans.
Unfortunately, in humans CHV-1 has a mortality rate somewhere in the neighborhood of 64-80% if left untreated. Infection in humans is rare, but most cases have involved contact with nonhuman primates. Immediate diagnosis and treatment with antiviral drugs has saved lives.
Since B virus has a high prevalence in conventional primate colonies, and animals can carry the disease without showing symptoms, it is best to assume that all primates are infected. Body fluids such as saliva and urine present the highest risk to workers. Caretakers are at special risk, since routine care and cage washing involves frequent contact with sharp edges on contaminated cages and production of aerosols from spraying animal waste.
In laboratories, all materials of non-human primates should be handled under very strict BSL-2 containment. Activities such as pipetting, opening containers and centrifuging urine, blood or mucosal swabs can present risk of contamination.
CHV-1 is fairly stable outside of the body (estimates suggest it can remain viable for up to 7 days at 37°C and weeks at 4°C). However, it can be inactivated by 70% ethanol, freshly diluted bleach (1:5), heating (50-60 C for >30 min), acidic pH's and/or detergent solutions.
Personnel working with NHP will be required to attend a special NHP training class offered by DLAS Veterinarians. Use of protective clothing, eye protection and respiratory protection are essential when working with NHP or their materials. Consistent caution and respect for the monkey is always a good idea. In addition, if you ever spot what looks like a cold sore on a monkey, be sure to contact a DLAS veterinarian before handling the animal or its cage. Any even potential exposure should be assessed and treated by a medical professional immediately. See incident response guidance and contact DLAS Veterinarians (x1-3421) and/or the Biosafety Office (x1-2663 or [email protected]) for assistance.
Further information about CHV-1 and measures for prevention, please see the following guidance:
. Public Health Service of Canada MSDS on CHV-1: http://www.phac- aspc.gc.ca/msds-ftss/msds81e.html
. Cohen JI, Davenport DS, Stewart JA, Deitchman S, Hilliard JK, Chapman LE and the B Virus Working Group (2002).
Georgia Regents University Biosafety Guide- June 2008 3-8 Recommendations for prevention of and therapy for exposure to B virus (cercopithecine herpesvirus 1). Clin Infect Dis. 35(10):1191-203. (A copy of this paper can be downloaded from GRU computers from the following URL: http://web.ebscohost.com/ehost/detail?vid=3&hid=5&sid=da4a1eb6- 61c4-475e-b22a-a5d1b9e3058d%40sessionmgr103 )
3.2.3.1.1.2 Other viral infections associated with NHP
Nonhuman primates can develop measles, usually following exposure to infected humans. Workers should be vaccinated against measles. Workers with measles should not work with nonhuman primates until they have recovered. Clinical signs in the animals are similar to those in humans, including a red rash on the face and hands. Transmission is usually by direct contact. More information on measles and measles virus can be viewed at: http://www.phac- aspc.gc.ca/lab-bio/res/psds-ftss/measles-rougeole-eng.php
Influenza virus can be transmitted between humans and nonhuman primates. Signs of disease for both humans and animals include fever and upper respiratory signs such as coughing, sneezing and nasoocular secretions. The disease is potentially fatal in immunocompromised individuals. Transmission is by aerosol. Prevention is achieved through the use of protective clothing and respiratory protection. More information about influenza can be found at the CDC informational URL: http://www.cdc.gov/flu/ .
Several pox viruses, including monkeypox and Yaba virus can cause painful nodular lesions in the skin of humans and nonhuman primates. Transmission is by direct contact, although infected tissues could present a risk to laboratory workers. Prevention of disease is through use of protective clothing. More information on monkey pox can be seen at: http://www.cdc.gov/ncidod/monkeypox/
Nonhuman primates may be carriers of filoviruses (http://www.cdc.gov/Ncidod/dvrd/spb/mnpages/dispages/filoviruses.htm) such as Ebola virus (http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/ebola- eng.php). These viruses cause an acute hemorrhagic fever in humans and nonhuman primates that is often fatal. Blood and body fluids are potential sources of the virus. Use of protective clothing and eyewear is preventative. Animals are screened upon entry into the country for filoviruses.
3.2.3.1.1.3 Gastrointestinal Diseases associated with NHPs
Bacterial agents such as Salmonella (http://www.phac-aspc.gc.ca/lab- bio/res/psds-ftss/msds135e-eng.php), Shigella (http://www.phac-aspc.gc.ca/lab- bio/res/psds-ftss/shigella-eng.php) and intestinal parasites such as Giardia lamblia (http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/msds71e-eng.php), Entamoeba histolytica (http://www.phac-aspc.gc.ca/lab-bio/res/psds- ftss/msds58e-eng.php), and Balantidium coli (http://www.phac-aspc.gc.ca/lab- bio/res/psds-ftss/msds15e-eng.php) can be carried by many species of nonhuman primates. Feces are the primary source of infections. Gloves, a laboratory coat and a respiratory mask should be worn when handling fecal samples or sanitizing caging. Symptoms of these diseases include diarrhea with or without blood, vomiting, and fever in both humans and nonhuman primates. Medical attention should be sought immediately.
Georgia Regents University Biosafety Guide- June 2008 3-9
3.2.3.1.1.4 Bacterial Respiratory Diseases associated with NHPs
Streptococcus pneumonia can cause an acute bacterial pneumonia in humans and nonhuman primates. Infected animals and lung or sputum samples can present a risk to workers. Sudden onset of fever, malaise and coughing are symptoms of bacterial pneumonia. Medical attention should be sought immediately.
Tuberculosis caused by Mycobacterium tuberculosis (http://www.phac- aspc.gc.ca/lab-bio/res/psds-ftss/msds103e-eng.php) infection causes an extremely rapidly fatal pneumonia in most Old World primate species, and chronic pneumonia in humans. Typically, infected humans present a much greater risk to the animals than animals do to humans. It would be unusual for the disease to be transmitted to humans, unless the animal is undergoing surgery or pathologic examination. Infected tissue samples can also present a risk to laboratory workers. Prevention of disease includes routine use of respiratory protection and protective clothing when working with tissues or when coming into close proximity to animals. Animals and human handlers should be screened every 6 months for disease. Animals should also be quarantined and screened on entry into the facility.
3.2.3.1.2 Birds Birds carry diseases such as psittacosis and an avian form of tuberculosis. Only inspected, properly quarantined birds should be used in research studies or teaching demonstrations. Mycological fecal contamination is also frequent. The causative agents for some common avian transmitted diseases are described below and the following links describe some of the potential illnesses associated with birds. Contact the Biosafety Office or Division of Laboratory Animals Services for further information.
• Chlamydia psittaci: http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/msds31e- eng.php • Histoplasma capsulatum: http://www.phac-aspc.gc.ca/lab-bio/res/psds- ftss/histoplasma-capsulatum-eng.php • Cryptococcus neoformans: http://www.phac-aspc.gc.ca/lab-bio/res/psds- ftss/cryptococcus-eng.php • Mycobacterium avium: http://www.phac-aspc.gc.ca/msds-ftss/msds102e.html
Transmission to humans occurs by exposure via the inhalation route for the fungal infections (Histoplasma, Cryptococcus) due to inhaling spores. Contact with tissues through cuts or scratches may also pose a risk. Another route of exposure may be surface contact while handling avian fecal specimens.
Those at risk include investigators, animal caretakers, laboratory personnel, or others who routinely handle birds, their tissues, and feces. Scratches or cuts involving birds or injuries from objects contaminated with body fluids or feces from birds require immediate first aid and medical attention.
Gloves, masks and a laboratory coat (or other dedicated protective clothing such as a scrub suit) should be worn when working with birds. In some cases protective eye wear is also indicated. Do not eat, drink, or apply cosmetics while working in an aviary, and always wash your hands after handling birds. Remember that unfixed tissues, body
Georgia Regents University Biosafety Guide- June 2008 3-10 fluids, and other materials derived from birds may also pose a risk. Guano (feces), hair and feathers may also exacerbate allergies.
3.2.3.1.3 Dogs (Canines) Dogs used in research have been vaccinated against rabies. However, it may be prudent to consider prophylactic immunization.
Bites and scratches may also pose serious problems through trauma and/or bacterial infection. Dogs may also have enteric bacteria such as Salmonella released in the feces so cage washers and any personnel who must clean bedding should wash hands with a disinfectant hand soap before leaving the facility. Dogs, like most mammals, can shed fur so anyone with allergies to fur, dander or animal bedding should wear personal protective clothing to minimize discomfort. Dogs may also carry biting insects, such as fleas, so personal protective equipment may also be used in this instance as well.
The following links describe some of the potential illnesses associated with dogs and may be found on-line:
• Brucella canis: http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/msds23e- eng.php • Campylobacter jejuni: http://www.phac-aspc.gc.ca/lab-bio/res/psds- ftss/msds29e-eng.php • Rabies: http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/rab-eng.php
Gloves, masks and a laboratory coat (or other dedicated protective clothing such as a scrub suit) should be worn when working with dogs. In some cases protective eye wear is also indicated. Do not eat, drink, or apply cosmetics while working in an animal use area, and always wash you hands after handling dogs. Remember that unfixed tissues, body fluids, and other materials derived from dogs may also pose a risk.
Bites or scratches involving dogs or injuries from objects contaminated with body fluids from dogs require immediate first aid and medical attention.
3.2.3.1.4 Cats (Felines) Bites and scratches may also pose serious problems through trauma and/or bacterial infection. Cats may also release microorganisms such as Salmonella and parasites such as Toxoplasma in the feces so cage washers and any personnel who must clean bedding should wash hands with a disinfectant hand soap before leaving the facility. Cats, like most mammals, can shed fur so anyone with allergies to fur, dander or animal bedding should wear personal protective clothing to minimize discomfort. Cats may also carry biting insects, such as fleas, so personal protective equipment may also be used in this instance as well.
The following links describe some of the potential illnesses associated with cats and may be found on-line:
• Bartonella henselae (Cat Scratch Fever): http://www.phac-aspc.gc.ca/lab- bio/res/psds-ftss/bartonella-henselae-eng.php • Rabies: http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/rab-eng.php • Toxoplasma gondii: http://www.phac-aspc.gc.ca/msds-ftss/msds153e.html
Georgia Regents University Biosafety Guide- June 2008 3-11 Bites or scratches involving these species or injuries from objects contaminated with body fluids from cats require immediate first aid and medical attention. Pregnant women need to be aware that toxoplasmosis, caused by infection with Toxoplasma gondii can cause problems with pregnancy, including abortion and should address these risks with the Occupational Health Office, Division of Laboratory Animal Services or Biosafety Office, as desired.
Gloves, masks and a laboratory coat (or other dedicated protective clothing such as a scrub suit) should be worn when working with cats. In some cases protective eye wear is also indicated. Do not eat, drink, or apply cosmetics while working in an animal use area, and always wash you hands after handling cats. Remember that unfixed tissues, blood, serum, urine, and other materials derived from cats may also pose a risk.
3.2.3.1.5 Rabbits (Lagomorphs) Rabbits raised in "clean" facilities are considered relatively free of zoonotic diseases unless they have been experimentally or accidentally infected with a human pathogen. However, those working with rabbits should also be aware of possible allergic reactions. These allergic reactions are often associated with cage cleaning due to the dust hazards of bedding and surface contact with urine and other waste materials.
The following links describe some of the potential zoonotic illnesses which may be associated with wild rabbits and may be found on-line: • Bordetella: http://www.phac-aspc.gc.ca/msds-ftss/msds19e.html • Pasteurella : http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/msds117e-eng.php • Tularemia (Francisella tularensis): http://www.phac-aspc.gc.ca/lab-bio/res/psds- ftss/msds68e-eng.php
Bites or scratches involving these rabbits or injuries from objects contaminated with body fluids from rabbits require immediate first aid and medical attention.
Gloves, masks and a laboratory coat (or other dedicated protective clothing such as a scrub suit) should be worn when working with rabbits. In some cases protective eye wear is also indicated. Do not eat, drink, or apply cosmetics while working in an animal use area, and always wash you hands after handling rabbits. Remember that unfixed tissues, blood, serum, urine and other materials derived from rabbits may also pose a risk. Bedding, hair and fur may also exacerbate allergies.
3.2.3.1.6 Rodents (mice, rats, hamsters, gerbils, guinea pigs) Rodents raised in "clean" facilities are considered relatively free of zoonotic diseases unless they have been experimentally or accidentally infected with a human pathogen. Note: those working with rodents should also be aware of possible allergic reactions. These allergic reactions are often associated with cage cleaning due to the dust hazards of bedding and surface contact with rodent urine proteins.
Wild rodents pose additional concerns. Wild-caught animals may act as carriers for such viruses as hantavirus and lymphocytic choriomeningitis (LCMV) depending on where they were captured. Additionally, each rodent species may harbor their own range of bacterial diseases, such as tularemia and plague. These animals may also have biting insect vectors which can act as a potential carrier of disease (mouse to human transmission).
The following links describe some of the potential zoonotic illnesses associated with rodents and may be found on-line:
Georgia Regents University Biosafety Guide- June 2008 3-12 • Lymphocytic Choriomeningitis Virus (LCMV) : http://www.phac- aspc.gc.ca/lab-bio/res/psds-ftss/lymp-cho-eng.php • Hantaviruses (Hemorrhagic fever with renal syndrome or Korean hemorrhagic fever): http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/hantavirus-eng.php • Tularemia (Francisella tularensis): http://www.phac-aspc.gc.ca/lab- bio/res/psds-ftss/msds68e-eng.php • Plague (Yersinia pestis): http://www.phac-aspc.gc.ca/lab-bio/res/psds- ftss/msds169e-eng.php
Bites or scratches involving these rodents or injuries from objects contaminated with body fluids from rodents require immediate first aid and medical attention.
3.2.3.1.7 Ferrets Commercially-raised laboratory ferrets are typically free of infections that could pose a risk to humans. Disease development from typical exposure to laboratory ferrets is not recognized as a significant public health risk. Risk of rabies is minor due to pre-arrival and on-site conditioning of ferrets. Zoonotic agents that can be transmitted by ferrets include:
• Salmonella spp. (http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/salmonella- ent-eng.php) • Campylobacter spp. (http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/msds29e- eng.php ) • Cryptosporidia (http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/msds48e- eng.php ) • Giardia spp. (http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/msds71e- eng.php ) • Leptospira (http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/msds95e-eng.php ) • Influenza (http://www.cdc.gov/flu/about/disease.htm )
Ferrets are very susceptible to influenza viruses and have served for years as an animal model in the laboratory. In ferrets flu is characterized by sneezing, fever, lethargy, mucoserous nasal discharge, conjunctivitis and photophobia. The course of the influenza infection usually lasts less than a week. The disease can be severe in young ferrets. Human cases of influenza have occurred from contamination by aerosols from infected ferrets. Similarly ferrets can be infected by humans shedding the virus.
Ferrets should not be allowed to roam freely, and their feces should be discarded in a hygienic manner. They also share parasites with dogs and cats (Toxocara, Dipylidium) as well as dermatophytosis (Microsporum canis, T. mentagrophytes).
Risk of exposure of workers to zoonotic materials may result from the following activities: changing animals from dirty (exposed to animals or their wastes) to clean cages; handling animals for injections, surgery, etc.; handling dirty (exposed to animals or their wastes) animal room supplies; interacting with people who have entered animal areas and have not changed clothes or showered; and eating and touching the face with contaminated hands (exposed to animals or their wastes). There is a moderate risk of injury from ferrets bites or scratches. Bites or scratches or other potential exposures or injuries involving ferrets or objects contaminated with body fluids from ferrets require immediate first aid and medical attention
3.2.3.1.8 Pigs/Swine
Georgia Regents University Biosafety Guide- June 2008 3-13 Swine harbor a range of parasites and diseases that can be transmitted to humans. These include:
• Trichinosis (http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/trichinella- eng.php) • Cysticercosis (http://www.cdc.gov/parasites/cysticercosis/index.html) • Brucellosis (http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/msds23e-eng.php) • Salmonella spp. (http://www.phac-aspc.gc.ca/msds-ftss/msds132e.html and http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/msds135e-eng.php) • Pathogenic E. coli (http://www.phac-aspc.gc.ca/msds-ftss/msds23e.html).
Pigs are also known to host large concentrations of parasitic ascarid worms (http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/msds10e-eng.php) in their digestive tract.
Pigs can be susceptible to pneumonia, usually caused by weather. Pigs have small lungs in relation to body size; for this reason, bronchitis or pneumonia can kill a pig quickly.
Pigs can be aggressive and pig-induced injuries are relatively common in areas where pigs are reared or where they form part of the wild or feral fauna. Their relatively large size and weight also pose a physical hazard for animal care givers and researchers.
Gloves, masks and a laboratory coat (or other dedicated protective clothing such as a scrub suit) should be worn when working with swine. In some cases protective eye wear is also indicated. Do not eat, drink, or apply cosmetics while working in an animal use area, and always wash you hands after working with animals. Remember that unfixed tissues, blood, serum, urine and other materials derived from swine may also pose a risk. Bedding, hay, dust and hair may also exacerbate allergies. Bites or scratches involving swine or injuries from objects contaminated with body fluids from swine require immediate first aid and medical attention
3.2.3.1.9 Large Hooved Mammals (Cows, Horses, Sheep, Goats) The size of hooved mammals poses additional concerns for researchers, due to the physical hazards of weight and strength of the animal. Hooved mammals may resist handling and may require multiple workers to administer medication or other functions.
With regard to pathogens, sheep are known to shed a rickettsia, Coxiella burnetii, that is the causative agent for Q-Fever. Ruminants and pigs may harbor their own range of bacterial pathogens and parasites, such as Salmonella, Campylobacter and Cryptosporidium. Skin conditions, such as Erysipelas and Orf may result after contact with pigs and sheep and goats, respectively. In addition, these animals may carry biting insect vectors who can act as a potential carrier of disease.
The following links describe some of the potential illnesses associated with hooved mammals, farm animals, and may be found on-line:
• Coxiella burnetii/ Q-fever (http://www.phac-aspc.gc.ca/lab-bio/res/psds- ftss/coxiella-burnetii-eng.php) • Salmonellosis (http://www.phac-aspc.gc.ca/msds-ftss/msds132e.html and http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/msds135e-eng.php) • Orf (“Sore Mouth” infection) http://www.cdc.gov/ncidod/dvrd/orf_virus/ • Pasteurella Pneumonia (http://www.phac-aspc.gc.ca/lab-bio/res/psds- ftss/msds117e-eng.php)
Georgia Regents University Biosafety Guide- June 2008 3-14 • Campylobacter jejuni (http://www.phac-aspc.gc.ca/lab-bio/res/psds- ftss/msds29e-eng.php) • Brucella spp./Brucelosis (http://www.phac-aspc.gc.ca/lab-bio/res/psds- ftss/msds23e-eng.php)
Bites or scratches involving these species or injuries from objects contaminated with body fluids from hooved mammals require immediate first aid and medical attention.
Gloves, masks and a laboratory coat (or other dedicated protective clothing such as a scrub suit) should be worn when working with hooved mammals. In some cases protective eye wear is also indicated. Do not eat, drink, or apply cosmetics while working in an animal use area, and always wash you hands after working with hooved mammals. Remember that serum, urine and other materials derived from hooved mammals may also pose a risk. Bedding, hay, dust and hair may also exacerbate allergies.
3.2.4 Amount of Material Present High quantities of infectious material pose a greater risk of spread than small quantities. Large volumes of cultures require additional considerations of the risk of spills, splashes, and laboratory logistics. These considerations should be factored into a comprehensive risk assessment.
Cultures of recombinant materials in volumes exceeding 10 liters have specific biosafety standards which should be followed, as described in Appendix K of NIH Guidelines (http://oba.od.nih.gov/oba/rac/Guidelines/APPENDIX_K.htm)
3.2.5 Experience of Staff GRU personnel receive basic biosafety training during the IBC-required initial training class and annual refresher training modules. However, specialized training in laboratory-specific operations is the responsibility of the Principal Investigator.
If a laboratory is utilizing technology which no one, including the PI, has previous experience in (e.g. recombinant viral vector systems or use of new equipment, such as a French Press), additional training should be sought by the Principal Investigator before embarking on these experiments. Often the Biosafety Office can be of assistance in providing additional training, or identifying appropriate trainers for the laboratory. Contact the Biosafety Office (x1-2663 or [email protected]) for assistance.
3.2.6 Mode of Transmission Infectious diseases may spread between people via different modes. Some, like the diseases associated with the most common bloodborne pathogens (HIV, Hepatitis B & C) require contact with blood via open wounds, needlestick or sexual transmission. This makes these diseases inherently more difficult to spread than those transmitted via an airborne route of transmission, such as influenza. The extent of risk to the public health and environment may factor in to the natural modes of transmission of diseases which may result from inadvertent infection of individuals.
3.2.7 Environmental Stability Because of their resistance to common disinfectant measures and their heat stability, biological agents such as endospore-forming species of bacteria (e.g., strains of Bacillus, Clostridium), and cyst-forming
Georgia Regents University Biosafety Guide- June 2008 3-15 protozoan parasites pose an additional risk than agents which are more easily decontaminated. See Section 7 on for further information on decontamination.
3.2.8 Institutional Public Image Some biological agents may present additional considerations based on the perceived risk that others may assess who are outside of the laboratory or the university. A good example of this might be a laboratory who wishes to work with the avirulent Sterne strain of Bacillus anthracis. Although this strain is commonly used as a vaccination in animals, and may be available through veterinary suppliers, and poses very little risk of infection in healthy adults, if someone became aware that a laboratory works with anthrax, a higher levels of risk may be perceived than actually exists. Anticipation and addressing these sorts of concerns prior to use of these agents may be necessary in order to maintain the institution’s public image.
Georgia Regents University Biosafety Guide- June 2008 3-16 4. RISK MANAGEMENT: BIOSAFETY LEVELS
Management of the risks associated with research involving biological materials is accomplished via a combination of Practices, Safety Equipment and Facilities. Biosafety Levels (BSLs) refer to the level of containment which is required and appropriate to contain the risk as assessed as described in Section 3, Risk Assessment. The CDC, WHO and NIH have established standards for four biosafety containment levels (BSLs) for work with all biohazardous materials in the publication Biosafety in Microbiological and Biomedical Laboratories (BMBL) http://www.cdc.gov/biosafety/publications/bmbl5/index.htm. These standards are reflected (although not always identical) to the four biosafety containment levels (BLs) described for work with recombinant DNA materials in Appendix G of the NIH Guidelines for Research with Recombinant DNA Molecules (“NIH Guidelines”) http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.htm. Both publications provide general descriptions of the combination of microbiological practices, laboratory facilities, and safety equipment as well as their recommended uses in order to contain biological agents which may be infectious to humans, or impact the environment.
Because working with hazardous materials in animals imposes additional risks to the laboratory worker or the environment and laboratory logistics, there are also standards for four biocontainment levels for research with biohazardous materials and animals or ABSLs (Section V of BMBL for all biohazardous material http://www.cdc.gov/biosafety/publications/bmbl5/BMBL5_sect_V.pdf ) or BL-Ns (in Appendix Q of NIH Guidelines for work with recombinant materials http://oba.od.nih.gov/oba/rac/Guidelines/APPENDIX_Q.htm ). Similarly, the American Committee of Medical Entomology of the American Society of Tropical Medicine and Hygiene has also developed arthropod containment levels (ACL1-4) for research with arthropods (http://www.astmh.org/SIC/acme.cfm).
NIH Guidelines also has developed specific biosafety containment levels for large scale cultures (>10 liters) BL-Large Scale in Appendix K (http://oba.od.nih.gov/oba/rac/Guidelines/APPENDIX_K.htm).
These Biosafety containment level standards are intended as a guideline for management of the risks as determined during the risk assessment. However, this is not a prescriptive, formulaic process. Mitigation and management methods should always provide prudent measures to contain the materials and protect exposure of the users to the materials based on the risks assessed. Therefore, since the risks in each laboratory are unique to the laboratory, the Standard Operating Procedures (SOPs) should be specific for the laboratory, agents, operations, and practices within the laboratory. Protective measures to block the routes of transmission, based on the operations involved and locations must be reviewed (See Figure 4, below for suggestions of appropriate mitigation methods to prevent exposure via the common routes of transmission within the laboratory). Keep in mind that on occasion, a risk may be present which may require an additional or alternative protective measure (e.g., development of a special practice or procedure, use of special equipment or restriction to a facility) to protect personnel and the environment from the biological agents proposed for use. These may go beyond those explicitly stated in the BMBL or NIH Guideline standards; however, it is the IBC’s responsibility (as per NIH Guidelines) to ensure the assessed risks involved in a proposed experiment have been reduced to an acceptable level based on the mandated comprehensive risk assessment. Therefore, the IBC may stipulate special containment measures as a condition of receiving approval.
Below is a summary of practices, equipment and facility requirements for agents assigned to basic biosafety levels 1–4 (BL 1–4) (Table 4). Additional information on biosafety levels may be found in Appendix C of this as well as in the BMBL at: http://www.cdc.gov/biosafety/publications/bmbl5/index.htm or NIH Guidelines at: http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.htm.
Only work at biosafety levels 1-2 is currently permitted at GHSUGRU. Most laboratories at the Georgia Health Sciences UniversityGeorgia Regents University work under BSL-2 containment levels. Work at BSL-3 containment levels will require special considerations, training and the standards will be documented in the GHSUGRU BSL-3 Biosafety Guide. No Biosafety level 4 work is allowed at GHSUGRU.
Georgia Health Sciences UniversityGeorgia Regents University Biosafety Guide- January 2012 4-1 Table 4. Summary of Recommended Biosafety Levels for Infectious Agents
Biosafety Agents Practices Safety Equipment Facilities (Secondary Level (Primary Barriers) Barriers) RG1; Not known to Standard Microbiological None required. Door required on lab cause disease in Practices. Handwash sink must be 1 healthy adults. available.
RG2; Associated BSL-1 practice plus: Class I or II BSCs or other BSL-1 plus: Autoclave or with human disease, • Limited access to lab physical containment devices other method for hazards are auto- • Biohazard warning used for all manipulations of decontamination available. inoculation, signs posted agents that cause splashes or ingestion, mucous • Sharps precautions aerosols of infectious membrane • materials; 2 Biosafety manual exposure. defining any needed waste & surface PPE: laboratory coats; decontamination or gloves; face protection as medical surveillance needed. policies
RG3; Indigenous or BSL-2 practice plus: Class II BCSs used for all BSL-2 plus: exotic agents with • Restricted access manipulations of agents (no • Physical separation from potential for aerosol • Decontamination of all open benchwork) access corridors transmission; waste before leaving • Self-closing, double- disease may have facility PPE: back-closing gowns or door access; serious or lethal • jump suits, shoe covers, • 3 Decontamination of lab Air exhausted from consequences. clothing before gloves; respiratory protection facility, not recirculated laundering; as needed • Negative airflow must be • Baseline serum maintained into sampling as laboratory appropriate • Sealed penetrations
RG4; Dangerous or BSL-3 practices plus: All procedures conducted in BSL-3 plus: exotic agents which • Clothing change Class III BSCs or Class I or II • Separate building or pose high risk of before entering; BSCs in combination with isolated zone; life-threatening • Shower on exit full-body, air-supplied, • Dedicated disease, aerosol- • All material positive pressure personnel supply/exhaust, vacuum, transmitted lab decontaminated on exit suit and decon systems; 4 infections; or related from facility • Double-redundancy of agents with facility systems unknown risk of • Other requirements transmission. outlined in BMBL
Adapted from the Office of Health and Safety, Centers for Disease Control and Prevention & Yale University
Georgia Health Sciences UniversityGeorgia Regents University Biosafety Guide- January 2012 4-2 Figure 4. Protection for the Routes of Transmission
Route of Transmission Protection Mucous Membranes Achieve Face Protection:
Through mucous membranes or the eyes . Wearing full-face shield or safety glasses and nose or mouth (splash, splatter). surgical mask . Working in a Biosafety cabinet or behind protective shields . Following good microbiological practices
Ingestion Prevent exposure via ingestion, by:
Mouth pipetting, eating, drinking, smoking in . Not eating, drinking, chewing gum, smoking the lab. in lab . Mechanical pipettors . No storage of food items or utensils in lab . Following good microbiological practices
Inhalation Avoid exposure to aerosols by:
Breathing in respirable sized aerosols . Working in a Biosafety Cabinet (<5µm), centrifuge leaks, spills, & . Using sealed rotors or safety caps for aerosol-generating procedures such as centrifuge buckets pipetting, homogenizing, etc. . Safety containment equipment . HEPA filtered respirator . Following good microbiological practices Percutaneous Avoid percutaneous exposures by:
Through intact or non-intact . Using extreme precautions with sharps skin via needlestick, puncture . Using needleless, retractable or safety sharp with a contaminated sharp systems object, animal scratch, bite, . Disposing sharps immediately in rigid through wounds, abrasions, or leakproof sharps container placed near site of use . Using animal restraints . Using cut/bite resistant gloves . Using sleeve covers, water proof bandages over any open wounds . Using double gloves . Using good work practices Contact (indirect transmission) Prevent indirect exposure by:
Via mucous membranes or non-intact . Decontamination of work surfaces skin from hands that have been in . Maintaining good personal hygiene (avoid contact with a contaminated surface touching your face or other unprotected skin (i.e. benches, phones, computers, with glove or non-gloved hands) equipment handles) or by failure to . Avoid touching non-contaminated areas (e.g. wash hands after working phones) with contaminated hands . Not applying cosmetics within the laboratory . Removing gloves and wash hands before exiting laboratory . Frequent glove changing
Georgia Health Sciences UniversityGeorgia Regents University Biosafety Guide- January 2012 4-3 4.1 LABORATORY PRACTICES
In this section, an attempt has been made to provide information regarding hazards involved with certain laboratory practices and methods for preventing them. Prevention is an important element to biohazard control, and it is recommended that anyone working in a laboratory read this section carefully. It is the responsibility of the Principal Investigator, Clinical Director and/or Instructional Course Designer or their designees to establish their laboratory-specific Standard Operating Procedures to address the risks within each laboratory. These should be documented as part of the Biosafety Protocol (BSP) Application process and these documents should provide a basis to begin laboratory-specific safety training for all laboratory personnel prior to initiation of work.
4.1.1 Human Factors and Attitudes in Relation to Laboratory Accidents
For the purpose of safety, an attitude can be defined as an accumulation of information and experience that predisposes an individual to certain behavior. Human factors and attitudes result in tendencies on the part of the individual to react in a positive or negative fashion to a situation, a person or an objective. Laboratory supervisors and Principal Investigators should understand the importance of attitudes and human factors in their own efforts to control biohazards in their laboratory. Some observations that may be of help to supervisors are listed below:
The lack of accident perception ability is often a significant factor in laboratory accidents.
Inflexibility of work habits, that tend to preclude last minute modification when an accident situation is recognized, plays a part in the causation of some laboratory accidents.
Working at an abnormal rate of speed is a significant causal factor.
Intentional violations of regulations are a frequent cause of accidents. This is termed excessive risk taking.
The performance of routine procedures such as diluting and plating cultures is the most frequent task being performed at the time of laboratory accidents.
Working when one is very tired is more likely to create a higher potential for accidents.
Working at a well-organized and uncrowded laboratory bench will help in the prevention of lab accidents.
Working alone or without supervision in a laboratory with higher hazard agents should be discouraged.
Each employee working with biohazardous agents must be constantly aware of the importance of the proper attitude in preventing accidents in the laboratory.
4.1.2 Practices Associated with Biosafety Levels 1 through 2+ Laboratories
4.1.2.1 Biosafety Level 1 (BSL-1/BL-1) As defined in: Section IV of the CDC BMBL: http://www.cdc.gov/biosafety/publications/bmbl5/BMBL5_sect_IV.pdf Appendix G-II-A for recombinant DNA BL-1: http://oba.od.nih.gov/oba/rac/Guidelines/APPENDIX_G.htm#_Toc7246561
Georgia Health Sciences UniversityGeorgia Regents University Biosafety Guide- January 2012 4-4 Keep laboratory door closed when experiments are in progress. There should be floor-to- ceiling physical separation from “laboratory areas” and “non-laboratory areas”. Therefore, any doors leading into non-laboratory areas, such as offices or hallways must be kept shut when experiments are in progress.
Use procedures that minimize aerosols. For example, pipette gently along the sides of tubes to prevent aerosol production. Open vacuum pressured containers/vials carefully using protective covers to avoid exposures to any aerosols that may arise from the back-pressure that may result.
Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human consumption must not be permitted in laboratory areas. Food must be stored outside the laboratory area in cabinets or refrigerators designated and used for this purpose.
Wear appropriate PPE: . Gloves must be worn to protect hands from exposure to hazardous materials. Glove selection should be based on an appropriate risk assessment. Alternatives to latex gloves should be available. . Laboratory gowns or coats should be worn over street clothes. . Protective eyewear should be worn when conducting procedures that have the potential to create splashes of microorganisms or other hazardous materials. Persons who wear contact lenses in laboratories should also wear eye protection.
Gloves should be changed when contaminated, their integrity have been compromised and/or when otherwise necessary. Periodic glove-changing is recommended. Be aware that some petroleum-based hand moisturizers can impact the integrity of latex gloves and should be avoided.
Gloves should be removed and hands washed after completing experimental procedures and before leaving the laboratory. Hand washing protocols must be rigorously followed. Soap should be used and hands washed for approximately 20 seconds prior to rinsing with warm water.
Dispose of used gloves with other contaminated laboratory waste after removal. Do not wash, save and/or reuse disposable gloves.
Do not mouth pipette. Use mechanical pipetting devices.
Avoid using hypodermic needles, scalpels, blades, glass, or other sharp items whenever possible. Consider alternatives or devices with safety features, for instance retractable blades, or substitute plastic for glass whenever feasible.
Disinfect work surfaces daily and immediately after a spill with an appropriate disinfectant (as documented in the laboratory SOPs). Use of taped-down benchkote paper is discouraged, since this is often not changed daily.
Decontaminate all biological wastes before discarding. Decontaminate other contaminated materials before washing, reusing, or discarding.
For off-site decontamination, package contaminated materials in closed, durable, leakproof containers before leaving the laboratory. See Section 4.1.5 for more information about appropriate transport procedures.
Ensure that all containers which contain biological waste are labeled with the biohazard symbol. This includes intermediary beakers or bins used on bench-tops to contain pipette tips or small microcentrifuge tubes which may have been used with biological materials.
Georgia Health Sciences UniversityGeorgia Regents University Biosafety Guide- January 2012 4-5
Control insect and rodent infestations.
Keep areas neat and clean.
The laboratory supervisor must ensure that laboratory personnel receive appropriate training regarding their duties, the necessary precautions to prevent exposures, and exposure evaluation procedures.
Personnel must receive annual updates or additional training when procedural or policy changes occur. Personal health status may impact an individual’s susceptibility to infection, ability to receive immunizations, or prophylactic interventions. Therefore, all laboratory personnel and particularly women of child-bearing age should be provided with information regarding immune competence and conditions that may predispose them to infection. Individuals having these conditions should be encouraged to self-identify to the institution’s healthcare provider for appropriate counseling and guidance.
4.1.2.2 Biosafety Level 2 (BSL-2/BL-2) As defined in: Section IV of the CDC BMBL: http://www.cdc.gov/biosafety/publications/bmbl5/BMBL5_sect_IV.pdf Appendix G-II-B for recombinant DNA BL-2: http://oba.od.nih.gov/oba/rac/Guidelines/APPENDIX_G.htm#_Toc7246566
Biosafety Level 2 containment practices are the same as those listed for Biosafety Level 1, with the following additions:
Keep laboratory door closed and facilities must be locked after work hours. There should be floor-to-ceiling physical separation from “laboratory areas” and “non-laboratory areas”. Therefore, any doors leading into non-laboratory areas, such as offices or hallways must be kept shut.
Only persons who have been informed of the research and its risks should be permitted to enter BSL-2 areas.
Keep plants and animals not used in BSL-2 experiment out of the laboratory.
Wear appropriate PPE (as documented in the laboratory SOPs): . Open wounds should be covered with occlusive bandages. . Gloves must be worn to protect hands from exposure to hazardous materials. Glove selection should be based on an appropriate risk assessment. Alternatives to latex gloves should be available. Double-gloving may be necessary for some procedures. . Laboratory gowns or coats must be worn within the laboratory and not removed from the laboratory. These should either be disposable or laundered by the institution— not taken home. . Protective eyewear should be worn when conducting procedures that have the potential to create splashes of microorganisms or other hazardous materials. Persons who wear contact lenses in laboratories should also wear eye protection. . Eye, face and respiratory protection should be used in rooms containing infected animals as determined by the risk assessment.
Use biological safety cabinets or similar containment equipment to contain aerosol-producing equipment.
Maintain a biological spill kit within the laboratory.
Georgia Health Sciences UniversityGeorgia Regents University Biosafety Guide- January 2012 4-6
Vacuum aspirator lines need to be properly maintained to prevent overflow. It is recommended that vacuum traps never be allowed to collect liquids past half-full to enable full decontamination prior to disposal. Vacuum traps outside of biosafety cabinets should be placed in secondary containment sufficient in size to contain the contents of the trap, should the trap implode under vacuum pressure. Vacuum lines should be protected with High Efficiency Particulate Air (HEPA) filters, or their equivalent. Filters must be replaced as needed.
Laboratory equipment should be routinely decontaminated by staff properly trained and equipped to work with the material, after spills, splashes, or other potential contamination. Equipment must be fully decontaminated before repair, maintenance, or removal from the laboratory.
Spills involving infectious materials must be contained, decontaminated, and cleaned up by staff properly trained and equipped to work with infectious material.
Report spills, accidents, potential exposures, near misses and disease symptoms related to laboratory acquired infection to the PI and the Biosafety Office. Additional reporting requirements may be required to comply with Human Resources and/or Campus Safety Committee requirements (See Section 6 for further information).
Laboratory personnel must be provided medical surveillance and offered appropriate immunizations for agents handled or potentially present in the laboratory. Some agents may require the collection and storage of serum samples from at-risk personnel prior to initiation of work within the laboratory. These measures must be documented in the laboratory Biosafety Manual and SOPs.
A laboratory-specific biosafety manual must be prepared and adopted as policy. The biosafety manual must be available and accessible to all personnel within the laboratory.
The laboratory supervisor must ensure that laboratory personnel demonstrate proficiency in standard and special microbiological practices before working with BSL-2 agents.
4.1.2.3 Biosafety Level 2+ (BSL-2+) Biosafety level 2+ (BSL-2+) is the designation utilized for those biohazard experiments that require practices that are more stringent than standard BSL-2 procedures. It is not strictly defined by the CDC or NIH, however, generally, BSL-3 practices are mandated in a space designed for BSL-2 work.
The BSL-2+ laboratory should be self-contained with all equipment required for the experiment located within the laboratory. A biohazard door sign listing the agent in use, emergency contact, and entry requirements is posted on the door while BSL-2+ work is in progress and the laboratory director should control access to the laboratory and restricts access to persons whose presence is required for program or support purposes. The laboratory doors should be kept closed when experiments are in progress. Access is especially restricted to those involved in the experiment. When work is completed and equipment has been decontaminated, the sign can be removed and the laboratory is returned to standard BSL-2 use.
Special precautions should be taken to limit the handling of any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels. The use of additional personal protective equipment should also be assessed based on the risks of the agents and operations within the laboratory.
Georgia Health Sciences UniversityGeorgia Regents University Biosafety Guide- January 2012 4-7 Laboratory and support personnel in BSL-2+ laboratories should receive appropriate training on the potential hazards associated with the work involved and the necessary precautions to prevent exposures. Personnel must demonstrate proficiency in safe microbiological practices and techniques. Special health counseling measures, and possibly baseline serum samples should be collected as appropriate and stored for all laboratory and other at-risk personnel.
Under BSL-2+ containment, all manipulations of biological material are conducted in a class II biological safety cabinet and secondary containment is utilized for centrifugation and other potential aerosol generating procedures. All potentially contaminated waste materials from the BSL-2+ laboratory should be decontaminated prior to disposal in the Stericycle Biohazard Waste boxes.
Additional requirements for work at BSL-2+ are listed in Appendix D. Please consult the Biosafety Office prior to initiating any work at BSL-2+.
4.1.3 Practices Associated with Activities Involving Animals Laboratory animal facilities are a special type of laboratory. Although many of the practices followed under ABSL (or BL-N) containment levels are often comparable to those used in vitro, the animal room can present unique problems since the activities of the animals themselves can present unique hazards not found in standard microbiological laboratories. Animals may generate aerosols, they may bite and scratch, and they may be infected with a zoonotic agent. Necropsies of infected animals also present a risk of exposure to personnel. The co-application of Biosafety Levels and the Animal Biosafety Levels are determined by a protocol driven risk assessment in order to appropriate contain the risks of the agents and animals used in an experiment.
The Institutional Biosafety Committee works in close conjunction with the Institutional Animal Care and Use Committee (IACUC) and the veterinarians in the Division of Laboratory Animals Services (DLAS) in order to ensure that the safety of human researchers and caretakers, the health of the animal colonies and humane treatment of research animals are ensured. These considerations not only impact the safety, but the integrity and ethics of the science occurring in the facilities. Considerations are given to the proper handling of animals which may have been experimentally infected as part of a research protocol, those which may naturally harbor zoonotic infectious agents (e.g., non-human primates), or those which may inadvertently have been infected with animal pathogens which may pose hazards for colony maintenance and integrity.
Animal Biosafety Levels as defined in the CDC BMBL (Section V) and the NIH Guidelines (Appendix Q) presuppose that laboratory animal facilities, operational practices, and quality of animal care meet applicable standards and regulations as described in: Institute of Laboratory Animal Research (ILAR), Commission on Life Sciences, National Research Council’s Guide for the Care and Use of Laboratory Animals: http://www.nap.edu/catalog.php?record_id=5140 U.S. Federal Laboratory Animal Welfare Regulations (9 C.F.R.) http://www.access.gpo.gov/nara/cfr/waisidx_07/9cfrv1_07.html
Training videos are available through the American Biosafety Association (ABSA) web site to provide guidance to animal handlers at various Animal Biosafety Levels http://www.absa.org/restraining.html .
In addition to the animal biosafety levels described in the CDC BMBL and NIH Guidelines, the USDA has developed facility parameters and work practices for handling agents of agriculture significance. These are described in Appendix D in the CDC BMBL http://www.cdc.gov/biosafety/publications/bmbl5/BMBL5_appendixD.pdf. USDA requirements are unique to agriculture because of the necessity to protect the environment from pathogens of economic or environmental impact. The importation, possession or use of the following agents are either prohibited or restricted by law or by USDA regulations or administrative policies. These agents of agricultural significance include:
• African horse sickness virus • Lumpy skin disease virus • Louping ill virus • Akabane virus • African swine fever virus
Georgia Health Sciences UniversityGeorgia Regents University Biosafety Guide- January 2012 4-8 • Malignant catarrhal fever virus (exotic • Classical swine fever virus strains or alcelaphine herpesvirus type 1) • Swine vesicular disease virus • Avian influenza virus (highly pathogenic) • Coccidioides immitis • Menangle virus • Teschen disease virus • Bacillus anthracis • Cochliomyia hominivorax (Screwworm) • Mycobacterium bovis • Theileria annulata • Besnoitia besnoiti • Coxiella burnetti (Q fever) • Mycoplasma agalactiae • Theileria lawrencei • Bluetongue virus (exotic) • Ephemeral fever virus • Mycoplasma mycoides subsp. mycoides, • Theileria bovis (small colony type) • Ehrlichia (Cowdria) ruminantium • Borna disease virus (heartwater) • Mycoplasma capricolum • Theileria hirci • Bovine infectious petechial fever agent • Eastern equine encephalitis virus • Nairobi sheep disease virus (Ganjam • Trypanosoma brucei virus) • Foot and mouth disease virus • Bovine spongiform encephalopathy prion • Trypanosoma congolense • Newcastle disease virus (velogenic • Francisella tularensis strains) • Trypanosoma equiperdum (dourine) • Brucella abortus • Goat pox • Nipah virus • Trypanosoma evansi • Brucella melitensis • Hemorrhagic disease of rabbits virus • Peste des petits ruminants virus (plague of • Trypanosoma vivax small ruminants) • Hendra virus • Brucella suis • Venezuelan equine encephalomyelitis • Rift Valley fever virus virus • Burkholderia mallei/Pseudomonas mallei • Histoplasma (Zymonema) farciminosum (Glanders) • Vesicular exanthema virus • Rinderpest virus • Infectious salmon anemia virus • Burkholderia pseudomallei • Vesicular stomatitis virus (exotic) • Sheep pox virus Japanese encephalitis virus • Camelpox virus • Wesselsbron disease virus • Spring Viremia of Carp virus
4.1.3.1 Animal Biosafety Level 1 (ABSL-1, BL-1N) As defined in: Section V of the CDC BMBL: http://www.cdc.gov/biosafety/publications/bmbl5/BMBL5_sect_V.pdf Appendix Q-II-A for recombinant DNA BL-1N: http://oba.od.nih.gov/oba/rac/Guidelines/APPENDIX_Q.htm
The animal facility director establishes and enforces policies, procedures, and protocols for institutional policies and emergency situations.
Each institute must ensure that research staff and animal handler safety and health concerns are addressed as part of the animal protocol review.
Prior to beginning a study animal protocols must also be reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) and the Institutional Biosafety Committee.
Personnel must have specific training in animal facility procedures and must be supervised by an individual with adequate knowledge of potential hazards and
Georgia Health Sciences UniversityGeorgia Regents University Biosafety Guide- January 2012 4-9 experimental animal procedures.
A safety manual specific to the animal facility is prepared or adopted in consultation with the animal facility director and appropriate safety professionals.
The safety manual must be available and accessible. Personnel are advised of potential hazards and are required to read and follow instructions on practices and procedures.
Appropriate medical surveillance program is in place, as determined by risk assessment. The need for an animal allergy prevention program should be considered.
Facility supervisors should ensure that medical staff is informed of potential occupational hazards within the animal facility, to include those associated with research, animal husbandry duties, animal care and manipulations.
Personal health status may impact an individual’s susceptibility to infection, ability to receive immunizations or prophylactic interventions. Therefore, all personnel and particularly women of child-bearing age should be provided information regarding immune competence and conditions that may predispose them to infection. Individuals having these conditions should be encouraged to self-identify to the institution’s healthcare provider for appropriate counseling and guidance.
Personnel using respirators must be enrolled in an appropriately constituted respiratory protection program.
A sign incorporating safety information must be posted at the entrance to the areas where infectious materials and/or animals are housed or are manipulated.
The sign must include the animal biosafety level, general occupational health requirements, personal protective equipment requirements, the supervisor’s name (or other responsible personnel), telephone number, and required procedures for entering and exiting the animal areas. Identification of specific infectious agents is recommended when more than one agent is being used within an animal room.
Advance consideration should be given to emergency and disaster recovery plans, as a contingency for man-made or natural disasters to prevent exposure of work personnel, accidental release/escape of animals or environmental contamination.
Protective laboratory coats, gowns, or uniforms are recommended to prevent contamination of personal clothing. These should not be worn outside of the facility, and non-disposable PPE must be decontaminated and cleaned by the institution—not taken home to launder.
Open wounds should be covered with occlusive bandages.
Gloves are worn to prevent skin contact with contaminated, infectious and hazardous materials, and when handling animals.
Gloves and personal protective equipment should be removed in a manner that minimizes transfer of infectious materials outside of the areas where infectious materials and/or animals are housed or are manipulated.
Persons must wash their hands after removing gloves, and before leaving the areas where infectious materials and/or animals are housed or are manipulated.
Eye and face and respiratory protection should be used in rooms containing infected
Georgia Health Sciences UniversityGeorgia Regents University Biosafety Guide- January 2012 4-10 animals, as dictated by the risk assessment.
When applicable, laboratory supervisors should adopt improved engineering and work practice controls that reduce the risk of sharps injuries. Precautions, including those listed below, must always be taken with sharp items. These include: o Needles and syringes or other sharp instruments are limited to use in the animal facility when there is no alternative for such procedures as parenteral injection, blood collection, or aspiration of fluids from laboratory animals and diaphragm bottles. o Disposable needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal. o Used disposable needles must be carefully placed in puncture-resistant containers used for sharps disposal. Sharps containers should be located as close to the work site as possible. o Non-disposable sharps must be placed in a hard-walled container for transport to a processing area for decontamination, preferably by autoclaving. o Broken glassware must not be handled directly. Instead, it must be removed using a brush and dustpan, tongs, or forceps. Plasticware should be substituted for glassware whenever possible.
Equipment containing sharp edges and corners should be avoided.
4.1.3.2 Animal Biosafety Level 2 (ABSL-2, BL-2N) As defined in: Section V of the CDC BMBL: http://www.cdc.gov/biosafety/publications/bmbl5/BMBL5_sect_V.pdf Appendix Q-II-B for recombinant DNA BL-2N: hthttp://oba.od.nih.gov/oba/rac/Guidelines/APPENDIX_Q.htm
In addition to the practices required by ABSL-1 containment, ABSL-2 requires that:
Access to the animal facility is restricted.
Personnel must have specific training in animal facility procedures, the handling of infected animals and the manipulation of pathogenic agents.
Personnel entering the facility must be supervised by individuals with adequate knowledge of potential hazards, microbiological agents, animal manipulations and husbandry procedures.
Procedures involving the manipulation of infectious materials, or where aerosols or splashes may be created, including animal necropsies, and injections of animals, should be conducted in BSCs or by use of other physical containment equipment.
Procedures involving a high potential for generating aerosols should be conducted within a biosafety cabinet or other physical containment device.
When a procedure cannot be performed within a biosafety cabinet, a combination of personal protective equipment and other containment devices must be used.
Consideration should be given to the use of restraint devices and practices that reduce the risk of exposure during animal manipulations (e.g., physical restraint devices, chemical restraint medications, etc).
Materials to be decontaminated outside of the immediate areas where infectious materials
Georgia Health Sciences UniversityGeorgia Regents University Biosafety Guide- January 2012 4-11 and/or animals are housed or are manipulated must be placed in a durable, leak proof, covered container and secured for transport. The outer surface of the container is disinfected prior to moving materials. The transport container must contain a universal biohazard label.
Equipment, cages, and racks should be handled in a manner that minimizes contamination of other areas. These should be periodically inspected to ensure that sharp edges are not present which may present a laboratory hazard.
Equipment must be decontaminated before repair, maintenance, or removal from the areas where infectious materials and/or animals are housed or are manipulated.
Spills involving infectious materials must be contained, decontaminated, and cleaned up by staff properly trained and equipped to work with infectious material.
Incidents that may result in exposure to infectious materials must be immediately evaluated and treated according to procedures described in the safety manual. All such incidents must be reported to the animal facility supervisor and the Biosafety Officer. Medical evaluation, surveillance, and treatment should be provided as appropriate and records maintained.
Persons having contact with the NHP should assess risk of mucous membrane exposure and wear appropriate protective equipment (e.g., masks, goggles, faceshields, etc.) as needed.
Respiratory protection is worn based upon risk assessment.
Sink traps are filled with water, and/or appropriate liquid to prevent the migration of vermin and gases.
Georgia Health Sciences UniversityGeorgia Regents University Biosafety Guide- January 2012 4-12 4.1.4 Tissue, Cell and Microbiological Culture Practices
4.1.4.1 Tissue/Cell Culture Practices Adhering to appropriate tissue culture techniques is intended to contain the risks of the biological agents used in an experiment, but also intends to improve the integrity of the science. The following measures are general guidelines for improved safety while performing cell culture techniques within a biosafety cabinet (BSC, also commonly called a “tissue culture hood”).
Wear long sleeved gowns with knit cuffs and long gloves when working in the biosafety cabinet. Maintain a clean lab coat reserved solely for cell culture work. Cover any open wounds with occlusive bandages prior to donning PPE.
Avoid causing unnecessary air disturbances in and around the BSC. Avoid moving ones hands in and out of the biosafety cabinet or sweeping side-to-side motions. Avoid talking during culture manipulations as aerosols may be drawn into the work area. BSCs should be placed in low traffic areas away from doors to prevent disturbance of the air curtain protecting the containment and sterility in the BSC.
Place all reagents and supplies inside the Biosafety cabinet before the experiment to reduce the disturbance of air within the BSC during use.
Work from a clean to a dirty side. Place all unused (clean) materials and reagents on one side of the work surface, and waste containers on the other (dirty) side.
Do not over-crowd your biosafety cabinets, which may interfere with proper laminar CLEAN DIRTY/WASTE air flow required for BSC function.
Do not block the grates in the front or the back of the BSC which would prevent proper laminar air flow required to maintain containment and sterility within the BSC. Work approximately 4 inches back from the front of the BSC work area.
Allow the BSC to run for fifteen (15) minutes prior to use and fifteen (15) minutes after use to purge the air within the BSC of any contaminants.
Clean all work surfaces, interior vertical surfaces and face shields before and after use with an appropriate disinfectant.
Do not use open flames inside the BSC. Heat currents generated from the flame may interfere with the laminar airflow of the BSC. In addition, the heat can damage the HEPA filters or the adhesives in the filter units or other components of the BSC. This can impact the warranty or UL ratings of the BSC. Lastly, flames pose a high flammability risk when used in the vicinity of alcohols, which are often used in tissue culture situations. Alternative devices or measures should be utilized, such as Glass bead sterilizer glass bead sterilizers, microincinerators, use of disposable instruments Microincinerator or having multiple packets of sterile instruments on-hand. If no appropriate substitute can be found for a flame, only the use on flame-on-demand devices, such as properly-used touch-plate microburners will be permitted. Gas and air balance
Touch-plate microburner
Georgia Health Sciences UniversityGeorgia Regents University Biosafety Guide- January 2012 4-13 should be set in the microburner such that only a small pilot light is visible most of the time; flame is only produced after depressing the plunger when needed.
Do not use volatile chemicals in unducted Class IIA2 Biosafety Cabinets. After HEPA filtration (which will not filter out chemicals), these BSC typically recirculate 70% of the air and exhaust 30% air from the BSC into the room. If volatile chemicals are used within the BSC, recirculation will serve only to concentrate the chemicals within the BSC, and pose a spark hazard; in addition, the chemicals will be inappropriately exhausted into the room posing health risks. Minute amounts of volatile chemicals can only be used in BSCs which are equipped with ducted exhaust ventilation—either via a thimble/canopy connection (a “ducted” Class IIA2 BSC); while somewhat larger amounts of volatile chemicals can be used in hard-ducted Class IIB2 BSCs. Please be aware, most BSCs on the GHSUGRU campus are unducted Class IIA2 types and therefore inappropriate for the use of volatile chemicals; please contact the Biosafety Office (x1- 2663 or [email protected]:[email protected] ) before using volatile chemicals in the BSC if you are not sure of what type of BSC you possess.
Radiological materials should not be used in unducted Class IIA2 Biosafety Cabinets unless protected using a charcoal filter box around the experiment. Contact the Radiation Safety Office for further information about charcoal protection devices for use in unducted BSCs. Radiological materials should not be used in these BSCs for similar reasons that volatile chemicals should not be used (see above); in addition, radiological material which adheres to dust particles may also contaminate the HEPA filters and lead to inadvertent exposures of BSC maintenance and certification personnel. Minute amounts of radiological materials can only be used in BSCs which are equipped with ducted exhaust ventilation—either via a thimble/canopy connection (a “ducted” Class IIA2 BSC); while somewhat larger amounts can be used in hard-ducted Class IIB2 BSCs. Please be aware, most BSCs on the GHSUGRU campus are unducted Class IIA2 types and therefore inappropriate for the use of radiological materials without additional protective measures.
Liquid wastes should be collected in order to decontaminate using an appropriate method which should be described in the laboratory SOPs. Typically, liquid wastes are collected in vacuum aspirators into which some disinfectant has been placed. To ensure appropriate decontamination, subsequent disinfection measures should be followed prior to disposal.
. Do not allow vacuum traps to become overfull (recommended not greater than half-full). This not only prevent liquids from being inadvertently drawn into the vacuum line, but will allow for full decontamination of the liquid wastes prior to disposal
. HEPA filters or equivalents should be placed in the vacuum lines of any laboratory requiring BSL-2 containment or higher.
In-line HEPA vacuum filters
. Do not leave pipettes in the ends of the vacuum aspirator hoses. After use, remove them from the hose and place in disinfection tray/container prior to disposal. Leaving pipettes within the hoses only presents additional exposure or
Georgia Health Sciences UniversityGeorgia Regents University Biosafety Guide- January 2012 4-14 contamination risks.
. Rinse vacuum tubing with disinfectant after use. This will prevent backflow of contaminated liquids within the vacuum line and subsequent contamination.
. If the vacuum traps are outside of the Biosafety Cabinet, place in sufficient secondary containment to hold the volume of liquid which may be spilled if implosion of the vacuum flask should accidentally occur.
Glassware/plasticware and other contaminated items should be disinfected or autoclaved before washing, reuse or disposal. Glassware should be thoroughly cleaned and rinsed, by washing repeatedly with tap water and distilled water.
Place dirty pipettes, tips and tubes in a decontamination tray or container in which disinfectant has been placed on the “dirty side” of the BSC rather than move hands in and out of the BSC
to dispose of these. This will avoid disrupting the protective air Pipette/decontamination tray curtain when hands are removed from the BSC. Discard empty tubes immediately into the disinfection tray or similar containment device; after the experiment, drain the disinfectant from the plastic wastes then dispose of the wastes in the biohazard waste containers.
Keep open tubes parallel to the airflow.
After transferring inoculums, always recap vials.
Do not place tubes on work surface.
Pipette gently along the sides of tubes to prevent production of aerosols
Photo courtesy of Yale Biosafety Spray and aerosol produced by expelling liquids from a pipette Work with one specimen at a time; recap before going to the next.
Autoclave verification should be performed routinely.
If a problem with contamination develops please contact the Biosafety Office (x1-2663 or [email protected]) for further assistance.
4.1.4.2 Microbiological Practices
In the absence of definite accidents or obvious spillage, it is not certain that the opening of plates, tubes and bottles of other microorganisms has caused laboratory infection. However, it is probable that among the highly infective agents some infections have occurred by this means. Particular care is required when opening plates, tubes, or bottles containing fungi, for this operation may release a large number of spores. Such cultures should be manipulated in a biological safety cabinet.
Georgia Health Sciences UniversityGeorgia Regents University Biosafety Guide- January 2012 4-15 To assure a homogenous suspension that will provide a representative sample, liquid cultures are agitated before a sample is taken. Vigorous shaking will create a heavy aerosol. A swirling action will generate homogenous suspension with a minimum of aerosol. When a liquid culture is re- suspended, a few minutes should elapse prior to opening the container to reduce the aerosol.
The insertion of a sterile, hot wire loop or needle into a liquid or slant culture can cause splattering and release of an aerosol. To minimize the aerosol production, the loop should be allowed to cool in the air or be cooled by touching it to the inside of the container or to the agar surface where no growth is evident prior to contact with the culture of colony.
Placing an inoculating loop, wire or needle directly into a flame after use can also cause splattering and release of an aerosol. Following use of inoculating loop or needle, it is preferable to sterilize the instrument in an electric or gas incinerator specifically designed for this purpose rather than heating in an open flame. These small incinerators have a shield to contain any material that may spatter from the loop or needle. Disposable inoculating loops are also commercially available. Rather than decontaminating them immediately after use with heat, they are discarded first into a disinfectant solution.
The practice of streaking an inoculum on rough agar results in aerosol production created by the vibrating loop or needle. This generally does not occur if the operation is performed on smooth agar. It is good safety practice to discard all rough agar poured plates that are intended for streaking purposes with a wire loop.
Water arising from syneresis in Petri dish cultures usually contains viable microorganisms and forms a film between the rim and lid of the inverted plate. Aerosols are dispersed when opening the plate breaks this film. Vented plastic Petri dishes, where the lid touches the rim at only three points, are less likely to pose this hazard. The risk may also be minimized by using properly dried plates, but even these (when incubated anaerobically) are likely to be wet after removal from an anaerobic jar. Filter papers fitted into the lids reduce, but do not prevent dispersal. If plates are obviously wet, they should be opened in the biological safety cabinet.
Less obvious is the release of aerosols when screw-capped bottles or plugged tubes are opened. This happens when a film of contaminated liquid, which may collect between the rim and the liner, is broken during removal of the closure. The practice of removing cotton plugs or other closures from flasks, bottles, centrifuge tubes, etc., immediately following shaking or centrifugation can generate aerosols and cause environmental contamination. The technique of shaking tissue cultures with glass beads to release viruses can create a virus-laden aerosol. Removal of wet closures, which can occur if the flask or centrifuge tube is not held in an upright position, is also hazardous. In addition, when using the centrifuge, there may be a small amount of foaming and the closures may become slightly moistened. Because of these possibilities, it is good safety practice to open all liquid cultures of infectious or hazardous material in a biological safety cabinet wearing gloves and a long sleeved laboratory garment.
Dried, infectious culture material may also collect at or near the rim or neck of culture tubes/flasks and may be dispersed into the air when disturbed. Containers of dry powdered hazardous materials should be opened in a biological safety cabinet.
4.1.4.3 Glass Ampules When a sealed ampule containing a lyophilized or liquid culture is opened, an aerosol may be created. Aerosol creation should be prevented or minimized; opening of ampules should be done in biological safety cabinets. When recovering the contents of an ampule, care should be taken not to cut the gloves or hands or disperse broken glass into eyes, face, or laboratory environment. In addition, the biological product itself should not be contaminated with foreign organisms or with disinfectants. To accomplish this, work in a biological safety cabinet and wear gloves. Nick the ampule with a file near the neck. Wrap the ampule in disinfectant wetted cotton. Snap the ampule
Georgia Health Sciences UniversityGeorgia Regents University Biosafety Guide- January 2012 4-16 open at the nick, being sure to hold the ampule upright. Alternatively, at the file mark on the neck of the ampule, apply a hot wire or rod to develop a crack. Then wrap the ampule in disinfected wetted cotton, and snap it open. Discard cotton and ampule tip into disinfectant. The contents of the ampule are reconstituted by slowly adding fluid to avoid aerosolizing the dried material. Mix contents without bubbling, and withdraw the contents into a fresh container. Some researchers may desire to use commercially available ampules prescored for easy opening. However, there is the possibility to consider that this may weaken the ampule and cause it to break during handling and storage. Ampules of liquid cultures are opened in a similar way.
Ensure that all hazardous fluid cultures or viable powdered infectious materials in glass vessels are transported, incubated, and stored in easily handled, nonbreakable leakproof secondary containers that are large enough to contain all the fluid or powder in case of leakage or breakage of the glass vessel. The secondary container must be labeled with a biohazard label bearing the name of the infectious material.
4.1.4.4 Cryovials and Cryopreservation in Liquid Nitrogen There is a foreseeable risk that when samples that have been stored in liquid nitrogen are removed from the liquid phase and warmed they might explode. Clearly there is potential for significant physical injury to anyone nearby and in many cases there might also be an associated infection risk. Therefore, all personnel working with liquid nitrogen storage vessels or near them should be made aware of the explosion and infection risks and appropriate control measures should be taken to protect personnel from these risks.
When plastics are placed in liquid nitrogen they become brittle and shrink due to the effects of the extremely low temperatures. This applies even to those plastics used for cryogenic vials sold specifically for storage of samples in liquid nitrogen. While the use of tubes with internal threads and gaskets will lower the risk of leakage, it is virtually impossible to achieve a leakproof vial once the specimen is placed in liquid phase liquid nitrogen. Inevitably, some seepage in to the vial will occur. When the vial is removed from liquid nitrogen for thawing, the quick warming and expansion of air within the vials can cause the tube to explode.
Therefore, cryopreserved materials should be stored in the vapor phase of liquid nitrogen in a cryotank. If it is placed in the liquid phase, commercially available plastic tubing may be sealed around the cryovial like a sausage skin. Special caution should be taken when removing cryovials from liquid nitrogen storage, and should include cryoprotective gloves to protect against liquid nitrogen burning, and full eye and face protection. Cryovials should be placed into a secondary container with a closed lid and into a Biosafety Cabinet as soon as possible to further protect the handler should the vial explode.
4.1.4.5 Vacuum Packed Tubes/Vials When a vacuum-sealed tube or vial (e.g., Vacutainer blood tubes or septum bottles) are opened, the lyophilized material or liquid culture may become aerosolized due to the sudden influx of air within the tube/vial. Protective measures to protect personnel from exposures to these aerosols should be incorporated into the laboratory Standard Operating Procedures. Ideally, opening of vacuum-sealed tubes/vials should be done in biological safety cabinets. Covering the lid or plug with an disinfectant-soaked gauze pad will also provide a barrier against exposures to the aerosolized material that may be generated upon opening. Commercial “cap” devices are also available to limit the spread of aerosols generated when opening tubes or vials.
4.1.4.6 Embryonated Eggs Harvesting cultures from embryonated eggs is a hazardous procedure and leads to heavy surface contamination of the egg trays, shells, the environment, and the hands of the operator. It is
Georgia Health Sciences UniversityGeorgia Regents University Biosafety Guide- January 2012 4-17 essential that operations of this type be conducted in a biological safety cabinet. A suitable disinfectant should be at hand and used frequently.
4.1.5 Transport of Biological Material on Campus (between Labs or Buildings) To prevent exposure to non-authorized personnel (with unknown health status) and potential environmental release and contamination, biological materials which must be transported between laboratory areas by authorized personnel only through common hallways or walkways must be properly packaged, contained and labeled. Here are the guidelines which should be followed:
Biological materials must be contained inside two leakproof containers prior to removal from the laboratory. The IBC standard for intramural transport is: • Inside a sealed, leakproof primary container. A container is considered “leakproof” if it can be filled with water, turned upside down without holding the lid in place and leakage does not occur.
• The primary container must be placed inside a sealed, leakproof, durable secondary container. The IBC standard for “durable” is puncture-resistant. Ziplock bags are not considered “durable” by this standard. Sealed 15 ml or 50 ml plastic centrifuge tubes or “burpable” household plastic storage containers (e.g., Tupperware®, Rubbermaid® or similar brands) are considered “durable.
• Absorbent material (e.g., paper towels) must be placed between the primary and secondary containers suitable for the volume transported.
• A biohazard sticker and label must be affixed on the outside of the secondary container with agent name, lab address and emergency contact phone number.
Utilize plastic containers whenever feasible. Avoid glass. If glass primary containers must be used, place containers within a sealed rigid plastic container with absorbent and padding to cushion vials during transport.
The outside of the primary container should be decontaminated before placing into the secondary container. The outside of the secondary container should be decontaminated before leaving the laboratory.
Biological materials may not be transported through non-GHSUGRU areas (e.g., the GHSUGRU HS hospital or Augusta Veterans Administration Hospital) without prior permission from these entities. Live animals must not be transported through non-GHSUGRU areas. Consider that the risk of transporting potentially infectious materials through hospitals is higher because of the poorer health status of individuals within the hospital.
Transport of biological materials in a private vehicle is discouraged. Keep in mind that transport of hazardous materials (which may include biological material, dry ice and liquid nitrogen) is against the terms of most private automobile insurance policies. Check with your insurer.
Any extramural transport of biological materials must comply with the IATA/DOT shipping standards as described in Section 10 of the GHSUGRU Biosafety Guide.
4.1.6 Housekeeping Well-defined housekeeping procedures and schedules are essential in reducing the risks associated with working with pathogenic agents and in protecting the integrity of the research program. This is particularly true in the laboratory operating under less than total containment concepts and in all areas used for the
Georgia Health Sciences UniversityGeorgia Regents University Biosafety Guide- January 2012 4-18 housing of animals, whether or not they have been intentionally infected. A well conceived and well executed housekeeping program limits physical clutter that could distract the attention and interfere with the activities of laboratory personnel at a critical moment in a potentially hazardous procedure, provides a work area that will not in itself be a source of physical injury or contamination, and provides an area that promotes the efficient use of decontaminates in the event of inadvertent release of an etiologic agent. Less immediately evident are the benefits of establishing among personnel of widely varying levels of education some concepts of the nature and sources of contamination.
4.1.6.1 Objectives of Housekeeping The objectives of housekeeping in the laboratory are to:
Provide an orderly work area conducive to the accomplishment of the research program.
Provide work areas devoid of physical hazards.
Provide a clean work area with background contamination ideally held to a zero level but more realistically to a level such that extraordinary measures in sterile techniques are not required to maintain integrity of the biological systems under study.
Prevent the accumulation of materials from current and past experiments that constitute a hazard to laboratory personnel.
Prevent the creation of aerosols of hazardous materials as a result of the housekeeping procedures used.
Procedures developed in the area of housekeeping should be based on the highest level of risk to which the personnel and integrity of the experiments will be subject. Such an approach avoids the confusion of multiple practices and retraining of personnel. The primary function, then, of routine housekeeping procedures is to prevent the accumulation of organic debris that may:
Harbor microorganisms potentially a threat to the integrity of the biological systems under investigation.
Enhance the survival of microorganisms inadvertently released in experimental procedures.
Retard penetration of decontaminants.
Be transferable from one area to another on clothing and shoes.
With sufficient buildup, become a biohazard as a consequence of secondary aerosolization by personnel and air movement
Cause allergenic sensitization of personnel (e.g., to animal dander).
Housekeeping in animal care units has the same primary function as that stated for the laboratory and should, in addition, be as meticulously carried out in quarantine and conditioning areas as in areas used to house experimentally infected animals. No other area in the laboratory has the constant potential for creation of significant quantities of contaminated organic debris than do animal care facilities.
4.1.6.2 Scope of Housekeeping In all laboratories, efforts to achieve total decontamination and to conduct a major cleanup of the biological materials are normally undertaken at relatively long time intervals. Routine
Georgia Health Sciences UniversityGeorgia Regents University Biosafety Guide- January 2012 4-19 housekeeping must be relied on to provide a work area free of significant sources of background contamination. The provision of such a work area is not simply a matter of indicating in a general way what has to be done, who will do it, and how often. The supervisor must view each task critically in terms of the potential biohazard involved, decide on a detailed procedure for its accomplishment, and provide instructions to laboratory personnel in a manner that minimizes the opportunity for misunderstanding.
The following list outlines a portion of the terms requiring critical review by the laboratory supervisor. It is not intended to be complete but is presented as an example of the detailed manner in which housekeeping in the laboratory complex must be viewed.
• Aisles • Hallways • Eyewashes • Supply Storage • Lab Entry and Exit Ways • Deep Freezer Chests • Bench Tops • Incubators • Floors • Waste Accumulations • Lab Equipment Cleanup • Dry Ice Chests • Biological Safety Cabinets • Insect and Rodent Control • Glassware • Work Surfaces • Refrigerators • Instruments • Cold Rooms
4.1.6.3 Assignment of Responsibilities Housekeeping in the laboratory is one avenue that leads to safely accomplishing the research program. It is important that housekeeping tasks be assigned to personnel who are knowledgeable of the research environment. The recommended approach to housekeeping is the assignment of housekeeping tasks to the research teams on an individual basis for their immediate work areas and on a cooperative basis for areas of common usage. Similarly, animal caretaker personnel should be responsible for housekeeping in animal care areas. The laboratory supervisor must determine the frequency with which the individual and cooperative housekeeping chores need be accomplished. The supervisor should provide schedules and perform frequent inspection to assure compliance. This approach assures that research work flow patterns will not be interrupted by a contracted cleanup crew; delicate laboratory equipment will be handled only by those most knowledgeable of its particular requirements; and the location of concentrated biological preparations, as well as contaminated equipment used in their preparation and application, will be known.
4.1.7 Standard Operating Procedure (SOP) Development Central to the concept of risk assessment and management is the development of laboratory-specific Standard Operating Procedures (SOPs). These function within the laboratory to train all personnel on the “rules of the laboratory”, and provide easy future reference for all staff members. These SOPs also serve as documentation to the Institutional Biosafety Committee that appropriate practices are in place to contain the biological risks posed in a particular experiment. All mitigation methods specific to the agents, locations and operations which are required to contain their risks should be documented.
At minimum, laboratory SOPs should address the following: Risk communication information. The risks of the agents and operations which may present possible routes of entry should be documented. Symptoms of any illnesses which may arise due to inadvertent exposure to the materials should also be documented, and instructions provided to personnel about communication of this information to any healthcare provider, should they symptoms present (even if the personnel was not knowingly exposed to the materials). Prophylactic measures (e.g., vaccinations) and possible post-exposure health measures should be
Georgia Health Sciences UniversityGeorgia Regents University Biosafety Guide- January 2012 4-20 documented.
Emergency Response Procedures. The procedures documenting the response of the personnel and supervisors should an incident, accident, exposure, spill, release or equipment failure involving biological materials should be documented. This includes spill response, centrifuge tube breakages, exposure responses, reporting requirements, etc.
Personal Protective Equipment (PPE) and Protective Equipment. The typical PPE to be worn by the personnel in the laboratory and any special PPE and/or equipment which must be used during particular agents (e.g., animal or biotoxin handling) or during particular operations which may present additional splash, spray or aerosol risks (see Section 3.2.3 for further information) must be documented and followed by personnel.
Disinfection/Decontamination/Disposal Procedures. Documentation should be provided to personnel related to the appropriate disinfection, decontamination agents and procedures for all surfaces (e.g., benchtops, stainless steel surfaces of equipment), equipment, liquid wastes, solid wastes, spills and instruments. Disposal procedures for sharps, solid waste, liquid waste and pathological waste (e.g., animal carcasses or anything identifiable as a body part) should also be documented. If autoclaves are used as a method of decontamination, autoclave maintenance and certification procedures and records should be documented. Any special deactivation procedures for biological toxins must also be included in the SOPs.
Standard Prudent Practices for Laboratories (e.g., no eating, drinking, gum chewing, mouth pipetting, hand-washing) as well as those particular to the PIs laboratory (e.g., closing the doors to inner laboratories).
Storage and Access Control measures. The procedures the laboratory is expected to follow to ensure no unauthorized personnel accesses the biological materials.
Transport/shipping procedures which should be followed by all personnel removing biological agents from the laboratory to transport to another GHSUGRU laboratory or shipping. This includes training requirements which may be required by these personnel.
Any training requirements of personnel.
A template has been developed to help assist researchers in starting to development of their laboratory SOPs: http://georgiahealth.edu/services/ehs/biosafe/ . This form is not intended to be comprehensive; PIs are encouraged to modify this form as appropriate for their laboratories. New or amended SOPs should be submitted to the Biosafety Office as part of the IBC review/approval process. The Biosafety Office encourages researchers to seek assistance from the biosafety office in SOP development; contact the office (x1-2663 or [email protected]@georgiahealth.edu).
4.2 LABORATORY EQUIPMENT Along with practices and facilities, proper use of laboratory equipment provides containment for work with biological materials. Equipment can refer to Personal Protective Equipment (PPE), as well as operational equipment, which are often used for the dual purposes of maintaining scientific integrity as well as maintaining safety. Laboratory staff should be trained by the Principal Investigator, Laboratory Director or Instructional Course Director in the proper use of laboratory equipment.
4.2.1 Personal Protective Equipment (PPE) Multidisciplinary research conducted in GHSUGRU laboratories requires that personal protective equipment (protective clothing and safety apparatus/equipment) be used to protect the researcher from contact with infectious, toxic and corrosive agents, excessive heat, cold, fire and other physical hazards.
Georgia Health Sciences UniversityGeorgia Regents University Biosafety Guide- January 2012 4-21 Suitable Personal Protective Equipment (PPE) also protects the experiment from contamination. The extent and kind of clothing and equipment to be selected for any particular activity depends upon the research operations and levels of risk associated with the research. While PPE is an important component of any biological safety program, PPE is used with the understanding that PPE serves as a second line of defense. Good laboratory techniques, procedures and appropriate laboratory equipment are the primary barriers against potential exposure to hazardous agents.
For additional information you are urged to consult the Biosafety Office (x1-2663 or [email protected]). In the event the Biosafety Office does not have a listing of the kind of protective devices you are seeking, efforts will be made to acquire the information needed.
4.2.1.1 Laboratory Clothing A commonly used PPE item within the laboratory is special clothing. Both reusable and disposable clothing is available. Whichever is used, it must be durable, designed to provide protection and prevent exposure of the skin to harmful agents, as well as be compatible with the methods of decontamination employed.
Laboratory clothing serves to protect the wearer, the experiment, and environment against contamination. If proper precautions are not taken, contaminated clothing may carry infectious materials outside the laboratory and into other work areas, cafeterias, or the home. Infectious agents can remain viable on cotton and wool fabrics and be disseminated from these fabrics.
Some additional points: Overt exposure to agents at all level of risk should be followed by immediate decontamination of the PPE and change into clean PPE to protect the worker, the experiments and the environment.
Provisions should be made for PPE to be provided to visitors and maintenance or security personnel, if applicable.
PPE worn within the laboratory should not be worn outside the facility to the library, cafeteria, or other places accessible to the public.
Personnel should be encouraged to use disposable facial tissues instead of personal handkerchiefs.
PPE should be placed in an appropriately designated area or container for storage, washing, decontamination or disposal.
All PPE should be decontaminated before being sent to the laundry or discarded. Treat contaminated areas of PPE with an appropriate disinfectant. Lab coats with extensive contamination may by placed in a biohazard bag and autoclaved.
Do not take PPE home to launder; select a laundry service that follows universal precautions.
Change PPE as soon as feasible whenever it is compromised, soiled or torn.
Wear appropriate sizes and keep an adequate supply of PPE available in the laboratory.
Wash hands whenever PPE is removed.
Do not touch door handles, elevator buttons, telephones, computers or other clean surfaces or items with gloved hands.
Georgia Health Sciences UniversityGeorgia Regents University Biosafety Guide- January 2012 4-22
Wear closed-toe shoes and long pants to guard against skin contamination or chemical exposure. Do not wear sandals or shorts in the laboratory.
4.2.1.1.1 Gloves Gloves should be comfortable and of sufficient length to prevent exposure of the wrist and forearm. Depending upon intended use, the composition and design of the glove may vary to provide the desired level of flexibility, strength, impermeability, and resistance to penetration by sharp objects, as well as protection against heat and cold. Quality assurance is an important consideration.
No one glove can be expected to be satisfactory for all intended uses. Gloves may be fabricated of cloth, leather, natural and synthetic rubbers, or plastics. New formulations of synthetic rubber and plastic continue to be developed as research makes varied and changing demands on the protective capabilities of gloves. Changing applications lead to improved capabilities of impermeability, strength, flexibility, tactile sense and control. Within even the modest laboratory, the glove applications may be such that no less than four or five types of protective gloves need to be stocked and used.
Disposable (single use) gloves provide a barrier between infectious agents and the skin. Glove use is a basic precept of preventing infectious agent transmission. Breaks in the skin barrier of the hand (damaged cuticles, scrapes, micro-cuts, dermatitis, etc.) are common.
Gloves shall be removed and hands washed before exiting the laboratory. Use the one glove method, or an appropriate secondary container, when transporting materials through common use areas.
The GHSUGRU Division of Environmental Health and Safety (EHS) can provide information on gloves needed for various tasks, such as working with animals, dry ice, heat, acids, etc. Consult the Division of EHS with details of your work to receive further information about the type and availability of gloves that will best meet your requirements.
Considerations for the selection and use of gloves: Gloves are not 100% leakproof; change gloves periodically and when soiled and always wash hands after removing gloves or other PPE.
Gloves will not prevent needle sticks or other puncture injuries.
Check gloves for visible tears before use.
Avoid wetting examination gloves as water or disinfectants will encourage wicking and leaking which may lead to exposure.
Do not reuse examination gloves; discard contaminated gloves in a biohazard bag immediately after use.
Double glove or use household utility gloves when cleaning spills. Household utility gloves may be decontaminated and reused (replace when compromised.)
Latex allergies among laboratory and clinic personnel are common and can become extremely severe (see Section 5.4.3.2, Latex Gloves and Related Allergies. Alternatives to latex should be considered.
Georgia Health Sciences UniversityGeorgia Regents University Biosafety Guide- January 2012 4-23
4.2.1.1.2 Procedure for Removing Gloves Gloves should be the last item of PPE removed as part of the de-gowning procedure. The exterior of the gloves should be considered contaminated and exposure to skin should be avoided. Grip the outside of one glove at wrist with the other gloved hand, pull glove off and gather in palm of gloved hand. Place index or middle finger of the ungloved hand on wrist of gloved hand, slide finger under the glove opening and pull glove off inside out.
When removing PPE, remove lab coat or solid front gown first, then remove gloves aseptically), remove face protection last to avoid touching your face with contaminated hands. If wearing double gloves, remove outer gloves before removing lab coat or solid front gown.
4.2.1.1.3 Shoes Shoes worn in the laboratory must have closed-toes. Special protective shoes (e.g., steel- toed shoes) may be required for certain work activities (e.g., when working in areas where there is a danger of foot injuries due to falling or rolling objects, or objects piercing the sole, and where feet are exposed to electrical hazards). When working with infectious agents it is advisable to wear shoe covers over street shoes; shoe covers should be removed and decontaminated (autoclaved) before disposal. For work in tissue culture laboratories it may be necessary to change from street shoes to specific laboratory shoes for protection of cultures from contamination.
In certain animal facilities, the GHSUGRU Division of Laboratory Animal Services (LAS) requires personnel to wear overshoes to protect the animals in containment areas. Similarly, people who work with animals and do cage washing are required to wear protective shoes. All personnel working in LAS facilities follow these and other Standard Operating Procedures established by GHSUGRU Division of Laboratory Animal Services.
4.2.1.1.4 Gowns, Lab Coats, Jumpsuits, Aprons and Other Protective Clothing Gowns, lab coats and jumpsuits protect the wearer’s clothing and skin from contamination. As with all PPE, the type of clothing needed depends on the task being performed and the degree of exposure anticipated.
Solid front wrap-around clothing offers better protection than pull-over type clothing or clothing with front closures. Lab coats are not 100% leakproof; change PPE when soiled, and always wash your hands after removing any PPE. Lab coats or other protective clothing will also not provide protection against needle sticks or other punctures.
Spills and splashes occur most often in the chest or lap area. The contaminated surface
Georgia Health Sciences UniversityGeorgia Regents University Biosafety Guide- January 2012 4-24 must be touched during removal of a front closing jacket or lab coat. The contaminated portion often ends up in the wearer’s face during removal of pullover clothing. Many workers prefer not to button up front closing jackets, which leaves street clothing exposed. If front closing jackets must be worn, strict measures shall be implemented to assure the clothing is closed at all times when performing procedures or tasks that may cause exposure.
Long-sleeved garments with snug fitting cuffs are preferred over open or short sleeves. Snug fitting cuffs prevent splashes, splatters and aerosols from making contact with exposed skin on the lower arms. Longer single-use gloves can be pulled over snug fitting cuffs to seal out any infectious materials.
Plastic, vinyl or rubber aprons are usually worn over other protective clothing when extra protection is desired. Aprons are necessary for protection against liquids spilling or splashing on clothing. It is recommended that appropriate aprons be worn to protect against the potential harmful effects of liquid waste. Aprons may also be used to provide protection from steam and hot water in locations such as animal handling facilities, autoclave rooms and laboratory glasswashing rooms.
4.2.1.1.5 Face and Eye Protection Protection of the face and eyes is of prime importance in laboratories due to the potential for foreign material, both liquid and solid, to splash on the head, face and eyes or contact lenses. A variety of face shields, head covers/hoods, protective goggles, and lenses are available from safety supply houses. The selection is dependent upon materials of construction, fit, comfort, and compatibility with the work and the overall facial area requiring protection.
Some of the considerations for selection and use of face and eye protection is indicated below: Face shields and hoods protect the face and the neck from flying particles and sprays of hazardous material; however, they do not provide basic eye protection against impacting objects.
Shields should cover the entire face, permit tilting back to clean the face if desired, and be easily removed in the event of an accident.
If an eye hazard exists in a particular operation or experiment, the soundest safety policy would be to require that eye or face protection, or both, be worn at all times by all persons entering or working in the laboratory.
Contact lenses do not provide eye protection; in fact, they may present more risk to the eyes by holding hazardous materials in contact with the eye for a longer period of time until they can be removed. It is recommended that contact lenses not be worn when working around chemicals, fumes, and other hazardous material and dust particles since these items may become trapped in the space between the contact lens and the cornea. When contact lenses are worn, eye protection, such as tight fitting goggles, must be worn.
4.2.1.1.6 Respiratory Protection Protection of the respiratory system is a major concern of any biological safety program because infectious organisms can readily enter the human body through the respiratory tract. The possibility of this occurring depends on the type and infectious dose of the particular organism. For some, as few as one to ten organisms, when inhaled, may cause
Georgia Health Sciences UniversityGeorgia Regents University Biosafety Guide- January 2012 4-25 infection. Particles with an effective aerodynamic diameter of between 0.5 and 5.0 µm (the respirable fraction) are most effective at penetration and retention in the deep pulmonary spaces. Particles larger than 5 micrometers are generally trapped in the upper respiratory tract and eventually cleared or swallowed.
Engineering controls, such as the use of biological safety cabinets, should always be considered as a first line of defense against respiratory infection when working with infectious organisms. Respirators should only be considered as a second line of defense after feasible engineering controls have been put into place and additional controls are still needed.
Respirators vary in design, application, and protective capability. Respirators can be placed into two categories: air purifying supplied air
By far, the most commonly used respirators in laboratories for protection against biological materials are air purifying respirators. These protect by purifying the existing breathing air through a filter (for particulates) or cartridge (for gases and vapors), or both. Standard air purifying respirators used at GHSUGRU are half-mask, full face, or (battery) powered air purifying respirators (PAPR). These rely on the proper cartridge selection to filter out the contaminant.
Dust masks that have been approved by the National Institute of Occupational Safety and Health (NIOSH) are also considered to be air purifying respirators. Approved dust masks will have one of the following designations – N95, N99, N100, R95, R99, R100, P95, P99, or P100. The selection of N-, R-, and P-series filters depends on the presence or absence of oil particles, as follows: If no oil particles are present in the work environment, use a filter of any series (i.e., N-, R-, or P-series). If oil particles (e.g., lubricants, cutting fluids, glycerine, etc.) are present, use an R- or P-series filter. Note: N-series filters cannot be used if oil particles are present. If oil particles are present and the filter is to be used for more than one work shift, use only a P-series filter.
Note: To help you remember the filter series, use the following guide: N for Not resistant to oil, R for Resistant to oil P for oil Proof
Selection of filter efficiency (i.e., 95%, 99%, or 99.97%) depends on how much filter leakage is acceptable. Higher filter efficiency means lower filter leakage. The NIOSH Guide to the Selection and Use of Particulate Respirators Certified under 42 CFR 84 can be found at: http://www.cdc.gov/niosh/docs/96-101/ . OSHA also has an online electronic tool for assisting in the proper selection of appropriate respirators: http://www.osha.gov/SLTC/etools/respiratory/respirator_selection.html .
Federal OSHA regulations (29 CFR 1910.134) require initial and annual training and fit- testing, and well as medical surveillance of all respirator wearers. If a respirator is to be used in the course of research with biological materials, the GHSUGRU IBC will require adherence to these OSHA standards: http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_id=12716&p_table=STA NDARD .
Please make sure that the GHSUGRU Office of Environmental Health and Occupational
Georgia Health Sciences UniversityGeorgia Regents University Biosafety Guide- January 2012 4-26 Safety (EHOS) is notified whenever the use of a respirator is being considered. Proper selection of cartridges and respirators is very important and should not be made without input from the EHOS. In addition, the EHOS office will assist in guiding researchers through the evaluation process and provide the required training and fit testing. The EHOS Office can be contacted at x1-2663. The Occupatonal Health Office must also be notified so that medical evaluation/surveillance and clearance can be issued prior to wearing the respirator.
4.2.1.2 Selection of PPE
Use the following PPE to minimize exposure via mucous membrane OR non-intact skin: For face protection, wear safety glasses and a mask, or a chin length face shield whenever splashing, splattering or droplets may be anticipated (any work with liquids on the open bench). An impact resistant face shield should be used when operating the autoclave or when working with cryogenic materials. Impact resistant face shields will protect the user’s face against splatters of hot liquids or broken glass or plastic fragments.
Gloves and a lab coat are worn to protect the skin and clothing from contact with potentially infectious materials. Wear gloves that are long enough to extend over the sleeves of the lab coat and cover wrists so no bare skin is exposed. Consider double gloving when working with cultures of infectious agents or handling spills. Thicker household utility gloves can be worn for cleaning blood or BSL-2 spills. Utility gloves can be decontaminated and reused until the integrity of the glove is compromised. Temperature resistant gloves should be worn to protect hands from physical damage when working with very hot (autoclave) or cold (liquid nitrogen tank, -70°C freezer) materials.
Sleeve covers may be worn over lab coat and gown sleeves to provide protection to the sleeves and wrists from contamination when working in the biological safety cabinet. Disposable sleeve covers have tight fitting grips at both ends.
Waterproof bandages are worn to cover any wounds or non-intact skin before gloving. It is preferred to double glove when skin is damaged or non-intact. Inform your supervisor of any severe skin conditions or wounds. Avoid working with Risk Group 2 or higher potentially infectious materials if non-intact skin cannot be adequately covered.
Solid front gowns provide more protection to clothing and skin than lab coats. Solid front gowns are worn for high hazard infectious agent work. The tight fitting cuffs of the gown help to minimize wrist contamination.
Impervious lab coats, gowns or aprons are worn when heavy contamination or soiling is likely.
Head covers are worn to protect the hair and scalp from splatter or droplets when working with heavy contamination or when contact with the head is likely. When choosing a head cover make sure it is impervious to liquids (some head covers are not impervious).
Shoe covers are worn over the shoes to protect shoes from contamination when working in heavily contaminated areas (such as large spills, crime scenes, morgues, cadaver dissection areas, surgical operation areas).
Gowns, head and shoe covers also help keep contaminants from entering the sterile area in clean rooms, surgical suites and barrier animal facilities.
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Use the following PPE to minimize exposure via cuts, slices, or scratches: Kevlar gloves and sleeves are cut resistant and will help guard against slices, scratches or cuts, but will not prevent direct puncture or needlestick injuries. Steel mesh gloves also protect against slices, cuts, and scratches but will not eliminate punctures. Neoprene and other abrasive resistant gloves are cut resistant, but significantly reduce dexterity.
Use the following PPE to minimize exposure via aerosols: HEPA filtered respirators (air purifying or powered air purifying) are worn to prevent exposure to potentially infectious aerosols when cleaning spills of concentrated infectious material or responding to centrifuge incidents. Employees who wear a respirator must enroll in the GHSUGRU Respiratory Protection Program through the EHOS (x1-2663) before using a respirator.
4.2.1.3 PPE Requirements Table Please see Table 4.2.1.4 for a guidance table for PPE
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Table 4.2.1.4. PPE Requirements Table
PPE Biosafety Level 1 Biosafety Level 2 Biosafety Level 2+/3 Gloves Recommended to prevent skin or clothing Required Required contact with biological materials. Note: work that may involve radioactive materials or chemicals will require the use of a lab coat and gloves Lab Coat Recommended to prevent skin or clothing Required Solid-front protective clothing such as back fastening contact with biological materials. Note: work gown with tight fitting cuffs must be worn to protect that may involve radioactive materials or street clothing, scrubs and/or skin from contact with chemicals will require the use of a lab coat and infectious agents. gloves Face Protection Wear protective eyewear and surgical mask or Face protection is not required when performing all chin-length face shield whenever splashing, work inside a biosafety cabinet (BSC). However if splattering or spraying is anticipated to prevent there is a potential for splashing, such as from a contact with mucous membranes of the eyes, nose dropped container during transport, face protection and mouth. Researchers may choose to augment must be worn. eye protection by performing experiments behind a protective splash shield Respiratory The GHSUGRU IBC may recommend respiratory The use of respiratory protective equipment such as a Protection protection on a case-by-case basis if the potential powered air purifying respirator (PAPR) will be for aerosol generation is high and alternate recommended or required by the GHSUGRU IBC containment devices, such as a biosafety cabinet, and/or the Biosafety Office on a case-by-case basis. cannot be used The use of PAPRs is required for response and cleanup of a BSL-2+ or BSL-3 spill. All those who may wear a respirator must be enrolled in the GHSUGRU Respiratory Protection Program. Other Other PPE, such as Tyvek coveralls, booties, sleeve Other PPE, such as Tyvek coveralls, booties, sleeve guards, plastic aprons, and household rubber gloves guards, plastic aprons, and household rubber gloves will be recommended on a case-by-case basis. will be recommended on a case-by-case basis. Generally, additional protective clothing is required Generally, additional protective clothing is required whenever there is a high potential for splashing of whenever there is a high potential for splashing of potentially infectious materials, such as organ potentially infectious materials, such as organ harvesting or large spill response and clean-up. harvesting or large spill response and clean-up.
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4.2.2 Biological Safety Cabinets (BSCs) The function of a BSC is dependent upon high efficiency particulate air (HEPA) filters within the plenum of the unit. The overall basis for HEPA filter function relies on the combination of multiple physical forces: inertial impaction (direct blocking of the particulates via the filter medium), diffusion (Brownian motion), Interception (“straining”) and electrostatic forces. Larger particulates are typically more efficiently blocked by interception and impaction; whereas smaller particles tend to be more efficiently retained by diffusion or electrostatic forces.
Together, these forces combine to enable these HEPA filters to remove a minimum of 99.97% of particles 0.3 µm in size. The overall efficiency of the HEPA filter is greater for removal of particles that are larger or smaller than 0.3 µm (see Figure, right, to review the relationship between HEPA filter efficiency relative to particle size). However, HEPA filters will not filter out gases and vapors.
HEPA filters have a limited life span (approximately 5-7 years) and are relatively fragile; they can be easily dislodged or damaged during moves or repairs of the unit. For this reason, Biosafety Cabinet function must be certified at least annually or after any move or repair. The Division of Environmental Health and Safety and the Laboratory Equipment Service (LES) office jointly run a program that monitors the annual performance of biological safety cabinets at least annually. Contact the EHOS office (x1-2663) or Laboratory Equipment Services (x1-6124) to request certification of a new, moved or repaired BSC. The BSC certification program conforms to guidelines established by the National Institutes of Health (NIH) and the Centers for Disease Control and Prevention (CDC) in their publication Primary Containment for Biohazards: Selection, Installation and Use of Biological Safety Cabinets http://www.cdc.gov/od/ohs/biosfty/bsc/bsc.htm and the Occupational Safety and Health Administration’s (OSHA) Bloodborne Pathogens Standard.
Biological safety cabinets (BSCs) are categorized by Classes: Class I units provide personnel protection only Class II units provide both personnel and product protection Class III are glove-box units which provide personnel protection and optional product protection The specifications for BSC function and types are defined by NSF ANSI Standard 49.
Most BSCs at GHSUGRU are Class II units, which means, when used properly, they provide a clean work environment for research or patient care activities while offering personnel and environmental protection. The BSC provides primary containment for infectious materials. The efficacy of BSCs depends upon the behavior of the operator and the orientation of the unit in the facility. Class II BSCs fall into four types (A1, A2, B1 and B2), the most common of which is the Class IIA2 BSC at GHSUGRU. Class IIA2 BSCs have the following characteristics:
100 feet per minute inward air face velocity 70% recirculated air, 30% exhausted (thru HEPA) (see airflow diagram, right) Potentially contaminated ducts and plenums are under negative pressure or surrounded by negative pressure ducts and plenums
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Class IIA2 cabinets can be either “ducted” or “unducted”. The overwhelming 30% majority of BSCs in GHSUGRU facilities are unducted Class IIA2 BSC. After HEPA filtration, the exhaust from a Class IIA2 Biosafety Cabinet can be expelled into the room, or it can be exhausted to an external area connected via a thimble or canopy connector through the buildings external exhaust system (i.e. a “ducted” Class IIA2 BSC). The great advantage of having a “ducted” Class IIA2 BSC is that it may be 70% used with minute quantities of volatile toxic chemicals and tracer amounts of radionuclides, since the air from the BSC is exhausted through properly functioning exhaust canopies. Radionuclides and volatile chemicals may not be used in “unducted” Class IIA2 BSCs unless charcoal filter containment units are placed within the unit to capture any potentially volatilized radionuclides prior to circulation within the BSC. Contact the Radiation Safety Office for further information about these charcoal devices. Keep in mind: if the BSC is ducted using a thimble connection, because this is not an air-tight connection, if failure were to occur in the building exhaust system during use, the BSC would still function to filter particulates, but will expel the air exhaust into the room and may pose a hazard if radionuclides or volatile chemicals are in use.
GHSUGRU also has a small handful of Class IIB2 BSCs in select areas on campus, which have the following characteristics: 100 feet per minute inward air face velocity 100% of the air is exhausted, no air recirculation occurs. Potentially contaminated plenums are under negative pressure or surrounded by negative pressure ducts and plenums. They are hard-connected to the building’s external exhaust system (i.e., they are always “ducted”) The typical exhaust requirements for these units are significantly higher than those of Class IIA2 units (~700-1200 CFM at 2” w.g.)
Because the function of a Class IIB2 BSC is intricately connected to the function of the building external exhaust system, any failure in the building exhaust system will compromise the function of a Class IIB2 BSC. Therefore, audible or visual alarm systems must be installed to alert any users in fluctuations in the building exhaust system. However, because exhausts from Class IIB2 units are connected via air-tight connection and exhausted externally from the building and no recirculation of air occurs within the BSC, there are fewer restrictions for volatile chemicals or radionuclide use in these BSCs.
BSCs isolate biohazards from personnel by confining the biohazardous material in the unit, primarily through the use of directional laminar airflow. The BSC removes aerosolized biohazardous material by moving air through high efficiency particulate air (HEPA) filters. The intake air coming from the front of the unit is drawn into the front grate of the BSC filtered through a HEPA filter before entering the BSC work area. Exhaust air also passes through a HEPA filter. Aerosols generated in the work area of the BSC are contained within the BSC.
Operating Procedures for Class II Biological Safety Cabinet (also see Section 4.1.4.1 for more information on Tissue/Cell Culture Practices):
If used, turn off UV light within the unit and switch on fluorescent light and blower. Disinfect all interior surfaces with 70% ethanol or suitable disinfectant. Place items required for procedure into cabinet; do not obstruct grills. Allow the unit to run approximately 15 minutes prior to use to purge contaminants from work area. Keep materials at least 4 inches inside work area. Work should proceed from clean to contaminated areas. After procedure, allow cabinet to run approximately 15 minutes before removing materials to
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purge contaminants from work area prior to shutting off motor. Wipe down all work surfaces with suitable disinfectant (as described in the laboratory SOPs) Turn off fluorescent light and blower if desired.
Many BSCs are equipped with germicidal ultraviolet (UV) lamps. Time of exposure, distance, presence of dust or debris and UV lamp intensity contribute to the germicidal effect of the UV lamp. The visible blue- violet glow of the UV lamp does not indicate there is germicidal effect; the efficacy of the UV lamp may decrease as the lamp grows older. The UV lamp needs to be cleaned periodically to remove dust. UV lamps may damage eyes, skin, and laboratory equipment. UV lamps should be turned off while the room is occupied. For these reasons, the Biosafety Office discourages the use of UV lamps inside of BSCs due to the potential damage which may result from UV lamp use, and the arguable reliability of UV decontamination over time. For further information related to the use of UV lamps in BSCs, please see the following references: Burgener, J (2006) Position Paper on the Use of Ultraviolet Lights in Biological Safety Cabinets. Applied Biosafety 11(4): 228-230. Meecham, P.J. and Wilson, C. (2006) Use of Ultraviolet Lights in Biological Safety Cabinets: A Contrarian View. Applied Biosafety 11(4): 222-227.
4.2.3 Laminar Flow Hoods/Clean Benches Laminar Flow Hoods (a.k.a. Clean Benches) differ from Biosafety Cabinets in that they offer only product protection, not personnel protection. They function by blowing HEPA filtered air through the metal baffle in the back of the unit directly across the work surface toward the user (see airflow diagram, right). While this provides a sterile work surface, this may increase the exposure risk of the individual using the unit to the materials on the work surface. Therefore, LFHs should not be used for work with hazardous materials. These function well for media preparation or similarly low hazard operations which may require sterile conditions.
4.2.4 Fume Hoods Unlike Biosafety Cabinets or Laminar Flow Hoods, chemical fume hoods offer personnel protection to the user, but no product or environmental protection. Air drawn from the front of the unit passes directly across the work surface of the fume hood, thereby potentially compromising the sterility of any materials exposed on the work surface (see airflow diagram, right). In addition, the exhaust air from the fume hood is not filtered in any way before expelling the air via the building’s exhaust systems. This may result in environmental contamination, and potential exposure of personnel who may be near the exhaust outtakes. However, typically fume hood exhausts are expelled at high velocity through an exhaust stack, reducing the latter risk. Fume hoods should be used for working with hazardous materials, such as biological toxins; however, not if sterility is needed.
4.2.5 Centrifuges All centrifugation of Risk Group 2 agents or higher shall be done using centrifuge safety buckets with safety caps or in sealed centrifuge tubes in sealed rotors. If a small centrifuge is used and centrifuge safety
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cups are not available, the centrifuge should be operated in the biological safety cabinet.
Each person operating a centrifuge should be trained on proper operating procedures. Keep a log book detailing operation records for centrifuges and rotors to assist in determining service requirements.
The following procedures for centrifugation are recommended safety measures to consider while using biological materials in centrifuges: Examine tubes and bottles for cracks or stress marks before using them. Fill and decant all centrifuge tubes and bottles within the biological safety cabinet. Centrifuge safety caps and buckets Never overfill centrifuge tubes as leakage may occur when tubes are filled to capacity. The maximum for centrifuge tubes is 3/4 full. Always cap tubes before spinning. Place all tubes in safety buckets with safety caps or in sealed rotors. Inspect the "O" ring seal of the safety bucket and the inside of safety buckets or rotors. Correct rough walls caused by erosion or adhering of matter and remove debris from the rubber cushions. Wipe exterior of tubes or bottles with disinfectant prior to loading into rotor or safety bucket. Wipe the exterior of the rotor or safety buckets before removal from Biosafety Cabinet. Never exceed safe rotor speed. Stop the centrifuge immediately if an unusual condition (noise or vibration) begins. Wait five to fifteen minutes after the run before opening the centrifuge. This will allow aerosols to settle in the event of a breakdown in containment. Decontaminate safety carriers or rotors and centrifuge interior after each use. Open safety buckets or rotors in a biological safety cabinet. If the rotor does not fit in the biological safety cabinet, use the fume hood. If construction of the centrifuge permits, the centrifuge chamber is to be connected to a vacuum pump with a HEPA filter installed between the centrifuge and the vacuum pump.
4.2.6 Vacuum Line Chemical Traps and Filters Considerations and Limitations of Vacuum Line Chemical Traps and Filters: Liquid wastes should be collected in order to decontaminate using an appropriate method which should be described in the laboratory SOPs. Typically, liquid wastes are collected in vacuum aspirators into which some full-strength disinfectant has been placed. When setting up a vacuum aspirator system, make sure the tubing inside the vacuum flasks extend far below the vacuum arm of the flask to prevent liquid wastes from being drawn through the flask’s vacuum arm and contaminating the vacuum line (see, e.g., “Collection flask” in figure below).
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Flexible vacuum tubing or Tygon® tubing used in aspirator systems should have walls thick enough to withstand the vacuum without collapsing, cracking or leaking. Periodically inspect and replace vacuum tubing which has become cracked over time. To ensure appropriate decontamination, subsequent disinfection measures should be followed prior to disposal. Do no allow vacuum traps to become overfull (recommended not greater than half-full). This not only prevents liquids from inadvertently drawn into the vacuum line, but will allow for full decontamination of the liquid wastes prior to disposal. HEPA filters or equivalents should be placed in the vacuum lines of any laboratory requiring BSL- 2 containment or higher. Vacuum line filters shall be examined and replaced if clogged or if liquid makes contact with the filter. Used filters shall be discarded in the medical waste stream. Do not leave pipettes in the ends of the vacuum aspirator hoses. After use, remove them from the hose and place in disinfection tray/container prior to disposal. Leaving pipettes within the hoses only presents additional exposure or contamination risks. Rinse vacuum tubing with disinfectant after use. This will prevent backflow of contaminated liquids within the vacuum line and subsequent contamination. If the vacuum traps are outside of the Biosafety Cabinet, place in sufficient secondary containment to hold the volume of liquid which may be spilled if implosion of the vacuum flask should accidentally occur
4.2.7 Syringes and Needles The hypodermic needle is a dangerous instrument. To lessen the chance of accidental injection, aerosol generation, or spills, the use of syringes should be avoided when alternate methods are available. For example, use a blunt needle or cannula on the syringe for oral or intranasal inoculations and never use a syringe and needle as a substitute for a pipette in making dilutions.
The following practices are recommended for hypodermic needles and syringes when used for parenteral injections: Use the syringe and needle in a biological safety cabinet only and avoid quick and unnecessary movements of the hand holding the syringe. Examine glass syringes for chips and cracks, and needles for barbs and plugs. This should be done prior to sterilization before use. Use needle-locking syringes only, and be sure that the needle is locked securely into the barrel. Replace glass syringes with plastic disposable syringes whenever possible. Whenever possible use safer needle systems. These might include retractable needle systems or shielded needle systems (see right). Wear latex or nitrile gloves for all manipulations with needles and syringes. Fill the syringe carefully to minimize air bubbles and frothing of the inoculum. Expel excess air, liquid and bubbles from a syringe vertically into a cotton pledget moistened with an appropriate disinfectant, or into a small bottle of sterile cotton. Do not use the syringe to forcefully expel a stream of infectious fluid into an open vial for the purpose of mixing. Mixing with a syringe is condoned only if the tip of the syringe is held below the surface of the fluid in the tube. If syringes are filled from test tubes, take care not to contaminate the hub of the needle, as this may result in the transfer of infectious material to the fingers. When removing a syringe and needle from a rubber-stoppered bottle, wrap the needle and stopper in a cotton pledget moistened with an appropriate disinfectant. If there is concern of the disinfectant contaminating sensitive experimental materials, a sterile pledget may be used and immediately discarded into a biohazard bag. When inoculating animals, position the hand that is holding the animal “behind” the needle or use
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a pair of forceps to hold the animal in order to avoid puncture wounds. Be sure the animal is properly restrained prior to the inoculation and be on the alert for any unexpected movements of the animal. Before and after injection of an animal, swab the injection site with an appropriate antiseptic. Discard syringes into an authorized sharps container, which should always be kept near the site of use. DO NOT bend, shear, recap or otherwise manipulate the needle. If recapping is unavoidable, use a one handed method. DO NOT discard syringes into biohazard bags.
4.2.8 Pipettes The following is excerpted from Laboratory Safety, Principles and Practices 2nd Ed., ASM Press. Never suction or pipette by mouth; always use some type of pipetting aid when pipetting infectious materials. Preferably, all activities should be confined to a biosafety cabinet. Mouth pipetting should be prohibited even with mouth pipetting devices that use an hydrophobic membrane filter that does not require fingers to touch the mouthpiece. This reusable pipetting device requires storage on the bench or other location between usage, which can result in contamination on the end piece that inserts into the mouth. Pipetting of toxic chemicals should be performed in a chemical fume hood. Infectious or toxic materials should never be forcefully expelled from a pipette. Mark-to-mark pipettes are preferable to other types because they do not require expulsion of the last drop. Infectious or toxic fluids should never be mixed by bubbling air from a pipette through the fluid. Infectious or toxic fluids should never be mixed by alternate suction and expulsion through a pipette. Gently discharge from a pipette as close as possible to the fluid or agar level, and the contents should be allowed to run down the wall of the tube or bottle whenever possible, not dropped from a height. Pipettes used for transferring infectious or toxic materials should always be plugged with cotton, even when safety pipetting aids are used. Avoid accidentally dropping infectious or toxic material from the pipette onto the work surface. Place a disinfectant dampened towel or other absorbent material on the work surface, and autoclave before discard or reuse. Plastic backed bench paper is suitable for this purpose. Contaminated pipettes should be placed horizontally into a pan or tray containing enough suitable disinfectant, such as hypochlorite, to allow complete immersion of the pipettes. Pipettes should not be placed vertically in a cylinder that, because of its height, must be placed on the floor outside the biosafety cabinet. Removing contaminated pipettes from the biosafety cabinet and placing them vertically in a cylinder provides opportunity for dripping from the pipette onto the floor, or the rim of the cylinder, thereby creating an aerosol, and the top of the pipettes often protrude above the level of disinfectant. Place discard pans for used pipettes within the biosafety cabinet. After suitable contact time, excess disinfectant can be carefully poured down the sink. The pan and pipettes can be autoclaved together, and replaced by a clean pan with fresh disinfectant.
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4.2.9 Blenders, Mixers, Sonicators and Cell Disruption Equipment Hazardous aerosols are created by most laboratory operations involving blending, mixing, stirring, grinding or disrupting biohazardous materials. Even the use of a mortar and pestle can be a hazardous operation. Other devices that may produce aerosols are ball mills, colloid mills, jet mills, tissue grinders, magnetic mixers, stirrers, sonic cleaning devices/sonicators, homogenizers, ultrasonic cell disintegrators, French Presses, and shakers.
Adequate decontamination is essential prior to sonic cleaning due to possible aerosol generation. Wherever sonicators are used in the cleaning process; such as in dishwashers, animal cage washers, etc.; all items should be sterilized prior to cleaning.
The laboratory practices generally required when using equipment that may generate aerosols with biohazardous materials are as follows: Operate blending, cell disruption, and grinding equipment in a biological safety cabinet. Use safety blenders designed to prevent leakage from the rotor bearing at the bottom of the bowl. In the absence of a leakproof rotor, inspect the rotor for leakage prior to operation. A preliminary test run with sterile water, saline, or methylene blue solution is recommended prior to use. If the blender is used with infectious material place a towel moistened with an appropriate disinfectant over the top of the blender. Sterilize the device and residual contents promptly after use. Glass blender bowls are undesirable for use with infectious material because of the potential for glass bowls to break. Blender bowls sometimes require supplemental cooling to prevent destruction of the bearings and to minimize thermal effects on the product. Before opening the safety blender bowl, permit the blender to rest for at least one minute to allow settling of the aerosol cloud. Grinding of infected tissues or materials with any open device is best done within a biological safety cabinet.
4.2.10 Lyophilizers Specimens snap-frozen in ampules are dried on a vacuum manifold or in a chamber-type drier at low negative pressure. If the glass neck of the ampule is sealed off while the ampoule is still under vacuum, it may cause implosion, either during the sealing or later when the evacuated ampule is being opened. To avoid this, after drying is completed, and before sealing is done, bring the pressure within the ampule back to normal by gradually introducing dry nitrogen, avoiding turbulent disturbance of the dry product.
The narrow or constricted neck of the ampoule is contaminated if the specimen is allowed to run down the wall of the neck during filling. Subsequently, when the ampule is sealed with a torch, the dried material on the wall becomes charred or partially decomposed; residues of this material may adversely affect the dried material when it is reconstituted. To avoid this, a syringe with a long cannula or a Pasteur-type pipette should be used to fill the vial. Do not allow the delivery end of the cannula or pipette to touch the neck of the vial.
All ampules used for freeze-drying of cultures, toxins, or other biohazardous material should be fabricated of Pyrex-type glass. This type of glass requires a high-temperature torch using an air-gas or oxygen-gas mixture for sealing. These hard glass ampoules are much less apt to form gas bubbles that burst inwardly during sealing under vacuum than the soft glass ampoules and are more resistant to breakage during
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handling and storage.
The filling of ampules and vials with infectious specimens, the subsequent freeze-drying, and sealing or closing of ampules and vials in the preparation of dry infectious specimens should be performed in a biological safety cabinet. The same is true for the preparation of ampules and vials containing liquid specimens not subject to freeze-drying.
Safety precautions to be taken will depend on the agents, equipment, and containment available. Therefore, before initiating this procedure, the principal investigator should work out the protocol for each machine in consultation with the Biological Safety Officer. All persons using the procedure must then follow the protocol.
4.2.11 Microtome/Cryostat Due to the very sharp blade and the nature of the materials used with the microtome/cryostat, training is essential in the use of the equipment and in the hazards of the materials used with the equipment. Users should be informed of the need to prevent cuts and scrapes as well as protect the eyes, nose, mouth and skin from exposure to the materials being used.
New personnel must be trained in the proper use and maintenance of the equipment, and demonstrate proficiency prior to use.
If using human tissue, microtome/cryostat users are required to attend Bloodborne Pathogens training. Fixatives take time to penetrate tissue; the fixatives may not inactivate pathogens deep in the tissue. Freezing and drying do not inactivate most pathogens, so, as with fixative use, the pathogens that may be present in the tissue should be considered capable of causing infection.
Microtome/cryostat users will likely need to also attend Chemical Safety Laboratory Personnel training due to the fixatives and dyes used in histology.
When purchasing new units the available safety features should be taken into consideration prior to deciding on a manufacturer or model. Some available safety features are: Auto-decontamination cycle Easy blade release for installing and changing blades. Retractable knife/blade to permit safe entry into chamber for cleaning, retrieving specimens, etc. Disposable blades.
Never retrieve samples, change blades, or clean equipment by hand with the blade in place; always use appropriate engineering controls (i.e. forceps, tweezers, dissecting probes, and small brushes).
Things to remember when using and maintaining microtomes/cryostats: Always keep hands away from blades. Use extreme caution when aligning blocks, the blocks may be close to the blades. If available, make sure block holder is in locked position when loading/aligning blocks. Use knife-edge protectors/guards. Do not leave knife-edges that may extend beyond microtome knife holder unprotected. Keep blocks wet when in the microtome to minimize airborne shavings during slicing. Use brushes to clean/brush equipment. Use engineering controls such as forceps when removing or changing the blade. Dislodge stuck blocks using mechanical means such as forceps and/or dissecting probes.
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Wear appropriate PPE such as a lab coat or gown, mask, safety glasses or goggles, surgical grade Kevlar gloves that provide dexterity and cut protection, and examination gloves to protect against biohazards. When changing blades, wear stainless steel mesh gloves to provide additional protection from cuts and scrapes. Avoid freezing propellants that are under pressure as they may cause splattering or droplets of infectious materials. Decontaminate equipment on a regular schedule using an appropriate disinfectant. Consider trimmings and sections of tissue as contaminated and discard in the appropriate waste stream. Do not move or transport microtome with knife in position. Do not leave knives out of containers when not in use. Do not leave motorized microtomes running unattended.
4.2.12 Fluorescence Activated Cell Sorters (FACS) and FACS analyzers The following is excerpted from Schmid, I, Lambert, C. Ambrozak D and Perfetto SP (2007) Standard Safety Practices for Sorting of Unfixed Cells. Current Protocols in Cytometry 3.6.1-3.6.20. John Wiley & Sons, Inc. :
In 1994 the International Society of Analytical Cytology (ISAC) first recognized the need to formulate safety guidelines for sorting and analysis of unfixed cells to provide laboratories with recommendations for practices to reduce the potential for biohazard exposure of instrument operators. These standards are periodically updated by ISAC, most recently in 2007 (See: http://www.isac- net.org/media/Biosafety_sorting_2007.pdf ).
Biological particles 0.1 μm to 60 μm in size (e.g., aerosols) have been found to be important in the spread of infectious diseases. Submicrometer particles formed through dehydration of small droplets (droplet nuclei) can contain inorganic material, organic material, or infectious agents and may stay suspended in air for prolonged periods of time. During inhalation, larger particles are deposited mainly into the nasal passages, 3- to 7-μm particles into the tracheal area and pharynx, and ≤3-μm particles into the lungs of the exposed individual. Droplets that fall out of suspension in air will land on surfaces, and pathogens they may contain can then be transmitted by exposure to broken skin or mucous membranes, or by ingestion.
Consequently, protection of all laboratory workers from exposure is critical, in particular during high-risk procedures such as droplet-based cell sorting using instruments with high system pressures.
Jet-in-air technology utilized for cell sorting involves a liquid stream carrying the cells through a nozzle vibrating at high frequency. At a given distance from the nozzle orifice the stream is broken into individual droplets. These droplets are then passed between high voltage plates. Droplets containing cells of interest with parameters preselected by the operator are electrostatically charged and deflected into sort sample receptacles. Overall droplet size depends on the instrument operating pressure and the size of the nozzle orifice and its vibration frequency. High-speed cell sorters utilize higher system pressures and sort frequencies and thus produce more smaller droplets compared to older instruments designed for lowspeed separations. All sorters also generate microdroplets, i.e., satellite droplets, 3 to 7 μm. Owing to the high fluid pressure produced in high-speed cell sorters large amounts of secondary aerosols of various and undefined droplet sizes can occur during instrument failures, for instance, when a partial clog in the nozzle causes a deflection in the fluid stream that is hitting a hard surface, e.g., the waste Aerosol produced after deflection from the waste catcher. This figure shows the amount and position of catcher. Droplets larger than 80 μm constitute the majority of droplets aerosols produced after deflecting from the side of the waste catcher. (Photo from: Perfetto, SP, Ambrozak generated during sorting and settle quickly out of the atmosphere; smaller DR, Koup RA, and Roederer M (2003) Measuring Containment of Viable Infectious Cell Sorting in droplets, however, may be aerosolized, particularly when they are elevated High-Velocity Cell Sorters. Cytometry Part A 52A:122–130.) by air currents. Because of the potential health risk to sorter operators and
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the environment if aerosols escape into the room, aerosol containment of a sorter, whether free standing or enclosed in a biological safety cabinet, must be verified routinely using appropriate testing methods, such as the use of highly fluorescent Glo-Germ® (5-µm melamine copolymer resin beads in a 5-ml volume of ethanol) under the same conditions as the cell sort.
Although FACS analyzers are typically a more contained unit that the FACS sorter, it is important to note that some functional FACS analysis measurements on cells (e.g., evaluation of calcium flux or membrane potential, certain apoptosis assays, cytokine assays, or live DNA or RNA staining) preclude cell fixation, and when performed on jet-in-air flow cytometers, can also expose operators or bystanders to potentially hazardous aerosols and sample splashes. Therefore, the safety practices outlined here apply whenever unfixed samples are run through a jet-in-air flow cytometer or a cell sorter that combines a flow cell with jet-in-air sorting.
When sorting any infectious or hazardous material, even if it is classified as BSL-2, it is critical to understand that droplet-based sorting procedures are considered BSL-3 practices.
It is therefore recommended that viable, unfixed samples that are potentially infectious be sorted at a minimum on a sorter which has been tested for aerosol containment located in a modified BSL-2 facility using practices and containment equipment recommended for BSL-3 by the CDC. However, because of the increased hazard of a sudden quick release of large amounts of fluid or aerosols into the environment, it is highly recommended that high-speed sorting be performed in a BSL-3 laboratory facility under complete BSL-3 containment.
All risks associated with materials being used or presented for sorting or analysis in any GHSUGRU FACS facility should be fully disclosed to the facility manager before use and documented on the PI’s Biosafety Protocol for risk assessment. Currently, the GHSUGRU core FACS facilities do not offer facilities capable of adequately containing BSL-2 materials when FACS sorting.
For further information on FACS Biosafety, please contact the Biosafety Office (x1-2663 or [email protected]@georgiahealth.edu) or the FACS core facility director. Also, you may wish to review the following references:
Schmid I, Lambert C, Ambrozak D, Marti GE, Moss DM and Perfetto SP (2007) International Society for Analytical Cytology Biosafety Standard for Sorting of Unfixed Cells. Cytometry Part A 71A: 414-437. Schmid I, Lambert C Ambrozak D and Perfetto SP (2007) Standard Safety Practices for Sorting of Unfixed Cells. Current Protocols in Cytometry 3.6.1-3.6.20. John Wiley & Sons, Inc. Perfetto SP, Ambrozak DR, Koup RA, Roederer M (2003) Measuring Containment of Viable Infectious Cell Sorting in High-Velocity Cell Sorters. Cytometry Part A 52A:122–130.
4.2.13 Miscellaneous Equipment (Waterbaths, Cold Storage, Shakers, etc.) Water baths and Warburg baths used to inactivate, incubate, or test infectious substances should contain a disinfectant. For cold water baths, 70% propylene glycol is recommended. Sodium azide should not be used as a bacteriostatic. It creates a serious explosion hazard.
Whenever possible, the use of dry heat/incubator blocks and dry incubators should be considered instead of water baths since this may reduce the risks of water intrusion upon the samples and contamination of water in baths or shakers. Dry heat blocks come with modular adapters designed to fit tubes of multiple sizes as well as rectangular 12-, 24- or 96-well plates.
Deep freezer, liquid nitrogen, and dry ice chests as well as refrigerators should be checked, cleaned out periodically to remove any broken ampules, tubes, etc. containing infectious material, and decontaminated.
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Use rubber gloves and respiratory protection during this cleaning. All infectious or toxic material stored in refrigerators or deep freezers should be properly labeled. If equipment failure occurs, immediate action to prevent specimen loss and contamination of equipment and facilities may be required. Security measures should be commensurate with the hazards.
The degree of hazard represented by contaminated liquid nitrogen reservoirs will be largely dependent upon the infectious potential of the stored microorganisms, their stability in liquid nitrogen, and their ability to survive in the airborne state. Investigations suggest that storing tissue culture cell lines in containers other than sealed glass ampoules might result in potential inter-contamination among cell lines stored in a common liquid nitrogen repository.
Care must be exercised in the use of membrane filters to obtain sterile filtrates of infectious materials. Because of the fragility of the membrane and other factors, such filtrates cannot be handled as non- infectious until culture or other tests have proved their sterility.
Shaking machines should be examined carefully for potential breakage of flasks or other containers being shaken. Screw-capped durable plastic or heavy walled glass flasks should be used. These should be securely fastened to the shaker platform. An additional precaution would be to enclose the flask in a plastic bag with or without an absorbent material.
No person should work alone while performing any extremely hazardous operation.
4.3 LABORATORY FACILITIES Laboratory Facility design and function is the third element, along with practices and equipment, which provides appropriate containment for work with biological materials. The unique design and construction of laboratory facilities contributes to the laboratory workers' protection, provides a barrier to protect persons outside the laboratory, and protects persons or animals in the community from infectious agents that may be accidentally released from the laboratory. Keep in mind, conversion of non-laboratory spaces into laboratory spaces is not always possible without significant monetary investment, since the design, ventilation requirements and furnishings within the non-laboratory areas may not comply with those required by the Board of Reagents or other safety standards.
Laboratory directors are 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 BSL-1 and BSL-2 facilities will be direct contact with the agents, or inadvertent contact exposures through contaminated work environments. Secondary barriers in these laboratories may include separation of the laboratory work area from public access, availability of a decontamination facility (e.g., autoclave), and hand washing facilities.
When the risk of infection by exposure to an infectious aerosol is present, higher levels of primary containment and multiple secondary barriers may become necessary to prevent infectious agents from escaping into the environment. Such design features include specialized ventilation systems to ensure 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 to isolate the laboratory. Design engineers for laboratories may refer to specific ventilation recommendations as found in the ASHRAE Laboratory Design Guide published by the American Society of Heating, Refrigerating, and Air- Conditioning Engineers (ASHRAE).
The Georgia Board of Reagents has issued general laboratory design criteria to which all facilities designed and built in the University Systems of Georgia system must adhere. However, additional considerations, based on the risk assessments of the biological, chemical and radiological materials agents to be used in the
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facility and anticipated operations should be incorporated into any laboratory construction or renovation project during the design phase of the project.
Animal facilities, in particular, present significant design and construction challenges. Small disruptions (loud or unusual noises, odors, illumination, etc.) can greatly impact animal research operations. From a biosafety perspective, special considerations should be made in determining the locations of these facilities, as well as the equipment and mechanical, electrical and plumbing (MEP) features in and around these facilities. These design issues can be particularly challenging for architects and engineers unfamiliar with these issues.
In addition, considerations should be made during design to ensure that the facility can be properly maintained without disruption of operations which may lead to inadvertent hazardous material exposure of maintenance personnel, laboratory personnel, ongoing research activities (including research animals), the environment or the surrounding community. Proper facility operation and maintenance is key to maintaining containment in any laboratory.
Some general biosafety considerations for design and operations of facilities are discussed below.
4.3.1 Physical Separation of Laboratory Spaces from Non-Laboratory Spaces A physical barrier must exist which distinguishes “laboratory areas” from “non-laboratory areas”. Basically, a room is either considered a laboratory or it is not. Laboratory areas are where hazardous materials may be handled. “Non-laboratory” areas may include offices, hallway areas, kitchens, or library, where unauthorized personnel (i.e., those who have not been properly informed of the hazards and provided precautionary medical screening) may be present and in which activities not permitted within the laboratory (e.g., food or drink consumption/storage, storage of utensils for food/drink consumption, application of cosmetics, gum chewing, etc.) are permitted.
All laboratories require floor-to-ceiling physical separation between these two areas. Cubicles, study carrels and/or artificially demarcated areas (e.g., taped-off areas) physically located within a laboratory do not satisfy the physical separation criteria. Therefore, all restrictions within laboratories (e.g., food/drink consumption and storage restrictions) apply to all desks physically located within laboratories which do not have floor-to-ceiling separation from the laboratory space. During laboratory design, accommodation for the needs of the laboratory research staff, particularly for separated areas for storage and consumption of food or drink, should be considered.
Also, consider that gloves must be removed and hands must be washed before exiting the laboratory areas, so hand-washing facilities near the exit doors is a convenient feature that should be considered during design of facilities.
4.3.2 Doors and Locks Doors leading from laboratories into non-laboratory areas (e.g., offices, hallways) should be kept closed after entering or exiting from the laboratory to maintain the physical barrier between laboratory and non- laboratory areas. Doors to hallways, offices or other “non-laboratory” area (as discussed in Section 4.3.1) should be kept closed. If doors open inward into laboratory spaces, consideration should be made to include a window in the door to prevent collisions into personnel handling biological (or other) hazardous materials. Ventilation grilles are not permitted for laboratory doors.
Laboratory doors in BSL-2 facilities should be self-closing and have locks to provide security for/from the materials stored and handled within the facility. Animal facility doors which open to the exterior should not only be self-closing, but also self-locking. Doors to areas where infectious materials and/or animals are housed should open inward and should be kept closed when experimental animals are present. These doors should never be propped open. Doors to cubicles inside of animal rooms may open outward or slide horizontally or vertically.
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4.3.3 Laboratory and Biological Material Security Measures should be taken to secure biological materials within the laboratory to prevent inadvertent exposure of personnel who have not been informed of the risks and received appropriate health screening/vaccinations, theft or inadvertent release. Whenever possible, accommodations for storage of higher risk agents (≥ Risk Group 2 and biological toxins) should be made within the most secure area of the PI’s laboratory rather than in common or shared storage rooms.
If materials must be stored in common/shared facilities, additional measures to secure the materials may be required, including freezer/cryotank locks or lockboxes within refrigerators or freezers.
Security for any Select Agent or Toxin (See Section 2.6.2.2) is even more critical and is required by Federal Law.
4.3.4 Windows Laboratory windows that open to the exterior are strongly discouraged. However, if a laboratory does have windows that open to the exterior, they must be fitted with screens.
In animal facilities, external windows are not recommended, since the presence of windows may impact facility security. Exterior windows in animal facilities should therefore be assessed by security personnel. However, if windows are present, they must be resistant to breakage, and where possible, sealed.
4.3.5 Floor Coverings, Walls and Ceilings Laboratory facilities in which biological materials are housed should be designed to be easily cleaned and decontaminated. Since organic material, including cloth and unvarnished wood, and porous materials are virtually impossible to properly disinfect, the use of these materials in construction or furnishing biological laboratories is discouraged. Carpets and rugs in laboratories are not permitted in any laboratory.
In animal facilities, all interior surfaces (walls, floors and ceilings) need to be assessed for cleanability. These surfaces need to be water-resistant, and floors must be slip-resistant, impervious to liquids and resistant to chemicals. Penetrations in floors, walls and ceiling surfaces should be sealed, to include openings around ducts, doors and door frames, to facilitate pest control and proper cleaning. Ceilings should be smooth, impervious to moisture, able to withstand cleaning with detergents and chemical disinfectants and be free of imperfect junctions.
4.3.6 Furnishings All furnishings within any laboratory must be capable of supporting anticipated work loads and uses and should be easily cleaned and disinfected. Since organic material, including cloth or unvarnished wood, and porous materials, such as unsealed cement, are virtually impossible to properly disinfect, the use of these materials in construction or furnishing biological laboratories is discouraged.
Chairs must be covered with a non-porous material that can be easily cleaned and decontaminated with an appropriate disinfectant. Sharp edges and corners should be avoided.
Spaces between benches, cabinets, and equipment should be accessible for cleaning. Storage of cardboard boxes, particularly on the floor, is discouraged because this may impede cleaning and disinfection efforts. In addition, cardboard may harbor insects or may become moldy (particularly when used in damp areas, such as cold rooms).
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In BSL-2 and ABSL-2 facilities, a method for decontaminating all laboratory wastes should be available in the facility (e.g., autoclave, chemical disinfection, incineration, or other validated decontamination method). No plants or animals which are not associated with the experiments in the laboratory are permitted in BSL-2 laboratories.
4.3.7 Lab Benches/Cabinetry Again, because they must be durable and cleanable, bench tops and cabinets in all laboratories must be impervious to water and resistant to heat, organic solvents, acids, alkalis, and other chemicals. Spaces between benches, cabinets, and equipment should be accessible for cleaning.
4.3.8 Exhaust Systems/Directional Airflow/HVAC Although there are no specific requirements on ventilation systems at BSL-1 or -2 facilities, the CDC/NIH recommends that in planning new facilities, considerations should be made in the mechanical ventilation systems to provide an inward flow of air without recirculation to spaces outside of the laboratory. This is often required by the Chemical Safety regulations, that are also often present in GHSUGRU laboratories.
In addition, any specific ventilation requirements related to the proposed use or equipment in the laboratory should also be addressed during laboratory design (e.g., compressed gas tanks, particularly tanks containing compressed cryogenic liquids, are permitted only in well-ventilated areas due to the potential asphyxiation risk associated with the rapid displacement of oxygen within an enclosed spaces).
Hazardous components of exhausted laboratory air can potentially contaminate the environment or inadvertently expose personnel (particularly maintenance personnel, who are more likely to be working on the ventilation systems on rooftops, etc.). This should also be considered when designing the exhaust system of these facilities. Stacked exhaust systems which expel the discharged air at high velocities and/or HEPA filtration should be considered based on the risks present.
Animal facilities have stricter requirements for ventilation to comply with the Guide for Care and Use of Laboratory Animals. No recirculation of exhaust air should occur in any animal facility and should be discharged to the outside without being recirculated to other rooms. While it is recommended that all animal rooms have inward directional airflow compared to adjoining hallways or rooms; this is required for compliance with ≥ABSL-2 standards. Ducted exhaust air ventilation systems are also an ABSL-2 standard. Ventilation system design should consider the heat and high moisture load produced during the cleaning of animal rooms, and especially the cage washing areas.
4.3.9 Plumbing/Eyewashes/Showers Handwash sinks should be present in each laboratory room to enable personnel to wash their hands just prior to exiting from the facility. In BSL-1 and BSL-2 containment laboratories, the sink may have a manual, hands-free or automated operation. The location of the handwash sink should be near the exit door, and additional sinks for hand-washing should be located in other appropriate locations within the facility, as well. For instance, if an animal facility has segregated areas where infectious materials and/or animals are housed or manipulated, a sink must also be available for hand washing at the exit from each segregated area.
Emergency safety showers are required in all animal facilities and within a 10 second walking distance from any location within a GHSUGRU laboratory, since hazardous chemicals are often present in the laboratories, in addition to biological materials.
BSL-2 laboratories and all animal facilities must also have eyewashes. GHSUGRU uses only plumbed twin-stream (binocular) eyewashes to enable compliance with ANSI standards for eyewashing. Contact the EHOS department at x1-2663 for recommendations of makes and models of eyewashes. Eyewashes should
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be flushed out regularly (weekly is recommended) by laboratory staff to prevent the growth of microorganisms within the water lines, which could expose personnel to further mucous membrane damage during emergency eyewash use.
Sink or floor drain traps are filled with water, and/or appropriate liquid to prevent the migration of vermin and gases into the laboratories. If floor drains are provided, the traps are filled with water, and/or appropriate disinfectant to prevent the migration of vermin and gases.
4.3.10 Biosafety Cabinets BSCs must be installed so that fluctuations of the room air supply and exhaust do not interfere with proper operations. BSCs should be located away from doors, windows that can be opened, heavily traveled laboratory areas, and other possible airflow disruptions.
HEPA filtered exhaust air from a Class II BSC can be safely re-circulated back into the laboratory environment if the cabinet is tested and certified at least annually and operated according to manufacturer’s recommendations.
BSCs can also be connected to the laboratory exhaust system by either a thimble (canopy) connection (e.g., a Class IIA2 BSC) or a direct (hard) connection (e.g., a Class IIB2 BSC). Provisions to assure proper safety cabinet performance and air system operation must be made and verified. All BSCs should be used according to manufacturer’s recommendation, to protect the worker and avoid creating a hazardous environment from volatile chemicals and gases. The choice of the most appropriate Class II BSC/exhaust system for the intended uses in the facility will likely hinge on anticipated use of volatile chemicals and radiological materials within the BSC. If the BSC is unducted, no work with radiological or volatile chemicals is permitted in the BSC; minute amounts are permitted in thimble-connected Class IIA2 BSCs; small amounts are permitted in hard-ducted Class IIB2 BSCs. However, laboratory designers are cautioned to consider the differences in the mechanical, electrical and HVAC demands of the different BSCs. Class IIB2 BSCs require significantly more static air pressure and energy to function than thimble-connected Class IIA2 BSCs. In addition, because the exhaust of the Class IIB2 BSCs is intricately linked to the HVAC exhaust system to which it is connected, they may be more prone to failure if exhaust fluctuations occur. Accurate specifications should be obtained and incorporated prior to final design.
4.3.11 Lighting, Air Ducts, Utility Pipes Adequate illumination is required for all activities in the laboratory. Care should be taken to avoid reflections and glare that could impede vision.
To facilitate cleaning efforts, all internal facility appurtenances, such as light fixtures, air ducts, and utility pipes, should be arranged to minimize horizontal surface areas to facilitate cleaning and minimize the accumulation of debris or fomites. This is a requirement of animal facility design.
Air ducts should not be located over lab benches, but should be positioned over aisles so airflow readings may be taken periodically to monitor the laboratory’s HVAC performance and air balance.
4.3.12 Additional Animal Facility Considerations Laboratory animal facilities pose unique challenges to balance biosafety and animal care and use issues. In general laboratory animal facilities requirements can be found in the Guide for the Care and Use of Laboratory Animals and Laboratory Animal Welfare Regulations; however, assessments should also be made based on biosafety, occupational health and safety risks to address potential hazards associated with the conduct of laboratory animal research.
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Facilities for laboratory animals used in studies of infectious or non-infectious disease should be physically separate from other activities such as animal production and quarantine, clinical laboratories, and especially from facilities providing patient care. Traffic flow that will minimize the risk of cross contamination should be incorporated into the facility design. Animal areas should be as restricted as possible. Special containment equipment or facility design may be required as determined by appropriate risk assessment.
In addition, provisions should be made for animal-specific operations. At ABSL-1, cages may be washed manually or preferably in a mechanical cage washer. At ABSL-2 containment, the cages should first be autoclaved or otherwise decontaminated prior to washing using a mechanical cage washer. The mechanical cage washer should have a final rinse temperature of at least 180°F. The cage wash area should also be designed to accommodate the use of high pressure spray systems, humidity, strong chemical disinfectants and 180°F water temperatures, during the cage/equipment cleaning process.
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5. MEDICAL SURVEILLANCE PROGRAM
All personnel who are authorized to work with biological materials in the execution of GRU’s research or educational missions are required to have access to Employee/Occupational health services.
The purpose of an Occupational Health program is to conduct periodic health assessments of personnel handling biological materials with particular attention devoted to factors or conditions associated with a particular biological agent a given individual might handle. For a particular person, the medical surveillance program might call for any of a number of precautionary measures, including immunizations, a periodic physical examination and collection of a serum specimen. Each laboratory should have the requirements for Occupational Health Screening and vaccinations as part of their Standard Operating Procedures. The purpose of the medical surveillance program is to:
recommend appropriate medical precautions to be followed, and
do periodic reassessment of employees to determine if medical conditions associated with employment are prevalent and, if so, to undertake definitive measures to alleviate them
The extent of medical surveillance for a given employee will vary greatly and be dependent upon:
the nature of the research project in which involved, the biological agents to which directly or potentially exposed, and certain additional factors relating to the current or previous health status of the individual
The CDC BMBL standard for Biosafety Level 2 places the responsibility on the laboratory supervisor to provide Occupational Health services and education pertaining to their health risks while working as indicated: “The laboratory supervisor must ensure that laboratory personnel receive appropriate training regarding their duties, the necessary precautions to prevent exposures, and exposure evaluation procedures. Personnel must receive annual updates or additional training when procedural or policy changes occur. Personal health status may impact an individual’s susceptibility to infection, ability to receive immunizations or prophylactic interventions. Therefore, all laboratory personnel and particularly women of child-bearing age should be provided with information regarding immune competence and conditions that may predispose them to infection. Individuals having these conditions should be encouraged to self- identify to the institution’s healthcare provider for appropriate counseling and guidance.” (CDC BMBL, Biosafety Level 2, A. Standard Microbiological Practices, 11: http://www.cdc.gov/biosafety/publications/bmbl5/BMBL5_sect_IV.pdf )
Occupational Health services are also a requirement of the OSHA Bloodborne pathogen standard (http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_table=STANDARDS&p_id=10051) 29 CFR 1910.1030 and maintain health records on all employees with potential occupational exposure to Bloodborne pathogens or other potentially infectious material.
GRU policies for seeking any appropriate health counseling and vaccination differ based on whether the laboratory personnel is an employee or a non-employee (which would include any student who is not an GRU employee). GRU contracts with the GRU Health, Inc. for Occupational Health services for GRU employees. A medical surveillance program should be offered by the employer to personnel engaged in biological research, which are conducted by the Occupational Health office (x1-3418) located at room 1174 FG Building, which is at 1515 Pope Avenue (see map, below). Non-GRU employee students who may be working with biological materials in the course of the education should seek health counseling and surveillance services with the Student Health Office (x1-3487) which is at 1040 AF (Pavilion II) Building off Laney Walker Blvd (see map, below). It is beholden upon GRU to offer any personnel who are not GRU employees (volunteers, visitors) who may be working with biological materials to further the GRU research or educational mission while at GRU the appropriate Occupational Health services prior to
Georgia Regents University Biosafety Guide- January 2012 5-1 authorizing them to work within the laboratory or clinic, or subsequent to an incident, exposure or accident involving biological materials. It is the responsibility of the Principal Investigator, Clinic Director and/or Instructional Course Director to map out how these personnel will receive the appropriate health counseling, vaccination and post-exposure follow-up health care as part of the laboratory SOPs
Occupational Health screening and medical surveillance should be provided without charge for any GRU employee whose job may result in potential exposure. As GRU has no university-wide health program, these costs are typically incurred at the departmental level. Individuals seeking Occupational Health Services should be provided by the authorized Occupational Health IDR for services prior to their appointment.
5.1 TUBERCULOSIS (TB) SCREENING Personnel who face occupational exposure to Tuberculosis (TB) should be enrolled in the University’s Tuberculosis Exposure Control Plan. The Occupational Safety and Health Administration has identified workers from the following areas as potentially exposed:
Laboratories that may handle M. tuberculosis or be exposed to patients or specimens from patients with tuberculosis Healthcare facilities Long term care facilities Correctional facilities Homeless shelters Substance abuse treatment facilities
Georgia Regents University Biosafety Guide- January 2012 5-2 New employees at risk must be tested for TB exposure by a tuberculin skin test (PPD) at time of hire (within 2 weeks of start date) to establish a baseline. All employees at risk must be PPD tested on an annual basis. Students who may encounter TB exposure as part of their educational operations should follow similar practices with the Student Health Services.
GRU personnel who have been exposed to active TB cases in the course of accomplishing their GRU- research or educational duties must report the incident and undergo an initial baseline TB test at time of exposure and a follow up test at 3 months post exposure. Please contact Occupational Health (x1-3418) or Student Health (x1-3487) to arrange for PPD testing. Contact the Biosafety Office in the Office of Environmental Health and Safety at x1-2663 or [email protected] for information on TB training.
In addition, any personnel working with non-human primates should be carefully monitored for potential exposures to tuberculosis. A TB infection causes an extremely rapidly fatal pneumonia in most Old World primate species which can devastate an entire NHP colony, while it typically manifests as chronic pneumonia in humans. Typically, infected humans present a much greater risk to the animals than animals do to humans. It is unusual for the disease to be transmitted to humans, unless the animal is undergoing surgery or pathologic examination. Infected tissue samples can also present a risk to laboratory workers. Prevention of disease includes routine use of respiratory protection and protective clothing when working with tissues or when coming into close proximity to animals. Animals and human handlers should be screened every 6 months for disease. Animals should also be quarantined and screened on entry into the facility.
5.2 IMMUNIZATIONS In certain situations, personnel engaged in particular research or educational activities would be immunized with appropriate vaccines, such as rabies, rubella and measles. Further information about adult vaccines can be found at the following URL: http://www.cdc.gov/vaccines/vpd-vac/adult-vpd.htm and the recommended schedule for adult vaccinations and boosters can be found at the following file: http://www.cdc.gov/vaccines/recs/schedules/adult-schedule.htm#print . Vaccines not commonly available will be obtained, whenever possible, for those engaged in specific research with potential exposure to the agent in question.
Vaccine Recommendations
Rabies Vaccine Recommended for all personnel entering laboratories or animal facilities with rabies vaccination entrance requirements.
Hepatitis B Vaccine Recommended for persons working with patients in a health care setting or persons working with human blood, body fluids, tissues or cell lines derived from human materials. MMR (Measles, Mumps, Rubella) Recommended for persons working with patients in a health care setting or persons working with human blood, body fluids, tissues or cell lines derived from human materials. Td (Tetanus, diphtheria) or TDap (Tetanus, Recommended for persons working with patients in a health care Diphtheria, Pertussis) setting or persons working with human blood, body fluids, tissues or cell lines derived from human materials.
Current tetanus vaccination is required of all those who work with laboratory animals, including researchers and animal caretakers. In addition, anyone whose job responsibilities associated with an animal facility may place them at risk of exposure to tetanus. Influenza Recommended for persons working with patients in a health care setting or persons working with human blood, body fluids, tissues or cell lines derived from human materials.
Georgia Regents University Biosafety Guide- January 2012 5-3 Varicella Zoster Vaccine Recommended for persons who have not previously had varicella zoster infection (i.e. chicken pox and/or shingles) who work with patients in a health care setting or persons working with human blood, body fluids, tissues or cell lines derived from human materials. Vaccinia (Smallpox) Vaccine Prior to working with vaccinia, employees are required to receive an independent medical counseling session from the Occupational Health regarding vaccinia immunization to discuss the risks and benefits of vaccinia vaccination. See 5.2.1, below, for further information. Contact the Biosafety Office (x1-2663 or [email protected]) to make arrangements.
In cases where infected animals are not housed in filter-top cages or other primary containment devices, vaccination shall be required for room entry. Arboviruses: Eastern and Western Equine Prior to working with arboviruses, employees are required to receive a Encephalitis Vaccines, Japanese Encephalitis medical evaluation and counseling from Occupational Health regarding Vaccine, Venezuelan Equine Encephalitis possible immunization. Vaccine, Yellow Fever Vaccine, Rift Valley Fever Vaccine
Lyme Disease Vaccine Recommended for persons working with the Lyme Disease agent or vectors in research laboratories, with animals, or in fieldwork.
Other vaccines such as Salmonella typhi To be determined by the Occupational Health Physician. (Typhoid),
In some cases, appropriate follow-up serum samples will be collected at periodic intervals to measure vaccine-induced antibodies when indicated.
5.2.1 Vaccinia Virus Use and Immunization Recommendations Recombinant vaccinia and other pox viruses are useful microbiological research tools for expression of exogenous proteins in a variety of cultured cell types. However, their use is not without risk to laboratory personnel. Naturally or experimentally infected laboratory animals are a potential source of infection to exposed unvaccinated laboratory personnel. Genetically engineered recombinant vaccinia viruses pose an additional potential risk to laboratory personnel, through direct contact or contact with clinical materials from infected volunteers or animals. Infectious vaccinia virus particles may be present in lesion fluids or crusts, respiratory secretions, or tissues of infected hosts and could lead to secondary infections of other individuals who may come in contact with these materials, such as family members of the laboratory personnel. Ingestion, parenteral inoculation, and droplet or aerosol exposure of mucous membranes or broken skin with infectious fluids or tissues, are the primary hazards to laboratory and animal care personnel. Serious ocular infections have been known to occur even in vaccinated individuals, see:
Lewis et al.(2006) Ocular vaccinia infection in a laboratory worker, Philadelphia, 2004. Emerging Infectious Diseases. 12(1): 134-7 Ruben FL & Lane JM. (1970) Ocular Vaccinia. Archives of Ophthalmology 84: 45-48.
Because of these risks, the IBC will expect that all manipulations of vaccinia strains be performed at BSL-2 containment in a biosafety cabinet. If work must be performed outside of a biosafety cabinet (e.g. animal surgery, microscopy), the following personal protective equipment must be used: gloves lab coat eye and mucous membrane protection [safety glasses or goggles which are ANSI certified and surgical mask or face shield]
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Multiple strains of vaccinia virus exist with varying levels of virulence for humans and animals. Depending on the strain used, vaccinia virus presents varying levels of health risk to laboratory personnel. Strains that are highly attenuated are typically unable to replicate or replicate poorly in human cells. On the other hand, non-highly attenuated strains of vaccinia have the ability to replicate in human cells and thus pose a risk to humans. Risks include localized skin infections and more severe, disseminated reactions to which immunocompromized individuals may be more susceptible. However, vaccination against vaccinia poses certain health risks to the individuals, and there are several contraindications for vaccination. The risks and benefits of vaccination and exposure to vaccinia in the laboratory sett to the laboratory personnel and those in close contact to the laboratory personnel should be discussed in a health counseling session with the Occupational Health Office.
The following information and recommendations are based on national guidelines issued by the CDC in Vaccinia (Smallpox) Vaccine Recommendations of the Advisory Committee on Immunization Practices. MMWR, Recommendations and Reports, 50, 2001 (http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5010a1.htm ):
Because of the inherent risks in the vaccination process, vaccination will likely not be recommended by the IBC for those working with the following highly attenuated strains, depending on the risk assessment of the Biosafety Protocol: Highly Attenuated Strain Biosafety Derived from: Level * MVA 2 Vaccinia virus (Ankara) NYVAC 1 Vaccinia virus (Copenhagen) TROVAC 1 Fowlpox virus ALVAC 1 Canarypox virus *Keep in mind: the eventual recommended biosafety level may increase depending on the presence and characteristics of a foreign protein expressed by a recombinant vaccinia virus or other aspects of the proposed experiment, as assessed by the IBC during its review process.
These recommendations were based upon the following considerations: Laboratory personnel who work with highly attenuated strains of vaccinia virus (e.g., MVA and NYVAC) or who work with the Avipoxvirus strains ALVAC and TROVAC do not require routine vaccinia vaccination.
The Occupational Safety Health Board of NIH no longer requires vaccinia vaccination for personnel manipulating MVA or NYVAC in laboratories using only those strains.
The Recombinant DNA Advisory Committee of the NIH reduced the biosafety level of NYVAC, TROVAC and ALVAC to level 1 based on accumulated attenuation data and biological properties of these strains.
Although there is no formal surveillance system in place, there have not been any reports of laboratory-acquired infection resulting from exposure to any of the above highly attenuated strains or recombinant vaccines derived from these strains in the literature or to the CDC.
Appropriate biosafety guidelines and infection control procedures should always be observed when working with viral material even if vaccination is not indicated.
The IBC is likely to highly recommend vaccinia vaccination to laboratory workers who directly handle cultures or animals infected with non-highly attenuated vaccinia virus strains, recombinant vaccinia viruses derived from non-highly attenuated vaccinia strains, or other orthopox viruses that can infect humans. Although recommended, the IBC will not require the vaccination, particularly considering the number of contraindications associated with vaccinations. However, the risks of working with vaccinia
Georgia Regents University Biosafety Guide- January 2012 5-5 without vaccination should be fully communicated to the laboratory staff members. Anyone who has a contraindication for vaccination (including: eczema, atopic dermatitis, immunosuppression and pregnancy, or having household contacts with any of these conditions) should also consider these contraindications for work with non-highly attenuated strains of vaccinia or orthopox viruses capable of human cell infection.
Non-highly attenuated strains WR (Western Reserve, mouse neuroadapted derivative) NYCBOH (strain used in vaccinia vaccine) Copenhagen Temple of Heaven Lister Other orthopox viruses Cowpox, Monkeypox
The vaccine, Dryvax®, is a live virus preparation of vaccinia virus manufactured by Wyeth and distributed by the CDC. It is administered with a bifurcated needle using a multi-puncture technique. Vaccinia immunization results in high seroconversion. The resulting immunity should provide protection to recipients against infections resulting from uncontrolled, inadvertent inoculation by unusual routes (e.g., the eye) with a substantial dose of virus of higher or unknown pathogenicity. Individuals with certain conditions are more likely to experience severe side effects or complications from the vaccine and should not be vaccinated. Contraindications include eczema, atopic dermatitis, immunosuppression and pregnancy, or having household contacts with any of these conditions. Revaccination every 10 years is recommended for people working with non-highly attenuated vaccinia strains; more frequent revaccination may be required for more virulent orthopox viruses. Laboratory personnel not directly handling cultures of vaccinia or animals infected with vaccinia, but working in the same lab where non-highly attenuated strains are being used should be offered medical screening for potential contraindications to vaccinia exposure. Other health-care workers (such as physicians and nurses) whose contact with these viruses is limited to contaminated materials (for example, dressings), but who adhere to appropriate infection control measures, are probably at lower risk for inadvertent infection than laboratory workers. However, because a theoretical risk of infection exists, vaccination may be considered for this group. A summary of published case reports of laboratory-acquired vaccinia virus infections is available in: Byers K (2005) Biosafety Tips-Biosafety Issues in Laboratory Experiments Using Vaccinia Viral Vectors. Applied Biosafety, 10(2): 118-122
Based on these guidelines, laboratory personnel for whom vaccination is recommended must receive mandatory confidential medical counseling before beginning work with the virus. These individuals must be counseled on the risks and benefits of the vaccine and medically screened for contraindications to vaccinia exposure or vaccination. During the counseling session, the risks and benefits of vaccinia immunization will be discussed and concerns or questions will be addressed. Following medical consultation the individual will either: sign a vaccinia vaccination consent form and receive the vaccine. (This will require follow-up visits to monitor the vaccination site), OR sign a declination form.
IBC approval may occur before all personnel listed on the Request for Vaccinia Vaccination form has been counseled and vaccinated. It is the Principal Investigator's responsibility to ensure that all personnel working directly with vaccinia have received counseling. Evidence of medical counseling must be documented in the laboratory biosafety manual.
Additional information about the Smallpox vaccine is available through the Centers for Disease Control and Prevention web site: Smallpox Fact Sheet: http://www.bt.cdc.gov/agent/smallpox/vaccination/facts.asp Smallpox Vaccine: What You Need to Know: http://www.bt.cdc.gov/agent/smallpox/vaccination/vaccine.asp
Georgia Regents University Biosafety Guide- January 2012 5-6
5.3 RESPIRATORY PROTECTION PROGRAM As described in Section 4.2.1.2, Federal OSHA regulations (29 CFR 1910.134) require initial and annual training and fit-testing, and well as medical surveillance of all respirator wearers (http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_table=STANDARDS&p_id=12716). The purpose of a medical evaluation program is to determine if employees can tolerate the physiological burden associated with respirator use, including: the burden imposed by the respirator itself (e.g., its weight and breathing resistance during both normal operation and under conditions of filter, canister, or cartridge overload); musculoskeletal stress (e.g., when the respirator to be worn is a SCBA); limitations on auditory, visual, and olfactory sensations; and isolation from the workplace environment. Since certain jobs and workplace conditions in which a respirator is used can also impose a physiological burden on the user, the medical evaluation must also consider the following factors: type and weight of the respirator to be worn; duration and frequency of respirator use; expected physical work effort; use of protective clothing and equipment to be worn; and temperature and humidity extremes that may be encountered. This information must be provided to the physican or other licensed health care professional (PLHCP) before the PLHCP makes a recommendation regarding an employee's ability to use a respirator.
The medical evaluation is designed to identify general medical conditions that place employees who use respirators at risk of serious medical consequences. Medical conditions known to compromise an employee's ability to tolerate respirator-, job-, and workplace-related physiological stress include: cardiovascular and respiratory diseases (e.g., a history of high blood pressure, angina, heart attack, cardiac arrhythmias, stroke, asthma, chronic bronchitis, emphysema); reduced pulmonary function caused by other factors (e.g., smoking or prior exposure to respiratory hazards); neurological or musculoskeletal disorders (e.g., ringing in the ears, epilepsy, lower back pain); impaired sensory function (e.g., perforated ear drums, reduced or absent ability to smell); and psychological disorders (e.g., claustrophobia and severe anxiety).
If a respirator is to be used in the course of research with biological materials, the GRU IBC will require adherence to these OSHA standards as a stipulation of IBC approval, since these represent prudent precautionary safety measures.
Please contact the GRU Office of Environmental Health and Occupational Safety (EHOS) (x1-2663) whenever the use of a respirator is being considered. EHOS staff will assist users in: Proper selection of cartridges and respirators Guiding researchers in the process to meet the appropriate medical evaluation requirements (which typically involves documenting annual respirator medical evaluation through the Occupational Health office) Providing the required annual training and respirator fit testing
5.4 MEDICAL RESTRICTIONS As previously described in Section 3.2.1 Risk Groups: Hazardous Characterizations of a Biological agent, all risk group and containment recommendations presented in the CDC/NIH BMBL, the NIH Guidelines, the WHO Biosafety Guidelines, etc., are based on the risks to healthy human adults. These recommendations do not account for individual health considerations, such as allergies, pregnancy, breast feeding, medication effects, a compromised immune system (due to illnesses or medical treatments such as steroids or chemotherapy) or other illnesses which may make individuals more susceptible to agents.
For this reason, for their own safety, any individual with special health concerns is strongly encouraged to discuss these with the Principal Investigator, Clinical Director or Instructional Course Director prior to initiation of work within the laboratory. In turn, PIs, Clinical Directors or Instructions Course Directors should offer the opportunity for the individual to seek Occupational Health Counseling through the Occupational Health Office to discuss their potential individual special risks.
Georgia Regents University Biosafety Guide- January 2012 5-7 5.4.1 Pregnancy It is recognized that exposure to certain infectious agents may adversely affect a fetus during pregnancy if the mother is infected with the agent. Therefore, if pregnancy is possible while you are working in an infectious disease laboratory or laboratory engaged in work with infectious agents you should consult your Principal Investigator or supervisor. The Occupational Health or Student Health Offices should also be made available for answering questions regarding the potential harm from the biological agents present within your laboratory.
Women who are pregnant or become pregnant are encouraged to inform their supervisors, Principal Investigators, Clinic Directors and/or Instructional Course Directors, who should, in turn, encourage them to seek appropriate health counseling through the Employee or Student Health Offices. Personnel are urged to discuss exposure issues with their supervisors or principal investigators regarding associated risks of research being conducted and pregnancy. Employee/Student Health will give advice about precautions that might be necessary.
The Occupational Health and Student Health Offices are resources for pregnant women to ask about any questions or concerns they may have regarding risks in their work environment. The Occupational Health and Student Health Offices may require additional information about the agents and on-going operations within the laboratory beyond what the laboratory personnel is able to offer. The Employee or Student Health Office may need to discuss these matters with the Principal Investigator, Clinic Director or Instructional Course Director, or they may contact the Biosafety Office (x1-2663 or [email protected]) to discuss the agents and operations documented in the laboratory’s Biosafety Protocol. The Occupational Health or Student Health Office may also act as a liaison between pregnant laboratory personnel and their respective supervisors or principal investigators. Please contact the Occupational Health (x1-3418) or Student Health Offices (x1-3487) for further information on reproductive and fetal pathogens.
5.4.1.1 Reproductive Biological Hazards The Occupational Health Physician will offer confidential counseling to any woman or man of childbearing age working with reproductive pathogens or other potentially infectious materials. Reproductive biological hazards include, but are not limited to the following:
Cytomegalovirus (CMV) Hepatitis B virus (HBV) Hepatitis E virus Human Immunodeficiency virus (HIV) Human parvovirus B19 Rubella (German Measles) Lymphocytic Choriomeningitis virus Toxoplasma gondii (Toxoplasmosis) Listeria monocytogenes Varicella-zoster virus (chicken pox) Coxiella burnetii (Q fever) Vaccinia virus
Whenever necessary, Occupational Health along with the Biosafety Office will offer an opportunity to review work procedures in the lab to ensure that potential exposure is minimized. Consideration for reassignment to other tasks that do not involve exposure to the reproductive hazard (generally with actual pathogens, not necessarily for only other potentially infectious materials such as blood or body fluids) should be given. Also, Principal Investigators actively working with reproductive hazards should explain these risks at the time of hire.
Georgia Regents University Biosafety Guide- January 2012 5-8 5.4.2 Work with Animals Occupational Health programs play an important part of both animal use regulations, as well as biosafety guidelines. Special hazards exist for workers who are exposed to animals, and therefore guidance is provided by the Institute for Laboratory Animal Research (ILAR) Commission on Life Sciences, National Research Council related to Occupational Health issues in:
Occupational Health and Safety in the Care of Research Animals http://www.nap.edu/catalog.php?record_id=4988
Occupational Health and Safety in the Care and Use of Nonhuman Primates http://www.nap.edu/catalog.php?record_id=10713#toc. Comment [L1]: Didn’t find an accurate link
5.4.3 Allergies
5.4.3.1 Allergies to Laboratory Animals (ALAs) Allergic reaction to animals is among the most common condition that adversely affects worker health. The estimated prevalence of allergic symptoms among workers exposed to animals is from 10% to 40%. Workers who are continually exposed to animal allergen tend to have progressively more frequent and severe symptoms, and an estimated 10% develop asthma. Studies have shown that about 50% of those with symptoms will eventually stop working with animals permanently or temporarily because of the discomfort involved in ALA.
Initial allergy symptoms are usually: Runny nose (allergic rhinitis), Itchy eyes (allergic conjunctivitis) Rashes (contact urticaria, atopy)
Symptoms usually evolve over a period of 1-2 years and may lead to acute anaphylaxis in a small number of patients.
Hence, it is critical that all workers seek to minimize their exposure to animal allergens and that Principal Investigators and Laboratory supervisors discuss these risks with their laboratory workers. Supervisors are also responsible to ensure that appropriate operating procedures have been established to prevent undue exposure of workers to animal allergens and provide Occupational Health/Student Health screening for workers who may be developing hypersensitivity to assess their risks of further work with animals. These risks should also be communicated to others in the laboratory to inform them of the potential consequences of exposure to their fellow laboratory staff member to prevent inadvertent exposure.
In rodents, the allergen protein is of urinary origin while in rabbits, the primary allergen is contained in the fur and dander and to a lesser degree in the saliva and urine. In guinea pigs, urine is the main allergen with dander, fur, and saliva contributing. The major cat allergen is produced in oil glands of the skin and coats the hair shafts. It is also present in saliva. Exposure to birds can cause rhinitis and asthma symptoms. Multiple bird proteins have been identified as allergens and can be found in serum and fecal droppings that contain serum. Fish proteins can be an inhalation allergen for those who are sensitized.
Prudent efforts to prevent allergen exposure and reduce the frequency of sensitization in animal workers require strict work practices and consistent use of PPE. Housing animals in filter-top cages, working in well-ventilated areas, and using ventilated hoods for soiled bedding disposal will minimize exposure to animal allergens.
The work area must be maintained clean to prevent inhalant and contact exposure. Procedures should be adopted that minimize release of airborne materials, including bedding dust and
Georgia Regents University Biosafety Guide- January 2012 5-9 antibiotic aerosols, and the contamination of hands, arms, body and face. Workers should adopt the use of PPE during each and every animal contact or allergen exposure.
Of particular importance is wearing a face mask to reduce inhalation and hand-to-face spread of allergens and covering all exposed skin (i.e. gloves, lab coat, sleeve protectors) to prevent allergen contact. In some cases, respirators may be recommended; in which case the employee must be enrolled in GRU’s respirator program, and undergo annual fit-testing as per OSHA standards (29 CFR 1910.134).
It is also important that once animal procedures are complete, all contaminated PPE and clothing are removed and properly disposed of to prevent repeated exposure while performing subsequent duties. Contact your supervisor, LAS supervisors or biosafety further information regarding PPE.
5.4.3.2 Latex Gloves and Related Allergies Allergic reactions to natural rubber latex have been increasing since 1987, when the Center for Disease Control recommended the use of universal precautions to protect against potentially infectious materials, bloodborne pathogens and HIV. Increased glove demand also resulted in higher levels of allergens due to changes in the manufacturing process. In additional to skin contact with the latex allergens, inhalation is another potential route of exposure. Latex proteins may be released into the air along with the powders used to lubricate the interior of the glove.
In June 1997, the National Institute of Occupational Safety and Health (NIOSH) issued an alert, “Preventing Allergic Reactions to Latex in the Workplace” (publication number DHHS (NIOSH) 97-135). The full text of this publication is available at the NIOSH web site, http://www.cdc.gov/niosh/topics/latex/.
NIOSH studies indicate that 8-12% of healthcare workers regularly exposed to latex are sensitized, compared to 1-6% of the general population. Latex exposure symptoms include skin rash and inflammation, respiratory irritation, asthma and shock. The amount of exposure needed to sensitize an individual to natural rubber latex is not known, but when exposures are reduced, sensitization decreases. NIOSH recommends the following actions to reduce exposure to latex:
If latex gloves must be used, choose reduced-protein, powder-free latex gloves Whenever possible, substitute another glove material (for instance, nitrile gloves) Wash hands with mild soap and water after removing latex gloves
5.4.3.3. Antibiotic Allergies Allergic reactions have been described to a large number of medicines, and those against antibiotics is one of the most common of these. Reactions to antibiotics can range from a rash or hives starting a few days after exposure to sudden onset of rashes, difficulty breathing, stomach upset and anaphylaxis soon after exposure. Because of the potential severity of these reactions, any personnel with known allergies to antibiotics should discuss their personal health risks in working in a laboratory with the Occupational Health or Student Health Offices.
Georgia Regents University Biosafety Guide- January 2012 5-10
In the laboratory setting, antibiotic allergies may impact the risks in two ways: 1. Should exposures occur to biological materials for which the medical treatment modality is administration of an antibiotic against which the laboratory personnel is allergic, an alternate post-exposure treatment plan should be made prior to exposure in conjunction with the Principal Investigator, Clinical Director and/or Instructional Course Director. These supervisors should be also be made aware of the potential for allergic reaction, so this can be communicated to health care providers in an emergency situation. 2. Biomedical laboratories often use antibiotics for research purposes. For instance, antibiotic selection is often used during culture operations in both microbiological and mammalian cell/tissue culture settings. The risks of exposure of allergic personnel to the antibiotic should be communicated to the Principal Investigator, Clinic Director and/or Class Instruction to: a. Enable development of any operating practices which would help limit the exposure any allergic personnel to the antibiotic. b. Enable communication of the exposure risks to others in the laboratory to consider the exposure risks to the allergic personnel.
5.4.3.4 Mold Allergies Some people are very sensitive to molds. For these people, exposure to molds can cause symptoms such as nasal stuffiness, eye irritation, wheezing, or skin irritation. Some people, such as those with serious allergies to molds, may have more severe reactions. Severe reactions may occur among workers exposed to large amounts of molds in occupational settings. Severe reactions may include fever and shortness of breath. Some people with chronic lung illnesses, such as obstructive lung disease, may develop mold infections in their lungs.
Molds can be found almost anywhere; they can grow on virtually any substance, providing moisture is present. There are molds that can grow on wood, paper, carpet, and foods. There is no practical way to eliminate all mold and mold spores in the indoor environment; the way to control indoor mold growth is to control moisture and humidity which may provide ideal conditions for mold growth. In buildings, conditions which can favor mold growth can include roof and plumbing leaks or floods, condensation, and excess humidity. In large buildings, mold and mildew are commonly found on the exterior wall surfaces of corner rooms in heating climate locations. In laboratories, mold is often found in areas where moist conditions are present and condensation is likely to occur such as cold rooms, inside refrigerators, or in thawed freezers or decommissioned warm rooms-- particularly when organic materials (like cardboard) are placed in these areas.
Because of the potential health consequences to personnel with mold allergies, care should be taken by all GRU personnel (i.e., not just laboratory staff), to take measures to prevent mold growth in GRU facilities. Prudent preventative measures include: Not storing organic materials which can serve as growth medium to mold in moist atmospheres. Do not store cardboard boxes in cold rooms, freezers or refrigerators. Inspect the building for signs of mold, moisture, leaks, or spills routinely. Clean up mold and eliminate sources of moisture as soon as possible. Fix the source of water problems or leaks to prevent mold growth. Reduce indoor humidity (to 30-60% ) to decrease mold growth by: venting bathrooms, dryers, and other moisture-generating sources to the outside; using air conditioners and de-humidifiers; increasing ventilation; and using exhaust fans whenever steam-producing operations occur (such as glassware or cage washing or shower facilities). Clean and dry any damp or wet building materials, floors/carpeting and furnishings within 24-48 hours to prevent mold growth. Clean mold off hard surfaces with water and detergent, and dry completely. Absorbent materials such as ceiling tiles, that are moldy, may need to be replaced.
Georgia Regents University Biosafety Guide- January 2012 5-11 Prevent condensation: Reduce the potential for condensation on cold surfaces (i.e., windows, piping, exterior walls, roof, or floors) by adding insulation. In areas where there is a perpetual moisture problem, do not install carpeting (i.e., by drinking fountains, by sinks, or on concrete floors with leaks or frequent condensation).
Further information about mold in the indoor environment, please see the EPA Mold Resources web page, at: http://www.epa.gov/mold/moldresources.html.
5.4.4 Other Restrictions Restrictions or recommendations will be made on an individual basis after discussion with the Occupational Health Physician and the employee's personal medical doctor. Examples of conditions that might warrant special precautions are HIV infection, immunosuppressive conditions and drug therapy that suppresses the immune system (e.g. steroid therapy). Therefore, if you are suffering from any of the above conditions, you must inform your physician and the Occupational Health or Student Health Offices about the situation.
5.5 MINORS IN LABORATORIES AND OTHER HAZARDOUS AREAS All risk group and containment recommendations presented in standard Biosafety Guidelines (CDC/NIH BMBL, the NIH Guidelines, the WHO Biosafety Guidelines, etc.), are based on the risks to healthy human adults and do not account for differential effects on immature bodies/systems. Persons under the age of 18 are prohibited from entering laboratory areas or other areas where hazardous materials or conditions may be present except when such entry is a pre-authorized, escorted, scheduled open house tour. During these times, no work with Risk Group ≥2 organisms or recombinant DNA will be permitted and the facility should be decontaminated to prevent inadvertent exposure to minors to any residual hazardous material while visiting.
Because of their biological, social, and economic characteristics, young workers have unique and substantial risks for work-related injuries and illnesses. For this reason, both the Federal and State Departments of Labor has issued special requirements for work of minors under the age of 18 years old http://www.dol.gov/dol/cfr/Title_29/. These laws specifically generally restrict minors under the age of 18 from working in hazardous areas http://www.dol.gov/dol/topic/youthlabor/hazardousjobs.htm, and specifically restrict any minor under the age of 18 from working in areas where this is exposure to radiation or storage/manufacture of explosives. CDC/NIOSH has also issued some recommendations for protecting the health and safety of minors while in occupational settings (see: http://www.cdc.gov/niosh/topics/youth/ and http://www.cdc.gov/niosh/docs/NIOSHRecsDOLHaz/default.html).
By definition, any laboratory is a potentially hazardous area, whether these hazards are presented by biological materials, chemicals, or radioactive substances. Few studies exist which adequately characterize potential differential health consequences of exposure of minors to these hazards. While the Biosafety Office recognizes that paid or volunteer employment within a laboratory can be valuable learning experience, minors under the age of 18 years should be restricted from working in these areas, unless provisions can be made and followed to address the following concerns:
Federal law explicitly prohibits any minor under the age of 18 from working in areas where exposure to radiation may occur or where explosive materials may be stored or used. This would limit the GRU laboratories in which a minor would be permitted to enter.
Risk communication and the comprehension of those risks and mitigation methods is a crucial safety measure within the biomedical laboratory. This may require a certain level of education background and maturity to understand. General biosafety training modules assume a base level of comprehension of biological and chemical concepts, and the Biosafety Office would not be
Georgia Regents University Biosafety Guide- January 2012 5-12 equipped to provide special training sufficient to compensate for the lack of education and experience.
Signed liability waivers, normally obtained from parents and guardians to permit minors to enter laboratories require that the parent or guardian be fully informed of the risks faced by their child in these areas. Again, the amount of education/training required to bring parents or guardians of minors who wish to work in a laboratory may greatly vary, and cannot be guaranteed.
Adequate supervision of the minor worker within the laboratory would have to be ensured. This means all activities engaged by the minor within the laboratory would have to be monitored at all times, which requires the physical proximity (i.e., within the supervisor’s line-of-sight) and attentiveness of the supervisor at all times while the minor is within the laboratory. This level of supervision is difficult to guarantee within the busy working laboratory.
Appropriate occupational health screening and vaccinations would be required of all personnel within the laboratory. Some vaccinations require multiple administrations over time, and completion of the vaccination series may not be possible within the time frame for the minor’s employment/volunteer opportunity. In addition, special health care provisions would have to be made for the minor in case of incident or potential exposure or illnesses which may have resulted from a potential exposure. If the minor is in the laboratory on a volunteer basis, and is not a GRU student, special provisions with the Occupational Health and Student Health Offices may be required to address the minor’s occupational health issues.
5.6 EMPLOYEE SERUM STORAGE Many infections do not result in an overt disease condition. Such infections are detected by development of antibodies to the agent in question. Therefore, Occupational Health may need to establish a program for persons engaged in BL3 research or working with non-human primates, which includes collection of pre- assignment serum. If an illness occurs which may be related to the agent the person is working with, additional serum samples may need to be collected.
Georgia Regents University Biosafety Guide- January 2012 5-13 6. ACCIDENTS, EXPOSURES, SPILL RESPONSE
Laboratory-specific SOPs should address any emergency response procedures, including those required if an accident, exposure, potential exposure, an illness which may have resulted from a possible laboratory exposure, release from primary containment or environmental contamination of any biologically hazardous material. Guidelines for the development of these SOPs are discussed below.
6.1 EMERGENCY PROCEDURES FOR EXPOSURE INCIDENTS An "exposure incident" is specific contact (eye, mouth, other mucous membrane, respiratory tract via inhalation, non-intact skin, or parenteral) with potentially infectious materials that results from the performance of an employee's duties or commission of the person’s responsibilities in conjunction with the research or educational mission of GRU. A person who sustains a known or potential exposure incident must remove their gloves and treat the affected area immediately by following the appropriate exposure incident response below.
6.1.1 Percutaneous Injury Wash the affected area with antiseptic soap and warm water for 15 minutes.
6.1.2 Splash to Face Flush affected area in eyewash continuously for 15 minutes.
6.1.3 Aerosol Exposure Do not inhale and immediately leave room. Remove Personal Protective Equipment (PPE) carefully. When removing PPE make sure to turn the exposed areas inward. Wash hands well with soap and water. Post a spill sign on the laboratory entry doors to prevent others from entering. The laboratory should remain evacuated for at least 30 minutes to allow for the droplets to settle and/or aerosols to be purged by the air exchange rate within the laboratory.
The PI must clear the laboratory before re-entry and spill clean-up to commence. For extensive BL2 contamination (i.e. an incident involving a centrifuge) or incidents involving BL2+ or BL3 agents, the Biosafety Office must be notified immediately (x1-2663) and will assume responsibility, in conjunction with the PI, to clear the laboratory for re-entry.
6.2 REPORTING INCIDENT The employee must report the incident to his/her supervisor. Because the health and safety issues of the injured personnel is of primary importance, if the injury is emergent, the supervisor should take (or make arrangements for the injured person to be taken) to the nearest emergency room and reporting requirements should be completed post-hoc.
The supervisor should take measures to ensure that additional personnel are restricted from areas to prevent further inadvertent exposures. Any incident, accident, exposure, possible exposure, illness which may have resulted from exposure, releases from primary containment or environmental contamination involving biological materials which occurred in the course of accomplishing the research and/or educational missions of the university should be reported as soon as possible to the GRU Biosafety Office (x1-2663 or [email protected]). It is the responsibility of the Biosafety Office to assist Principal Investigators, Clinical Directors and/or Instructional Course Directors in completion of any funding agency-specific, public health or environmental safety reporting requirements and to review the incident
Georgia Regents University Biosafety Guide- January 2012 6-1 with the supervisor and the injured person to discuss whether alterations in the laboratory’s or institution’s Standard Operating Procedures would be prudent to prevent future similar occurrences.
Injured personnel and their supervisors should also keep in mind that the IBC and Biosafety Office are primarily concerned with ensuring that the appropriate prevention, treatment and post-exposure follow-up measures are implemented to ensure the health and safety of the exposed personnel and the environment and prevention of future incidents. Other GRU offices may require the injured person and/or their supervisor to report incidents, accidents or exposures for other purposes, such as completion of GA State Workman’s Compensation requirements and/or to maintain campus safety statistics. Therefore, it is recommended that PIs, Clinic Directors and Instructional Course Directors review these requirements and incorporate these additional reporting requirements in any laboratory SOP to assist laboratory personnel in completion of all reporting requirements. Please see the following GRU Policies and Procedures for guidance:
For GRU Employees: GRU Policy & Procedure 1.4.17: http://www.georgiahealth.edu/policies/pdf/1417.pdf For Persons who are not GRU employees (including students): GRU Policy & Procedure 3.0.03: http://www.georgiahealth.edu/policies/pdf/3003.pdf
For quick reference, here is some contact information and URLs of some reporting forms that may be required as part of development of laboratory reporting SOPs for compliance with these other GRU policies. (These are provided merely to assist PIs, Clinic Directors and Instructional Course Designers in the development of their post-exposure follow-up laboratory SOPs): • GA State Department of Administrative Services (DOAS): o Phone number: 1-877-656-7475 o Pertinent forms may include: • WC-1 form Employer's First Report of Injury http://www.caes.uga.edu/intranet/coextopr/pdfs/wcform.pdf • DOAS “Incident Notice Only” form: http://doas.ga.gov/StateLocal/Risk/DOCS_Risk/Incident_Notice.pdf
• GRU Human Resources Benefits Section o Phone number: x1-3770 o Fax: x1-1572 o Location: HS-1111, Annex Building o Pertinent forms may include: • GRU Human Resources Employee’s Report of Accident/Injury form (MC-419) http://policy.georgiahealth.edu/2010/08/24/accident-injury- reporting-policy/
• GRU Public Safety Office x1-2911
6.3 MEDICAL ASSISTANCE In non-emergent cases, employees are urged to follow the medical assistance instructions provided to them by the DOAS and their health care provider after they have received proper available first aid at site of exposure. For certain exposures such as non-human primate bites, scratches or splashes, tick or insect bites, or exposure to infectious agents, the employee will be advised to be evaluated by the Occupational Health Office. In situations when Occupational Health is not available or if more extensive treatment is required, the employee will likely be referred to the nearest Emergency Room, and asked to follow up with visit(s) with the Occupational Health Office (see Section 5, Medical Surveillance for a map showing nearby health facility locations).
Personnel who have been potentially exposed on the job will be provided with post-exposure evaluation and follow-up at no cost to employees who experience "exposure incidents". The post-exposure
Georgia Regents University Biosafety Guide- January 2012 6-2 monitoring periods are dependent on the type of exposure. This time period is related to the various incubation periods of the infectious agents.
Employees can obtain copies of their Occupational Health records by contacting the GRUHS Occupational Health Office at FG-1174 (x1-3418). GRU must retain medical records for your duration of employment plus 30 years.
6.4 SPILL CLEAN-UP PROCEDURES This section is intended to outline the basic procedures for dealing with some of the biological spills that may be encountered in a research laboratory. All lab personnel should refer to the relevant spill response procedures before initiating their experiments.
6.4.1 Composition of a Basic Biohazard Spill Kit Microbiological and biomedical research laboratories should prepare and maintain a biological spill kit. A spill kit is an essential safety item for labs working with agents which require Biosafety Level 2 or higher containment and for groups working with large volumes (> 1 liter) under Biosafety Level 1 containment. The following items should be included in the spill kit:
Concentrated household bleach (< 1 year old) A spray bottle for making 10% bleach solutions Forceps, disposable broom/dust pan, and/or other mechanical devices for handling sharps or removing solid objects within the spill Paper towels or other suitable absorbent (diapers, disposable shop towels) Biohazard bags for the collection of contaminated spill clean-up items Utility gloves and medical examination gloves Face protection (eye wear and mask, or full face shield) Signage for warning others to avoid the area
Additional personal protective equipment, such as a disposable cover-gown or disposable booties may also be recommended equipment in some spill clean-up situations
Although household bleach is recommended as a standard disinfectant in the spill kit, other suitable disinfectants may be used provided the disinfectant is effective against the agents in use at the appropriate dilutions and contact time. Disinfectants should be registered with the Environmental Protection Agency as tuberculocidal for compliance with the Occupational Health and Safety Administration Bloodborne Pathogens Standard (29CFR 1910.1030).
Representatives from the Biosafety Office are available if you have any questions regarding biological spill response procedures or decontamination (x1-2663 or [email protected]).
6.4.2 Spills Occurring Outside of Laboratory Areas Because laboratory facilities are designed to contain hazards within the confines of the laboratory, where all who are likely to encounter the material have knowledge of the risks, any spill outside of the laboratory area poses a particular risk for exposure of the general public or environmental contamination. Therefore, the procedures for addressing a spill which occurs outside the confines of a laboratory are:
Attend to any injuries or exposures Alert others to avoid the area to prevent contamination of additional personnel and environment Contact the Biosafety Office (x1-2663) immediately to assist in spill clean-up. If after hours, contact Public Safety Office (x1-2911), who has the 24/7 on-call numbers for EHS staff members.
Georgia Regents University Biosafety Guide- January 2012 6-3
6.4.3 Spills within the Laboratory Areas Because laboratory facilities are designed to contain hazards within the confines of the laboratory, spills within the laboratory are generally not as potentially problematic as those which occur outside the laboratory. Of course, the overall risk will depend on the agents, operations and personnel involved in the spill and clean-up measures.
6.4.3.1 Biosafety Level 1 (BSL1) Spills
Immediate Procedures • Notify others in the area, to prevent contamination of additional personnel and environment. • Remove any contaminated clothing and wash exposed skin with soap and water.
Clean-up of BSL1 Spill • Wearing gloves, lab coat, and face protection, cover spill with paper towels, pour concentrated disinfectant around the spill allowing it to mix with spilled material. Allow suitable contact time, at least 15 min. • Pick up any pieces of broken glass with forceps or other mechanical device(s) (not with your hands!) and place in a sharps container. • Discard all disposable materials used to clean up the spill into a biohazard bag. • Wash hands with soap and water.
6.4.3.2 Biosafety Level 2 (BSL2) Spills
Immediate Procedures • Avoid inhaling airborne material, while quickly leaving the room. Notify others to leave. • Close door, and post with a warning sign. • Remove contaminated clothing, turning exposed areas inward, and place in a biohazard bag. • Wash all exposed skin with soap and water. • Inform Supervisor, and, if assistance is needed, consult the Biosafety Office (x1- 2663).
Clean-up of BL2 Spill • Allow aerosols to disperse for at least 30 minutes before reentering the laboratory. Assemble clean-up materials (disinfectant, paper towels, biohazard bags, and forceps). • Put on protective clothing (lab coat, face protection, utility gloves, and booties if necessary). • Depending on the nature of the spill, it may be advisable to wear a HEPA filtered respirator instead of a surgical mask. • Cover the area with disinfectant-soaked towels, and then carefully pour disinfectant around the spill. Avoid enlarging the contaminated area. Use more concentrated disinfectant as it is diluted by the spill. Allow at least a 30 minute contact time. • Pick up any sharp objects with forceps or other mechanical devices (not your hands!) and discard in a sharps container. • Soak up the disinfectant and spill using mechanical means, such as an autoclavable broom and dustpan, since there may be sharps under the paper towels, and place the materials into a sharps container. Smaller pieces of glass
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may be collected with cotton or paper towels held with forceps. If no sharps were involved in the spill discard the materials into an autoclave bag. • Wipe surrounding areas (where the spill may have splashed) with disinfectant. • Spray the area with freshly prepared 10% household bleach solution and allow to air-dry (or wipe down with disinfectant-soaked towels after a 30-minute contact time). • Place all contaminated paper towels and any contaminated protective clothing into a biohazard bag and autoclave or dispose in the appropriate Stericycle waste box. • Wash hands and exposed skin areas with soap and water.
6.4.3.3 Blood Spills For blood or other material with a high organic content and low concentration of infectious microorganisms: • Wear gloves, eye protection, and a lab coat. • Absorb blood with paper towels and place in a biohazard bag. Collect any sharp objects with forceps or other mechanical device and place in a sharps container. • Using a detergent solution, clean the spill site of all visible blood. • Spray the spill site with 10% household bleach and allow to air-dry for 30 minutes. • After the 30 minute contact time, wipe the area down with disinfectant-soaked paper towels. • Discard all disposable materials used to decontaminate the spill and any contaminated personal protective equipment into a biohazard bag. • Wash your hands with soap and water.
6.4.3.4 Spill in a Biological Safety Cabinet • Leave the biological safety cabinet blower on and begin cleanup immediately. • While wearing PPE (gloves and gown) cover the spill area with paper towels or disinfectant soaked paper towels. Do not place your head in the cabinet to clean the spill, keep your face behind the viewscreen. • If necessary, flood the work surface as well as the drain pans and catch basins below the work surface, with disinfectant. Be sure the drain valve is closed before flooding the area under the work surface. • Wipe cabinet walls, work surfaces, and inside the viewscreen with disinfectant. • Lift the front exhaust grill and work surface; wipe all surfaces with disinfectant. Be sure no paper towels or soiled debris are blown into the area under the spill tray • If the work surface, as well as drain pans and catch basins under the work surface, have been flooded with disinfectant soak up the disinfectant in the work surface. Place a container under the drain valve and drain the disinfectant under the work surface into the container. • Wipe the areas under the work surface to remove residual disinfectant. • Wash hands and exposed skin with soap and water. • Autoclave all cleanup materials and protective clothing. • Notify your PI or supervisor. • If the spill overflows the drain pan/catch basin under the wok surface into the interior of the biological safety cabinet notify the Biosafety Office (x1-2663). A more extensive decontamination of the biological safety cabinet may be required.
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6.4.3.5 Centrifuge Spill • Always use sealed safety safety-cap sealed buckets or sealed rotors with O-rings. Examine O-ring and replace if worn, cracking or missing. Check tubes and bottles for cracks and deformities before each use. • Wait five minutes before opening the centrifuge following the end of a run with potentially hazardous biological material if using safety caps or sealed rotors. If a spill is identified after the centrifuge lid is opened, carefully close the lid and evacuate the laboratory and close the laboratory door. Remain out of the laboratory for at least 30 minutes. Post a sign on the laboratory door indicating there is a biohazard spill and do not enter. • Remove any contaminated protective clothing and place into a biohazard bag. Wash hands and any exposed skin surfaces with soap and water. • Notify your supervisor and the Biosafety Office (x1-2663 or [email protected]).
After 30 minutes... • Enter the lab with personal protective equipment and spill cleanup materials. Full-face protection, lab coat and utility gloves should be worn. • Transfer rotors and buckets to a biological safety cabinet. Immerse rotor/buckets in 70% ethanol or a non-corrosive disinfectant effective against the agent in use. Allow at least a one hour contact time. Intact tubes may be wiped down and placed into a new container. Handle any broken glass with forceps and discard into a sharps container. • Carefully retrieve any broken glass from inside the centrifuge using forceps and discard into a sharps container. Smaller pieces of glass may be collected with cotton or paper towels held with forceps. Carefully wipe the inside of the centrifuge with disinfectant. • Spray the inside of the centrifuge with disinfectant and allow to air dry. If bleach is used, follow by wiping with 70% ethanol to remove any corrosive residues. • Place contaminated items and disposable personal protective equipment in an autoclave bag and autoclave. • Wash hands with soap and water.
6.4.3.6 Spill of a Biohazardous Radioactive Material A biohazardous spill involving radioactive material requires emergency procedures that are different from the procedures used for either material alone. Use procedures that protect you from the radiochemical while you disinfect the biological material.
Before any clean up, consider the type of radionuclide, characteristics of the microorganism, and the volume of the spill. Contact the GRU Radiation Safety Office (x1-2663) for isotope cleanup procedures.
Immediate Procedures • Avoid inhaling airborne material, while quickly leaving the room. Notify others to leave. • Close door, and post a warning sign. • Remove contaminated clothing, turning exposed areas inward, and place in a biohazard bag labeled with a radioactive materials label or a radioactive waste container labeled with a biohazard label. • Wash all exposed skin with soap and water; follow with a three-minute water rinse.
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• Inform supervisor and Radiation Safety Office of spill, and monitor all exposed personnel for radiation. If assistance is needed in handling the microorganism, contact the Biosafety Office at x1-2663.
Clean-Up of a Biohazardous Radioactive Material • Allow aerosols to disperse for at least 30 minutes before reentering the laboratory. Assemble clean-up materials (disinfectant, autoclavable containers, forceps, towel, and sponges), and confirm with the Radiation Safety representative that it is safe to enter the lab. • Put on protective clothing (gown, surgical mask, gloves, and shoe covers). Depending on the nature of the spill, it may be advisable to wear a HEPA-filtered respirator instead of a surgical mask. • Cover the area with disinfectant-soaked towels, and carefully pour disinfectant around the spill. Avoid enlarging the contaminated area. Use more concentrated disinfectant as it is diluted by the spill. Allow at least 30 minutes contact time.
Do Not use bleach solutions on iodinated material, radioiodine gas may be released. Instead, use an alternative disinfectant such as an iodophor or phenolic.
• Handle any sharp objects with forceps. Wipe surrounding areas, where the spill may have splashed, with disinfectant. • Soak up the disinfectant and spill, and place the biologically decontaminated waste, along with all protective clothing contaminated with radioactive materials, into an approved radioactive waste container and label it according to Radiation Safety Guidelines. • Contaminated protective clothing must also be biologically decontaminated prior to disposal as radioactive waste.
Do Not autoclave the waste unless this action is approved by the Radiation Safety Officer. If waste can not be autoclaved, add additional disinfectant to ensure biological decontamination of all the materials.
• Wash hands and exposed skin areas with soap and water; monitor personnel and spill area for residual radioactive contamination. • If skin contamination is found, repeat decontamination procedures under the direction of the Radiation Safety Officer. • If the spill area has residual activity, determine if it is fixed or removable and handle accordingly. • Discarding items contaminated with radioactive materials: • Place the contaminated item(s) on absorbent paper. • Spray disinfectant (freshly prepared 10% household bleach) on the contaminated areas and allow 20 minute contact time. • Wrap the item(s) inside the paper and dispose of as radioactive waste. • Wash hands with soap and water.
6.5 INVESTIGATION OF LABORATORY ACCIDENTS The Division of Environmental Health and Safety (EHS), in cooperation with the Principal Investigator and his or her staff, will conduct the necessary investigation of a laboratory accident. The goal of the investigation is the prevention of similar accidents as well as obtaining information concerning the circumstances and number of employees who have been exposed to the agent in question. In addition,
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EHS, in consultation with the Occupational Health and Human Resources might institute further steps to monitor the health of those who may have been exposed to the agent in question.
It should be emphasized that the reporting of accidents to the Principal Investigator or laboratory supervisor is the responsibility of the employee who has the accident. The Principal Investigator or the laboratory supervisor should then report to the Biosafety Office (x1-2663 or [email protected]).
We encourage Principal Investigators and employees to also report incidents that did not result in an exposure (“near misses”) to the Biosafety Office. Evaluation of near misses can lead to alternative work practices and implementation of engineering controls to minimize future incidents.
Whenever an injury involves a sharp and human material (body fluid, tissue, cell line, etc.) the Biosafety Office must perform an investigation to determine if a safe sharps device is available to prevent future occurrences of the injury. If safe sharps devices are available, they must be evaluated by the biosafety office in conjunction with the Group or Department. The incident must also be recorded on the University’s Sharps Injury Log, maintained by the Human Resources Office. The confidential log will include the type and brand of device involved in the incident; the Department or work area where the exposure incident occurred; and an explanation of how the incident occurred.
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7. DECONTAMINATION OF BIOLOGICALS
7.1 DECONTAMINATION METHODS Decontamination methods fall into three main categories: heat, application of chemical decontaminants (including vapors and gases) and physical methods (such as filtration or irradiation).
7.1.1 Heat The application of heat, either moist or dry, is recommended as the most effective method of sterilization. Steam at 121°C under pressure in the autoclave is the most convenient method of rapidly achieving sterility under ordinary circumstances. Dry heat at 160°C to 170°C for periods of two to four hours is suitable for destruction of viable agents on an impermeable non-organic material such as glass, but is not reliable in even shallow layers of organic or inorganic material that can act as insulation. Incineration is another use of heat for decontamination. Incineration serves as an efficient means of disposal for human and animal pathological wastes.
7.1.1.1 Autoclave Use and Maintenance Moist heat causes the denaturation of proteins at lower temperatures and shorter times than dry heat. One of the most effective physical decontamination controls is steam sterilization (autoclave) which generates moisture and high temperature pressurized steam within a sealed chamber. Autoclaves can sterilize all items that are heat stable. In gravity autoclaves, a cycle of 250°F (121°C) at 15 to 18 pounds per square inch (psi) of pressure for one hour may be required for decontamination. In the newer vacuum autoclaves, decontamination may require a cycle of 270°F (132°C) at 27 to 30 psi for 45 minutes.
The hazards of handling hot solids and liquids are reasonably familiar. Laboratory personnel should be cautioned that steam under pressure, such as that found in autoclaves, could be a source of scalding jets if the equipment is misused. When preparing to use an autoclave, check the door seal gaskets each time to ensure these are intact prior to using the autoclave. Also, before use, check to make sure the drain at the bottom of the autoclave is unobstructed. Prepare to autoclave loads of manageable size only. Do not overfill autoclaves.
Fluids treated by steam under pressure may be superheated if removed from the sterilizer too soon after treatment; which may cause a sudden and violent boiling of contents from the containers that can splash scalding liquids onto personnel handling the containers. Therefore slow exhaust cycles should be used to autoclave liquids. In addition, bottles with liquids should allow for the liquid expansion during autoclaving; “head room” should be left in the vessels before autoclaving. Similarly, to avoid over-pressurization will occur inside of vessels with liquids if gas is not able to freely enter/exit the bottle during the cycle. Therefore, only loosely fit covers or caps on these vessels prior to autoclaving.
For solid materials, keep in mind that steam must be in contact with the materials to efficiently sterilize them. Therefore autoclave bags should be left partially opened and/or some additional water should be placed on the inside of the bag prior to autoclaving.
Because of the air and liquid exchange inside of bags or vessels, other hazardous materials should not be included in autoclave loads. Mixed waste—either chemicals (such as phenol: chloroform) or radiological material mixed with biological materials should never be autoclaved due to the chemical and radiological hazards present.
In addition, some materials should never be autoclaved. Nitrocellulose materials (tubes/filters), for instance, can explode under autoclave temperatures. Make sure any plastic materials that are autoclaved are guaranteed “autoclavable” by the manufacturer. Most plastics will melt inside the
Georgia Regents University Biosafety Guide- January 2012 7-1 autoclave and produce irritating odors.
Always place materials to be autoclaved inside of a metal or autoclavable plastic autoclave tray to prevent spillage of agar or melted plastics into the bottom of the autoclave. (Note: most plastics are not autoclavable unless specially formulated, so check the manufacturer’s specifications to ensure your plastics are autoclavable!). Melted and re-solidified agar or plastic plugs the drain at the bottom of the autoclave, which prevents proper function of the autoclave and often requires maintenance to repair.
Wear closed toe shoes, pants, lab coat, face shield and long sleeved insulated gloves when operating an autoclave. A heavy, rubberized insulated apron is further recommended for those who autoclave frequently. Allow time for loads to cool before removing them from autoclaves after a run. Take proper precautions when first opening the door; first crack the seal on the door sufficiently to allow the initial burst of steam to escape, then leave the door open about ½ inch to vent the autoclave for about 5-10 before fully opening the door.
All autoclaves should be on a preventative maintenance program and certified regularly to ensure proper function. Heat-sensitive autoclave tape can be used to ensure that an autoclave got warm; however, this is insufficient to tell the user that the If the autoclave is appropriate temperature and pressure were maintained over a sufficient Prior to not functioning period of time to provide full decontamination. Please note: GRU does autoclaving correctly and spores not have a campus-wide autoclave certification and maintenance germinate program. Responsibility for certifying and maintaining autoclaves falls to the owner—the department or Principal Investigator who owns the autoclave. Autoclaves should be certified using bioindicators for steam autoclaves. Typically, these are vials containing spores of Geobacillus stearothermophilus in growth media with an colored indicator. To certify that an autoclave is functioning properly, a vial is placed in the middle of a typical autoclave load (attaching a string to the vial prior to placement allows for later retrieval). The autoclave load is run as usual, and the vial is retrieved from the load after autoclaving. The vial is cultured to determine whether the spores are capable of germinating, which would Bioindicators cause a color change in the indicator. Germination of the spores would indicate that the autoclave is not decontaminating the loads sufficiently, and maintenance should be performed on the autoclave.
If you experience any problems or unusual occurrences during autoclave use, please report these to your supervisor and/or building/department manager to enable them to contact the autoclave maintenance provider. For autoclaves attached to “house” steam lines, ensure that steam pressure is sufficient to operate the autoclave prior to contacting external repair offices. Insufficient steam pressure should be reported to the Facilities Management Office (x1-2434) for repair.
7.1.2 Chemical Decontaminants In general, chemical decontaminants find their most practical use in surface decontamination and, at sufficient concentration, as decontaminants of liquid wastes for final disposal in sanitary sewer systems. Further information about the characteristics of the specific chemicals used for disinfection can be found in Section 7.2.
7.1.2.1 Liquid Chemical Decontaminants There are many misconceptions concerning the use of liquid decontaminants. This is due largely to a characteristic capacity of such liquids to perform dramatically in the test tube and to fail miserably in a practical situation. Such failures often occur because proper consideration was not given to such factors as temperature, contact time, pH, the presence and state of dispersion, penetrability and reactivity of organic material at the site of application. Small variations in the
Georgia Regents University Biosafety Guide- January 2012 7-2 above factors may make large differences in the effectiveness of decontamination. For this reason even when used under highly favorable conditions, complete reliance should not be placed on liquid decontaminants when the end result must be sterility.
There are many liquid decontaminants available under a wide variety of trade names. In general, these can be categorized as halogens, acids and alkalis, heavy metal salts, quaternary ammonium compounds, phenols, aldehydes, ketones, alcohols, and amines. Unfortunately, the more active the decontaminant, the more likely it will posses undesirable characteristics such as corrosivity. In addition, some of the chemical disinfectants will require disposal as chemical waste after disinfection.
None is equally useful or effective under all conditions for all infectious agents. Particular care should be observed when handling concentrated stock solutions of disinfectants. Personnel assigned to the task of making up use-concentrations from stock solutions must be informed of the potential hazards and trained in the safe procedures to follow and appropriate personal protective equipment to use as well as the toxicity associated with ocular, skin and respiratory exposure.
7.1.2.2 Vapors and Gases A variety of vapors and gases possess decontamination properties. The most useful of these are formaldehyde, vapor-phase hydrogen peroxide (VHP), chlorine dioxide and ethylene oxide. When these can be employed in a closed system and under controlled conditions of temperature and humidity, excellent decontamination can result. Vapor and gas decontaminants are primarily useful in decontaminating biological safety cabinets and associated air-handling systems and air filters; bulky or stationary equipment that resists penetration by liquid surface decontaminants; instruments and optics that may be damaged by other decontamination methods; and rooms, buildings and associated air-handling systems.
Only those who have received appropriate training in vapor and gas decontamination should attempt these operations. They should avoid inhalation of vapors of any of these vapors of gasses. Stock containers of these products should be capable of confining these vapors and should be kept in properly ventilated chemical storage areas. In preparing use-dilutions and when applying them, personnel should control the operations to prevent exposure of others and wear respiratory protection as necessary.
Mutagenic potential has been attributed to ethylene oxide; toxic and hypersensitivity effects are well-documented for formaldehyde. Ethylene oxide use is very limited and is generally used in surgical and clinical areas.
Use of vapor and gas disinfection methods is monitored closely by the GRU Division of Environmental Health and Safety. Please contact EHS at x1-2663 for information regarding the exposure monitoring program.
7.1.3 Physical Methods Alternative measures for decontamination which rely on physical properties of decontaminants are often used as precautionary measures, such as HEPA filtrations on BSC or exhaust systems, rather than a primary mode of disinfection. The function of HEPA filters was discussed earlier, in Section 4.2.2, Biosafety Cabinets, and will not be repeated here. Most HEPA filters remove a minimum of 99.97% of particulates from air.
Gamma-wave irradiation is another method used for decontamination; however, this often requires a high- energy irradiator, currently making it impractical as an everyday method for decontamination of most materials at GRU.
Georgia Regents University Biosafety Guide- January 2012 7-3 UV irradiation is a somewhat controversial method of disinfection for surfaces. While it is an effective measure, time of exposure, distance, presence of dust or debris and UV lamp intensity can all affect the germicidal effect of the UV lamp. Users should recognize that the visible blue-violet glow of the UV lamp does not indicate there is germicidal effect. The UV lamp needs to be cleaned periodically to remove dust. UV lamps may damage eyes, skin, and laboratory equipment and shut off while the room is occupied.
Because users tend to over-rely on UV irradiation for surface decontamination in lieu of thorough chemical surface decontamination and the compounding factors which may impede proper decontamination using UV irradiation, the Biosafety Office discourages the use of UV lamps. For further information related to the use of UV lamps for surface decontamination, please see the following references: Burgener, J (2006) Position Paper on the Use of Ultraviolet Lights in Biological Safety Cabinets. Applied Biosafety 11(4): 228-230. Meecham, P.J. and Wilson, C. (2006) Use of Ultraviolet Lights in Biological Safety Cabinets: A Contrarian View. Applied Biosafety 11(4): 222-227.
7.2 CHARACTERISTICS OF CHEMICAL DECONTAMINANTS
Chemicals with decontaminant properties are, for the most part, available as powders, crystals, or liquid concentrates. These may be added to water for application as surface decontaminants, and some, when added in sufficient quantity, find use as decontaminants of bulk liquid wastes. Chemical decontaminants that are gaseous at room temperature are useful as space-penetrating decontaminants. Others become gases at elevated temperatures and can act as either aqueous surface or gaseous space-penetrating decontaminants.
Inactivation of microorganisms by chemical decontaminants may occur in one or more of the following ways: Coagulation and denaturation of proteins Lysis Binding to enzymes or inactivation of an essential enzyme by oxidation, binding, or destruction of enzyme substrate
The relative resistance to the action of chemical decontaminants may be altered substantially by such factors as: concentration of active ingredient, duration of contact, pH, temperature, humidity, and presence of extrinsic organic matter. Depending on how these factors are manipulated, the degree of success achieved with chemical decontaminants may range from minimal inactivation of target microorganisms to an indicated sterility within the limits of sensitivity of the assay system employed. Ineffectiveness of a decontaminant is due primarily to the failure of the decontaminant to contact the microorganisms rather than failure of the decontaminant to act. If an item is placed in a liquid decontaminant, tiny bubbles are visible on the surface of the item. The area under the bubbles is dry and microorganisms in these dry areas will not be affected by the decontaminant. If there are spots of grease, rust or dirt on the item, microorganisms under these protective coatings will not be contacted by the decontaminant. Scrubbing an item when immersed in a decontaminant is helpful. A decontaminant should have, and most do have, incorporated surface-active agents.
7.2.1 Properties of Some Common Decontaminants
7.2.1.1 Chlorine/Hypochlorites This halogen is a universal decontaminant active against a broad spectrum of microorganisms, including bacterial spores. Chlorine combines with protein and rapidly decreases in concentration in the presence of protein. Free available chlorine is the active element. Chlorine solutions must be prepared frequently because of its instability in water.
Georgia Regents University Biosafety Guide- January 2012 7-4 Sodium hypochlorite is usually used as a base for chlorine decontaminants. An excellent decontaminant can be prepared from household or laundry bleach. These bleaches usually contain 5.25%, or 52,500 ppm, available hypochlorite. If diluted 1 to 10, the resulting solution will contain 0.525% or 5,250 ppm of available hypochlorite. The addition of a 0.7% (v/v) of nonionic detergent can improve the disinfection properties.
Because hypochlorite is a strong oxidizing agent, it can be corrosive to metals. Therefore, caution should be taken to prevent etching of stainless surfaces cleaned with bleach solutions.
7.2.1.2 Alcohols Ethyl or isopropyl alcohol in a concentration of 70-85% by weight is often used; however, both lose effectiveness at concentrations below 50% and above 90%. Alcohols denature proteins and are somewhat slow in germicidal action. However, alcohols are effective decontaminants against lipid-containing viruses. A contact time of ten minutes is generally employed in efficacy tests with disinfectants. Due to the high evaporation rate of alcohols, repeated applications may be required to achieve the required ten-minute contact time for decontamination. Because of this, the OSHA Bloodborne Pathogens Standard does not recognize alcohol as an effective decontaminant for surfaces.
Isopropyl alcohol is generally more effective against vegetative bacteria; ethyl alcohol is a more virucidal agent.
Alcohols are also very flammable, so precautions should be taken to prevent exposure to spark or flame sources.
7.2.1.3 Aldehydes (Formaldehyde, Glutaraldehyde) Aldehydes are commonly-used fixative agents in laboratories; however, during the process of fixation, materials are decontaminated. Formaldehyde for use as a decontaminant is usually marketed as a solution of about 37% concentration referred to as formalin, or as a solid polymerized compound called paraformaldehyde. Glutaraldehyde is commonly used in concentrations of 2-4%.
Formaldehyde at a concentration of 5% active ingredient is an effective liquid decontaminant. A glutaraldehyde-based commercial disinfectant (Cidex®) is used in some hospital settings. Aldehyde disinfectants lose considerable activity at refrigeration temperatures, and the pungent, irritating odors and health risks make formaldehyde solutions difficult to use in the laboratory. The use of Cidex® has been restricted in the U.K. because of the carcinogenic effects. All materials disinfected with aldehydes must be disposed as chemical wastes to adhere to EPA standards.
Formaldehyde vapor generated from solution is an effective space decontaminant for buildings or rooms, but in the vapor state in the presence of water tends to polymerize on surfaces to form paraformaldehyde, which is persistent and unpleasant. Heating paraformaldehyde to depolymerize it can liberate formaldehyde gas. In the absence of high moisture content in the air, formaldehyde released in the gaseous state forms less polymerized residues on surfaces and less time is required to clear treated areas of fumes than is the case in the vapor state.
7.2.1.4 Phenols Phenol itself is not often used as a decontaminant. The odor is somewhat unpleasant and a sticky, gummy residue remains on treated surfaces. This is especially true during steam sterilization. Although phenol itself may not be in widespread use, phenol homologs and phenolic compounds are basic to a number of popular decontaminants, such as the original Lysol® and Amphyl®.
Georgia Regents University Biosafety Guide- January 2012 7-5 Phenolic compounds are effective decontaminants against some viruses, fungi, and vegetative bacteria, including rickettsiae. Phenolics are not effective in ordinary use against bacterial spores.
7.2.1.5 Quaternary Ammonium Compounds (a.k.a. “Quats”) After 40 years of testing and use, there is still considerable controversy about the efficacy of the Quats as decontaminants. These cationic detergents are strongly surface-active and are effective against lipid-containing viruses and often vegetative gram positive bacterial however, they have variable activities against gram negative bacteria and fungi, and are not very effective against non- lipid enveloped viruses. The Quats will attach to protein so that dilute solutions will quickly loose effectiveness in the presence of proteins. Quats tend to clump microorganisms and are neutralized by anionic detergents such as soap. They have the advantages of being nontoxic, odorless, stable, non-staining, non-corrosive to metals, and inexpensive. Many common household and laboratory disinfectants are Quats (e.g., Roccal®, Germex®, Coverage Plus®).
7.2.1.6 Iodine The characteristics of chlorine and iodine are similar. One of the most popular groups of decontaminants for laboratory use are the iodophors, including Wescodyne®, Betadyne® and Providone®.
The range of dilution of Wescodyne® recommended by the manufacturer is 1 oz. in 5 gal. of water (25 ppm available iodine) to 3 oz. in 5 gal. of water (75 ppm available iodine). The small amount of free iodine available in this range can rapidly be taken up by extraneous protein that may be present. Clean surfaces or clear water can be effectively treated with 75-ppm available iodine, but difficulties may be experienced if any appreciable amount of protein is present. For iodophors such as Wescodyne®, it is critical that the manufacturer’s written instructions are followed. Higher concentrations of iodophores are actually less effective, as the iodine is bound to itself or the carrier molecule. For washing the hands or for use as a sporicide, it is recommended that Wescodyne® be diluted 1 to 10 in 50% ethyl alcohol (a reasonably good decontaminant itself.) This will give 1,600 ppm of available iodine, at which concentration relatively rapid inactivation of any and all microorganisms will occur.
7.2.1.7 Peroxygens Peroxide-based disinfection has become more popular in recent years with the increasing popularity of vapor-phase hydrogen peroxide generators, which are an effected area decontamination method. Liquid peroxygen disinfectants, such as Virkon-S® are also available as surface decontamination methods. Peroxygens have broad-spectrum disinfectant properties and are generally effective against vegetative bacteria, viruses and some spores. Peroxygens have variable efficacy in the presence of organic matter. However, peroxygens can be incompatible with some materials (such as Aluminum, Copper, Zinc, Brass, Natural rubber and some plastics), which should be considered when selecting disinfectants.
7.2.2 Selecting Chemical Disinfectants No single chemical disinfectant or method will be effective or practical for all situations in which decontamination is required. Selection of chemical disinfectants and procedures must be preceded by practical consideration of the purposes for the decontamination and the interacting factors that will ultimately determine how that purpose is to be achieved. Selection of any given procedure will be influenced by the information derived from answers to the following questions:
What is the target organism(s)? What disinfectants, in what form, are known to, or can be expected to, inactivate the target organism(s)? What degree of inactivation is required?
Georgia Regents University Biosafety Guide- January 2012 7-6 In what menstruum is the organism suspended (i.e. simple or complex, on solid or porous surface, and/or airborne)? What is the highest concentration of organisms anticipated to be encountered? Can the disinfectant, either as a liquid, vapor, or gas, be expected to contact the organism and can effective duration of contact be maintained? What restrictions apply with respect to compatibility of materials? What is the stability of the disinfectant in use concentrations, and does the anticipated use situation require immediate availability of the disinfectant or will sufficient time be available for preparation of the working concentration shortly before its anticipated use? Will conditions permit safe use of some disinfectants (e.g., is ventilation sufficient to safely use aldehydes)?
The primary target of decontamination in the laboratory is the organism(s) under investigation. Laboratory preparations or cultures usually have titers in excess of those normally observed in nature. Inactivation of these materials presents other problems since agar, proteinaceous nutrients, and cellular materials can effectively retard or chemically bind the active moieties of chemical disinfectants. Such interference with the desired action of disinfectants may require higher concentrations and longer contact times than those shown to be effective in the test tube. Similarly, a major portion of the contact time required to achieve a given level of agent inactivation may be expended in inactivating a relatively small number of the more resistant members of the population. The current state of the art provides little information with which to predict the probable virulence of these more resistant cells. These problems are, however, common to all potentially pathogenic agents and must always be considered in selecting disinfectants and procedures for their use.
Organisms exhibit a range of resistance to chemical disinfectants. In terms of practical decontamination, most vegetative bacteria, fungi, and lipid-containing viruses are relatively susceptible to chemical disinfection. The non-lipid-containing viruses and bacteria with a waxy coating, such as tubercule bacillus, occupy a mid-range of resistance. Spore forms and unconventional (slow) viruses are the most resistant.
A disinfectant selected on the basis of its effectiveness against organisms on any range of the resistance scale generally will be effective against organisms lower on the scale. Therefore, if disinfectants that effectively control spore forms are selected for routine laboratory decontamination, it can be assumed that any other organism generated by laboratory operations, even in higher concentrations, would also be inactivated.
Pertinent characteristics and potential applications for several categories of chemical disinfectants most likely to be used in the biological laboratory are summarized in the table on the following pages. Practical concentrations and contact times that may differ markedly from the recommendations of manufacturers of proprietary products are suggested. It has been assumed that microorganisms will be afforded a high degree of potential protection by organic menstruums. It has not been assumed that a sterile state will result from application of the indicated concentrations and contact times. It should be emphasized that these data are only indicative of efficacy under artificial test conditions. Individual investigators should conclusively determine the efficacy of any of the disinfectants. It is readily evident that each of the disinfectants has a range of advantages and disadvantages as well as a range of potential for inactivation of a diverse microflora. Equally evident is the need for compromise as an alternative to maintaining a veritable “drug store” of disinfectants.
7.2.3 Characteristics of Some Liquid Disinfectants Table The following tables, prepared by the Iowa State University Center for Food Security and Public Health are provided for reference on properties of various chemical disinfectants.
Georgia Regents University Biosafety Guide- January 2012 7-7
Georgia Regents University Biosafety Guide- January 2012 7-8
Georgia Regents University Biosafety Guide- January 2012 7-9 7.3 DEACTIVATION OF BIOLOGICAL TOXINS The following information is compiled directly from the CDC/NIH BMBL: Appendix I (http://www.cdc.gov/biosafety/publications/bmbl5/BMBL5_appendixI.pdf) and the Georgia State University Biosafety Manual: Further reference material can be found at the URL, above.
Toxin stability varies considerably outside of physiological conditions depending upon the temperature, pH, ionic strength, availability of co-factors and other characteristics of the surrounding matrix. Literature values for dry heat inactivation of toxins can be misleading due to variations in experimental conditions, matrix composition, and experimental criteria for assessing toxin activity. Moreover, inactivation is not always a linear function of heating time, and some protein toxins possess a capacity to re-fold, and partially reverse inactivation caused by heating. In addition, the conditions for denaturizing toxins in aqueous solutions are not necessarily applicable for inactivating dry, powdered toxin preparations.
General guidelines for laboratory decontamination of selected toxins are summarized in Tables 7.3A, B and C, but inactivation procedures should not be assumed to be 100% effective without validation using specific toxin bioassays. Many toxins are susceptible to inactivation with dilute sodium hydroxide (NaOH) at concentrations of 0.1-0.25N, and/or sodium hypochlorite (NaOCl) bleach solutions at concentrations of 0.1-0.5% (w/v). Use freshly prepared bleach solutions for decontamination; undiluted, commercially available bleach solutions typically contain 3-6% (w/v) NaOCl. Special care should be taken while deactivating acute biological toxins to protect the handler, but also to ensure thorough decontamination. See Tables 7.3A, B and C for further guidance on disinfectant procedures.
To chemically decontaminate toxins, perform all operations in a fume hood or biosafety cabinet with the sash at the lowest reasonable sash height for safe and effective work. Place plastic-backed absorbent paper (e.g. Benchkote or diaper) on the work surface of the fume hood or BSC to protect the surfaces. Wear long-sleeved protective clothing (lab coat, gown), gloves and eye protection while decontaminating toxins. Place toxin into solution in a non-glass primary container, which can be placed in a secondary container, such as a beaker or rack. Slowly dispense equal volume of the concentrations of NaOCl and/or NaOH or similar disinfectant to deactivate the toxin. Do not replace the cap on the primary container and allow for a minimum of 30 minutes exposure time or as recommended in the tables below.
Depending upon the toxin, contaminated materials and toxin waste solutions can be inactivated by incineration or extensive autoclaving, or by soaking in suitable decontamination solutions (See Table 7.3B). Autoclaving should not be used for destruction of any low molecular weight toxins (e.g., mycotoxins, marine and reptile venoms). To autoclave, the cap on the primary container should be loosened in a fume hood or BSC to allow for steam penetration, then placed in secondary containers before autoclaving at 121°C for 1 hour on liquid cycle (slow exhaust). Allow time for the materials to cool before handling and dispose materials as of as toxic waste.
Contaminated or potentially contaminated protective clothing and equipment should be decontaminated using suitable chemical methods or autoclaving before removal from the laboratory for disposal, cleaning or repair. If decontamination is impracticable, materials should be disposed of as toxic waste.
Georgia Regents University Biosafety Guide- January 2012 7-10 Table 7.2A PHYSICAL INACTIVATION OF SELECTED TOXINS (Reproduced from Appendix I, CDC/NIH BMBL http://www.cdc.gov/biosafety/publications/bmbl5/BMBL5_appendixI.pdf )
TOXIN STEAM DRY HEAT FREEZE-THAW GAMMA- AUTOCLAVE (10 MIN) IRRADIATION Botulinium Yesa > 100°Cb Noc Incompleted neurotoxin (BoNT) Staphylococcal Yese > 100°C; refoldsf Nog Incomplete Enterotoxin Ricin Yesi > 100°Ci Noj Incompletek Microcystin Nol > 260°Cm Non ND Saxitoxin Nol > 260°Cm Non ND Palytoxin Nol > 260°Cm Non ND Tetrodotoxin Nol > 260°Cm Non ND T-2 mycotoxin Nol > 815°Cm Non ND Brevetoxin (PbTx-2) Nol > 815°Cm Non ND
Table 7.2 A Notes: ND indicates “not determined” from available decontamination literature aSteam autoclaving should be at ≥ 121°C for 1 h. For volumes larger than 1 liter, especially those containing Clostridium botulinum spores, autoclave at ≥ 121°C for 2 h to ensure that sufficient heat has penetrated to kill all spores. bExposure to 100°C for 10 min. inactivates BoNT. Heat denaturation of BoNT as a function of time is biphasic with most of the activity destroyed relatively rapidly, but with some residual toxin (e.g., 1-5%) inactivated much more slowly. cMeasured using BoNT serotype A at -20°C in food matrices at pH 4.1-6.2 over a period of 180 days. dMeasured using BoNT serotype A and B with gamma irradiation from a 60Co source. eProtracted steam autoclaving, similar to that described for BoNT, followed by incineration is recommended for disposal of SE- contaminated materials. fInactivation may not be complete depending upon the extent of toxin re-folding after denaturation. Biological activate of SE can be retained despite heat and pressure treatment routinely used in canned food product processing. gSE toxins are resistant to degradation from freezing, chilling or storage at ambient temperature. Active SEB in the freeze-dried state can be stored for years. iDry heat of ≥100°C for 60 min in an ashing oven or steam autoclave treatment at >121°C for 1 h reduced the activity of pure ricin by >99%. Heat inactivation of impure toxin preparations (e.g., crude ricin plant extracts) may vary. Heat-denatured ricin can undergo limited refolding (<1%) to yield active toxin. jRicin holotoxin is not inactivated significantly by freezing, chilling or storage at ambient temperature. In the liquid state with a preservative (sodium azide), ricin can be stored at 4°C for years with little loss in potency. kIrradiation causes a dose-dependent loss of activity for aqueous solutions of ricin, but complete inactivation is difficult to achieve; 75 MRad reduced activity 90%, but complete inactivation was not achieved even at 100MRad. Gamma irradiation from a laboratory 60Co source can be used to partically inactivate aqueous solutions of ricin, but dried ricin powders are significantly resistant to inactivation by this method. lAutoclaving with 17 lb pressure (121-132°C) for 30 min failed to inactivate LMW toxins. All burnable waste from LMW toxins should be incinerated at temperatures in excess of 815°C (1,500°F). mToxin solutions were dried at 150°C in a crucible, placed in an ashing oven at various temperatures for either 10 or 30 min, reconstituted and tested for concentration and/or activity; tabulated values are temperatures exceeding those required to achieve 99% toxin inactivation. nLMW toxins are generally very resistant to temperature fluctuations and can be stored in the freeze-dried state for years and retain toxicity.
Please see: CDC/NIH BMBL, Appendix I (http://www.cdc.gov/biosafety/publications/bmbl5/BMBL5_appendixI.pdf) for references and further information.
Georgia Regents University Biosafety Guide- January 2012 7-11 Table 7.2B CHEMICAL INACTIVATION OF SELECTED TOXINS (Reproduced from Appendix I, CDC/NIH BMBL http://www.cdc.gov/biosafety/publications/bmbl5/BMBL5_appendixI.pdf )
TOXIN NaOCl (30 NaOH (30 NaOCl + NaOH OZONE MIN) MIN) (30 MIN) TREATMENT Botulinium >0.1%a >0.25 N ND Yesb neurotoxin Staphylococcal >0.5%c >0.25 N ND ND Enterotoxin Ricin >1.0%d ND >0.1% + 0.25 Ne ND Microcystin ≥0.1%e ND 0.25% + 0.25 Ne ND Saxitoxin ≥0.1%e ND 0.25% + 0.25 Ne ND Palytoxin ≥0.5%e ND 0.25% + 0.25 Ne ND Tetrodotoxin ≥0.5%e ND 0.25% + 0.25 Ne ND T-2 mycotoxin ≥2.5%f ND 0.25% + 0.25 Ne ND Brevetoxin (PbTx-2) ≥2.5%e,f ND 0.25% + 0.25 Ne ND
Table 7.2 b Notes: ND indicates “not determined” from available decontamination literature aSolutions of NaOCl (≥0.1%) or NaOH (>0.25 N) for 30 min inactivate BoNT and are recommended for decontaminating work surfaces and spills of C. botulinum or BoNT. Chlorine at a concentration of 0.3-0.5 mg/L as a solution of hypochlorite rapidly inactivates BoNT (serotypes B or E tested) in water. Chlorine dioxide inactivates BoNT, but chloramines are less effective. bOzone (>2 mg/L) or powdered activated charcoal treatment also completely inactivate BoNT (serotypes A, B tested) in water under defined condition. cSEB is inactivated with 0.5% hypochlorite for 10-15 min. dRicin is inactivated by a 30 min exposure to concentrations of NaOCl ranging from 0.1-2.5%, or by a mixture of 0.25% NaOCl plus 0.25 N NaOH. In general, solutions of 1.0% NaOCl are effective for decontamination of ricin from laboratory surfaces, equipment, animal cages, or small spills. eThe minimal effective concentration of NaOCl was dependent on toxin and contact time; all LMW toxins tested were inactivated at least 99% by treatment with 2.5% NaOCl, or with a combination of 0.25% NaOCl and 0.25N NaOH. fFor T-2 mycotoxin and brevetoxin, liquid samples, accidental spills, and nonburnable waste should be soaked in 2.5% NaOCl with 0.25% N NaOH for 4 h. Cages and bedding from animals exposed to T-2 mycotoxin or brevetoxin should be treated with 0.25% N NaOCl and 0.025 N NaOH for 4 h. Exposure for 30 min to 1.0% NaOCl is an effective procedure for the laboratory (working solutions, equipment, animal cages, working area and spills) for the inactivation of saxitoxin or tetrodotoxin.
Alternate methods of chemical decontamination: 1 N sulfuric or hydrochloric acid did not inactivate T-2 mycotoxin and only partially inactivated microcystin-LR, saxitoxin, and brevetoxin (PbTx-2). Tetrodotoxin and palytoxin were inactivated by hydrochloric acid, but only at relatively high molar concentrations. T2 was not inactivated by exposure to 18% formaldehyde plus methanol (16 h), 90% Freon-114+ 10% acetic acid, calcium hypochlorite, sodium bisulfate, or mild oxidizing. Hydrogen peroxide was ineffective in inactivating T-2 mycotoxin. This agent did cause some inactivation of saxitoxin and tetrodotoxin, but required 16 h contact time in the presence of ultraviolet light.
Please see: CDC/NIH BMBL, Appendix I (http://www.cdc.gov/biosafety/publications/bmbl5/BMBL5_appendixI.pdf) for references and further information.
Georgia Regents University Biosafety Guide- January 2012 7-12
Table 7.3C. Inactivation Procedures for Select Agent Toxins
7.4 DEACTIVATION OF PRIONS Work with neural tissues presents the possible remote risk for the presence of prions. Prions are characterized by resistance to conventional inactivation procedures including irradiation, boiling, dry heat, and chemicals (formalin, betapropiolactone, alcohols). While prion infectivity in purified samples is diminished by prolonged digestion with proteases, results from boiling in sodium dodecyl sulfate and urea are variable. Likewise, denaturing organic solvents such as phenol or chaotropic reagents such as guanidine isothiocyanate have also resulted in greatly reduced but not complete inactivation. The use of conventional autoclaves as the sole treatment has not resulted in complete inactivation of prions.
Formalin-fixed and paraffin-embedded tissues, especially of the brain, remain infectious. Some investigators recommend that formalin-fixed tissues from suspected cases of prion disease be immersed for 30 min in 96% formic acid or phenol before histopathologic processing, but such treatment may severely distort the microscopic neuropathology.
The safest and most unambiguous method for ensuring that there is no risk of residual infectivity on contaminated instruments and other materials is to discard and destroy them by incineration. Current recommendations for inactivation of prions on instruments and other materials are based on the use of sodium hypochlorite, NaOH, Environ LpH and the moist heat of autoclaving with combinations of heat and chemical being most effective (See Table 7.4). Unfortunately, these solutions are corrosive and require suitable personal protective equipment and proper secondary containment. These strong corrosive solutions require careful disposal in accordance with local regulations.
Precautions in using NaOH or sodium hypochlorite solutions in autoclaves NaOH spills or gas may damage the autoclave if proper containers are not used. The use of containers with a rim and lid designed for condensation to collect and drip back into the pan is recommended. Persons who use this procedure should be cautious in handling hot NaOH solution (post-autoclave) and in avoiding potential exposure to gaseous NaOH, exercise caution during all sterilization steps, and allow the autoclave, instruments, and solutions to
Georgia Regents University Biosafety Guide- January 2012 7-13 cool down before removal. Immersion in sodium hypochlorite bleach can cause severe damage to some instruments.
Further information about biosafety measures for handling prions and the risks of prion diseases can be found in Section VIII-H of the CDC/NIH BMBL: http://www.cdc.gov/biosafety/publications/bmbl5/BMBL5_sect_VIII_h.pdf .
Table 7.4 PRION INACTIVATION METHODS FOR REUSABLE INSTRUMENTS AND SURFACES
1. Immerse in 1 N NaOH, and heat in a gravity displacement autoclave at 121°C for 30 minutes. Clean and sterilize by conventional means. 2. Immerse in 1 N NaOH or sodium hypochlorite (20,000 ppm) for 1 hour. Transfer into water and autoclave (gravity displacement) at 121°C for 1 hour. Clean and sterilize by conventional means. 3. Immerse in 1N NaOH or sodium hypochlorite (20,000 ppm) for 1 hour. Rinse instruments with water, transfer to open pan and autoclave at 121°C (gravity displacement) or 134°C (porous load) for 1 hour. Clean and sterilize by conventional means. 4. Surfaces or heat-sensitive instruments can be treated with 2N NaOH or sodium hypochlorite (20,000 ppm) for 1 hour. Ensure surfaces remain wet for entire time period, then rinse well with water. Before chemical treatment, it is strongly recommended that gross contamination of surfaces be reduced because the presence of excess organic material will reduce the strength of either NaOH or sodium hypochlorite solutions. 5. Environ LpH (EPA Reg. No. 1043-118) may be used on washable, hard, non-porous surfaces (such as floors, tables, equipment, and counters), items (such as non-disposable instruments, sharps, and sharp containers), and/or laboratory waste solutions (such as formalin or other liquids). This product is currently being used under FIFRA Section 18 exemptions in a number of States. Users should consult with the State environmental protection office prior to use.
Georgia Regents University Biosafety Guide- January 2012 7-14 8. BIOLOGICAL WASTE MANAGEMENT
8.1 PURPOSE OF BIOLOGICAL WASTE MANAGEMENT PROGRAM This section is intended to guide MCG personnel in the safe and legal way to dispose of biological waste. Our program is designed to protect the people who handle, transport and dispose of your waste; protect the Environment; protect the public perception of the university and minimize GRU’s regulatory liability.
Some people believe they can save money by working around this program. These attempts are counter productive. They may place other people and the University at risk. The costs associated with one injury, or violation fines can easily exceed annual operational costs. We would much rather hear and consider your suggestions for program improvement than have you implement unauthorized procedures.
The biological waste management program does not supersede the requirements for radioactive and/or hazardous chemical waste programs. Radioactive or hazardous chemical wastes shall be disposed of through the radioactive waste stream or the hazardous chemical waste stream respectively. In fact, in mixed waste situations (biological/chemical or biological/radiological), the waste disposal requirements of the chemical or radiological waste disposal procedures will take precedence over the biological, particularly, since biological wastes are more capable of being decontaminated/deactivated prior to placing the waste in the chemical or radiological waste streams.
The Division of Environmental Health and Safety is continually working behind the scenes to improve this program and control its cost. Direct any questions or suggestions to the Environmental Health and Safety Office at x1-2663. Call if you have questions about unusual situations or anything not covered in this guide.
8.2 DEFINITION OF BIOMEDICAL WASTE “Biological waste”, “biomedical waste” and “biohazardous wastes” are terms commonly applied interchangeably on GRU’s campus. These materials must be excluded from the general waste stream unless appropriately decontaminated or deactivated as per the Georgia Environmental Protection Division (EPD) as defined in the Georgia EPD biomedical waste regulations Chapter 391-3-4-.15 http://rules.sos.state.ga.us/docs/391/3/4/15.pdf. These include:
1. Waste Cultures and Stocks of Microorganisms or Etiologic Agents Including: a) Cultures and stocks of infectious agents or microorganisms from facilities assigned to Biosafety Levels 1 through 3 (BL1, BL2, BL3).
b) Cultures of specimens from medical and pathological laboratories.
c) Disposable containers, materials, and supplies that may have been contaminated during the manipulation and transportation of microbial cultures and stocks. This includes culture dishes and devices used to transfer, inoculate, and mix cultures.
d) Wastes from the production and handling of biological materials (including all tissue culture materials).
e) Live and attenuated vaccines.
2. Human Pathological Wastes Pathological waste consists of all recognizable human tissues and body parts (except teeth) which are removed during surgery, obstetrical procedures, autopsy, and laboratory procedures. This also includes blood and blood products, exudates secretions,
Georgia Regents University Biosafety Guide-January 2012 8-1 suctionings, and other dialysate; cerebrospinal, synovial, pleural, peritoneal, and pericardial fluids and other bodily fluids; and their respective containers.
3. Waste Human Blood and Blood Products and Their Containers Including: a) Waste human blood and blood products (e.g. blood plasma, platelets, red or white corpuscles, and other derived licensed products such as interferon, etc.). b) Items contaminated with human blood or blood products. c) Items caked with dried human blood or blood products. d) Intravenous bags.
4. Used Sharps Waste This category includes used hypodermic needles, syringes (with or without the attached needles), pasteur pipettes, disposable plastic pipettes, scalpel blades, razor blades, blood vials, test tubes, needles with attached tubing, broken plastic culture dishes, unbroken glass culture dishes, and other types of broken and unbroken glassware that was in contact with biological material including microscope slides and coverslips.
5. Unused Sharps Waste Unused hypodermic needles, suture needles, syringes, and scalpel blades. This includes those which have never been in contact with any biological materials.
6. Waste Animal Carcasses, Body Parts, and Bedding All animal carcasses, bedding and body parts are to be disposed through the Division of Laboratory Animal Services (LAS). As per LAS policies any animal bedding, caging or carcasses which have been purposely infected or known to have been exposed to Risk Group 2 agents shall be autoclaved prior to disposal in Biowaste containers.
All animal carcass and body parts will be stored in the appropriate DLAS freezers and placed in the Stericycle® waste carts for transport to Stericycle® and incineration.
7. Isolation Wastes Isolation wastes are defined as biological wastes and discarded materials contaminated with blood, excretion, exudates, or secretions from humans or animals isolated due to infection with Class 4 microbial agents. If a human or animal is known to be infected with a Risk Group 3 or 4 agent, contact the Biological Safety Officer (x1-2663) immediately.
8. Chemotherapy waste Any disposable material which has come in contact with cytotoxic/antineoplastic agents (agents toxic to cells) and/or antineoplastic agents (agents that inhibit or prevent the growth and spread of tumors or malignant cells) during the preparation, handling, and administration of such agents. Such waste includes, but is not limited to, masks, gloves, gowns, empty IV tubing bags and vials, and other contaminated materials. Liquid waste containers must first be classified as empty which means a quantity that it is not subject to other federal or state waste management regulations prior to being handled as biomedical waste (typically less than 3%).
9. Discarded medical equipment and parts This excludes expendable supplies and materials, which have not been decontaminated, and that were in contact with infectious agents.
In general, biological waste at GRU can be divided into five categories: 1. Solid biomedical waste (including sharps) 2. Pathological waste* 3. Liquid biomedical waste
Georgia Regents University Biosafety Guide-January 2012 8-2 4. Chemotherapy agent wastes* 5. Mixed biological waste (bio/rad or bio/chem.) *
*These wastes must be incinerated and may not be placed in biohazardous waste cardboard cartons unless a special incineration sticker has been applied. For proper disposal information, contact the Biosafety Office or the EHOS office at x1-2663.
8.3 SOLID BIOMEDICAL WASTE Following collection from the laboratories and clinics, all solid biological waste generated by GRU is congregated for transport in a truck for eventual transportation by a professional biomedical waste handling company (as of June 2008, that contractor is Stericycle®). Stericycle® will transport the waste containers to their facilities for decontamination and disposal. Except for the material in the animal waste carts, those demarcated as chemotherapy waste, and mixed waste (e.g., radiologically contaminated biohazardous waste decayed to background levels), most of the biological waste at GRU is autoclaved and land-filled by Stericycle®.
Even though the waste is transported away from GRU for decontamination and disposal, GRU is still liable for the Environmental repercussions of inappropriate transport or disposal of any waste generated at GRU; therefore it is crucial that all personnel who may handle biologicals understand the appropriate waste disposal procedures:
Liquids or soggy materials should NOT be placed inside the Stericycle red-bag lined boxes. Although the red bags are of high grade, there is still a potential for these biohazardous wastes to leak from the bags before or during transport. This not only exposes GRU and/or Stericycle staff to this material, but transport of uncontained biohazardous waste constitutes a violations of the U.S. Department of Transportation regulations and can result in steep fines.
No chemicals or radiological materials should be placed in the general biomedical waste stream due to the potential risk posed to the health and safety of both GRU and Stericycle® handlers, but also to the risks of contaminating the landfill into which this waste is placed.
Biohazardous waste, by definition, is hazardous material and must remain secured at all times. Biohazard waste boxes or containers should not be left in unsecured areas (e.g., in hallways or on loading docks) where non-trained personnel or personnel with unknown health status may encounter them. They should be removed from the laboratories and clinics and directly transferred and secured in the Stericycle® truck. Placing this waste in secondary accumulation areas (e.g., closets within buildings) is not permitted. Care should be taken to not allow the boxes to become wet or damaged or exposed to vermin.
Never remove a leaking biohazard waste box from a laboratory. Notify the laboratory staff and/or the Biosafety Office to enable the staff to safely re-package the wastes prior to removal. If leakage is noticed after removal from the laboratory, the Environmental Services staff should contact the Environmental Health and Safety Office (x1-2663) immediately, and provide relevant information to identify the laboratory of the waste’s origin.
Because it is significantly more costly to dispose of biomedical waste than regular trash, items which are not biological waste or potentially contaminated with biological materials should NOT be placed in the biohazardous waste containers. This includes pipette wrappers, notebook papers, paper towels, which have not been in contact with any biological material. However, if one is unsure whether a particular item is contaminated with biological material, one should default to the biohazardous waste container. Since food items are not permitted in the laboratory, soda cans, candy wrappers, etc. should never be found in biohazard waste containers.
Georgia Regents University Biosafety Guide-January 2012 8-3 Biohazardous waste bags/boxes/sharps containers should NOT be used for alternate purposes other than for collection of biohazardous wastes. These are provided by the biohazardous waste contractor and should be used expressly for collection of biohazardous wastes.
8.3.1 Proper Solid Biohazardous Waste Disposal Procedures
The GRU Environmental Services (a.k.a. housekeeping) staff is responsible for providing the appropriate biohazardous waste containers and removing these items from the laboratory and clinics. To request additional biohazardous waste containers, or to request waste pick-ups, please contact x1-2434.
Biowaste boxes, red bags, sealing tape and animal carts are delivered in each arriving empty truck from Stericycle®. Environmental Services staff is responsible for removing these materials from the truck prior to loading any biohazard waste containers into the trailer for disposal. Supplies should be stored in the Environmental Services supply areas throughout campus.
The GRU Environmental Services Office will provide and handle the four different types of authorized biohazardous waste containers, shown below. Alternate waste containers need to be provided by the laboratory and placed into one of the authorized Stericycle® waste containers by the laboratory staff for removal by the Environmental Services staff.
The biohazard “red bag” box No liquids or soggy materials should be placed in these containers. No loose sharp items should be placed in these containers which would puncture the red bags. These containers have a 50 lb. maximum weight limit. Any material of higher hazard (≥BSL-2) should be decontaminated prior to placement into these containers to prevent inadvertent contamination of those who may close the bag as per IBC approval agreements. The box should be closed and removed before the contents leak or spill over the edge. Do not overfill boxes.
When preparing these boxes, Environmental Services Staff should use at least three (3) strips of packing tape to secure the bottoms of the boxes, each overlapping the edges by at least six (6) inches on either side. One strip of tape should be placed across the seam of the box; the other two strips should be placed perpendicular to the first strip of tape to secure it. The box is turned upright, and the top flaps bent backwards. Each box should be lined with one red biohazard plastic bag, folded over the box flaps, as shown above.
After filling, while wearing appropriate PPE, the laboratory staff should carefully gather the edges of the bag together to avoid production of aerosols, and grasp the bag approximately 10 inches below the edges and twist the bag closed. The twisted bag should be secured with several pieces of packing tape.
After the bags are securely closed, before removal from the laboratory, while wearing PPE (gloves, eye protection, coat/gown) the Environmental Services staff should tape the top box flaps closed with three
Georgia Regents University Biosafety Guide-January 2012 8-4 strips of tape, overlapping each edge by at least six (6) inches (similarly to the way the bottom of the box was closed). They should place a Stericycle® label (white labels designate the box to be autoclaved and landfilled; yellow labels designate the box for incineration). The label should include the date the waste was removed from the laboratory and the building and room number where the waste originated. The boxes should be removed by Environmental Services staff and placed on the Stericycle® truck for transport to their facilities for decontamination and disposal.
Special dollies are made to facilitate transport of vendor biowaste cartons. Ordering information (as of March 2008) is: Stericycle: Phone number: 866-783-7422-4-2-1 Catalog number 1818V2 (lid and dolly) Cost: $91.30 Please note: the purchaser must provide departmental payment information at the time of order. These purchases will not be invoiced to GRU with biowaste manifests.
Should laboratory staff notice waste that requires disposal, please contact Environmental Services (x1- 2434).
The Sharps Containers: There are three varieties of sharps containers available upon request through GRU Environmental Services (x1-2434). These are shown below:
The Large Sharps Container The Standard/Small Sharps Container with Slotted Lid
Each of these containers all comply with OSHA BBP standard requirements dictated for sharps containers. These are: closable, puncture-resistant, leakproof on the sides and bottom, labeled and/or color-coded. In addition, to comply with these standards these containers need to be maintained properly within the laboratory. They should be: Easily accessible and located close to work areas where sharp materials are used (place containers near sharps; do not walk across rooms handling sharps to dispose into the containers). The containers must be maintained upright. These containers need to be replaced routinely. Do not allow these containers to become overfull. As a rule, no The 7.5 gallon Biohazard Waste sharps container should be allowed to become >2/3 full. Bucket Many sharps containers have handy “Full” arrow marks on the containers to remind users to replace the containers once they have reached ~2/3 full.
Georgia Regents University Biosafety Guide-January 2012 8-5 Sharp items which should be placed in these containers include anything which is capable of puncturing the biohazard waste bags, including hypodermic needles, syringes (with or without the attached needles), pasteur pipettes, disposable plastic pipettes, scalpel blades, razor blades, blood vials, test tubes, needles with attached tubing, broken plastic culture dishes, unbroken glass culture dishes, and other types of broken and unbroken glassware, including microscope slides and coverslips, that was in contact with biological material.
In addition, hypodermic needles, suture needles, syringes, and scalpel blades which have never been in contact with biological materials should also be placed in these sharps wastes containers, since they pose a risk of injury to any staff member who may come in contact with these. In addition, some of these items (e.g., syringes without needles) may raise a public perception issue should they be placed in the general waste stream. Therefore, these should all be disposed in the sharps biomedical waste containers.
Because these containers have leak-proof sides and bottoms, soggy items and small amounts of liquids (e.g., a few ml of blood remaining in a tube) can be disposed in these containers. However, larger volumes of liquids should be handled as liquid waste (described in Section 8.5, below).
Environmental Services is responsible for closing and removing these containers from the laboratories. These are typically placed UPRIGHT in the red bag/boxes prior to sealing the box as described above.
8.3.2 Non-contaminated glass Unbroken or non-broken glass which has not been contaminated with any hazardous materials should be collected in solid-sided 5-gallon white plastic buckets with fitted lids or closed heavy duty cardboard cartons before disposal in the general waste stream.
8.3.3 Improper Solid Biohazardous Waste Disposal Environmental Services Staff members are to handle only the four authorized waste containers above. They are not to: Lift any biohazard (red) bag from any waste receptacle (lifting only increases the Environmental Services staff members’ potential exposure risk to biohazardous aerosols which may be generated while lifting or materials which may leak from the bag should the bag puncture). Combine the contents of multiple waste containers in order to compile one full container for disposal (combining only increases the risk of exposure to biohazardous aerosols, but also risks contaminating floors and surrounding areas). Leave any waste container unsecured or unprotected outside of the laboratory/clinic. Leave the Stericycle® truck unsecured. Accumulate wastes in any secondary waste collection sites (e.g., closets). The waste should be removed from the laboratory or clinic and taken to the Stericycle® truck.
Please note: like all sharps containers, the 7.5 gallon biohazard buckets are not intended to be re-usable waste receptacles. Bag liners are not to be used in conjunction with these buckets, and they should always be used with their fitted plastic lids.
Georgia Regents University Biosafety Guide-January 2012 8-6 Laboratory staff may choose to use alternate intermediary biohazard waste containers provided that these containers are: Clearly demarked as biohazardous waste with color-coded labels (e.g., by using red bags or biohazard stickers). The waste in these containers are removed as promptly after use and not allowed to linger in these intermediary waste containers. Capable of being decontaminated and are decontaminated often. No sharps are disposed in these intermediary containers (sharps must be directly placed into authorized sharps containers). The waste is transferred to the authorized waste receptacle by the laboratory staff (not the Environmental Services staff).
A word of caution about the use of lifting biohazard bags: The act of lifting a bag containing biohazardous materials can increase ones risks of exposure to biohazardous materials. First, the act of lifting may generate aerosols; therefore staff should be fully protected with PPE (at least lab coat, gloves and eye protection), and the bag should be tightly closed before lifting. In addition, any pointed items that may penetrate the bag (e.g., pipettes, micropipette tips) should not be placed in bags that are to be lifted, since they may also increase the exposure risks if the bag were to be punctured during lifting. Care should be taken by any staff member lifting a bag. These should be placed in the red bag/box for eventual disposal.
8.4 PATHOLOGICAL WASTES Any item which is identifiable as a human or animal body part or an animal carcass needs to be disposed pathological waste. These items must be appropriately labeled for incineration. At GRU, the majority of the pathological waste is generated through animal waste disposal procedures. Therefore, it is critical that all animal carcasses be packaged and labeled according to LAS/IACUC compliant procedures and placed in one of the necropsy freezers (e.g., in CB-1344, CA-1105, CA-1126, CA-1135, BG-1135A, CL-1119 or CL-1133) or in the cold storage room (CB-3100). Contact LAS (x1-3421) for further information about labeling procedures. These wastes will be placed in the animal waste carts and placed on the Stericycle® trailer just prior to departure. Contact the Division of Environmental Health and Safety (x1-2663) if your have any other questions related to pathological waste disposal.
8.5 LIQUID BIOLOGICAL WASTES Liquids which are derived from biological organisms (e.g., blood) or have been exposed to biological materials (e.g., tissue culture media, cell extracts) must be decontaminated prior to disposal. Liquid waste decontamination and disposal methods must be documented in each laboratory’s or clinic’s Standard Operating Procedures (SOPs).
The sanitary sewer was designed for the disposal of certain liquid wastes. Use of the sanitary sewer reduces the chance for leaks or spills during transport and reduces disposal costs. Whenever possible, decontamination of liquids by autoclaving or use of chemicals which can be disposed in the sewer system (namely, bleach), is highly recommended (See Section 7, Decontamination). Remember to rinse sink with copious water after disposal of decontaminated/deactivated biological materials.
Other chemical disinfectant methods may require subsequent disposal of the wastes through the chemical waste stream. Similarly, if the biological wastes are contaminated with chemicals or radiological materials,
Georgia Regents University Biosafety Guide-January 2012 8-7 disposal procedures will have to be modified to comply with disposal requirements for these materials (see Section 8.7, Mixed Waste).
Caution must be paid to disposal of any material which may clog or clog sewer disposal pipes. This would include large amounts of blood or agar. Disinfection of large amounts of blood may be accomplished by treatment with Isolyzer Plus®. This chlorine-based granular product solidifies and disinfects the blood or body fluids so they may be placed in general (clear), not biowaste (red) waste bags. Agar should be allowed to solidify prior to disposal in the regular waste stream (if not used in conjunction with biological materials), or placed in solid-sided waste containers, such as the 7.5 gallon biohazard waste bucket (if potentially contaminated with biological materials).
8.6 CHEMOTHERAPY WASTES Chemotherapy agents need to be segregated from the general biohazardous waste, as per Georgia EPD Chapter 391-3-4-.15 http://rules.sos.state.ga.us/docs/391/3/4/15.pdf. While these agents are often used in clinical therapy, many of these agents may also be used for in vitro and in vivo animal research applications (e.g., Actinomycin D, Mitomycin-C, Bleomycin, GM-CSF, Interleukin-2, INF-α, Gleevec) and must be disposed as chemotherapy waste. A list of chemotherapy agents can be found at: http://www.chemocare.com/bio/
According to GA EPD, chemotherapy wastes include: “Any disposable material which has come in contact with cytotoxic/antineoplastic agents (agents toxic to cells) and/or antineoplastic agents (agents that inhibit or prevent the growth and spread of tumors or malignant cells) during the preparation, handling, and administration of such agents. Such waste includes, but is not limited to, masks, gloves, gowns, empty IV tubing bags and vials, and other contaminated materials. The above waste must first be classified as empty which means such quantity that it is not subject to other federal or state waste management regulations prior to being handled as biomedical waste.”
(Note: "Empty" is generally defined as containing less than 3% by weight of the total capacity of the container).
Stock solutions of these chemicals and items that are heavily contaminated are disposed of through the Chemical Hazardous Waste Program. Call the Chemical Safety Office (x1-2663) for guidelines concerning the disposal of chemical hazardous waste.
8.7 MIXED WASTE “Mixed waste” are those wastes which are contaminated with more than one type of hazardous material. For the purposes of this Guide, focus will be on those materials which are contaminated with biologicals and either chemical or radiological materials.
8.7.1 Biological/Chemical Mixed Waste Many chemicals serve to disinfect biological materials through lysis, dehydration or protein-crosslinking. Common chemical fixatives (formalin, glutaraldehyde) may serve to decontaminate a biological material, leaving only the chemical waste disposal issues to address. Whenever possible, in biological/chemical mixed waste situations, efforts should be made to determine whether the chemicals have adequately decontaminated the biologicals. If they have not, seek assistance from the Biological and Chemical Safety Offices (x1-2663) to determine whether a chemically-compatible decontamination method can be devised which would then allow these items to be disposed as chemical waste. These should be documented in your laboratory SOPs prior to initiation of any experiment which would produce the mixed waste.
Items contaminated with ethidium bromide, diaminobenzidine (DAB), phorbol, or phenol-chloroform mixtures should not be mixed with other medical waste. These are already chemically decontaminated and
Georgia Regents University Biosafety Guide-January 2012 8-8 do not autoclave or bleach. These items should be segregated into their own containers and disposed as chemical waste.
Do not dispose of biological items which contain mercury in the biowaste. Biological items which may contain mercury (e.g., extracted teeth which contain mercury amalgams fillings) should first be decontaminated using a broad-spectrum chemical disinfectant (never autoclave mercury!) and disposed as mercury waste through the EHOS office (x1-2663).
8.7.2 Biological/Radioactive Mixed Waste All Radioactive wastes are required to be disposed through the Radiation Safety Office (x1-9826). Any biological materials that can be decontaminated with bleach, first, should be decontaminated and the pH of the resultant waste adjusted by the addition of non-hazardous buffering agents (sodium bicarbonate, Tris) prior to disposal as radioactive waste.
Radioactive sharps waste should be disposed of in sharps containers to which Radioactive warning labels have been clearly affixed. These should be disposed through the Radiation Safety Office.
Animal carcasses, tissue/parts, and excreta containing/contaminated with radioactive materials shall be handled and collected by those with proper radioactive material training and exposure badging (contact the Radiation Safety Office x1-9826 for further information).
8.8 REQUISITIONS FOR NEW OR ADDITIONAL BIOHAZARDOUS WASTE CONTAINERS Extra Stericycle box-bag units or authorized sharps containers can be obtained from Environmental Services at no cost to your laboratory (x1-2434 to make these requests). Any alternate required intermediary waste containers (small biohazard bags, small sharps containers) must be provided by the laboratory director.
If an unusual situation arises which requires disposal of large amounts (>15 gallons) of soggy materials or liquid materials, please contact the Environmental Health and Safety Office (x1-2663) for assistance.
8.9 REQUISITION FOR BIOHAZARDOUS WASTE PICK-UP The Environmental Services staff (a.k.a. Housekeeping) is responsible for removal of any full authorized waste containers. If laboratory staff members notice containers that are full and require removal, please contact the Environmental Services Office at x1-2434.
Georgia Regents University Biosafety Guide-January 2012 8-9 9. PLACARD SIGNS AND LABELS
Signs and labels which identify laboratories and equipment in which biological materials may be encountered are requirements of: • OSHA Bloodborne Pathogen standards (29 CFR 1910.1030) http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_table=STANDARDS&p_id=10051 • The State of Georgia’s Bloodborne Pathogen Act (O.C.G.A. § 31-12-13 http://www.lexis- nexis.com/hottopics/gacode/) • The CDC Biosafety in Microbiological and Biomedical Laboratories (BMBL) http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm • The NIH Guidelines for Research with Recombinant DNA Molecules (NIH Guidelines) http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.htm.
According to the CDC BMBL and the NIH Guidelines BSL-1 and BSL-2 standards, the laboratory supervisor is expected to limit access to the laboratory and establish policies and procedures whereby only persons who have been advised of the potential hazard and meet any specific entry requirements (e.g., immunization) may enter the laboratory or animal rooms. The Principal Investigator has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory.
Therefore, the purpose of the door placard is to advise all persons entering the laboratory of the potential hazards the specific entry/exit requirements that must be met as per the laboratory’s policies and procedures.
9.1 DOOR PLACARDS Entryways into any laboratory or clinic area in which biological materials are handled must be labeled with a biohazard door placard (see Figure 9.1). These signs are available through the Biosafety Office (x1-2663 or [email protected]). Biohazard door placards must be fluorescent orange-red (or predominantly so) with lettering or symbols in a contrasting color and include the following information:
1. The universal biohazard symbol with the legend “BIOHAZARD”. 2. The building and room number. 3. The name of the responsible investigator, laboratory director, or clinic director. This person must have knowledge of the types of biological material, the hazards they pose, and the appropriate laboratory incident emergency response standard operating procedures. 4. The name of an alternate contact person who may be contacted in the case of an emergency. 5. The daytime phone numbers and emergency after- hours contact numbers for the responsible investigator, laboratory or clinic direction and those of the alternate contact person. 6. The Biosafety Containment Level (BSL) of the room. (See Section 4 for more information). Figure 9.1 Example of required Biohazard Door Placard 7. A description of the biological hazards within the laboratory. 8. The appropriate PPE that must be worn by all personnel within the laboratory.
Georgia Regents University Biosafety Guide- January 2012 9-1
9. Any additional entry requirements or special provisions for entry as established by the PI in the laboratory SOPs (e.g. required vaccination, respirators, authorizations, etc.) as described in NIH Guidelines and/or the CDC BMBL.
9.2 LABELS AND COLOR-CODING Inside any facility in which biological materials are handled, warning labels shall be affixed to containers of medical waste, refrigerators, freezers, incubators, and centrifuges which may contain biohazardous material for any period of time without supervision of laboratory personnel. Labels of various sizes are available in the Biosafety Office upon request (x1-2663 or [email protected]). Other equipment such as waterbaths, sonicators, and biological safety cabinets do not require a permanent biohazard label if decontaminated after each use. In these situations, a biohazard labels should be temporarily posted on the equipment while in use with human blood, other potentially infectious materials, or an infectious agent.
Warning labels shall also be affixed to other containers used to store or transport biological materials. This includes temporary bench-top waste containers (e.g., beakers used to collect pipette tips) or transport containers. Labels required must be fluorescent orange-red (or predominantly so) with lettering or symbols in a contrasting color, have the international biohazard symbol and bear the legend "Biohazard". Labels shall be affixed as close as feasible to the container by string, wire, adhesive, or any other method that prevents their loss or unintentional removal.
The use of warning labels may be waived if: (1) waste is placed in red bags or red containers; (2) containers of blood, blood components, or blood products are labeled as to their contents and have been released for transfusion or other clinical use within GRU HS facilities as per GRU HS policies; or (3) individual primary containers of biological materials are placed inside a labeled secondary container during storage, transport, shipment or disposal.
9.3 LABELING EQUIPMENT SENT OUT FOR REPAIR OR DISPOSAL Contaminated and potentially contaminated equipment sent out for repair or disposal must be decontaminated as thoroughly as possible as per the laboratory standard operating procedures (SOPs). Affix a tag to the equipment indicating when the equipment was decontaminated, what disinfectant was used, and the name of the person who performed the decontamination.
Thorough decontamination of highly technical or sensitive equipment or equipment with limited access to contaminated areas may not be possible. Decontaminate the equipment to the best degree possible (flushing lines or wiping down the exterior) and affix a label to the equipment before sending it out for repair. The label must indicate what portions of the equipment may remain contaminated, with which agent(s) and include the biohazard symbol as well as the legend "Biohazard". The label must convey this information to all affected workers (service representatives, manufacturer, etc.). A sample of this tag appears at left and in Appendix J.
Georgia Regents University Biosafety Guide- January 2012 9-2
GUIDE TO SHIPPING BIOLOGICAL SUBSTANCES AND SUPPORT MATERIALS (SECTION 10 – GRU BIOSAFETY GUIDE)
The following Section is an adapted version of materials originally developed by Andy Glode and David R. Gillum at the University of New Hampshire. The GRU Biosafety Office wishes to thank Mr. Glode and Mr. Gillum for their work in producing the original document and their generosity in sharing their document with GRU.
This guide includes information about how to properly classify, package, mark and label your biological materials for shipment or extramural transport. This Section also describes the training requirements necessary to ship biological materials and dry ice. Requirements for intramural transport are discused in Section 4.1.5 of the GRU Biosafety Guide. Information on the regulations and procedures for transport/shipping of live animals can be found through the Division of Laboratory Animal Services.
Shipped/transported biological specimens, infectious agents and other biological materials are regulated by governmental and non-governmental, consensus development organizations. Penalties for non-compliance with the rules are significant and could result in the following fines:
• Up to $250,000 and up to a year jail sentence for individuals. • Up to $500,000 per incident for organizations.
Several agencies regulate the shipment and transport of biological materials including:
• International Air Transport Association (IATA). • US Department of Transportation (DOT). • US Public Health Service (PHS). • Occupational Health and Safety Administration (OSHA). • United States Postal Service (USPS).
Infectious substances and other dangerous goods must always be transported according to the appropriate regulations. Carrying dangerous goods by hand, for example in a vial in your pocket or in luggage, is strictly prohibited. IATA and DOT regulations cover your checked luggage, materials you carry on, or materials you carry in your pockets when you board an airplane. Persons who violate regulations are subject to fines and criminal prosecution.
IATA regulations are commonly encountered since they regulate materials transported by air and are generally the most restrictive. For these reasons, this guide pays special attention to IATA protocols; however the DOT standards often reflect those of IATA and also pertain to ground transportation of your materials.
10.1 TRAINING REQUIREMENTS
Federal rules require that anyone wishing to ship biological materials or dry ice must first have shipping training. If you intend to package biological materials or dry ice for shipment, you must complete a special training module, and provide documentation for training to the GRU Biosafety Office. GRU must be able to document training certification for anyone shipping dangerous goods during regulatory audits. Training consists of:
Georgia Regents University Biosafety Guide - 2013 10-1 1. Read this section of the Biosafety Guide. This document will provide familiarity with the general provisions relating to the regulations and will direct you to obtain more detailed training in the requirements applicable to shipping biological materials and/or dry ice.
2. Have a current Biosafety training and bloodborne pathogen training. This training ensures that you are familiar with hazards presented by infectious materials, proper handling and emergency response procedures.
3. Complete an DOT/IATA-compliant shipping training module. This must provide you with a certificate of completion and a copy of this certificate must be submitted to the Biosafety Office. This training is required once every two years. Currently, the Biosafety Office has created online shipping training module that can be accessed through the Desire2Learn, learning Management System. However, the Biosafety Office may accept alternate acceptable forms of training, as long as the content of the class can be documented and complies with the DOT/IATA requirements.
4. Document your intent to ship or transport biological materials. The IBC will ask that you document your intent to ship or transport biological materials in your Biosafety Protocol or by generating a shipping Standard Operating Procedure. For shipment of certain High Consequence Dangerous Goods, development of safety and security plans are required by law.
Shipping regulations change frequently so it is necessary to repeat training certification every two years.
10.2 SHIPPING OVERVIEW Follow these steps when shipping biological materials and dry ice.
1. Classify your materials for shipment/transport. See Section 10.3. 2. Package, mark, and label your material(s) appropriately. See Section 10.4. 3. Fill out the Shipper’s Declaration for Dangerous Goods form. See Section 10.5. 4. If you are shipping any Federally regulated materials, including Select Agents, special regulations may apply and/or permits may be required. Consult Section 10.6. 5. If you plan on importing or exporting biological materials, permits may be required. Consult Section 10.7.
10.3 SHIPMENT TYPE For shipment purposes, biological material will fit into one of the following categories:
• Unregulated biological material; • Category A infectious substances; • Category B infectious substances; • Patient specimens; • Genetically modified organisms and microorganisms; or • Regulated Medical/Clinical Waste
Read each material section carefully to determine how to classify a material. If you are shipping a biological material that cannot cause disease, infectious substance regulations do not apply, unless sent by mail (see Section 10.9). Refer to the classification guide to assist with classification of materials (Figure 10.3). Note: All specimens or packaging containing dry ice or liquid nitrogen must be shipped properly (see Other Packaging Requirements, Section 10.4.2). All samples preserved with flammable or corrosive materials, such as ethanol or formalin, must receive consultation and pre-authorization from the GRU Chemical Safety Office (x1-2663) and be shipped appropriately (also please see Section 10.4.2). Materials which are both biological and radioactive require consultation with and pre-authorization from the Radiation Safety Office (x1-9826).
Georgia Regents University Biosafety Guide - 2013 10-2
10.3.1 Unregulated Biological Material The materials listed below are technically not subject to IATA or DOT infectious substance shipping regulations. However, guidance for shipping these materials (e.g., patient specimens, biological products) are found in subsequent sections and these may require a permit for shipment abroad. Please check with the Biosafety Office if you have any questions about these materials. All shipments of blood and blood products must be labeled with a biohazard symbol. • Substances which do not contain infectious substances or which are unlikely to cause disease in humans or animals; • Non-infectious biological materials from humans, animals or plants. Examples include non-infectious cells, tissue cultures, blood or plasma from individuals not suspected of having an infectious disease, DNA, RNA, or other genetic elements; • Substances containing microorganisms, which are non-pathogenic to humans or animals; • Substances that have been neutralized or inactivated such that they no longer pose a health risk; • Environmental samples which are not considered to pose a significant risk of infection; • Dried blood spots*; • Fecal occult blood screening tests*; • An infectious substance, other than a Category A infectious substance (See Section 10.3.2.1), contained in a patient sample being transported for research, diagnosis, investigational activities, or disease treatment and prevention, or a biological product, when such materials are being transported by a private or contract carrier in a motor vehicle used exclusively to transport such materials; • Blood or blood components which have been collected for the purpose of transfusion or the preparation of blood products to be used for transfusion or transplantation*; • Tissues or organs intended for use in transplantation*; • A material with a low probability of containing an infectious disease or where the concentration of the infectious substance is at a level naturally occurring in the environment so it cannot cause disease when exposure to it occurs. Examples of these materials include foodstuffs and environmental samples (such as water or a sample of dust or mold); or • A biological product, including an experimental or investigational product or component of a product, subject to federal approval, permit, review or licensing requirements such as those required by the Food and Drug Administration (see, e.g., http://www.fda.gov/importeddrugs/ or http://www.fda.gov/RegulatoryInformation/Guidances/ucm125789.htm ), or the US Department of Agriculture* (see, e.g., http://www.aphis.usda.gov/import_export/index.shtml and http://www.aphis.usda.gov/vs/ncie/fac_imp.html for guidance).
* When mailing these items with the USPS, follow packaging guidelines for non- regulated items. See Section 10.9.
Georgia Regents University Biosafety Guide - 2013 10-3 Figure 10.3 The Classification Guide
Is your sample expected to YES contain any human, animal or NO plant pathogens?
Is your sample: Is your sample a direct patient • Dried blood spots? specimen and packaged as an exempt NO • Fecal occult blood screening tests? human or animal specimen? YES • Blood, blood components, organs or tissues for purposes of transfusion or transplantation or for the preparation of products for the YES purpos of transfusion or transplantation? NO Is your sample a genetically modified NO organism or micro-organism? Is your sample on the indicative examples of Category A Infectious YES Substances (See Table 10.3.2.1) OR Is your sample capable of causing YES permanent disability, life threatening or fatal disease? Does your sample affect humans only or humans & animals? NO NO
Does your sample YES Is your sample YES affect animals only? Medical waste?
YES NO
UN 2814 UN 2900 Category A Category A UN 3373 UN 3291 UN 3245 Infectious Infectious Biological Patient specimens Biomedical Genetically modified substance, substance, substance, (PI 650 Not waste, n.o.s. organisms and micro- Not affecting afecting Category B recommended) regulated (PI 650) organisms regulated humans animals (PI 650) See Section See Section (PI 913) (PI 602) (PI 602) See Section 10.3.3 10.3.6 See Section 10.3.5 See Section See Section 10.3.2.2 10.3.2.1 10.3.2.1
Note: This flow chart only refers to IATA classificaztion of biological materials and does not cover other hazards &/or considerations for: •Special permits for transfer (See Section 10.6) •Dry ice (See Section 10.4.2.2) •Liquid Nitrogen (See Section 10.4.2.3) •Radiological hazards (Refer to Radiation Safety Office) •Chemical hazards (Refer to Chemical Safety Office)
Georgia Regents University Biosafety Guide - 2013 10-4
10.3.2 Infectious Substances Infectious substances are materials known to be, or are reasonably suspected to contain, an animal or human pathogen. A pathogen is a virus, microorganism (including bacteria, plasmids, or other genetic elements), proteinaceous infectious particle (prion) or recombinant microorganism (hybrid or mutant) that is known or reasonably expected to cause disease in humans or animals. Microorganisms that are unlikely to cause human or animal disease are not subject to biological shipping regulations.
10.3.2.1 Category A Infectious Substances Category A infectious substances are capable of causing permanent disability, life threatening or fatal disease in humans or animals when exposure to them occurs. Category A infectious substances are shipped as infectious substances, affecting humans (UN2814), or infectious substances affecting animals (UN2900). Indicative examples of Category A infectious substances are listed in Table 10.3.2.1.
10.3.2.1.1 Packaging The triple packaging concept (explained in Section 10.4) applies to Category A infectious substances. Purchase packaging compliant with IATA Packing Instruction 602 as detailed in the IATA Dangerous Goods Regulations (DGR), which is available in the Biosafety Office. See Table 10.3.2.1.1 for a list of packaging suppliers. Make sure to specify if you are shipping a refrigerated sample (ice packs or dry ice). The maximum quantity of infectious substance that can be shipped by air in one package is 4 L or 4 kg. The maximum quantity that may be shipped via passenger aircraft is 50 mL or 50 g.
Georgia Regents University Biosafety Guide - 2013 10-5 Table 10.3.2.1. Indicative Examples of Category A Infectious Substances
UN # and Proper Shipping Name Microorganism - Bacillus anthracis cultures - Japanese Encephalitis virus cultures - Brucella abortus cultures - Junin virus - Brucella melitensis cultures - Kyasanur Forest disease virus - Brucella suis cultures - Lassa virus - Burkholderia mallei - Pseudomonas mallei - Glanders cultures - Machupo virus - Burkholderia pseudomallei - Pseudomonas pseudomallei cultures - Marburg virus - Chlamydia psittaci - avian strains cultures - Monkeypox virus - Clostridium botulinum cultures - Mycobacterium tuberculosis cultures - Coccidioides immitis cultures - Nipah virus
- Coxiella burnetii cultures - Omsk hemorrhagic fever virus - Crimean-Congo hemorrhagic fever virus - Poliovirus cultures - Dengue virus cultures - Rabies virus cultures - Eastern equine encephalitis virus cultures - Rickettsia prowazekii cultures - Escherichia coli, verotoxigenic cultures - Rickettsia rickettsia cultures - Ebola virus - Rift Valley fever virus
humans - Flexal virus - Russian spring-summer encephalitis virus cultures UN 2814 UN - Francisella tularensis cultures - Sabia virus - Guanarito virus - Shigella dysenteriae type 1 cultures - Hantaan virus - Tick-borne encephalitis virus cultures - Hantavirus causing hemorrhagic fever with renal syndrome - Variola virus - Hendra virus - Venezuelan equine encephalitis virus - Hepatitis B virus cultures - West Nile virus cultures - Herpes B virus cultures - Yellow fever virus cultures - Human immunodeficiency virus cultures - Yersinia pestis cultures Infectious substance affecting - Highly pathogenic avian influenza virus cultures - African swine fever virus cultures - Avian paramyxovirus Type 1 – Velogenic Newcastle disease virus cultures - Classical swine fever virus cultures - Foot and mouth disease virus cultures - Lumpy skin disease virus cultures
- Mycoplasma mycoides - Contagious bovine pleuropneumonia cultures - Peste des petits ruminants virus cultures - Rinderpest virus cultures - Sheep pox virus cultures - Goatpox virus cultures
animals - Swine vesicular disease virus cultures UN 2900 UN Infectious - Vesicular stomatitis virus cultures substance affecting
* This list is not exhaustive. New or emerging pathogens not on the list may meet the criteria to be included in Category A.
Georgia Regents University Biosafety Guide - 2013 10-6 Table 10.3.2.1.1. Manufacturers of Shipping Containers for Infectious Substances and Dry Ice
Air Sea Atlanta All-Pak, Inc. CARGOpak Corporation 1234 Logan Circle Corporate One West 3215-A Wellington Court Atlanta GA 30318 1195 Washington Pike Raleigh, NC 27615 Phone: 404-351-8600 Bridgeville, PA 15017 Phone: 800-266-0652 http://www.airseaatlanta.com Phone: 800-245-2283 http://www.cargopak.com http://www.all-pak.com
DG Supplies, Inc. EXAKT Technologies, Inc. HAZMATPAC, Inc 5 Boxal Drive 7416 N Broadway Ext., Suite E 5301 Polk St., Bldg 18 Cranbury, NJ 08512 Oklahoma City, OK 73116 Houston, TX 77023 Phone: 800-347-7879 Phone: 800-923-9123 Phone: 800-347-7879 http://www.dgsupplies.com http://www.exaktpak.com http://www.hazmatpac.com
Inmark, Inc. JIT Certified, Inc. Polyfoam Packers Corporation 220 Fisk Drive S.W. 1740 Fenpark Drive 2320 S. Foster Avenue Atlanta, GA 30336-0309 Fenton, MO 63026 Wheeling, IL 60090 Phone: 800-646-6275 Phone: 800-962-8636 Phone: 888-765-9362 http://www.inmarkinc.com http://www.jitcertifed.com http://www.polyfoam.com
SAF-T-PAK, Inc. Source Packaging of New England, Therapak Corporation 10807 - 182 Street Edmonton, Alberta, Inc. 1440 Arrow Highway, Unit A Canada, T5S 1J5 405 Kilvert St. Irwindale, California 91706 Phone: 800-814-7484 Warwick, RI 02886 Phone: 888-505-7377 http://www.saftpak.com Phone: 800-200-0366 http://www.therapak.com http://www.sourcepak.com
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10.3.2.1.2 Labeling The outer container of a Category A infectious substance shipment must display the following information:
. Sender and recipient’s full name and address; . Infectious substance label (see below); . “UN2814, Infectious substance, affecting humans” and net quantity or “UN2900, Infectious substance, affecting animals” and net quantity; . The text “Person responsible: [name, phone number]” You must provide a 24 hr/day, 7 days/week contact for a person who knows what material has been shipped and emergency response information. This should be documented in your shipping/transport SOP; . Class 9 label (see below), including UN1845 and net weight, if packaged with dry ice; and . Cargo Aircraft Label (see below), when shipping over 50 mL or 50 g.
Infectious substance Class 9 Label Cargo Aircraft Label Label
DANGER
10.3.2.2 Category B Infectious Substances Category B infectious substances are materials that are infectious, but do not meet the standard for inclusion in Category A. Category B infectious substances are assigned to UN3373.
10.3.2.2.1 Packaging The basic triple packaging concept applies to Category B infectious substances. Purchase packaging that complies with IATA Packing Instruction 650. See Table 10.3.2.1.1 for a list of some packaging suppliers. Be sure to specify if the shipment is a refrigerated sample (e.g., ice packs or dry ice) or will be sent at ambient (room) temperature.
For Category B infectious substances, the maximum quantity of liquid per primary receptacle is 1 liter and outer packaging must not contain more than 4 L or 4 kg.
10.3.2.2.2 Labeling The outer container of a Category B infectious substance shipment must display the following information:
UN3373 Label
Georgia Regents University Biosafety Guide - 2013 10-8 . The sender and recipient’s full name and address; . The words “Biological Substance, Category B”; . UN3373 label (see right); . The text “Person responsible: [name, phone number]” You must provide a 24 hr/day, 7 days/week contact for a person who knows what material has been shipped and emergency response information. This should be documented in your shipping/transport SOP; and . Class 9 label (see Section 10.2.2.1.2), if packaged with dry ice.
10.3.3 Patient Specimens Patient specimens that have a minimal likelihood of containing pathogens are exempt from many shipping requirements. Professional judgment is used to determine if a specimen contains pathogens and should be based on the patient’s medical history, symptoms, local conditions and individual circumstances.
If there is more than a “minimal likelihood” that a patient specimen contains pathogens, it must be shipped as a Category A infectious substance (UN2814 or UN2900) or a Category B infectious substance (UN3373).
Patient specimens unlikely to contain pathogens must be prepared for shipment as follows:
10.3.3.1 Packaging • Leak-proof primary container; • Leak-proof secondary packaging; • Fragile primary containers must be wrapped or separated to prevent breakage; • Absorbent material must be placed between the primary and secondary containers to absorb entire contents so that no liquid release will reach the outer packaging; and • Outer packaging must be durable enough for its intended use with at least one side 100 mm x 100 mm or larger.
10.3.3.2 Labeling The outer package must be marked with “Exempt human specimen,” or “Exempt animal specimen.”
10.3.3.3 Dried blood Special guidance has been provided by the CDC for shipment of dried blood spot specimens which vary from those above. In particular, these should not be packaged in airtight, leak-proof plastic bags because the lack of air exchange in the inner environment of a sealed plastic bag causes heat buildup and moisture accumulation that can damage the dried blood spot test substances. In addition, various chemicals that can adversely affect the test substances in the dried blood spots could leach from these plastics and thus cause incorrect analytical test results. See: http://www.cdc.gov/od/ohs/biosfty/driblood.htm for further information.
10.3.4 Biological Products Biological products are derived from living organisms and manufactured for use in the prevention, diagnosis, treatment or cure of diseases in humans or animals and are certified by the USDA, FDA or other national authority.
Examples of biological products include certain viruses, therapeutic serums, toxins, antitoxins, vaccines, blood, and blood products. Materials which contain incidental blood products, such as fetal calf serum, may also be regulated, particularly internationally. For further information and guidance for shipping biological materials, see:
Georgia Regents University Biosafety Guide - 2013 10-9 Food and Drug Administration Guidelines Importation: http://www.fda.gov/ora/import/ Exportation: http://www.fda.gov/RegulatoryInformation/Guidances/ucm125789.htm US Department of Agriculture Importation/exportation URL: http://www.aphis.usda.gov/import_export/index.shtml Materials which do not require permits: http://www.aphis.usda.gov/biotechnology/submissions.shtml
Biological products transported for final packaging, distribution, or use by medical professionals are not subject to biological shipping regulations (although international shipments may have importation or exportation standards, See Section 10.7). Biological products that do not meet these criteria must be assigned to UN2814, UN2900 or UN3373, as appropriate.
10.3.5 Genetically Modified Organisms or Microorganisms Genetically modified organisms (GMO) or microorganisms (GMMO) are organisms and microorganisms in which genetic material has been purposely altered through genetic engineering in a way that does not occur naturally. GMOs and GMMOs that are not infectious but that can alter animals, plants or microorganisms in a way that is not normally the result of natural reproduction are considered a miscellaneous hazard (Class 9) and are assigned to UN3245. GMOs and GMMOs that are infectious must be assigned to UN2814, UN2900 or UN3373.
10.3.5.1 Packaging These materials are packed for shipment in the same way as Category A infectious substances, except there are no testing requirements for the packaging; this packaging variation is IATA Packing Instruction 913. Packages designed for Packing Instruction 913 may not be available from most vendors. In this case, use packages compliant with Packing Instruction 602.
The maximum allowable quantity per primary receptacle is 100 mL or 100 g. There is no maximum net quantity per package.
10.3.5.2 Labeling The outer container of a GMO or GMMO assigned to UN3245 must display the following information:
. The sender and recipient’s full name and address; . Class 9 label (See Section 10.3.2.1.2); and . Genetically modified microorganisms, UN3245, and net quantity.
10.3.6 Regulated Medical Waste Regulated Medical Waste (also sometimes referred to as Regulated Biomedical Waste or Clinical Waste) is defined differently by many state and federal agencies. Under DOT rules, regulated medical waste (RMW) is a waste or reusable material suspected or known to contain an infectious substance, and is generated in the diagnosis, treatment, immunization, or biomedical research of humans or animals. Regulated Medical Waste (RMW) is assigned UN 3291 and is generally packaged consistently with IATA packing instruction 650 (See Section 10.3.2.2, Category B Infectious Substances for further information about packing instruction 650), although some special exceptions exist in the DOT regulations (49 CFR 173.137 (c) and (d) and 179.197).
GRU currently packages all RMW in boxes and carts provided by Stericycle® (See Section 8, Waste Management); which comply with the regulations for packaging and labeling of RMW. Typically, these are transported from GRU campus by Stericycle® personnel who have to adhere to the requirements for
Georgia Regents University Biosafety Guide - 2013 10-10 transport of these materials, and are generally not transported by most GRU personnel in vehicles on public roads. However, special considerations should be made should occasion arise that any regulated medical wastes are to be transported, offered for transport or shipped if these wastes are to be:
Transported via vehicles traveling on public thoroughfares (e.g., to GRU or to the Stericycle® truck from remote campus locations) Any material transported under the Regulated Medical Wastes must be in a vehicle dedicated for RMW transport if it may contain agents of Risk Group 2 or higher.
Transported outside of campus in containers other than the large Stericycle® boxes or carts (e.g., in sharps containers), which may not have the required markings. These should be appropriately packaged and labeled prior to transport.
Please contact the Biosafety Office (x1-2663, [email protected]) to declare your intention to transport RMW and for further guidance prior to transport.
10.4 PACKAGING BIOLOGICAL MATERIALS Potentially hazardous biological materials must be packaged to withstand leakage of contents, shocks, temperature, pressure changes and other conditions that can occur during ordinary handling in transportation. Packaging your material(s) appropriately is accomplished by purchasing certified packaging. Refer to Table 10.3.2.1.1 for vendors that can supply certified packaging for biological materials. When ordering, specify what type of material(s) you will be shipping: Category A infectious substances, Category B infectious substances, etc. Different categories have slightly different packaging needs, but all follow the basic triple packaging requirements described below.
10.4.1 Triple Packaging Biological materials must be packaged according to the triple packaging principle. The three elements of triple packaging include: primary receptacle, leak-proof secondary container, and durable outer container. Infectious substances in Category A and B, patient specimens and genetically modified microorganisms must be packaged in this way, with slight variations. An example of triple packaging is illustrated in Figure 10.4.1.
The primary container holds the biological material; it must be leak-proof. It must be labeled with the name of the contents. A leak-proof seal, such as a heat seal, skirted stopper or metal crimp, is required. If the container has a threaded lid, it must be secured with waterproof tape (e.g., Parafilm, etc.). Petri plates cannot be used as primary receptacles. Lyophilized substances can only be shipped in flame sealed glass ampoules or rubber stopped glass vials with metal seals. Packaging purchased for shipping infectious substances usually does not include the primary container.
The secondary container holds one or more primary containers, and must also be leak-proof. Secondary containers for all Category A and liquid Category B infectious substances must meet specific pressure test standards when shipping liquids. Containers purchased from commercial vendors are designed to meet the necessary standards. If you are shipping any liquid, there must be enough absorbent material in the secondary container to absorb all of the liquid in the primary receptacle(s). If multiple primary containers are used, they must be wrapped to prevent contact between them so they do not break during transport.
The outer container must be rigid and have one side that is at least 100 mm X 100 mm, in order for required markings and labels to fit. The outer package must be of adequate strength for its capacity, mass, and intended use. An itemized list of package contents must be included between the outer and secondary container. The outer package should be marked to identify hazardous contents, including the proper shipping name, UN number and net quantity for each substance, if required.
Georgia Regents University Biosafety Guide - 2013 10-11
Figure 10.4.1
Source: Transporting Infectious Substances Safely, US DOT Document PHH50-0079-0706.
10.4.2 Other Packaging Requirements
10.4.2.1 Overpacks An overpack can be used to combine several triple packages into one large package. This may be done to save on shipping charges when shipping multiple samples. Each triple package inside the overpack must be properly marked and labeled. The outside of the overpack must bear the same markings and labels as the triple packages within including hazard labels and proper shipping names. The outer container of the overpack must also be marked with the word, “Overpack.”
10.4.2.2 Dry Ice
10.4.2.2.1 Hazard Identification Dry ice is classified by DOT and IATA as a “miscellaneous” hazard, class 9. Dry ice is considered hazardous during transportation for three reasons:
Explosion hazard: Dry ice releases a large volume of carbon dioxide gas as it sublimates. If packaged in a container which does not allow for release of the gas, it may explode, causing potential injury and/or property damage.
Suffocation hazard: a large volume of carbon dioxide gas emitted in a confined space may create an oxygen-deficient atmosphere.
Contact hazard: Dry ice is a cryogenic material that causes severe frostbite upon contact with skin..
Packaging dry ice properly will minimize the risk to personnel transporting the material. The explosion hazard will be eliminated with a package designed to vent gaseous carbon
Georgia Regents University Biosafety Guide - 2013 10-12 dioxide. Suffocation and contact hazards will be greatly reduced by labeling the package correctly, so those who come in contact with it will be aware of the contents.
10.4.2.2.2 Packaging Dry Ice There are five basic requirements for all shipments of dry ice:
a. Gas venting: packages must allow for release of carbon dioxide gas. Dry ice must never be sealed in a container with an airtight seal such as a jar with a threaded lid or a plastic cooler. When transporting in a vehicle, the box should not be placed inside the passenger compartment to prevent carbon dioxide accumulation within the vehicle. b. Package integrity: a package containing dry ice must be of adequate strength for intended use. It must be strong enough to withstand the loading and unloading normally encountered in transport. It must also be constructed and closed in order to prevent any loss of contents that might be caused by vibration of by changes in temperature, humidity, or altitude. c. Package materials: do not use plastics that can be rendered brittle or permeable by the temperature of dry ice. This problem can be avoided by using commercially available packages intended to contain dry ice (see Table 10.3.2.1.1). d. Waybill: the waybill (also referred to as the airbill) must include the statement “Dry ice, 9, UN1845, [number of packages] X [net weight in kilograms]” FedEx has a check box on their waybill to satisfy this requirement (see Figure 10.4.2.2.2A). Airborne Express requires a slightly different format (see Figure 10.4.2.2.2B). Check with your courier to make sure you have made the proper notation on their paperwork. e. Labeling: the outermost container must be labeled with a hazard class 9 label, UN1845m and net weight of dry ice in kilograms. See Figure 10.4.2.2.2C, below. This must be a specific size (5” x 5”). A full-scale printable version is included in Appendix K.
Figure 10.4.2.2.2A. FedEx Waybill which properly documents 1 box containing 6 kg of dry ice.
Georgia Regents University Biosafety Guide - 2013 10-13
Figure 10.4.2.2.2B. AirBorne Express waybill which properly documents 1 box containing 5 kg of dry ice.
If shipping biological materials with your dry ice, you must comply with the requirements for both shipping of biological materials and dry ice.
When shipping biological materials and dry ice together: a. Dry ice must be placed outside the secondary packaging (See Figure 10.4.2.2D) b. Secure your samples in such a way that when the dry ice sublimates, they will not move freely inside the insulated box. This can be accomplished by wedging your samples in place with cardboard or Styrofoam. Fragile containers such as glass tubes or vials should be wrapped in cushioning material. Figure 10.2.2.2C Dry ice label (not to size) c. A Shipper’s Declaration for Dangerous Goods is not required for shipments in which dry ice is the only hazardous material; however, dry ice is included on declarations for shipments that include other hazardous materials such as infectious substances.
Other issues that you should consider when shipping with dry ice: a. Refer to your package manufacturer’s recommendations. Make arrangements with your cosignee to make sure your package will be received on its intended delivery date. Take into account local holidays or closings that might delay package receipt. b. Minimize the volume of air to which the dry ice is exposed in order to slow the rate of sublimation. If there is any air space after you fill the package with dry ice, fill it with packing peanuts or other material to reduce the volume of air space. c. Shipments are generally recommended to contain 5-10 pounds (2.27-4.54 kg) of dry ice per 24 hours.
Georgia Regents University Biosafety Guide - 2013 10-14 d. Dry ice shipments can be made with FedEx and DHL. UPS and the U.S. Postal Service have extremely restrictive policies concerning shipments of hazardous materials. Do not ship dry ice with UPS or with the U.S. Postal Service.
Material packaged in primary and secondary packages 10.4.2.3 Liquid Nitrogen (as if for ambient shipping– See Biological materials can be shipped refrigerated with liquid Figure 10.4.1) nitrogen in cryogenic dry shippers, which are insulated packages containing refrigerated liquid nitrogen fully absorbed in a porous material. The dry shippers, themselves, do not contain DRY ICE hazardous materials, do no allow for the build-up of pressure within the container and will not permit the release of any Cooler (with lid refrigerated liquid nitrogen regardless of the dry shipper’s which allows for gas venting) orientation. Properly used, the dry shippers, alone, do not contain free liquid nitrogen, and are not subject to hazardous material regulations by the DOT or IATA. Dry shippers are capable of maintaining cryogenic temperatures normally Outer box associated with liquid nitrogen for approximately 24 hours (depending on the manufacturer) without risk of liquid nitrogen spilling. However, be aware, improperly filled dry shippers present a risk of liquid nitrogen leakage and are subject to regulation should spillage occur.
Follow the manufacturer’s instructions for filling the dry shipper. Some general practices when filling the dry shipper are: Figure 10.2.2.2D Packaging for Dry Ice a. Wear insulated gloves made for handling liquid nitrogen and a face shield. b. Add the liquid nitrogen slowly since a significant volume of nitrogen gas will form as the cold liquid contacts the warm surfaces. c. When the liquid level reaches the neck of the dry shipper, stop filling. Replace the cap and set the dry shipper aside for the period specified by the manufacturer to allow the liquid nitrogen to saturate the absorbent. d. Repeat steps a-c until the liquid level no longer drops on standing. Special packing regulations apply to shipments containing nitrogen. Contact the Biosafety Office at x1- 2663 or [email protected] if you need to ship materials with liquid nitrogen.
Some manufacturers have empty and full weights for their dry shippers. Dry shippers that will not achieve their full weight may indicate a problem with the absorbent’s ability to hold the nitrogen. This may prevent the dry shipper from maintaining the proper cryogenic temperatures during shipment and may damage your samples. Contact the manufacturer to determine if the dry shipper is safe to use.
When you are ready to ship, follow the following steps: a. Remove all free liquid nitrogen from the dry shipper before transport. b. Wear insulated gloves and a face shield when emptying the dry shipper. c. Do not pour liquid nitrogen on to the floor since it could splash on your shoes or legs and cause severe burns. It is recommended to pour the excess liquid nitrogen back into a large liquid nitrogen dewar. d. Hold the dry shipper upside down until the liquid stops flowing. e. Stand the dry shipper upright for the period specified by the manufacturer. f. Repeat steps a-e, above as many times as necessary to remove remaining liquid nitrogen. g. Place your canes of material into the dry shipper and replace the cap. h. Place the dry shipper into the case supplied by the manufacturer.
Georgia Regents University Biosafety Guide - 2013 10-15 Make sure that the materials you are transporting are not hazardous chemicals such as samples frozen in propane, ethane, halocarbon or other hazardous gas. If you are shipping biological materials, your specimens will still have to comply with the IATA/DOT standards for biologicals, as described above.
Special packing regulations apply to shipments containing liquid nitrogen. Contact the Chemical Safety Office at x1-2663 if you need ship or transport materials with liquid nitrogen.
10.4.2.4 Samples Preserved in Fixative Special consultation with both the Biosafety and Chemical Safety Offices are required before shipping materials which may be preserved in chemical, such as formalin or ethanol. However, below are reference guidelines for shipping materials preserved in aqueous solutions of formalin or ethanol. Packages prepared according to these guidelines must not contain any materials other than those described (i.e. containers holding formalin- or ethanol-preserved specimens and related absorbent or packaging materials). Laboratory or sampling equipment, unrelated documents, or other goods must be packaged and shipped in separate boxes.
10.4.2.4.1 In Aqueous Formaldehyde Solutions (Formalin) <25% Aqueous formaldehyde solutions of less than 25% are considered hazardous materials when shipped by air. This is because formalin can cause eye, skin, and respiratory tract irritation. Formaldehyde is regulated by OSHA as a carcinogen. Additionally, exposure to formaldehyde solutions may cause an allergic respiratory reaction. Be sure to review the MSDS before handling or shipping any hazardous material.
Proper packaging shipments of formalin will minimize the chance of leakage during transportation. Properly labeling and documenting these shipments will communicate the hazard to transport workers who may be exposed to the formaldehyde in the event of a leak.
Formaldehyde solutions are assigned to hazard class 9, packing group II. As such, each inner packaging may not contain more than 30 ml. Each outer package may contain not more than 500 ml. At this amount, these are considered “excepted quantities”.
Packaging for excepted quantities must have three basic components: a. Inner (primary) packaging, such as a vial, tube, jar, etc. Do not completely fill inner packagings; allow 10% head-space for liquid expansion. Liquids must not completely fill inner packagings at a temperature of 55°C (130°F). Closures of inner packagings must be held securely in place with tape, wire, metal crimps, or other positive means. b. Intermediate (secondary) packaging, such as a ziplock or other plastic bag. Use good quality bags that are well sealed. Intermediate packaging must contain enough absorbent material to absorb all contents and must not react with formaldehyde. Use two plastic bags: put the absorbent and the inner container(s) in the first bag and seal it well with tape. Then seal this bag in another bag for added protection. c. Outer packaging, such as a cardboard box. Formaldehyde solutions may not be shipped in envelopes, Tyvek sleeves, or other non-rigid mailers. The dimensions of the outer box must be at least 100 m (~4 inches) on two sides.
Georgia Regents University Biosafety Guide - 2013 10-16 Labels and Marks on the outer packaging must include the following: a. Dangerous Goods in Excepted Quantities Label, See Figure 10.4.2.4A. This label must be filled out with the signature, title, name and address of the shipper and the date. It must be affixed to the outer container on a vertical side. For formaldehyde solutions of less than 25%, check the box for Class 9 material and enter “UN 3334” as the applicable UN number. The printable label may be found in Appendix K and must be printed in color. Its overall dimensions must be at least 100 mm x 100 mm (~4 in. x 4 in).
b. Name and Address: The outer container must display the name and address of the shipper and consignee.
Many printer inks run when exposed to small amounts of water, such as rain or snow. Therefore, it may be necessary to fully cover each label you have affixed to the box with clear plastic tape. Also, when re-using shipping boxes, completely obliterate all unnecessary labels and marks.
Figure 10.4.2.4A. Dangerous Goods in Excepted Quantities Label (not to scale)
Package Tests must be performed and documented to ensure package compliance. A representative example of packaging used for excepted quantities of formaldehyde solutions must pass a drop test and compressive load test without any breakage or leakage of any inner packaging and without any significant reduction in package effectiveness. Perform the following tests on representative example of your packaging and keep a record of the results.
Drop Test. Drop a representative package from a height of 1.8 m (5.9 feet) directly on to a solid unyielding surface: • One drop flat on bottom; • One drop flat on top • One drop flat on the long side; • One drop flat on the short side; and • One drop on a corner at the junction of three intersecting edges.
Compressive Load Test. Apply a force to the top surface of a representative package for a duration of 24 hours, equivalent to the total weight of identical packages if stacked to a height of 3 meters.
Georgia Regents University Biosafety Guide - 2013 10-17 Proper documentation is required for all shipments of hazardous materials. Incorrect documentation is the most common cause of package refusal. If using documentation for couriers other than FedEx and DHL, contact EHS for assistance.
For domestic shipments with FedEx Express, fill out the standard US waybill. Fill out the form completely and be sure to include the following information: In section 6, Special Handling, check the box “Yes, Shipper’s Declaration not required.” On the top of the form above the FedEx tracking number, include the statement, “Dangerous Goods in Excepted Quantities”. See example in Figure 10.4.2.4B.
For DHL shipments, under the “Nature and Quantity of Goods” box on the air waybill, include the words “Dangerous Goods in Excepted Quantities”.
Figure 10.4.2.4B Example of FedEx waybill for excepted quantity shipment
10.4.2.4.2 In Aqueous Ethanol Solutions of 55-100% Ethanol solutions of 55-100% are considered hazardous materials when shipped by air. This is because ethanol is a flammable liquid (NFPA rating = 3), and its vapor can travel a considerable distance to an ignition source and “flash back”. Contact of ethanol with strong oxidizers, peroxides, strong alkalis, and strong acids may cause fires and explosions. Be sure to review the manufacturer MSDS before handling or shipping any hazardous material.
Georgia Regents University Biosafety Guide - 2013 10-18
Ethanol solutions are assigned to hazard class 3, packing group II. As such, each inner packaging may not contain more than 30 ml. Each outer package may contain no more than 500 ml.
Packaging, labeling requirements, package tests and documentation are similar to those indicated above for formaldehyde solutions (See Section 10.4.2.4.1) with the exception that for ethanol solutions of 55-100%, the Dangerous Goods in Excepted Quantities Label (as seen in Figure 10.4.2.4A for formaldehyde solutions) should have the Class 3 material box checked and “UN 1170” as the applicable UN number.
10.5 SHIPPER’S DECLARATION FOR DANGEROUS GOODS A Shipper’s Declaration for Dangerous Goods must be completed when shipping a Category A infectious substance assigned to UN2814 or UN2900or a GMO or GMMO assigned to UN3245. A declaration is not required for shipments in which dry ice is the only hazardous material. A declaration is not required for shipments of Category B infectious substances assigned to UN3373. Improperly completed declarations are the most common cause of package refusal.
Refer to the Shipper’s Declaration for Dangerous Goods (Figure 10.5) for an explanation of each section:
A. Shipper: Enter your full name, address and telephone number. B. Consignee: Enter full name and address of recipient. When shipping infectious substances, include the text, “Person responsible:” [then you must provide a 24 hr/day, 7 days/week contact for a person who knows what material has been shipped and emergency response information. This should be documented in your shipping/transport SOP]. C. Transport Details: Indicate here if your shipment is restricted to cargo aircraft only (if it is more than 50 ml or 50 g of an infectious substance). Airport of departure and airport of destination will be filled out by the carrier, leave blank. D. Shipment Type: Cross out “radioactive” to indicate you are shipping a non-radioactive substance. E. UN or ID Number: Enter appropriate UN number as found in Table 10.5. F. Proper Shipping Name: Enter the proper shipping name exactly as it appears in Table 10.5. G. Class or Division: Enter appropriate hazard class as found in Table 10.5. H. Packing Group: For dry ice, enter “III” in this column. Biological materials are not assigned packing groups. I. Quantity and Type of Packaging: Enter the net quantity for each material here. Use only metric units. At the bottom of this column, indicate the number and type of packages used (usually, “All packed in one fibreboard box.”). If using an overpack, indicate here with “Overpack Used.” J. Packing Instructions: Enter appropriate packing instruction number. Refer to Table 10.5. K. Authorization: Leave this column blank. L. Additional Handling Instructions: You must provide an emergency contact name and phone number which can be reached 24 hours per day, 7 days per week to provide emergency response information should a question develop about your package during transport. The statement “Emergency Contact: [fill in contact name; phone number]” must be provided. M. This section is self-explanatory. Sign and date each copy of your Shipper’s Declaration.
A blank Shipper’s Declaration for Dangerous Goods is available in Adobe PDF format at http://www.unh.edu/ehs/shipping. Please note the following:
• Declarations must be typewritten or computer-generated; handwritten declarations will not be accepted. • Declarations must be printed in color to display the red-striped border. • Always print at least four copies: provide three to the carrier and keep one for your records.
Georgia Regents University Biosafety Guide - 2013 10-19 • Remember to sign and date each copy. • Regulations require that you must retain your copy for 2 years.
A completed sample declaration can be found in Figure 10.5B. Contact the Biosafety Office x1-2663 or [email protected] with any questions regarding the Shipper’s Declaration.
Table 10.5. Summary of Shipping Information.
Max. Net Max. qty. Max. Net Hazar Packing Packing qty./pkg. Proper Shipping UN per qty./pkg. Shipment Type d Group Instructi for Name Number primary for Cargo Class (PG) on (PI) Passenger receptacle Aircraft Aircraft Category A infectious Infectious substance, Liquids: 4 L 50 ml or 50 4 L or 4 substance, affecting UN2814 6.2 - 602 affecting humans Solids: 4 kg g kg humans and possibly animals Category A infectious Infectious substance, Liquids: 4 L 50 ml or 50 4 L or 4 substance, affecting UN2900 6.2 - 602 affecting only Solids: 4 kg g kg animals animals (not humans)
Category B Biological Liquids: 1 L 4 L or 4 infectious substance, Category UN3373 6.2 - 650 4 L or 4 kg Solids: 4 kg kg substance B
Dry Ice or Dry Ice UN1845 9 III 904 N/A 200 kg 200 kg Carbon Dioxide, solid
Non-infectious, transducing Genetically genetically modified UN3245 9 - 913 No limit No limit No limit modified organism microorganisms or microorganism
Clinical waste, Biomedical waste Regulated medical UN3291 6.2 II 650 No limit No limit No limit Regulated medical waste, n.o.s. waste
Dangerous Aqueous Goods in formaldehyde Aviation regulated Excepted UN3334 9 II 30 ml - 500 ml solutions of less liquid, n.o.s. Quantities instructions than 25% (IATA 2.7)
Dangerous Aqueous ethanol Goods in solutions (55- Ethanol solution UN1170 3 II Excepted 30 ml - 500 ml 100%) Quantities (IATA 2.7)
Georgia Regents University Biosafety Guide - 2013 10-20
Figure 10.5A. Shippers Declaration
Georgia Regents University Biosafety Guide - 2013 10-21 Figure 10.5B. Example of Completed Shippers Declaration
Georgia Regents University 1120 15th St., CB-4100 Augusta, GA 30912
Ben Thompson (706)555-1212
Ben Thompson (706)555-1212
Augusta, GA, March 30, 2008
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10.6 REGULATED AGENTS WHICH MAY REQUIRE SPECIAL PERMITS FOR TRANSFER
10.6.1 CDC/USDA Select Agents and Toxins The U.S. Department of Health and Human Services has developed a list of biological agents (see Section 2.6.2.2) that have the potential to pose a severe threat to public health. Special regulations apply to the use and transfer of these materials, including registration with the GRU Institutional Biosafety Committee and the Centers for Disease Control and Prevention (CDC) or United States Department of Agriculture (USDA). If you are planning to, or currently possess, use or transfer any of the select agents and have not registered, contact the Biosafety Officer at x1-2663 ([email protected] ). Specific shipping restrictions apply to these agents which are not discussed in this document.
10.6.2 Agricultural Pests, Pathogens and Biological Agents The USDA/APHIS requires permits to transfer any plant or agricultural animal pest or pathogens. In addition, permits may be required to export or import the agents shown below. Further information can be obtained from the URL and phone number shown below:
APHIS Agricultural Permits http://aphisweb.aphis.usda.gov/ppq/permits/ http://www.aphis.usda.gov/import_export/index.shtml Material which do not require permits instruction: http://www.aphis.usda.gov/vs/ncie/fac_imp.html Telephone: 1-877-770-5990 EXPORT/IMPORT • Arthropods (insects and mites) • Livestock • Arthropods inhabiting dung or of medical/veterinary significance • Moths • Bees and bee related articles • Noxious weeds • Biological materials containing animal material • Nursery stocks (including seeds) • Butterflies • Parasitic plants • Cell cultures of bovine or other livestock origins • Plant pathogens • Cut flowers • Predators and parasitoids of arthropods • Earthworms • Prohibited material for research purposes • Endangered species • Rice and rice related articles • Endangered species of wild fauna and flora • Seeds • Entomopathogens • Snails and slugs • Farm animals • Soil • Foreign cotton and covers • Sugarcane products and by-products (including parts of the • Fruits and vegetables sugarcane plant) • High consequence livestock pathogens and toxins • Tissue culture materials of bovine or other livestock origins • Indian corn or maize, broomcorn and related plants • Weed biocontrol • Infectious agents of livestock • Wildlife • Khapra beetle products • Wood products • Live arthropods for display or educational purpose
Georgia Regents University Biosafety Guide - 2013 10-23 10.6.3 Agents of Vectors of Human Disease CDC permits are required when shipping any infectious agent known or suspected to cause disease in humans, unsterilized specimens of human or animal tissues (including blood and other fluids), or biological vectors of infectious animals, bats, insects, arthropods and snails. This includes the materials in the table below; further information can be obtained at the URL and phone numbers below.
CDC Permit to Import or Transport Agents or Vectors of Human Disease [http://www.cdc.gov/od/ohs/biosfty/imprtper.htm] Telephone: 1-404-498-2260
INFECTIOUS SUBSTANCES • It is impractical to list all of the several hundred species of infectious substances. In general, an import permit is needed for any infectious substance known or suspected to cause disease in man.
BIOLOGICAL MATERIALS • Unsterilized specimens of human and animal tissues (such as blood, body discharges, fluids, excretions or similar material) containing an infectious agent requires a permit in order to be imported.
VECTORS • Animals: Any animal known or suspected of being infected with an organism capable of causing disease transmissible to man may require a CDC permit. Importation of live turtles of less than 4 inches in shell length and all nonhuman primates requires an importation permit issued by the Division of Quarantine. • Bats: All live bats require an import permit from the CDC and the U.S. Department of Interior, Fish and Wildlife Services. • Insects or Arthropods: All live fleas, flies, lice, mites, mosquitoes, or ticks require a CDC import permit, regardless of infection status. Permits are required for adult forms, as well as eggs, larvae, pupae, and nymph stages. Any other living insect or arthropod, known or suspected of being infected with any disease transmissible to man requires a CDC import permit. • Snails: Any snail species capable of transmitting a human pathogen require a permit from the Centers for Disease Control.
10.6.4 Department of Commerce- Bureau of Industry and Security (BIS) Regulated Agents A permit may be required from the Commerce Department, when exporting infectious agents of human, plant, and animal diseases, including genetic material, and products which might be used for culture of large amounts of agents (Commerce Control List Supplement No. 1 to Part 774 Category 1, pages 54 - 59). In fact, in some instances, permits may be required to domestically ship commerce-controlled materials to certain foreign nationals or U.S. nationals who may fall on U.S. Governmental prohibition lists. In fact, these same individuals may even require a permit in order to access to these materials or their information while at GRU (known as “deemed exports”). See the list below for the biological agents which fall on the Commerce Control List. For further information on export compliance, view the URL below and contact the GRU Legal Office for further guidance.
Georgia Regents University Biosafety Guide - 2013 10-24
Commerce Department – Bureau of Industry and Security (BIS) [http://www.bis.doc.gov/index.htm] HUMAN PATHOGENS and TOXINS Bacteria Viruses • Bacillus anthracis • Chikungunya virus • Brucella abortus • Congo-Crimean haemorrhagic fever virus • Brucella melitensis • Dengue fever virus • Brucella suis • Eastern equine encephalitis virus • Burkholderia mallei (Pseudomonas mallei) • Ebola virus • Burkholderia pseudomallei (Pseudomonas pseudomallei) • Hantaan virus • Chlamydia psittaci • Hendra virus (Equine morbillivirus) • Clostridium botulinum • Japanese encephalitis virus • Clostridium perfringens, epsilon toxin producing types • Junin virus • Enterohaemorrhagic Escherichia coli, serotype O157 and other verotoxin • Kyasanur Forest virus producing serotypes • Lassa fever virus • Francisella tularensis • Louping ill virus • Salmonella typhi • Lymphocytic choriomeningitis virus • Shigella dysenteriae • Machupo virus • Vibrio cholerae • Marburg virus • Yersinia pestis • Monkey pox virus • Murray Valley encephalitis virus • Nipah Virus Toxins • Omsk haemorrhagic fever virus • Oropouche virus • Abrin • Powassan virus • Aflatoxins • Pulmonary and renal syndrome-haemorrhagic fever viruses (Seoul, • Botulinum toxins Dobrava, Puumala, Sin Nombre) • Cholera toxin • Rabies virus cultures • Clostridium perfringens toxins • Rift Valley fever virus cultures • Conotoxin • Rocio virus • Diacetoxyscirpenol toxin • South American haemorrhagic fever virus (Sabia, Flexal, Guanarito) • HT-2 toxin • St. Louis encephalitis virus • Microcystin (Cyanginosin) • Tick-borne encephalitis virus (Russian Spring-Summer encephalitis virus) • Modeccin toxin • Variola virus • Ricin • Venezuelan equine encephalitis virus cultures • Saxitoxin • Western equine encephalitis virus • Shiga toxin • White pox • Staphylococcal aureus toxins • Yellow fever virus • T-2 toxin • Tetrodotoxin • Verotoxin • Volkensin toxin • Viscum Album Lectin 1 (Viscumin)
Rickettsiae • Bartonella quintana (Rochalimea quintana, Rickettsia quintana) • Coxiella burnetii • Rickettsia prowasecki • Rickettsia rickettsii ANIMAL PATHOGENS and TOXINS Bacteria Mycoplasma mycoides Viruses • African horse sickness virus • Newcastle disease virus • African swine fever virus • Peste des petits ruminants virus • Avian influenza virus (certain highly pathogenic strains – see the Export • Porcine enterovirus type 9 (swine vesicular disease virus) Administration Regulations for more information) • Porcine herpes virus (Aujeszky’s disease) • Bluetongue virus • Rinderpest virus • Foot and mouth disease virus • Sheep pox virus • Goat pox virus • Swine fever virus (Hog cholera virus) • Lumpy skin disease virus • Teschen disease virus • Lassa virus • Vesicular stomatitis virus
Georgia Regents University Biosafety Guide - 2013 10-25
Commerce Department – Bureau of Industry and Security (BIS) - CONTINUED [http://www.bis.doc.gov/index.htm] GENETIC ELEMENTS/GENETICALLY MODIFIED ORGANISMS • Genetic elements that contain nucleic acid sequences associated with the • Genetically modified organisms that contain nucleic acid sequences pathogenicity of controlled microorganisms. associated with the pathogenicity of controlled microorganisms. • Genetic elements that contain nucleic acid sequences coding for any • Genetically modified organisms that contain nucleic acid sequences coding controlled “toxins” or “sub-units of toxins.” for any controlled “toxins” or “sub-units of toxins.” • Technical Note: Genetic elements include, inter alia, chromosomes, genomes, plasmids, transposons, and vectors, whether genetically modified or unmodified.
PLANT PATHOGENS Bacteria Fungi • Xanthomonas albilineans • Colletotrichum coffeanum var. virulans (Colletotrichum kahawae) • Xanthomonas campestris pv. citri including strains referred to as Xanthomonas • Cochliobolus miyabeanus (Helminthosporium oryzae) campestris pv. citri types A,B,C,D,E or otherwise classified as Xanthomonas • Magnaporthe grisea (pyricularia grisea/pyricularia oryzae) citri, Xanthomonas campestris pv. aurantifolia or Xanthomonas campestris pv. • Microcyclus ulei (Dothidella ulei) Citrumelo. • Puccinia graminis (Puccinia graminis f. sp. tritici) • Puccinia striiformis (Puccinia glumarum)
10.6.5 FDA Import Permits All food (except most meat and poultry), drugs, biologics, cosmetics, medical devices, and electronic products that emit radiation require a permit or registration before importation into the United States. See: http://www.fda.gov/ora/import/ for more information about FDA import permits.
Export permits requirement information can be found at: http://www.fda.gov/RegulatoryInformation/Guidances/ucm125789.htm
10.6.6 Fish and Wildlife Service Permits A permit may be required for transporting fish, wildlife, endangered species, or materials found in the list below.
Fish and Wildlife Service Permit Station [http://www.fws.gov/international/permits/antiques.html] Telephone: 1-800-770-0150 EXPORT • African elephant ivory • Ginseng • Animals • Marine mammals • Artificially propagated plants • Museum specimens • Asian elephant ivory • Personal pet • Biological samples • Plants • Captive-born export • Raptors • Circuses/traveling animal exhibitions • Trophies by taxidermist • Goldenseal • Wildlife IMPORT • African elephant • Marine mammals • African elephant ivory • Museum specimens • African leopard • Personal pet • Argali • Plants • Asian elephant ivory • Polar bears • Biological samples • Scientific and zoological breeding or display • Birds • Sport hunted trophy • Bontebok • White rhinoceros
Georgia Regents University Biosafety Guide - 2013 10-26 • Circuses/traveling animal exhibitions • Wildlife
10.7 INTERNATIONAL SHIPMENTS Shipping and receiving animals and animal-derived materials, infectious or biohazardous agents, biological toxins, and genetically modified organisms may require the approval of federal agencies, both domestic and foreign. Regulations that govern the transfer of biological materials help to minimize or eliminate the possible threats to public health and agriculture. In addition, the Departments of Commerce, Treasury and State regulate exportation based on special considerations on the material’s economic impact, commercial value, ecological impact and/or military dual-use.
Some countries, couriers and airlines restrict the importation or transportation of some hazardous materials (e.g., dry ice). It is advisable for the shipper to determine these restrictions prior to shipment/transport. Contact the Biosafety Office for assistance x1-2663 or [email protected].
Packages shipped internationally generally require increased preparation time due to the additional paperwork required for such packages. An import/export permit may be required when shipping biological materials internationally (See Section 10.6). Check the following U.S. governmental agencies for permits and additional information.
10.7.1 Exporting from the United States Depending on the nature of the shipment, a U.S. export permit may be required when sending your package. Additionally, an import permit may be required in the country where the package is being shipped. If your shipment requires an export permit, it must be completed and approved by the appropriate government agency prior to shipment. Typically, a copy of the import permit of the country of destination is included in the shipping documentation and should be obtained from the consignee prior to shipment. For more information on whether your shipment requires an export permit, please contact the GRU Biosafety Office x1-2663 or [email protected] .
Note: Packages may be opened and inspected when leaving the United States or at any time by any inspection service provided by other countries. In order to assure that your package is safely delivered to its intended destination, always consider the following:
1. If necessary, obtain an export permit from the appropriate governmental organization prior to shipment. 2. Package and label the material according to the guidelines listed in this manual. 3. Include a courtesy letter with the shipment describing the contents in detail including information about whether the material is infectious. Copies of importation paperwork from the consignee should be included, if required.
10.7.2 Importing into the United States All shipments entering the United States are processed by the U.S. Bureau of Customs and Border Protection. An import permit may be required to deliver the package even if a permit is not required by the originating country. Check with the appropriate governmental organization prior to shipment of the material.
Note: Packages may be opened and inspected upon entry into the United States. In order to assure that your package is safely delivered to its intended destination, always consider the following:
If necessary, obtain an import permit from the appropriate governmental organization prior to shipment.
1. Package and label the material according to the guidelines listed in this manual.
Georgia Regents University Biosafety Guide - 2013 10-27 2. Consider including a courtesy letter with the shipment.
The importer is legally responsible for assuring that foreign personnel package, label, and ship the infectious materials according to USPHS and IATA regulations. Shipping labels containing the universal biohazard symbol, the address of the importer, the permit number, and the expiration date are also issued to the importer with the permit. The importer must send the labels and one or more copies of the permit to the shipper. The permit and labels inform the U.S. Customs and Border Protection and U.S. Division of Quarantine personnel of the package contents.
10.8 SHIPPING COMPANY RESTRICTIONS Some shipping companies may have requirements that are more restrictive than those discussed in this document. Consider the following information before planning a shipment.
10.8.1 DHL DHL will accept shipments made according to IATA or DOT regulations. Shipments made according to instructions in this manual will be acceptable to DHL.
10.8.2 FedEx FedEx Express and FedEx Ground will accept shipments prepared according to instructions in this manual. FedEx will not accept any material considered to be in Risk Group 4. A Risk Group 4 pathogen is one that usually causes serious human or animal disease and that can be readily transmitted from one individual to another, directly or indirectly, and for which effective treatments and preventive measures are not usually available.
10.8.3 United Parcel Service (UPS) UPS will not accept shipments of Category A materials. UPS will accept shipments of UN3373 and exempt patient specimens.
10.8.4 United States Postal Service (USPS) The USPS has highly restrictive regulations concerning the shipment of hazardous materials by mail. Category A materials may not be mailed with the USPS. USPS will accept shipments of UN3373 and exempt patient specimens. For more information, refer to Section 10.9.
10.9 UNITED STATES POSTAL SERVICE MAILINGS The United States Postal Service (USPS) does not allow Category A infectious substances to be mailed. Follow the procedures below when mailing Category B substances, exempt patient specimens and non-regulated items.
10.9.1 Mailing Category B Substances Follow packaging and labeling requirements listed in Section 10.3.2.2 and note the following variations:
• Shipments of both liquid and solid substances must be packaged in a pressure tested primary or secondary container; and • Category B substances may be mailed as First-Class, Priority, or Express mail.
Georgia Regents University Biosafety Guide - 2013 10-28 10.9.2 Mailing Exempt Human and Animal Specimens Follow packaging and labeling requirements listed in Section 10.3.3 and note the following variations:
• Inner containers and the total volume per package are limited to 500 mL or 500 g; • Outer packaging must me rigid; and • Exempt specimens must be mailed as First-Class, Priority, Express, or Package Services mail.
10.9.3 Mailing Non-Regulated Materials According to USPS regulations, specific packing instructions apply when mailing non-regulated materials. The following are examples of non-regulated biological materials:
• Biological products not containing Category A or Category B substances; • Blood or blood products collected for transfusion or preparation of blood products; • Tissues or organs intended for transplantation; • Dried blood spots; and • Dried specimens for fecal occult blood detection.
Quantity limits and form of substance (liquid or solid) determine the packaging requirements for non- regulated materials. Refer to the appropriate category below to determine how to package you material.
10.9.3.1 Non-Regulated Liquid Substance, Not Exceeding 50 ml Primary container and total package contents may not exceed 50 ml. Primary receptacle must be leak-proof and properly sealed. Include cushioning and enough absorbent to absorb entire contents of liquid. Enclose the primary container(s) in a leak-proof secondary container (e.g. plastic bag). Label primary or secondary container with a biohazard symbol. No other labeling is required. Secondary container may serve as the outer container.
10.9.3.2 Non-Regulated Liquid Substance, Exceeding 50 ml Primary container must not exceed 50 ml; total package may not exceed 500 ml. Package in triple packaging. Include cushioning and enough absorbent to absorb entire contents of liquid. Label primary or secondary container with a biohazard symbol. No other labeling is required.
10.9.3.3 Non-Regulated Dry Substance Primary container must be sift-proof and must be enclosed in a sift-proof secondary container. Label primary or secondary container with a biohazard symbol. No other labeling is required. Secondary container may serve as the outer container.
10.10 TRANSPORT AS AIRLINE BAGGAGE Hazardous materials should never be carried in the passenger compartment of an airplane. Do not even attempt to carry these aboard an airline. Although occasionally, limited non-infectious biological materials and small amounts of dry ice may be transported as checked baggage on some passenger flights, these must not only comply with DOT/IATA and other standards as described above, but must adhere to each airlines policies and prerogative to carry such materials. Some airlines may refuse to carry these. Because of this, the practice is highly discouraged and it is recommended that GRU personnel make arrangements to ship their materials prior to departure rather than attempt to check their baggage. However, in the exceptional situations where shipment is not possible, those wishing to transport non-infectious biological materials and/or dry ice as checked baggage must contact the
Georgia Regents University Biosafety Guide - 2013 10-29 airline(s) well ahead of time to confirm that the airline’s policies will permit this and any special limitations/instructions they may have for preparing such packages. All packages must comply with the IATA/DOT packing, marking and labeling standards, and comply with any special permit standards as described in earlier sections. Those who intend on transporting such materials in this manner must have documented shipping training and declare their intentions to carry these materials on a passenger airline on their IBC-approved Biosafety Protocols and SOPs and fully disclose the nature of these materials to airline and TSA personnel.
10.11 TRANSPORT IN GROUND VEHICLES USDOT regulations do not apply to private or contract motor carriers used exclusively to transport biological materials, diagnostic specimens or biological products; however, other standards (e.g., permits) may still be required. Medical or clinical equipment and laboratory products may be transported aboard the same vehicle provided they are properly packaged and secured against exposure or contamination. Note, in order for the vehicle to be “dedicated,” the vehicle can not be utilized for other purposes at the same time (e.g., patient or passenger transport, food transport). Although no specific packing instructions are required by law, packages should be prepared in accordance with the packaging guidelines outlined in Section 10.4 and the carrier’s specifications, if applicable.
All those transporting materials must be licensed drivers and comply with all applicable driving laws and accepted safety standards (e.g., seat belt use).
Intention to transport biological materials or dry ice via vehicles by GRU personnel should be fully disclosed in each IBC-approved Biosafety Protocol and SOPs prior to transport. These SOPs should include a description of the steps which should be taken if one is involved in a motor vehicle accident en route. These include: • Call for emergency assistance, if needed • Let all emergency response teams know that you are transporting potential biohazards • Notify your supervisor to contact the shipper and recipient of the sample status • Arrange for alternate transportation if you are not able to get to your destination
10.11.1 Automobiles Special considerations should be made for the locations and security of the package in a passenger vehicle. During transport, the vehicle must be dedicated to the purpose. Biological materials should not be transported in any area where food/beverages are transported and these areas should be fully decontaminated prior to transport of food/beverages. Packages with dry ice or liquid nitrogen should never be transported in the passenger compartment of the vehicle due to the suffocation hazards (see Section 10.4.2.2). Materials should be secured from theft. Any material transported in an open truck must be secured to prevent loss via jostling of the vehicle during transport.
Transport via privately-owned vehicle is discouraged as many private insurance companies do not cover this activity. Check with your insurance company to verify the terms of your policy prior to transport.
10.11.2 Public Modes of Transportation (e.g., Shuttles busses) Transport of biological materials or other hazardous materials is not permitted in passenger compartments of vehicles used for public transportation, such as shuttle busses.
10.11.3 Courier Services Several commercial courier services are available to transport patient (diagnostic) specimens or biological products. Although USDOT regulations do not apply to private or contract motor carriers used exclusively to transport patient (diagnostic) specimens or biological products, and therefore no specific packing instructions are required by law,
Georgia Regents University Biosafety Guide - 2013 10-30 those wishing to offer their packages of biological materials to a commercial carrier for transport must prepare shipments in accordance to the courier’s standards and must fully declare the nature of the materials which are being offered for transport. Often, these will be similar to that described in Section 10.4.
Georgia Regents University Biosafety Guide - 2013 10-31 FOR BIOSAFETY OFFICE USE ONLY: For assistance completing this form, (Date Stamp) contact: Biosafety Officer Danielle Daniely, Ph.D. Email: [email protected] BSP Application #: Phone: 706-721-2918
Review Track: (Check Box) Assistant Biosafety Officer
Full IBC Review Laura Meyer, MA Email: [email protected] Expedited Clinical Subcommittee Review Phone: 706-721-7458
Expedited Basic Subcommittee Review Return by email to: Administrative Review Environmental Health & Safety - Biosafety Email: [email protected]
Environmental Health and Safety Biosafety Protocol (BSP) Application
Notes and Instructions: • BSPs must be approved by the Institutional Biosafety Committee (IBC) or the Biological Safety Office (Administrative Review) prior to the initiation of research. • Approval is valid for the duration of the research proposed in this application, but MUST be amended as needed and renewed annually using the Biosafety Protocol Amendment Application. • Whenever you amend your Animal Use Protocol (AUP) or Institutional Review Board (IRB) Protocol to add/delete agents, change personnel/location, or modify a procedure, you must amend your BSP using the Biosafety Protocol Amendment Application. • Access the IBC webpage for submission deadlines, meeting dates and locations for BSP review. • An example application and a checklist are available on the IBC webpage to assist you in completing this application. • Complete this form electronically (must be typed) and save as BSP_PI name (First, Last). Example: BSP_JohnDoe • Submit the electronic documents to the [email protected] email account. To authenticate, the PI must send from his/her University email account/mailing address or the preparer must copy the PI in the email.
Required additional forms: Standard Operating Procedures (SOPs) - These are a list of rules and procedures which are expected to be followed by those working in your laboratory, and therefore should address the specific hazards and risks in your laboratory.
A template to help you get started in developing some basic SOPs is available online at: http://www.gru.edu/research/ibc/apps.php
General Information: Principal Investigator (PI): Department:
Office Phone Number: Emergency Phone Number:
Laboratory Phone Number: Email Address:
Fax number: Campus Address:
Emergency Laboratory Contact Emergency Contact Office Phone Number Emergency Phone Number for (Other than the PI): (If different from lab number) : Emergency Contact: Protocol Information: BSP Title:
Mark all sections below that are applicable to your protocol. Applicable Sections Go to those sections and answer all questions. Administrative Mandatory Complete Section 1 Recombinant and Synthetic Nucleic Acid Molecules (e.g., bacterial/mammalian Yes No If “yes”, complete Section 2 expression plasmids, replication incompetent viral vectors, chemically synthesized nucleic acid molecules) Human & Non-Human Primate Material (e.g., blood, fluids, tissues, Yes No If “yes”, complete Section 3 primary/established cell lines) Microorganisms/Potentially Infectious Material (e.g., viruses, bacteria, yeast, Yes No If “yes”, complete Section 4 fungi, parasites, prions)
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Whole Animals/Animal Material (e.g., introduction of biologicals/chemicals into Yes No If “yes”, complete Section 5 animals, use of animal cell lines and/or tissues) Biological Toxins (e.g., cholera toxin, pertussis toxin, diphtheria toxin, tetrodotoxin) Yes No If “yes”, complete Section 6 Human gene transfer/therapy (e.g., DNA Vaccines) Yes No If “yes”, complete Section 7 Nanoparticles (e.g., use of Jet-Pei or Poly-L-Lysine to form nano-sized particles) Yes No If “yes”, complete Section 8 Arthropods (e.g., insects, spiders, crabs, lobsters, shrimp) Yes No If “yes”, complete Section 9 Plants (e.g., toxic/transgenic plants) Yes No If “yes”, complete Section 10 Investigator’s Assurance Mandatory Complete Section 11
SECTION 1: ADMINISTRATIVE Description of research: 1.1 Outline the overall goal(s) of the projects to be covered by this application. Describe recombinant/synthetic nucleic acid molecules and biohazardous materials and procedures (experimental set-up). Use separate paragraphs for multiple projects. Use non-technical language to enable all Institutional Biosafety Committee members (those with non-science backgrounds) to understand the research project and assess the risks.
List grant/study titles associated with Grant/Info.Ed./HAC/CC Funding Funding Dates: this application: RI#: Agency: 1.2 1. 2. List additional BSPs required to cover the biological agents and manipulations, operations, personnel and locations described in the grant/study title shown above. BSP Approval Describe how these BSPs will “dovetail” (i.e. which BSP BSP #: PI listed on BSP: Date: "covers" which portion of the grant/study title): 1. 2. List all locations where work pertaining to this application will be performed in the table below:
Note: Collaborating Institutions/Companies should be listed below. If collaborating with a PI outside of GRU, the Biosafety Office may request a copy of their Biosafety/IBC approval letter. 1.3 List Biological Agents Building Code (e.g. Recombinant DNA; Animals or animal cell lines, and Room tissues, fluids or organs; Human or Non-Human Primate Number cell lines, tissue, fluids or organs, Biosafety Facility: (e.g., cold rooms, (Address for Potentially infectious or infected material, Toxins of Level tissue culture) Off-site areas): biological origin, Microbial pathogens) (BSL) : GRU Health Sciences Campus GRU Summerville Campus GRU Animal Facilities GRU Core Facilities GRMC Hospital/Clinics VA Medical Center Off-site Clinic/Laboratory Other (list):
Personnel: List all individuals supervising or physically working on the research proposed in this application or that may be exposed to the research materials including the PI, collaborators, technicians, post docs, graduate students, work-study students, volunteers, etc. If extra space is needed, multiple individuals can be listed together if they will have the same responsibilities; however, list the experience for each.
Note: Clinical Sub-investigators that are NOT supervising those conducting research and do NOT handle the samples, but only perform “standard of care” duties should not be listed below. 1.4 Does the person If yes, where was have experience with this experience Job/ materials listed in If yes, which materials? (e.g., obtained and Name Position Title this applications: lentivirus, human tissues, bacteria) number of years? Yes No Yes No
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Yes No Yes No Yes No 1.5 Training Requirements: Have all personnel listed on this protocol completed Initial Biosafety Training (didactic – required Yes No* once) or an Annual Biosafety Refresher Training Module (online – required annually during November compliance month after completing the initial training)?
Have all personnel listed on this protocol who may have occupational exposure to human blood, Yes No* other potentially infectious materials (OPIM) of human origin (e.g., cells/cell lines, unfixed tissues) or Not Applicable human bloodborne pathogens (BBP) completed BBP Training (online – required annually as per OSHA)? Yes No* Have all personnel in your laboratory who plan to ship, transport, export, pack, label, mark, or Not Applicable complete/sign paperwork for shipping biological materials (including diagnostic/clinical specimens, cell cultures, tissues, genetically modified organisms and/or infectious agents, toxins of biological origin) or items on dry ice, liquid nitrogen or in fixative completed Shipping Biological Substance and Support Materials Training (online – required by DOT and IATA)?
*If no, or unsure, contact the Biosafety Office at [email protected] to be assigned the training or for verification of training. 1.6 Medical Surveillance and Vaccinations: Vaccines or other tests/evaluations may be required for the work being conducted in this application. (Check all that apply):
Hepatitis B Vaccine or signed Waiver (required for all individuals having occupational exposure to human blood, other potentially infectious materials (OPIM) of human origin (cells/cell lines, unfixed tissues) or human bloodborne pathogens (BBPs), required as per OSHA. Seasonal Influenza Vaccine Vaccinia Respirator evaluation and annual fit test (e.g., N95, PAPR, full/half face respirators) Other (list):
Contact Employee Health and Wellness, 706-721-3418, to schedule an appointment for the services indicated above. 1.7 Laboratory/Agent-Specific SOPs: Do you have laboratory/agent specific Standard Operating Procedures (SOPs)? Yes No*
*If no, see the Biosafety webpage for a guidance template to create your laboratory/agent-specific SOPs. A copy of these SOPs must be submitted with this application and be available in your laboratory Biosafety Binder.
SECTION 2: RECOMBINANT DNA AND SYNTHETIC NUCLEIC ACID MOLECULES Section I-B. Definition of Recombinant and Synthetic Nucleic Acid Molecules
In the context of the NIH Guidelines, recombinant and synthetic nucleic acids are defined as:
(i) molecules that a) are constructed by joining nucleic acid molecules and b) that can replicate in a living cell, i.e., recombinant nucleic acids; (ii) 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 (iii) molecules that result from the replication of those described in (i) or (ii) above.
These activities are regulated by the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules
See the following for assistance in Risk Group classification and recommended Biosafety Level usage: • American Biosafety Association Risk Group Guide: http://www.absa.org/riskgroups/index.html • Public Health Agency of Canada MSDSs: http://www.phac-aspc.gc.ca/msds-ftss/ • NIH Guidelines for Recombinant DNA Research: http://oba.od.nih.gov/rdna/nih_guidelines_oba.html • CDC’s Biosafety in Microbiological and Biomedical Laboratories (BMBL): http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm
Note: Your answers to the questions in this section will determine the level of review that your experiments require. 2.1 Does your application involve recombinant and/or synthetic nucleic acid Yes – Complete this Section
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molecules? No – Skip Section 2 & Go to Section 3 “Non-exempt” Recombinant DNA “Exempt” 2.2 Experiments which require IBC approval prior to Experiments that require IBC approval Recombinant DNA experiments that do not require IBC initiation: simultaneous with initiation: approval, but require registration with the Biosafety Office: 1. Deliberate transfer of a drug trait to a microorganism 1. Experiments using rDNA/synthetic nucleic 1. Those synthetic nucleic acids that: (1) can neither replicate not known to acquire it naturally (if it could acids containing < 2/3 of the genome of a nor generate nucleic acids that can replicate in any living cell compromise the use of the drug to control disease eukaryotic virus, demonstrated to be free (e.g., oligonucleotides or other synthetic nucleic acids that agents in humans, animals or agriculture). (Note: this of helper virus or complementing helper do not contain an origin of replication or contain elements would likely exclude most sub-cloning procedures virus components may be contained at known to interact with either DNA or RNA polymerase), and using antibiotic selectable markers in E. coli K12 BSL1. (2) are not designed to integrate into DNA, and (3) do not derivatives) Section III-E-1 Yes No produce a toxin that is lethal for vertebrates at an LD50 of Section III-A-1 Yes No less than 100 nanograms per kilogram body weight. 2. Whole Plants, except for those that fall in Section F-1 Yes No 2. Cloning of DNA encoding toxic molecules lethal to Sections under III-A, B, D, or F. vertebrates at an LD50 of <100 µg/kg body weight. Section III-E-2 Yes No 2. Those that are not in organisms, cells, or viruses and that Section III-B-1 Yes No have not been modified or manipulated (e.g., encapsulated 3. Creation of transgenic or knockout rodents into synthetic or natural vehicles) to render them capable 3. Human gene transfer/therapy experiments; for which BSL-14 containment is of penetrating cellular membranes. Section III-C-1 Yes No appropriate Section F-2 Yes No Section III-E-3 Yes No 4. Introduction of rDNA/synthetic DNA in risk group 2, 3. Those that consist solely of the exact recombinant or 3, 4 or restricted agents (e.g., viral vectors or 4. All experiments not specified in this chart synthetic nucleic acid sequence from a single source that biological materials treated with viral vectors) . exists contemporaneously in nature. Section III-D-1 Yes No Section F-3 Yes No
5. Cloning of DNA from all Risk Group4 2, 3, 4 or 4. Those that consist entirely of nucleic acids from a agents into non-pathogenic prokaryotic or lower prokaryotic host, including its indigenous plasmids or eukaryotic host-vector systems (e.g., cloning an HIV viruses when propagated only in that host (or a closely gene into bacteria) related strain of the same species), or when transferred to Section III-D-2 Yes No another host by well-established physiological means. Section III-F-4 Yes No 6. Experiments using more than 2/3 of the genome of infectious animal or plant viruses or defective viruses 5. Those that consist entirely of nucleic acids from a grown in the presence of helper virus or eukaryotic host including its chloroplasts, mitochondria, or complementing helper virus components (e.g., plasmids (but excluding viruses) when propagated only in Adeno-associated virus in conjunction with that host (or a closely related strain of the same species). Adenovirus) Section III-F-5 Yes No Section III-D-3 Yes No 6. Those that consist entirely of DNA segments from different 7. Recombinant DNA/synthetic nucleic acid species that exchange DNA by known physiological experiments involving whole animals, including processes, though one or more of the segments may be a transgenic or knockout rodent experiments requiring synthetic equivalent. BSL24 containment; or transplantation of genetically Section III-F-6 Yes No engineered cells into animals. Section III-D-4 Yes No 7. Those genomic DNA molecules that have acquired a transposable element, provided the transposable element 8. Whole plants (e.g., genetically engineered plants) does not contain any recombinant and/or synthetic DNA. Section III-D-5 Yes No Section III-F-7 Yes No
9. Large scale DNA projects (>10 liter cultures at any 8. rDNA containing less than 1/2 of an eukaryotic viral moment in time). genome propagated in cell culture (with the exception of expression of DNA from Risk Group4 2, 3, 4 or restricted Section III-D-6 Yes No 5 agents ); 10. Influenza Viruses Appendix C-I Yes No Section III-D-7 Yes No 9. rDNA work involving E. coli K12 derivatives, S. cerevisiae, Kluyeromyces, and B. subtilis /lichenformis host-vector systems (with the exception of expression of DNA from Risk Group 2, 3, 44 or restricted agents). Appendix C-II, C-III, C-IV Yes No
10. The purchase or transfer of transgenic rodents that require BSL1 containment. Appendix C-VII Yes No
11. Breeding of different strains of transgenic rodents. (1) Both parental rodents can be housed under BL1 containment; and (2) neither parental transgenic rodent contains the following genetic modifications: (i) incorporation of more than one-half of the genome of an exogenous eukaryotic virus from a single family of viruses; or (ii) incorporation of a transgene that is under the control of a gammaretroviral long terminal repeat (LTR); and(3) the transgenic rodent that results from this breeding is not expected to contain more than one-half of an exogenous viral genome from a single family of viruses. Appendix C-VIII Yes No
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2.3 Use the table below to describe your rDNA/synthetic experiments Tropism (i.e. what species Backbone of cells can the Vector Source (e.g. What is the Name of virus infect?) Will you Technical bacterial, largest fraction Packaging Cell Ecotropic expose Name (e.g. yeast, MLV, of the Is the line(s) or (Rodents) humans, pKLO.1, MSCV, HIV, Full Name and eukaryotic viral vector Helper Amphotrophic animals, pcDNA) or FIV, Vaccinia, Abbreviation of genome designed to plasmids used (mammals) plants, synthetic Adenoviral, Inserted DNA Product contained in be in co-transfect Pantropic (all arthropods, nucleic acid AAV, and source Produced (e.g. Anticipated Effect the rDNA replication ion to produce animals including or cells to name Plasmids) (species/strain) protein, siRNA) of the Insert? molecules? competent? viral particles insects, birds, fish) the rDNA? Anti-apoptotic <1/2 Yes No Ecotropic Humans Growth Factor >1/2 but Amphotrophic Animals Tumor Inducer <2/3 Pantropic (list): Cytokine Inducer >2/3 Plants Oncogene N/A (list): Tumor Inhibitor Arthropods Cytokine Inhibitor (list): Toxic Cells Other (list): (list): Anti-apoptotic <1/2 Yes No Ecotropic Humans Growth Factor >1/2 but Amphotrophic Animals Tumor Inducer <2/3 Pantropic (list): Cytokine Inducer >2/3 Plants Oncogene N/A (list): Tumor Inhibitor Arthropods Cytokine Inhibitor (list): Toxic Cells Other (list): (list): Anti-apoptotic <1/2 Yes No Ecotropic Humans Growth Factor >1/2 but Amphotrophic Animals Tumor Inducer <2/3 Pantropic (list): Cytokine Inducer >2/3 Plants Oncogene N/A (list): Tumor Inhibitor Arthropods Cytokine Inhibitor (list): Toxic Cells Other (list): (list): Anti-apoptotic <1/2 Yes No Ecotropic Humans Growth Factor >1/2 but Amphotrophic Animals Tumor Inducer <2/3 Pantropic (list): Cytokine Inducer >2/3 Plants Oncogene N/A (list): Tumor Inhibitor Arthropods Cytokine Inhibitor (list): Toxic Cells Other (list): (list):
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2.4 What host organism will you use for DNA propagation? List the species and strain (i.e., E.coli DH5α)
2.5 How will you purify the recombinants and what measures will you take to avoid aerosol production during purification?
2.6 Will the recombinant (vector + insert) be purchased from a commercial vendor, provided by a collaborator and/or created/packaged in the laboratory?
2.7 Have you provided restriction/vector maps for each vector listed above to the Biosafety Office? Example:
Yes No*
*If not, email the maps to [email protected] to be distributed with your application for review. 2.8 What regions/genes of the viral genome are deleted or altered (if any) to produce the viral vector? (i.e. what is the basis of vector attenuation or replication incompetence, if any)
2.9 Will you be assaying for the production of wild-type/helper/replication competent viral particles? Yes* No *If yes, describe methods and stage in your experiment at which these assays will be performed.
2.10 Will you handle more than 10 liters of culture of this agent(s) at any one time?
*If yes, special precautions may be required for large-scale cultures involving recombinant DNA. These can Yes* No be reviewed at:http://oba.od.nih.gov/rdna/nih_guidelines_new.htm#_Toc331174152m Provide answers to Appendix K, with this application. 2.11 List any procedure that will be performed with this material which may be associated with increased potential for exposure (i.e. generation of splashes, sprays or aerosols from centrifugation, sonication, homogenization, vortexing, FACS, use of sharps (needles or glass).
2.12 What are the signs/symptoms of exposure to this material?
(Note: All accidents/injuries where exposure to recombinant DNA or synthetic nucleic acids should be reported to the Biosafety Office after seeking medical attention, if necessary). SECTION 3: HUMAN AND NON-HUMAN PRIMATE MATERIAL 3.1 Does your protocol involve the use of organs or tissues from living or dead humans or non-human primates, cell lines (including established cell lines), Yes – Complete this Section blood, blood products and body fluids, including cell cultures purchased No – Skip Section 3 & Go to Section 4 from commercial sources? Use this section to describe the types of human/non-human primate materials that will be used in your research
Human and non-human primate materials must be handled using BSL2 facilities, practices and equipment. Per OSHA requirements, all individuals with occupational exposure to any materials listed in the table below must complete Bloodborne pathogen training. Training is completed online at: http://www.usg.edu/ehs/training/pathogens/ The OSHA – Bloodborne Pathogen Standard and Letters of Interpretation can be found on the Biosafety webpage. 3.2 Origin Material(s): Human Teeth Feces Sputum Non-human Blood Semen Breast Milk primate Sweat Vaginal Secretions Peritoneal Fluid Tears Nasal Secretions Synovial Fluid Urine Pleural Fluid Cerebrospinal Fluid Amniotic Fluid Pericardial Fluid Bones Gingival Fluid Tissues (list): Embryonic Stem Cells Are these stem cells listed in the NIH human embryonic stem cell line registry? Yes No The NIH human embryonic stem cell line registry is available at the following link: http://grants.nih.gov/stem_cells/registry/current.htm
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Induced Pluripotent Stem Cells (iPSCs) Methods of induction: Mesenchymal Stem Cells (MSCs)
Source(s) of the materials above: Commercial Vendor (list): Collected Specimens (list collection site in Section 1.3) Provided by a collaborator (list Collaborator Name and Institution): Other (list): Cells/Cell lines (i.e.HEK239) (list them below) Established Cells/Cell Line Primary (Commercial) Established in Genetically Potentially Will these be Name (Fresh) e.g., ATCC the Laboratory Engineered Tumorigenic cultured? Yes No Yes No Yes No Yes No Yes No Yes No Blood-derived products (i.e. red blood cells, plasma) (list):
Other (list): 3.3 Will you handle more than 10 liters of culture of this agent(s) at any one time? Yes* No
*If yes, special precautions may be required for large-scale cultures involving recombinant DNA. These can be reviewed at:http://oba.od.nih.gov/rdna/nih_guidelines_new.htm#_Toc331174152m Provide answers to Appendix K, with this application. 3.4 Did this human material originate outside of the United States? Yes* No *If yes, a CDC Etiologic Agent Import Permit (www.cdc.gov/od/eaipp/) may be required. The Biosafety Office can assist you in determining permit requirements.
If permit(s) have already been obtained, submit a copy to [email protected] 3.5 Do these materials contain known pathogens? (i.e. blood samples from HIV positive patients) Yes* No
*If yes, list the known pathogen(s) and the signs and symptoms of exposure: 3.6 Are you and your laboratory staff aware of the common signs and symptoms of exposure to Yes No bloodborne pathogens present in human and non-human primate materials (i.e. fever, flu-like symptoms, fatigue, nausea)?
(Note: All accidents/injuries where exposure the human/non-human primate materials should be reported to the Biosafety Office after seeking medical attention, if necessay). 3.7 List any procedure that will be performed with this material which may be associated with increased potential for exposure (i.e. generation of splashes, sprays or aerosols from centrifugation, sonication, homogenization, vortexing, FACS, use of sharps (needles or glass).
3.8 Explain any type of treatment the material has undergone prior to receipt (i.e. formaldehyde fixation, testing for viruses).
3.9 Will you be introducing these materials into animals? Yes* (also complete Section 5) No 3.10 Will you be introducing these materials into humans? Yes* No
*If yes, answer the following questions below: a. Method of delivery? b. Health status of the patients/patient population? c. Frequency of administration? d. The anticipated effect of the introduction of the agent upon the patient (if known)? e. The expected persistence of the agents after administration (if known)? f. How long after administration will you obtain specimens to be analyzed? g. What types of specimens will be obtained? h. What types of analyses will be performed? (e.g. behavioral analyses, in vivo instrumentation, blood tests, analyses of tissue biopsies?) i.What biosafety precautions will be taken to avoid inadvertent exposure to other patients, researchers, or health care
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providers? j.Indicate any safety tests or pathogen screening which will be performed on these cells/tissues prior to delivery into humans? k. Have these agents been passaged through animals or other cells/cell lines? 3.11 Do you obtain human blood from volunteers (for volunteer blood donation)? Yes No
3.12 Do you have IRB approval? Yes* No** Pending *If yes, what is your application/IRB#: Not Required **If no, contact Angela Randazzo, for instructions on submitting an application Email: [email protected] Phone: 706-721-3110 3.13 Do you conduct research in the Augusta VA facility? Yes* No
*If yes, answer the following questions: Does your research involve GRU personnel, or transfer to GRU property? Yes* No Have you applied for and/or received VA Biosafety approval for your research? Yes* No Pending
3.14 Will you be exposing any of the material listed above to chemotherapeutic/antineoplastic agents (i.e. BrdU, STZ, Tamoxifen)? Yes* No *If yes, list the agent: Note: There may be special safety and handling requirements for these agents, contact Ken Erondu, Chemical Safety Officer, for assistance at [email protected] 3.15 Will you be exposing live human subjects, non-human primates, human cells, or non-human primate cells to recombinant/synthetic DNA? Yes* No
*If yes, make sure you complete Section 2 – Recombinant DNA and Synthetic Nucleic Acid Molecules SECTION 4: MICROORGANISMS/POTENTIALLY INFECTIOUS MATERIAL Does your protocol involve microorganisms/potentially infectious material Yes – Complete this Section 4.1 (i.e. viruses, bacteria, fungi, prions, parasites)? No – Skip Section 4 & Go to Section 5 Will you introduce recombinant/synthetic DNA to any microorganism /potentially infectious agent, use recombinant/synthetic DNA to change the genetic make-up of any microorganism/potentially infectious agent, or use DNA from any microorganism/infectious agent to perform any recombinant 4.2 Yes* No DNA experiments?
*If yes, make sure you complete Section 2 – Recombinant DNA and Synthetic Nucleic Acid Molecules 4.3 Signs and symptoms of infection from exposure to this material:
4.4 List any procedure that will be performed with this material which may be associated with increased potential for exposure (i.e. generation of splashes, sprays or aerosols from
centrifugation, sonication, homogenization, vortexing, FACS, use of sharps (needles or glass)
4.5 Will you be introducing this material into animals? Yes* (also complete Section 5) No 4.6 Will you be introducing this material into humans? Yes* (also complete Section 3 ) No 4.7 How long from the time of infection, will the agent(s) inactivated or lysed? 4.8 Will these experiments result in acquisition of new characteristics of these infectious agents, such Yes* No as altered virulence or infectivity, or changes in resistance/susceptibility to drug therapy or changes in host range?
*If yes, please describe:
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4.9 List each microorganism/potentially infectious agent to be used in this protocol • For Risk Group Classification, link to the Risk Group Database or Appendix B – NIH Guidelines • For a list of Select Agents/Toxins, link to the National Select Agent Registry • The Risk Group an agent is placed in is not the Biosafety Level (BSL) suitable for containing the agent. Risk Is this a Agent Name Group Select (Genus, (see Agent? Is the agent Species & Type (e.g. above (see above Is the agent This agent can spread replication Has this materials been genetically Strain) virus, bacteria) link) link) hazardous to: via: competent? Virulence modified? Yes No Humans Blood Yes No Yes No Animals Feces Plants Saliva/Nasal If yes, list modification: Other: Droplets Direct Contact Other: Yes No Humans Blood Yes No Yes No Animals Feces Plants Saliva/Nasal If yes, list modification: Other: Droplets Direct Contact Other: Yes No Humans Blood Yes No Yes No Animals Feces Plants Saliva/Nasal If yes, list modification: Other: Droplets Direct Contact Other: Yes No Humans Blood Yes No Yes No Animals Feces Plants Saliva/Nasal If yes, list modification: Other: Droplets Direct Contact Other: Yes No Humans Blood Yes No Yes No Animals Feces Plants Saliva/Nasal If yes, list modification: Other: Droplets Direct Contact Other: 4.10 What are the signs/symptoms of exposure to this material?
(Note: All accidents/injuries where exposure to microorganisms should be reported to the Biosafety Office after seeking medical attention, if necessary).
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SECTION 5: WHOLE ANIMALS/ANIMAL MATERIAL 5.1 Does your protocol involve working with animals or animal materials? Yes – Complete this Section No – Skip Section 5 & Go to Section 6 5.2 Does your protocol involve working with animals that are field caught? Yes* No
*If yes, describe: 5.3 Do you have Institutional Animal Care and Use Committee (IACUC) approval? Yes* No** Pending *If yes, what is your AUP#: Not Required **If no, contact Jenny Whitlock, IACUC Compliance Coordinator for instructions on submitting an AUP Email [email protected] Phone: 706-721-0198 5.4 Will you be exposing any animals to chemotherapeutic/antineoplastic agents (i.e. BrdU, STZ, Tamoxifin)? Yes* No
*If yes, list the agent(s): Note: There may be special safety, handling and training requirements for these agents, contact Ken Erondu, Chemical Safety Officer, for assistance at [email protected], 706-721-2663 5.5 List each species/strain of laboratory animal that will be used in your research Animal Species/Strain Max (List same species Dose/Animal Specify Route in one row, different Biological Agent (i.e. and Frequency of Shedding/ Are there special species in separate vectors, human cell Housing Post of Method of Delivery Excretion of housing/handling row) lines, microorganisms) Introduction of Agent Administration (Check all that apply) Agent procedures: Conventional ABSL-1 Stereotactic Injection Urine Disposable Cages Conventional ABSL-2 IP Injection Saliva Microisolator Cages Barrier ABSL-1 IV Injection Feces All work done in a Biosafety Barrier ABSL-2 IM Injection Blood Cabinet ABSL-3 SQ Injection Wound Animals handled by only NHP Housing/Facilities Intranasal Other: research staff Other: Oral None Use of Safety Engineered Ocular Unknown Sharps Other: Cages labeled with Hazard Other: Conventional ABSL-1 Stereotactic Injection Urine Disposable Cages Conventional ABSL-2 IP Injection Saliva Microisolator Cages Barrier ABSL-1 IV Injection Feces All work done in a Biosafety Barrier ABSL-2 IM Injection Blood Cabinet ABSL-3 SQ Injection Wound Animals handled by only NHP Housing/Facilities Intranasal Other: research staff Other: Oral None Use of Safety Engineered Ocular Unknown Sharps Other: Cages labeled with Hazard Other: 5.6 Do you intend to perform any safety tests or pathogen screening prior to introduction of biological agents into animals (i.e. viral assays of cells Yes* No or helper viral assays or MAP testing) or monitoring for agents after introduction of the agents into animals?
*If yes, describe: 5.7 What is the anticipated effect of introduction of the agent(s) described above on the animal (if known)?
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5.8 What is the expected persistence of the biological/chemical agent (i.e. cells, toxin, infectious agent) after administration (if known)?
5.9 At what stage of your experiments will the infectious agent(s) be inactivated or lysed? 5.10 List the animal cells, cell lines, tissues or organs that you plan to utilize in your research?
Cells/Cell Established Species Line/Tissues/Org Primary (Commercial) Established in Genetically Potentially Will these be of Origin ans Name (Fresh) e.g., ATCC the Laboratory Engineered Tumorigenic cultured? Yes No Yes No Yes No Yes No Yes No Yes No 5.11 Procedures – List any procedure that will be performed with this material which may be associated with increased potential for exposure (i.e. cage changing, necropsies, injections, inoculations).
5.12 Signs and symptoms from exposure to this material (including via animal bite/scratch):
(Note: All animal-related accidents/injuries should be reported to the Biosafety Office after seeking medical attention, if necessary). 5.13 Is there special Personal Protective Equipment (PPE) that should be worn by laboratory personnel and animal care staff when handling these animals? Yes* No
*If yes, describe: 5.14 Will you be creating transgenic animals, breeding transgenic animals, exposing animals to Yes* No recombinant DNA, or purchasing/obtaining transgenic animals from a commercial vendor or collaborator?
*If yes, make sure you complete Section 2 – Recombinant DNA and Synthetic Nucleic Acid Molecules and answer the following questions:
1. List the genes that will be inserted or knocked out by stable introduction of rDNA/synthetic nucleic acids into the germ-line: 2. Describe the marking system that will be used to identify the transgenic animals: SECTION 6: BIOLOGICAL TOXINS 6.1 Does your protocol involve biological toxins (i.e. tetrodotoxin cholera toxin, pertussis toxin, diphtheria toxin, botulinum toxin)? Yes – Complete this Section
No – Skip Section 6 & Go to Section 7 Note: Select Agents are in Bold above. More information and a complete list can be found on the Select Agent Program Website. 6.2 Will you be performing experiments where you clone toxin molecules with Yes* (also complete Section 2) No an LD 50 of 100 ng/kg or less? 6.3 Will you be introducing this material into animals? Yes* (also complete Section 5 ) No 6.4 Will you be introducing this material into humans? Yes* (also complete Section 3) No 6.5 List each biological toxin in the table below: Maximum Is the toxin a Where is it stored? Quantity on HHS/USDA Select Toxin LD50 Hand Building/Room# Location Agent or Toxin? Flammable Cabinet Yes* No Refrigerator Freezer Locked Cabinet Locked Box Other: Flammable Storage Yes* No Cabinet Refrigerator Freezer Locked Cabinet 11
Locked Box Other: 6.6 *If you are working with a toxin that is a Select Agent, are you working with it within the Yes* No permissible amounts (see below)?
Toxins Amount Abrin 100 mg Botulinum neurotoxins 0.5 mg Short, paralytic alpha conotoxins 100 mg Diacetoxyscirpenol (DAS) 1000 mg Ricin 100 mg Saxitoxin 100 mg Staphylococcal Enterotoxins (Subtypes A, B, C, D, and E) 5 mg T-2 toxin 1000 mg Tetrodotoxin 100 mg 6.7 What are the signs and symptoms of exposure to this material:
(Note: All accidents/injuries where exposure to toxins should be reported to the Biosafety Office after seeking medical attention, if necessary). 6.8 Procedures – List any procedure that will be performed with this material which may be associated with increased potential for exposure (i.e. generation of splashes, sprays or aerosols from centrifugation, sonication, homogenization, vortexing, FACS, use of sharps (needles or glass)
6.9 What is the method of destruction or inactivation for the toxins listed: 6.10 Is there an antidote available for this toxin? Yes* No
*If yes, list: SECTION 7: HUMAN GENE TRANSFER/THERAPY 7.1 Does your protocol involve human gene therapy/transfer? Yes – Complete this Section No – Skip Section 7 & Go to Section 8 7.2 Provide the Federal Drug Administration (FDA) Investigational New Drug (IND) number and date that the application was submitted.
7.3 Does this protocol fit the following criteria for exemption from the NIH/OBA requirements for protocol submission, review and reporting process as described below (from Appendix M-VI-A of the NIH Guidelines): Human studies in which induction or enhancement of an immune response to a vector-encoded microbial immunogen is the major goal, such an immune response has been demonstrated Yes No in model systems, and the persistence of the vector-encoded immunogen is not expected.
• If “yes”, please address questions and provide material as described in Section A, below. • If “no”, please provide material as described in Section B, below. 7.4 Section A: For DNA Vaccine Human Gene Therapy Protocols which fall under the Appendix M-VI-A exception: a. Describe below what evidence exists to suggest that the vector-encoded immunogen expression is not expected to persist in the patient? b. Attach responses to Appendices M-II through M-V of NIH Guidelines, Description of the Proposal, Informed Consent, Privacy and Confidentiality, and Special Issues. Responses to Appendices M-II through M-V may be provided either as an appendix to the clinical protocol or incorporated in the clinical protocol. If responses to Appendices M-II through M-V are incorporated in the clinical protocol, each response must refer to the appropriate Appendix M-II through M-V. 7.5 Section B: Human Gene Transfer protocols which do not fall under the Appendix M-VI-A exception: a. Attach a copy of the complete protocol submitted to NIH OBA, as described in Appendix M-I-A b. Attach a copy of the written response from NIH OBA to the protocol submission that either: i. Indicates the submission does not present characteristics that warrant public RAC review and discussion; or ii. Provides a summary of the RAC’s key comments and recommendations after public review. 7.6 Include a copy of your Human Gene Therapy SOP along with this Schedule.
SECTION 8: NANOPARTICLES 8.1 Does your protocol involve the use/creation of nanoparticles? Yes – Complete this Section No – Skip Section 8 & Go to Section 9 8.2 List each nanoparticle in the table below:
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Description (including structure, hazards, etc. – Description of laboratory procedures Nanoparticle Name attach involving the Nanoparticle literature if necessary) 1. 2. What are the signs and symptoms of exposure to this material: 8.3 (Note: All accidents/injuries where exposure to nanoparticles should be reported to the Biosafety Office after seeking medical attention, if necessary). SECTION 9: ARTHROPODS 9.1 Does your protocol involve the use of arthropods? Yes – Complete this Section No – Skip Section 9 & Go to Section 10 9.2 Will you be using, creating, or breeding transgenic arthropods or exposing arthropods to recombinant DNA? Yes* No
*If yes, make sure you complete Section 2 – Recombinant DNA and Synthetic Nucleic Acid Molecules 9.3 Indicate the arthropods that will be used?
9.4 Will this work involve the importation, movement and/or field release of genetically engineered (GE) arthropods? Yes* No *If yes, a USDA/APHIS/PPQ permit may be required, see http://www.aphis.usda.gov/permits/ for more information. The Biosafety Office can assist you in determining permit requirements.
If permit(s) have already been obtained, submit a copy to [email protected] SECTION 10: PLANTS Yes No – Skip Section 10 10.1 Does your protocol involve the use of plants? & Go to Section 11 10.2 Will you be creating transgenic plants, exposing plant to recombinant DNA, transgenic arthropods, or transgenic microorganism/infectious agents? Yes* No
*If yes, make sure you complete Section 2 – Recombinant DNA and Synthetic Nucleic Acid Molecules 10.3 Indicate the plants that will be used?
10.4 Will this work involve the importation, movement and/or field release of genetically engineered (GE) plants?
*If yes, a USDA/APHIS/PPQ permit may be required, see Yes* No http://www.aphis.usda.gov/import_export/index.shtml for more information. The Biosafety Office can assist you in determining permit requirements.
If permit(s) have already been obtained, submit a copy to [email protected] SECTION 11: INVESTIGATOR’S ASSURANCE 11.1 Please review each of the following terms of this agreement prior to electronically signing this agreement, below.
1. I attest that the information contained in the attached application and supplements is accurate and complete. I also understand that, should I use the project described in this application as a basis for a funding proposal, I am responsible for ensuring that the description of the procedures in the funding proposal is identical to those contained in this application. 2. I have read and understand my responsibilities as a Principal Investigator outlined ion Section IV-B-7 of the NIH Guidelines and agree to comply with these responsibilities. 3. I agree that biological waste will be disposed of within the biohazardous waste stream. Any potentially infectious material, including but not limited to human or non-human primate fluids or unfixed tissues, and/or infectious recombinant DNA, will be decontaminated prior to disposal in the GRU Biohazardous waste containers. 4. I agree that all shipping of biological materials, dry ice and other dangerous goods will be done in accordance with IATA/DOT regulations. Shipping and transport will be performed only by those who can document training in hazardous materials shipping requirements and procedures. 5. I agree that intramural transport of biological materials will be done in a sealed, primary container inside of a sealed, leak proof durable secondary container. 6. I agree that if use of a Biosafety Cabinet is required by this application to control exposures to biological agents and aerosols shall be tested annually or after any move or adjustments. It is my responsibility to maintain the integrity of safety equipment. 7. I agree that safety caps/sealed rotors will used when centrifuging biological specimen, or if unavailable, the lid will not be opened until 15 minutes have passed after the samples have stopped spinning to allow aerosols to settle. 8. I agree that entry ways to areas where biological material is used or stored will be posted with “Biohazard” placard. 9. I agree to prevent unauthorized removal of biological material, biological material will be secured when not in use. 13
10. I agree that all containers of biological material will be properly labeled. 11. I will ensure that before entering my laboratory, any person is advised of the potential hazards. 12. I agree to accept responsibility and accountability that all laboratory personnel are familiar with and trained to employ the proposed safety precautions, appropriate emergency procedures, and the practices and techniques described in this BSP. 13. I will ensure that all personnel listed in this application complete all Georgia Board of Regents and IBC training requirements. 14. I agree to immediately report the following to the Biological Safety Officer (x-12663), followed by completion of the incident report found on the Biosafety webpage: • A violation of these conditions or any other policy governing the use of biological material under this authorization. • Any unauthorized transfer, disposal, or release of biological material including release to the sanitary sewer system, air or as solid waste. • The release or transfer of contaminated or potentially contaminated equipment to facilities for non- biological material use. • The use of biological material by unauthorized personnel. • Lost, stolen, or unaccountable biological materials. • Spills of biological materials or contamination of personnel with biological materials. • Any exposures, potential exposures, releases from primary containment, or equipment failure that may result in personnel exposure or environmental contamination. 15. I understand that the IBC may be obligated to report any non-compliance to Biosafety guidelines or regulations to my funding agencies (e.g. NIH) or Federal authorities. 16. I will not carry out the work described in the attached application until it has been approved by the Institutional Biosafety Committee (IBC) and/or the Biological Safety Office. 17. I agree to amend this protocol to include any changes in agents, personnel, locations, applications or major equipment (e.g. biosafety cabinets, autoclaves) prior to implementation of the changes. 18. I will annually verify that the research associated with this protocol is currently active, and that no unauthorized changes have been made to the protocol. 19. This authorization may be terminated at any time by the Institution Biosafety Committee or suspended by the Biosafety Officer if the conditions of this authorization are violated or if necessary to preclude harm to staff, facilities, or the environment. 20. The PI may terminate this authorization at any time by notifying the Biological Safety Officer and completing an amendment form. The PI shall notify the Biosafety Officer in a timely manner of the anticipated termination of biological material use.
11.2 Principal Investigator Date (By electronically entering your name, you are indicating verification that all items are accurate and you agree to ensure compliance with the above items.) ***Please save this form as BSP_PI name and submit electronically to [email protected]***
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Appendix B Classification of Human Etiologic Agents on the Basis of Hazard
This section has been reprinted from the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines), October 1997.
This appendix includes those biological agents known to infect humans as well as selected animal agents that may pose theoretical risks if inoculated into humans. Included are lists of representative genera and species known to be pathogenic; mutated, recombined, and non-pathogenic species and strains are not considered. Non-infectious life cycle stages of parasites are excluded.
This appendix reflects the current state of knowledge and should be considered a resource document. Included are the more commonly encountered agents and is not meant to be all-inclusive. Information on agent risk assessment may be found in the Agent Summary Statements of the CDC/NIH publication, Biosafety in Microbiological and Biomedical Laboratories (see Sections V-C, V-D, V-E, and V-F, Footnotes and References of Sections I through IV. Further guidance on agents not listed in Appendix B may be obtained through: Centers for Disease Control and Prevention, Biosafety Branch, Atlanta, Georgia 30333, Phone: (404) 639-3883, Fax: (404) 639-2294; National Institutes of Health, Division of Safety, Bethesda, Maryland 20892, Phone: (301) 496-1357; National Animal Disease Center, U.S. Department of Agriculture, Ames, Iowa 50010, Phone: (515) 862-8258.
A special committee of the American Society for Microbiology will conduct an annual review of this appendix and its recommendation for changes will be presented to the Recombinant DNA Advisory Committee as proposed amendments to the NIH Guidelines.
Appendix B - Table 1. Basis for the Classification of Biohazardous Agents by Risk Group (RG) Risk Group 1 (RG1) Agents that are not associated with disease in healthy adult humans
Risk Group 2 (RG2) Agents that are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available
Risk Group 3 (RG3) 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)
Risk Group 4 (RG4) Agents that are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available (high individual risk and high community risk)
Appendix B-I. Risk Group 1 (RG1) Agents RG1 agents are not associated with disease in healthy adult humans. Examples of RG1 agents include asporogenic Bacillus subtilis or Bacillus licheniformis (see Appendix C-IV-A, Bacillus subtilis or Bacillus licheniformis Host-Vector Systems, Exceptions), Escherichia coli-K12 (see Appendix C-II-A, Escherichia coli K-12 Host-Vector Systems, Exceptions), and adeno-associated virus types 1 through 4.
Those agents not listed in Risk Groups (RGs) 2, 3 and 4 are not automatically or implicitly classified in RG1; a risk assessment must be conducted based on the known and potential properties of the agents and their relationship to agents that are listed.
Appendix B-II. Risk Group 2 (RG2) Agents RG2 agents are associated with human disease that is rarely serious and for which preventive or therapeutic interventions are often available.
Appendix B-II-A. Risk Group 2 (RG2) - Bacterial Agents Including Chlamydia ♦ Acinetobacter baumannii (formerly Acinetobacter calcoaceticus)
Georgia Regents University Biosafety Guide-June 2008 B-1 ♦ 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 in Appendix B-III-A (RG3) ♦ 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 (RG1) ♦ Legionella including L. pneumophila ♦ Leptospira interrogans - all serotypes ♦ Listeria ♦ Moraxella ♦ Mycobacterium (except those listed in Appendix B-III-A (RG3)) 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 ♦ Streptobacillus moniliformis ♦ Streptococcus including S. pneumoniae, S. pyogenes ♦ Treponema pallidum, T. carateum ♦ Vibrio cholerae, V. parahemolyticus, V. vulnificus ♦ Yersinia enterocolitica
Appendix B-II-B. Risk Group 2 (RG2) - Fungal Agents ♦ Blastomyces dermatitidis ♦ Cladosporium bantianum, C. (Xylohypha) trichoides ♦ Cryptococcus neoformans ♦ Dactylaria galopava (Ochroconis gallopavum) ♦ Epidermophyton ♦ Exophiala (Wangiella) dermatitidis ♦ Fonsecaea pedrosoi ♦ Microsporum
Georgia Regents University Biosafety Guide-June 2008 B-2 ♦ Paracoccidioides braziliensis ♦ Penicillium marneffei ♦ Sporothrix schenckii ♦ Trichophyton
Appendix B-II-C. Risk Group 2 (RG2) - Parasitic Agents ♦ Ancylostoma human hookworms including A. duodenale, A. ceylanicum ♦ Ascaris including Ascaris lumbricoides suum ♦ Babesia including B. divergens, B. microti ♦ Brugia filaria worms including B. malayi, B. timori ♦ Coccidia ♦ Cryptosporidium including C. parvum ♦ Cysticercus cellulosae (hydatid cyst, larva of T. solium) ♦ 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 ♦ Onchocerca filaria worms including, O. volvulus ♦ Plasmodium including simian species, P. cynomologi, P. falciparum, P. malariae, P. ovale, P. vivax ♦ Sarcocystis including S. sui hominis ♦ Schistosoma including S. haematobium, S. intercalatum, S. japonicum, S. mansoni, S. mekongi ♦ Strongyloides including S. stercoralis ♦ 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
Appendix B-II-D. Risk Group 2 (RG2) - 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 reference source (see Section V-C, Footnotes and References of Sections I through IV) ♦ Bunyaviruses • Bunyamwera virus • Rift Valley fever virus vaccine strain MP-12
Georgia Regents University Biosafety Guide-June 2008 B-3 • Other viruses as listed in the reference source (see Section V-C, Footnotes and References of Sections I through IV) ♦ 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 reference source (see Section V-C, Footnotes and References of Sections I through IV) ♦ Hepatitis A, B, C, D, and E viruses ♦ Herpesviruses - except Herpesvirus simiae (Monkey B virus) (see Appendix B-IV-D, Risk Group 4 (RG4) -Viral Agents) • Cytomegalovirus • Epstein Barr virus • Herpes simplex types 1 and 2 • Herpes zoster • Human herpesvirus types 6 and 7 ♦ Orthomyxoviruses • Influenza viruses types A, B, and C • Other tick-borne orthomyxoviruses as listed in the reference source (see Section V-C, Footnotes and References of Sections I through IV) ♦ Papovaviruses • All human papilloma viruses ♦ 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 - all types except Monkeypox virus (see Appendix B-III-D, Risk Group 3 (RG3) – Viruses and Prions) and restricted poxviruses including Alastrim, Smallpox, and Whitepox (see Section V-L,Footnotes and References of Sections I through IV) ♦ Reoviruses - all types including Coltivirus, human Rotavirus, and Orbivirus (Colorado tick fever virus) ♦ Rhabdoviruses • Rabies virus - all strains • Vesicular stomatitis virus - laboratory adapted strains including VSV-Indiana, San Juan, and Glasgow ♦ Togaviruses (see Alphaviruses and Flaviviruses) • Rubivirus (rubella)
Appendix B-III. Risk Group 3 (RG3) Agents RG3 agents are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available.
Appendix B-III-A. Risk Group 3 (RG3) - Bacterial Agents Including Rickettsia ♦ Bartonella
Georgia Regents University Biosafety Guide-June 2008 B-4 ♦ Brucella including B. abortus, B. canis, B. suis ♦ Burkholderia (Pseudomonas) mallei, B. pseudomallei ♦ Coxiella burnetii ♦ Francisella tularensis ♦ Mycobacterium bovis (except BCG strain, see Appendix B-II-A, Risk Group 2 (RG2) - Bacterial Agents Including Chlamydia), 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
Appendix B-III-B. Risk Group 3 (RG3) - Fungal Agents ♦ Coccidioides immitis (sporulating cultures; contaminated soil) ♦ Histoplasma capsulatum, H. capsulatum var. duboisii
Appendix B-III-C. Risk Group 3 (RG3) - Parasitic Agents None
Appendix B-III-D. Risk Group 3 (RG3) - Viruses and Prions ♦ Alphaviruses (Togaviruses) - Group A Arboviruses • Semliki Forest virus • St. Louis encephalitis virus • Venezuelan equine encephalomyelitis virus (except the vaccine strain TC-83, see Appendix BII-D (RG2)) • Other viruses as listed in the reference source (see Section V-C, Footnotes and References of Sections I through IV) ♦ Arenaviruses • Flexal • Lymphocytic choriomeningitis virus (LCM) (neurotropic strains) ♦ 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 reference source (see Section V-C, Footnotes and References of Sections I through IV) ♦ Poxviruses • Monkeypox virus ♦ Prions • Transmissible spongioform encephalopathies (TME) agents (Creutzfeldt-Jacob disease and kuru agents)(see Section V-C, Footnotes and References of Sections I through IV, for 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
Appendix B-IV. Risk Group 4 (RG4) Agents RG4 agents are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available.
Appendix B-IV-A. Risk Group 4 (RG4) - Bacterial Agents
Georgia Regents University Biosafety Guide-June 2008 B-5 None
Appendix B-IV-B. Risk Group 4 (RG4) - Fungal Agents None
Appendix B-IV-C. Risk Group 4 (RG4) - Parasitic Agents None
Appendix B-IV-D. Risk Group 4 (RG4) - Viral Agents ♦ Arenaviruses • Guanarito virus • Lassa virus • Junin virus • Machupo virus • Sabia ♦ 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) ♦ Paramyxoviruses • Equine morbillivirus ♦ Hemorrhagic fever agents and viruses as yet undefined
Appendix B-V. Animal Viral Etiologic Agents in Common Use The following list of animal etiologic agents is appended to the list of human etiologic agents. None of these agents is associated with disease in healthy adult humans; they are commonly used in laboratory experimental work.
A containment level appropriate for RG1 human agents is recommended for their use. For agents that are infectious to human cells, e.g., amphotropic and xenotropic strains of murine leukemia virus, a containment level appropriate for RG2 human agents is recommended.
♦ Baculoviruses ♦ Herpesviruses • Herpesvirus ateles • Herpesvirus saimiri • Marek's disease virus • Murine cytomegalovirus • Papovaviruses • Bovine papilloma virus • Polyoma virus • Shope papilloma virus • Simian virus 40 (SV40) ♦ Retroviruses • Avian leukosis virus • Avian sarcoma virus • Bovine leukemia virus
Georgia Regents University Biosafety Guide-June 2008 B-6 • Feline leukemia virus • Feline sarcoma virus • Gibbon leukemia virus • Mason-Pfizer monkey virus • Mouse mammary tumor virus • Murine leukemia virus • Murine sarcoma virus • Rat leukemia virus
Appendix B-V-1. 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.
Georgia Regents University Biosafety Guide-June 2008 B-7
Georgia Regents University Biosafety Guide-June 2008 B-8 Appendix C Biosafety Levels Only work at biosafety levels 1,2 and 3 is allowed at GRU. There are no biosafety level 4 facilities for biosafety level 4 work allowed at GRU.
The CDC and NIH have established biosafety guidelines that are found in the CDC/NIH publication Biosafety in Microbiological and Biomedical Laboratories (BMBL). The following is reprinted from that publication. For additional information please contact the Biosafety Office at x1-2663 or [email protected].
PRINCIPLES OF BIOSAFETY 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.
Primary containment, the protection of personnel and the immediate laboratory environment from exposure to infectious agents, is provided by both good microbiological technique and the use of appropriate safety equipment. 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 infected 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 hazards that will or may be encountered and that specifies practices and procedures designed to minimize or eliminate risks. Personnel should be advised of special hazards and should be required to read and to follow the required practices and procedures. A scientist trained and knowledgeable in appropriate laboratory techniques, safety procedures, and hazards associated with handling infectious agents must direct laboratory activities.
When standard laboratory practices are not sufficient to control the hazard associated with a particular agent or laboratory procedure, additional measures may be needed. The laboratory director is responsible for selecting additional safety practices, which must be in keeping with the hazard associated with the agent or procedure.
Laboratory personnel, safety practices, and techniques must be supplemented by appropriate facility design and engineering features, safety equipment, and management practices.
SAFETY EQUIPMENT (PRIMARY BARRIERS) 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 of the CDC/NIH publication. Class I and Class II biological safety cabinets are primary barriers that offer significant levels of protection to laboratory personnel and 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, bouffant head covers, 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 BARRIERS) The design of the facility is important in providing a barrier to protect persons working inside and outside of the laboratory within the facility, and to protect persons or animals in the community from infectious agents that may be accidentally released from the laboratory. Laboratory management is responsible for providing facilities commensurate with the laboratory's function and the recommended biosafety level for the agents being manipulated.
The recommended secondary barrier(s) will depend on the risk of transmission of specific agents. For example, the exposure risks for most laboratory work in Biosafety Level 1 and 2 facilities will be direct contact with the agents, or inadvertent contact exposures through contaminated work environments. Secondary barriers in these laboratories may include separation of the laboratory work area from public access, availability of a decontamination facility (e.g., autoclave), and hand-washing facilities.
As the risk for aerosol transmission increases, higher levels of primary containment and multiple secondary barriers may become necessary to prevent infectious agents from escaping into the environment. Such design features could include specialized ventilation systems to assure 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. 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.
BIOSAFETY LEVEL 1 practices, safety equipment, and facilities are appropriate for undergraduate and secondary educational training and teaching laboratories, and for other facilities in which work is done with defined and characterized strains of viable microorganisms not known to cause disease in healthy adult humans. Bacillus subtilis, Naegleria gruberi, and infectious canine hepatitis virus are representative of those microorganisms meeting these criteria. Many agents not ordinarily associated with disease processes in humans are, however, opportunistic pathogens and may cause infection in the young, the aged, and immunodeficient or immunosuppressed individuals. Vaccine strains which have undergone multiple in vivo passages should not be considered avirulent simply because they are vaccine strains.
Biosafety Level 1 represents a basic level of containment that relies on standard microbiological practices with no special primary or secondary barriers recommended, other than a sink for hand-washing.
BIOSAFETY LEVEL 2 practices, equipment, and facilities are applicable to clinical, diagnostic, teaching and other facilities in which work is done with the broad spectrum of indigenous moderate-risk agents present in the community and associated with human disease of varying severity. With good microbiological techniques, these agents can be used safely in activities conducted on the open bench, provided the potential for producing splashes or aerosols is low. Hepatitis B virus, the 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 BSL2 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.
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 which realistically address the issue of the infective hazard of clinical specimens. Except in extraordinary circumstances (e.g., suspected hemorrhagic fever), the initial processing of clinical specimens and identification of isolates can be done safely at Biosafety Level 2, the recommended level for work with bloodborne pathogens such as hepatitis B virus and HIV. The containment elements described in Biosafety Level 2 are consistent with the Occupational Exposure to Bloodborne Pathogens Standard from the Occupational Safety and Health Administration (OSHA), that requires the use of specific precautions with all clinical specimens of blood or other potentially infectious material (Universal Precautions). Additionally, other recommendations specific for clinical laboratories may be obtained from the National Committee for Clinical Laboratory Standards.
Biosafety Level 2 recommendations and OSHA requirements focus on the prevention of percutaneous and mucous membrane exposures to clinical material. Primary barriers such as biological safety cabinets 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. Appendix D BL2+ Work Practices
Biosafety Level 2 Plus (BL2+) is the designation utilized for those biohazard experiments that require practices that are more stringent than standard BL2 procedures. Generally, BL3 practices are mandated in a space designed for BL2 work. It is preferred that the BL2 laboratory be self-contained; that is, all equipment required for the experiment should be located within the lab. A sign is posted on the door while BL2+ work is in progress, and access is restricted to those involved in the experiment. When work is completed, and equipment has been decontaminated, the sign is removed and the lab returns to standard BL2 or BL1 usage.
BL3 practices require that all work be conducted under physical containment. Therefore, all manipulations with BL2+ material are conducted within a Class II biological safety cabinet and secondary containment is utilized for centrifugation and other potential aerosol generating procedures. The following notes further describe the requirements for work at BL2+.
Personal Protective Equipment (PPE) ♦ Dedicate PPE for the experiment. PPE worn for BL2+ work should not be worn in other areas. Remove before leaving the laboratory. ♦ Wear a lab coat or solid-front gown, preferably with a knit or grip cuff. ♦ Double glove for all work within the biological safety cabinet (BSC). Remove the outer pair before exiting the BSC, and don a new pair each time you reenter the BSC. ♦ Ensure that your gloves extend over the sleeve of your lab coat. An opening at the wrist will allow aerosols generated within the BSC to contaminate your wrist and forearm, extending hand-washing to your elbow. ♦ Sleeve covers can be worn to ensure coverage of the wrist and will also minimize contamination of the sleeves of your lab coat. ♦ Face Protection (mask and eyewear, can also be worn and will protect mucous membranes from exposure in the event a spill outside the BSC during transfer of material to and from the incubator. It will also help to prevent you from touching your eyes, nose and mouth when working within the BSC. ♦ Remove PPE before leaving the laboratory. Placing a coat hook within the BL2+ area will facilitate this requirement. Remove your outer gloves first, then your lab coat or gown, followed by the inner gloves. Take your face protection off last. Don’t touch your face with gloved hands. Remove gloves and other clothing aseptically, from the inside out, and avoid touching the contaminated outer side of the glove. ♦ Decontaminate reusable PPE as soon as feasible after it has been contaminated. Small areas can be spot treated with a suitable disinfectant, such as 1-10% household bleach. Lab coats can also be autoclaved, or sent to a laundry facility equipped to handle biohazardous PPE. Disposable PPE can be placed within a biohazard bag, treated and discarded as biomedical waste. ♦ Wash your hands with soap and water after removing PPE and before leaving the laboratory.
Work Practices In the Biological Safety Cabinet (BSC) ♦ Perform all work within a BSC. This includes discarding waste within the BSC. Moving your hands in and out of the BSC will disrupt the protective air curtain at the front access opening. ♦ Place all items required for the experiment within the BSC before starting work. ♦ Wipe items down with disinfectant prior to placement within the BSC. ♦ Segregate clean areas from contaminated areas within the BSC (by at least 12-14”). ♦ Keep the front and rear grilles clear when working within the BSC. Avoid blocking the rear grille. Don’t store items on top of the BSC. Remind fellow researchers to minimize traffic and work behind the operator, as this may interfere with cabinet airflow. Depending on the location of the BSC within the room, opening and closing the room door can significantly interfere with BSC airlfow. ♦ Avoid the use of a flame within the BSC. In addition to presenting a fire hazard, an open flame can disrupt airflow and possibly damage the paper filter located above the work surface. If the use of flame is absolutely necessary, use a burner with a pilot light that provides a flame only when depressed and releases after contact. Never leave an open flame (burner or pilot light) unattended in your BSC. ♦ Store tissue culture flasks in the incubator within small secondary trays to help minimize contamination. Trays will also facilitate transfer to and from the BSC. ♦ Keep your hands away from your face (face protection helps to minimize the potential for this route of exposure). ♦ Avoid the use of glass Pasteur pipettes or needles and syringes. Substitute plastic for glass whenever feasible. Alternatives to glass Pasteur pipettes include: plastic pipettes, plastic transfer pipettes, plastic gel loading pipette tips and pipette tip extendors, aspirators, and flexible plastic aspiration pipettes. Some researchers will either score and break the end off of a 1 ml or 5 ml plastic pipette or remove the wool plug and use for aspirating cultures. ♦ If the use of sharps cannot be avoided, maintain a sharps container in the immediate vicinity of use. Discard intact needles and syringes immediately after use. Use a one-handed disposal method (keep a hand behind your back or by your side, don’t place on or near the opening of the sharps container). Never recap, bend, break or otherwise manipulate sharps by hand. If you must remove the needle from the syringe, use the small opening on the top of the needlebox for this purpose. Forceps, tweezers, or small pliers may also be utilized. ♦ Protect the house vacuum system or pump from contamination by installing a trap and filter system. Use a primary collection flask containing disinfectant, followed by an overflow flask, which leads through a HEPA or hydrophobic filter. ♦ Collect all waste within the BSC. Smaller biohazard waste bags may be utilized along with beakers or shallow trays containing disinfectant for the collection and disinfection of pipettes and other contaminated items. Waste can also be collected within the BSC in the following manner. • Horizontal collection: Horizontal trays containing disinfectant allow total immersion of pipettes. • Vertical collection: Beakers containing disinfectant can be used if disinfectant is drawn up inside the pipette and allowed to run down the interior wall upon disposal into the beaker. • Bags: Bags have the potential for creating aerosols when moved. At BL2+, seal autoclave bags within the cabinet and place within a second bag. Carefully add water to the primary bag before sealing (25 ml for smaller bags, 200 ml for larger bags). The addition of water will help to generate steam within the bag during the autoclave cycle. ♦ Wipe items down with disinfectant prior to removal from the BSC. ♦ Wipe down BSC with disinfectant after use (work surface, grilles, sides, back and inside front view screen). ♦ Decontaminate liquid waste with household bleach diluted 10% against the volume of the waste. Allow at least a 30 minute contact time for full decontamination. ♦ Transport waste to autoclave in a leakproof container.
Centrifugation ♦ Use sealed rotors or safety buckets as secondary containment for centrifugation. ♦ Load and unload the rotor or safety buckets within the BSC. ♦ Don’t overfill primary containers, limit to < ¾ full. Wipe exterior of tube with disinfectant before loading. ♦ Seal rotor or bucket and wipe down with disinfectant, remove outer gloves, and transport to the centrifuge. ♦ Post a sign on centrifuge that includes the biohazard symbol, name of the agent with Biosafety Level, and your name. ♦ Wait 2-5 minutes after the run to allow aerosols to settle in the event of a spill. Transport sealed rotor or safety bucket to cabinet to complete your experiment. Don new pair of outer gloves. ♦ Decontaminate the rotor or safety bucket by spraying with 70% ethanol and allowing to air dry. Wipe the throw line within the centrifuge with disinfectant and remove your biohazard sign. In the event of a spill during centrifugation, follow the spill response procedures outlined in the the laboratory SOPs. ♦ Avoid the use of microfuge, which is difficult to contain. If you cannot avoid using a microfuge, use a model that has built in secondary containment (a sealed rotor) along with microfuge tubes equipped with a O-ring seal. You can also operate your microfuge in the rear of your BSC (don’t perform any work within the BSC while the microfuge is in operation, and wait 2-5 minutes after the run before opening the microfuge).
Labels ♦ Post a biohazard sign at the entry to the BL2+ laboratory. ♦ Ensure that any specific entry requirements (vaccination), the name of the agent, the Biosafety Level, and the name of an emergency contact person ARE posted on the door placards. ♦ Place the BL2 wall notice (not a door sign) inside your laboratory to remind researchers of the core safety practices. ♦ Label equipment housing the agent (incubators, freezers) with the universal biohazard symbol and agent name.
Transport of Biohazards on Campus (between labs or buildings): ♦ Must have two leakproof containers, including the following: • a sealed primary container • a sealed secondary container • absorbent (paper towels) between the primary and secondary containers suitable for the volume transported • a biohazard sticker on the outside of the secondary container with agent name • lab address and phone number on the outside of the secondary container ♦ Utilize plastic containers whenever feasible. Avoid glass. ♦ Sealed plastic (not glass) primary vials can be transported within sealed, labeled plastic bags. ♦ If glass primary containers must be used, place containers within a sealed rigid plastic container with absorbent and padding to cushion vials during transport. ♦ Decontaminated the outside of the primary container before placing into the secondary container. ♦ Decontaminate the secondary container before leaving the laboratory
Hand-washing ♦ Wash hands whenever PPE is removed and before leaving the laboratory. ♦ Wash with soap and warm water for at least 15 seconds. Since the contact time of most soaps is quite extensive for actual decontamination, mechanical friction from scrubbing and water dilution are essential for complete cleaning. ♦ No glove is 100% leakproof. ♦ Never wet or handwash your gloves with water or disinfectant, as this will encourage wicking and increase permeability of the protective barrier.
Spills and Exposure Incidents ♦ All researchers must be familiar with the applicable exposure response procedures before initiating their experiments. ♦ Review the attached Biosafety Spill and Incident Response SOPs before starting work.
Appendix E: Standard Operating Procedure Development Template
Avialable in MS Word format at: http://www.GRU.edu/research/ibc/apps.htm
This template should be considered a starting point to initiate the SOP development process. It covers only the basic standard issues addressed in most biological laboratory settings; however, this should be edited and/or modify to address the risks within each laboratory or clinic.
Special considerations to document practices such as extramural transport, possession, use or handling of biological toxins or any specific issues relevant to the laboratory should be added.
LABORATORY STANDARD OPERATING PROCEDURE DEVELOPMENT GUIDANCE TEMPLATE Please edit and complete as necessary to address Biosafety Risks within your laboratory
Date:
Principal Investigator:
Department:
Room(s) + BSL of each room:
Phone:
______1. Access to the laboratory is limited to staff, or other persons with permission of the Principal Investigator, when work with BSL-2 pathogens is being conducted. Access is limited according to attached procedures. 2. All laboratory personnel must be screened by Employee Health before working with potentially infectious materials, including fresh, unfixed, human or non-human primate specimens, uncharacterized cell lines, etc. Any available vaccinations which would reduce the risks associated with exposure to any of the agents in the protocol must be offered to all personnel or a signed waiver must be obtained by the PI. 3. Persons who have increased risk of infection, or for whom infection may have serious consequences, must not be allowed to enter laboratory when work with infectious agents is in progress without permission of the Principal Investigator. 4. All persons entering the laboratory must be advised of potential hazards. Laboratory workers must be trained and made aware of the hazards and appropriate safety precautions before working with any of the biological agents. 5. Spills and accidents that result in overt exposures to infectious materials must be immediately reported to the Principal Investigator and appropriate medical evaluation must be provided. 6. Laboratory staff must not eat, drink, smoke, handle contact lenses, chew gum, apply cosmetics in laboratory. 7. Food or drink for human consumption or utensils or cups must stored outside laboratory work area in refrigerators designated for that purpose only. 8. Laboratory staff and all other persons working with infectious substances must wear gloves. Contaminated gloves must be changed IMMEDIATELY. Under NO CIRCUMSTANCES will gloves be reused. 9. Laboratory staff must wash hands after handling infectious materials, after removing gloves, and before leaving the laboratory. 10. Face protection (goggles, mask, face shield, or other splatter guard) must be used for all procedures when such procedures could produce splashes or sprays of infectious or other hazardous materials or when microorganisms are manipulated outside the biological safety cabinet. See attached procedures for more detailed PPE requirements. 11. Protective clothing must be removed and left in laboratory before going to non-laboratory areas (cafeteria, library, administrative areas). 12. Protective clothing must be either disposed of in laboratory or laundered by institution. (NEVER taken home!) 13. Only mechanical pipetting devices must be used in the laboratory. 14. All sharps must be placed in red (approved) sharps containers. 15. Sharps containers must be turned in when ¾ full. 16. All procedures must be performed to minimize creation of splashes or aerosols according to attached procedures. 17. A Biosafety Cabinet must be used for all procedures with potential for creating infectious aerosols or splashes, or whenever handling high concentrations of infectious materials. 18. Biological Safety Cabinet must have a current (annual) certification. 19. Biological Safety Cabinet must not be used until it is recertified after relocation, any movement, or dislocation. 20. Centrifuges used for high concentrations or large volumes of infectious agents must have sealed rotor heads or centrifuge safety cups. Safety cups must be opened only in a Biosafety cabinet. 21. All infectious animal carcasses must be disposed of by placing in a plastic bag and placing in infected animal carcass freezer or according to attached procedures. 22. All non-liquid contaminated cultures, stocks, plasticware and other biologically contaminated waste must be disinfected and placed in biomedical waste containers for disposal according to attached procedures. 23. All infectious liquids must be decontaminated or disinfected prior to being poured into drain according to attached procedures. 24. All work surfaces must be decontaminated at the completion of work, at the end of the day, or after any spill or splash of viable material according to attached procedures. 25. Laboratory equipment, surfaces and other contaminated materials must be decontaminated with an effective disinfectant on a routine basis, after work with infectious materials is finished, and especially after overt spills, splashes, or other contamination according to attached procedures. Equipment must be decontaminated before removal from the laboratory (for repair maintenance or other purposes). Decontamination protocol is attached. 26. Laboratory doors must be locked when the laboratory is not occupied. 27. Spaces between benches, cabinets, and equipment must be kept accessible for cleaning. 28. Eyewash must be flushed at least monthly and kept clear of obstructions. 29. All personnel must know the location of the nearest safety shower and must ensure that area under the shower is kept free of obstructions. 30. Any personnel with overt exposure will be taken to Employee Health and/or the ER for immediate (<1 hour) post- exposure medical intervention and monitoring. 31. Any overt exposures of personnel to biological agents will be reported to the PI and the BSO as soon as possible.
SOP ADDENUM- DETAILED LABORATORY-SPECIFIC PROTOCOLS
1. Storage and access control of cells or cultures include the following. Consider storage in all locations, including freezers, cryotanks, cold rooms.
Yes No Agents are stored inside well-labeled tubes/vials. Yes No Agents are stored inside well-labeled storage boxes. Yes No Biohazard labels are clearly affixed on any storage device clearly identifying the hazards inside Yes No Storage refrigerators/freezers/cryotanks are in rooms whose access is limited to authorized personnel. Yes No Refrigerators are kept locked and accessible only to authorized personnel Yes No Freezers are kept locked and accessible only to authorized personnel Yes No Cryotanks are kept locked and accessed only by authorized personnel Yes No Lockboxes are utilized and accessible only to authorized personnel Yes No Policies and procedures are in place whereby only persons who have been advised of the potential hazard and meet any specific entry requirements (e.g., immunization) are authorized to enter the laboratory or have access to agents. Yes No Others (please specify)
2. Procedures employed which may result in the generation of aerosols, splashes and/or sprays of biological material and safety precautions that should be followed by personnel performing these procedures are as follows:
Centrifugation Sonication Vortexing Homogenization Opening Vacuum vials Syringe filtration Placing biological materials under pressure (through small bore hoses or needles) Animal cage changing Intranasal inoculation Necropsy of infected animals Fluorescence activated cell sorting Fluorescence activated cell analysis Other (please specify):
Safety precautions (e.g. use of Biosafety Cabinets, shields, respirators, containment devices):
3. Transport procedures for transport of agent(s)* between locations (i.e. through hallways and other non-laboratory areas). Note: For BSL2 agents, consider transporting agents inside a sealed, leakproof primary container inside a well-labeled sealed, leakproof, durable secondary container. *If transporting animals, please describe procedure. *If transporting any biological agent off-campus, please refer to Biosafety Schedule L for transport guidelines.
4. Disinfection/decontamination and disposal of viable agents or recombinant DNA or items which have come in contact with these agents. See: http://www.GRU.edu/research/ibc/disinfectants for guidance in selecting appropriate disinfectants for the agents in this protocol.
LIQUID WASTES (check all that apply):
Disinfection/Decontamination Method Treatment Final Disposal (after disinfection indicated in left column)) time (eg. 30 Check all that apply. If “other”, please specify below min) None necessary N/A In sharps container As chemical waste Other Autoclaving Into Sewer In sharps container As chemical waste Other Chemical Methods: Bleach (freshly diluted to final 10% (v/v)) Into Sewer In sharps container As chemical waste Other Other chlorine products (Clidox®) As chemical waste Other Iodine/iodophors (eg. Wescodyne®) As chemical waste Other Alcohols (e.g. final 70% (v/v) EtOH or IPA) As chemical waste Other Phenolic agents (e.g. Biozide®, Vesphene®) As chemical waste Other Quaternary Ammonium Agents (e.g. Roccal®, As chemical waste Other Coverage Plus®, Cavicide®, Lysol®) Aldehydes (e.g. 2-4% glutaraldehyde, 4% As chemical waste Other formaldehyde, Cidex®) Peroxygens (e.g. Virkon®) As chemical waste Other Other, please specify:
SOLID WASTES (Plasticware, glassware, tubes, tissues) (check all that apply):
Disinfection/Decontamination Method Treatment Final Disposal (after disinfection indicated in left column) time (eg. 30 Check all that apply. If “other”, please specify below min) All biological waste must be disposed as Biohazardous Waste None required N/A In Biohaz. box In biohaz. sharps container Other Autoclaving In Biohaz. box In biohaz. sharps container Other Chemical Methods Bleach (freshly diluted to final 10% (v/v)) In Biohaz. box In biohaz. sharps container Other Aldehydes (e.g. fixation with 2-4% In Biohaz. box In biohaz. sharps container Other glutaraldehyde, 4% formaldehyde) Peroxygens (e.g. Virkon®) In Biohaz. box In biohaz. sharps container Other Other, please specify:
ANIMAL TISSUES/CARCASS WASTE (check all that apply):
Disinfection/Decontamination Method Treatment Final Disposal (after disinfection indicated in left column) time (eg. 30 Check all that apply. If “other”, please specify below min) Autoclaving prior to removal from laboratory In LAS animal disposal freezer In Biohaz. waste box Other None, transported to LAS as per protocol below N/A In LAS animal disposal freezer In Biohaz. waste box Other
Other, please specify:
5. Work surfaces, instruments, equipment will be decontaminated using the following method(s). Consider both benchtop surfaces and stainless steel surfaces (such as those in biosafety cabinets), as well as in equipment (such as centrifuges) and instruments (check all that apply):
Check all that apply Disinfection/Decontamination Method Contact time Frequency Benchtops Stainless Equipment Instruments (e.g. 30’) (e.g. daily and after Surfaces spills) Autoclaving Chemical Methods: Bleach (freshly diluted to final 10% (v/v)) Bleach (freshly diluted to final 10% (v/v)), + rinse with 70% Alcohol Other chlorine products (Clidox®) Iodine/iodophors (eg. Wescodyne®) Alcohols (e.g. final 70% (v/v) EtOH or IPA) Phenolic agents (e.g. Biozide®, Vesphene®) Quaternary Ammonium Agents (e.g. Roccal®, Coverage Plus®, Cavicide®, Lysol®) Aldehydes (e.g. 2-4% glutaraldehyde, 4% formaldehyde, Cidex®) Peroxygens (e.g. Virkon®) Other, please specify:
6. Spills involving biological material will be handled in your laboratory using the following procedures (check all that apply):
(a) Describe which measures will be employed to handle spills in your laboratory: Large spills or high-hazard spills will be reported to PI and BSO immediately for assistance Spills in public (non-laboratory) areas will be reported to PI and BSO immediately Spills involving high hazard agent(s) (e.g. Select Agents) will be reported to the PI and BSO immediately Any injured individuals will be removed from areas and medical attention and follow-up will be provided All personnel in area will be advised of hazard and told to avoid the area Spill will be covered with copious absorbent material (like paper towels) The spill will be decontaminated with an appropriate disinfectant for the agent(s) spilled for an appropriate contact period (see below) After decontamination, absorbent material will be disposed as biohazardous waste After decontamination, the surface will be cleaned to remove residual disinfectant Spills in equipment (e.g. Biosafety cabinets or centrifuges) will be disinfected according to written procedures on the GRU Emergency Response Flip charts which have been posted in my laboratory (b) Spills will be disinfected using the following disinfectant and contact time:
Disinfection/Decontamination Method Contact time (e.g. 30’) Autoclaving Chemical Methods: Bleach (freshly diluted to final 10% (v/v)) Bleach (freshly diluted to final 10% (v/v)), + rinse with 70% Alcohol Other chlorine products (Clidox®) Iodine/iodophors (eg. Wescodyne®) Alcohols (e.g. final 70% (v/v) EtOH or IPA) Phenolic agents (e.g. Biozide®, Vesphene®) Quaternary Ammonium Agents (e.g. Roccal®, Coverage Plus®, Cavicide®, Lysol®) Aldehydes (e.g. 2-4% glutaraldehyde, 4% formaldehyde, Cidex®) Peroxygens (e.g. Virkon®)
7. Protective equipment which will be utilized to minimize exposure of personnel to agents while in the laboratory:
(check all that apply): Gloves (nitrile or latex) Cuffed wrists on gowns Safety glasses Surgical mask Double Gloves (nitrile or latex) Sleeve protectors Goggles N95 Respirator Lab coat Scrubs Face shield PAPR Respirator Tyvek coveralls Shoe covers/booties Plexiglass shield Aprons Rear-closing gowns Biosafety Cabinet Safety caps/Sealed rotors on Rubber boots Centrifuge Other (please specify)
8. Protective equipment which will be utilized to minimize exposure of personnel to agents while working with animals (if applicable):
Agents will be delivered into animals using which BSL containment practices and standards: BSL-1 BSL-2 BSL-3
Please indicate Personal Protective Equipment (PPE) and other safety equipment to be utilized while delivering agents into animals (check all that apply): Gloves (nitrile or latex) Cuffed wrists on gowns Safety glasses Surgical mask Double Gloves (nitrile or latex) Sleeve protectors Goggles N95 Respirator Lab coat Scrubs Face shield PAPR Respirator Tyvek coveralls Shoe covers/booties Plexiglass shield Aprons Rear-closing gowns Biosafety Cabinet Rubber boots Other (please specify): ______
After delivery of agents into animals: After delivery of agents, animals will be housed in (check all that apply): Separate cages from other animals Well-labeled cages indicating possible hazards to animal staff Cages with filter-top lids Microisolator cages Conventional/ABSL-1 facilities ABSL-2 facilities ABSL-3 facilities Live animals will not be returned to LAS facilities after delivery Other (please specify): ______
How long after delivery of agents will animals be analyzed and/or sacrificed (indicate time range)? ______
Appendix F Human Gene Transfer Clinical Trials Proposed clinical trials involving human gene transfer require registration and approval from both campus and federal agencies before initiation. NIH defines human gene transfer as the “the deliberate transfer of recombinant DNA, or DNA or RNA derived from recombinant DNA, into human subjects.” The GRU Institutional Biological Safety Committee requirements for human gene therapy protocols are detailed below. Federal requirements (NIH and FDA) for these experiments are described in significant detail in Appendix M of the NIH Guidelines for Research Involving Recombinant DNA, May, 1999, and in the Code of Federal Regulations, 21 CFR, Part 312 (FDA Points to Consider).
GRU/GRMC Standards which must be met: GRU Institutional Review Board (IRB) x1-1478 (through the Office of Human Research Protection) GRU Institutional Biological Safety Committee (IBC) x1-2663 (through Biosafety Representatives) GRMC(or other hospital/clinic) Epidemiology/Infection Control Office
Federal Registration and Approval: NIH Office of Biotechnology Activities (301) 496-9838 http://www4.od.nih.gov/oba/apndxm.htm Appendix M (Human Gene Therapy)
FDA Center for Biologics Evaluation and Research http://www.fda.gov/cber/ind/ind.htm 21 CFR Part 312
Note: Approval from the GRU IRB and IBC are required prior to submission to the the FDA. In accordance with the update in the NIH Guidelines for Research Involving Recombinant DNA, Appendix M, GRU’s IRB and IBC cannot approve a HGT protocol until it has been reviewed by the NIH Recombinant DNA Advisory Committee. Upon notification from the NIH Office of Biotechnology Activities that the RAC has completed its review, the IRB and IBC will be eligible to complete their respective reviews and approve if warranted.
Appendix M-1 of the NIH Guidelines includes the submission requirements and addresses for both the NIH Office of Biotechnology Activities and FDA Center for Biologics Evaluation and Research. A copy of the 21 CFR, Part 312 detailing the FDA IND Content and Format requirements can be downloaded directly from the FDA web address listed above.
Application for Human Gene Transfer Clinical Trials at GRU To initiate the review of a proposed human gene transfer clinical trial, please submit a description of your protocol in the format described in Appendix M of the attached NIH Guidelines for Research involving recombinant DNA, May, 1999. To obtain a copy of the NIH Guidelines, access the NIH OBA web site or contact the Biosafety Office at x1-2663 or [email protected]. Send a copy of your completed Appendix M to:
The GRU Institutional Biological Safety Committee C/O Biosafety Office Division of Environmental Health & Safety 1405 Goss Lane, CI-1006 Augusta, GA 30912-7521
Contact person: Biosafety Officer, 706-721-2663
Only complete protocols will be sent to Committee members for review. Specifically, we’ll need: Scientific abstract Non-technical abstract Your responses to NIH Guidelines Appendix M-II through M-V Your response to Adverse Event reporting requirements detailed in Appendix M-VII A copy of your clinical protocol (your IND Submission) A copy of the Investigator’s Brochure A copy of the Informed Consent Document Curricula vitae (2 pages) for each key professional in biographical sketch format The proposed location for vector production and description of the Good Manufacturing or Good Clinical Practices that will be utilized to prepare the vector A copy of the Certificate of Analysis (CoA) for sterility for each lot of vector made at GRU or sent to the University for this experiment
Additional responsibilities of the Principal Investigator conducting a rDNA experiment are detailed in Section IV-B- 7, Roles and Responsibilities of the NIH Guidelines. The full set of PI responsibilities can be accessed at http://www4.od.nih.gov/oba/sect4.htm
Adverse Events All adverse events must be reported in an annual data summary that is prepared for the Yale IRB and the IBC, the FDA, the NIH Office of Biotechnology Activities, and your sponsor. Any Serious Adverse Events (SAE’s) must be reported by telephone within 24 hours followed by a written report within 10 days. This report must be on file with the GRU IRB and IBC, the NIH OBA, the FDA, and NIH Office for Protection from Research Risks if applicable within 15 days. Please note that SAE’s must be reported whether related to the protocol or not. SAE’s shall not be designated as confidential, either in whole or in part, and the SAE reports shall be stripped of patient identifiers, such as name, address, contact information, social security numbers, and date of birth. If the SAE occurs after the trial and deemed related to the HGT trial, it must be reported within 15 days of the date of determination.
The NIH OBA reporting form can be downloaded from their website at http://www.nih.gov/od/oba.
GRU Institutional Review Board (IRB)--Human Assurance Committee (HAC) or Chesapeake Research Review, Inc (CCRI) An GRU-authorized IRB must approve all experiments involving human subjects prior to initiation. Please contact the Office of Human Research Protection (OHRP) office at x1-1478 for information on their requirements.
Appendix G Sources of Contamination If contamination is experienced in the laboratory, the following items may be sources of the contamination. For additional assistance please contact the Biosafety Office at x1-2663 or [email protected].
Personal items, such as coats, hats, storm boots or overshoes, umbrellas, purses, etc., do not belong in the laboratory. These articles should be stored elsewhere. Nonspecific contamination by environmental organisms from humans, animals, equipment, containers for specimens or supplies, and outside air is a complication that may affect or invalidate the results of an experiment. Human sources of this type of contamination are evaluated as follows: Sneezing, coughing and talking. Sneezing, variously reported to generate as many as 32,000 or 1,000,000 droplets below 100 microns in diameter; coughing, which produces fewer and larger droplets; and talking, which has been reported to average only 250 droplets when speaking 100 words, show great differences between persons in regard to the number of microorganisms aerosolized. As a general rule, it may be said that these actions by normal healthy persons may play a less important role in transmission of airborne infection to humans or experimental materials than does liberation of microorganisms from human skin. Dispersal of bacteria from human skin. There is a tremendous variation in the number of bacteria shed from the skin by a clothed subject. For instance, in one study, the number varied from 6,000 to 60,000 per minute. These bacteria were released on skin scales of a size that could penetrate the coarse fabric used for the laboratory and surgical clothing in the test. Dispersal of skin bacteria was several times greater from the area below the waist than from upper parts of the body. Effective reduction is accomplished by use of closely-woven or impervious clothing fitted tightly at the neck, wrists, and ankles to prevent the clothing from acting as bellows that disperses air carrying skin scales laden with bacteria. Such clothing sometimes is too warm to work in. The purpose of this summary is to alert laboratory personnel to the existence of this source of contamination. Prolific dispersal of bacteria occurs from infected abrasions, small pustules, boils, and skin disease. Washing of lesions with germicidal soap will greatly decrease the number of organisms on the skin and dispersal into the air. Healthy nasal carriers who generate aerosolized staphylococci usually can be identified by the presence of heavy contamination of their fingers, face, and hair. This point may be useful in investigating the source of staphylococcal contamination of cell lines. Footwear. In moderate and high-risk situations, shoes reserved only for laboratory use have been recommended as a precaution against transporting spilled infectious agents outside the laboratory. In experiments during which reduction of potential contamination of experimental materials is important, laboratory-only shoes can also reduce the microbial load brought into the laboratory each day by street shoes. Shoes are efficient transporters. In one study, there were 4 to 850 times as many bacteria per square centimeter on the laboratory footwear as on the floor itself.
Personal Work Practices Food, candy, gum, and beverages for human consumption will be stored and consumed only outside the laboratory. Smoking is not permitted in the laboratory, GRU buildings or on GRU campus. Shaving and brushing of teeth are not permitted in the laboratory. Razors, toothbrushes, toiletry supplies and cosmetics are permissible only in clean areas, and should never be used until after showering or thorough washing of the face and hands. A beard may be undesirable in the laboratory in the presence of actual or potential airborne contamination, because it retains particulate contamination more persistently than clean-shaven skin. A clean-shaven face is essential to the adequate facial fit of a facemask or respirator when the work requires respiratory protection. Develop the habit of keeping hands away from mouth, nose, eyes, face and hair. This may prevent self-inoculation. For product protection, person with long hair should wear a suitable hair net or head cover that can be decontaminated. This has long been a requirement in hospital operating rooms and in facilities where biological pharmaceutical products are manufactured. A head cover also will protect the hair from fluids, splashes, from swinging into Bunsen burner flames and Petri dishes, as well as reduce facial contamination caused by habitual repetitive manual adjustment of the hair. Long flowing hair and loose flapping clothing are dangerous in the presence of open flame or moving machinery. Rings and wristwatches also are a mechanical hazard during operation of some types of machines. Contact lenses do not provide eye protection. The capillary space between the contact lenses and the cornea may trap any material present on the surface of the eye. Caustic chemicals trapped in this space cannot be washed off the surface of the cornea. If the material in the eye is painful or the contact lens is displaced, muscle spasms will make it very difficult, if not impossible, to remove the lens. For this reason, contact lenses must not be worn by persons exposed to caustic chemicals unless safety glasses with side shields, goggles or full face shield are worn to provide full protection. Plants, cut flowers, an aquarium, and pets of any kind are undesirable sources of yeast, molds and other potential microbial contaminants of biological experimental materials. Books and journals returnable to the institutional library should be used only in the clean areas as much as possible. When change rooms with showers are provided, the employer should furnish skin lotion. When employees are subject to potential occupational infection, the shower and/ or face/handwashing facilities should be provided with germicidal soap. Personal cloth handkerchiefs should not be used in the laboratory. Disposable cleansing tissues should be available for use instead. Hand washing for personal protection: • This should be done promptly after removing protective gloves. Tests show it is not unusual for microbial or chemical contamination to be present despite use of gloves, due to unrecognized small holes, abrasions, tears, or entry at the wrist. • Throughout the day, at intervals dictated by the nature of the work, the hands should be washed. Presence of a wristwatch discourages adequate washing of the wrist. • Hands should be washed after removing soiled protective clothing, before leaving the laboratory area, before eating and smoking. The provision of hand cream by the employer encourages this practice. • A disinfectant wash or dip may be desirable in some cases, but its use must not be carried to the point of causing roughening, desiccation or sensitization of the skin. Anyone with a fresh or healing cut, abrasion, or skin lesion should not work with infectious materials unless the injured area is completely protected, such as with waterproof bandages and double gloving. Persons vaccinated for smallpox may shed vaccinia virus during the phase of cutaneous reaction. Therefore, vaccination requires permission of the appropriate supervisor, because two weeks absence may be necessary before returning to work with normal cell cultures or with susceptible animals, such as the normal mouse colony. Use of surgeon’s mask of gauze or filter paper is of little value for personal respiratory protection. It is designed to prevent escape of droplets from the nose or mouth. If use of biohazards demands respiratory protection, contact the Biosafety Office(x1-2663 or [email protected]) for assistance. Appendix J Biosafety Notice for Decontamination of Equipment
BIOSAFETY NOTICE
The exterior and interior of this piece of equipment were decontaminated and are free of any Biological Hazards. This notice does not apply to radiation or chemical hazards (if any).
This equipment is released for (please circle one):
Service/Repair Relocation Discard/Surplus
Decontamination performed by:
Chemcial or disinfection used:
Date of decontamination:
Location of equipment:
Laboratory/clinic telephone number:
Note: The following areas indicated below on this equipment may still be contaminated with biological hazards and a biohazard warning label has been attached near the contaminated area(s):
Georgia Regents University Biosafety Guide-June 2008 J-1
Georgia Regents University Biosafety Guide-June 2008 J-2 Appendix K Shipping Labels
All of the shipping labels on the following pages should be printed clearly in full size on white paper (except the Cargo Aircraft Only label, which should be printed on orange paper background). Any color in the labels is required.
Georgia Regents University Biosafety Guide-June 2008 K-1
Appendix L Map of GRU Campus
Georgia Regents University Biosafety Guide-June 2008 L-1 ACKNOWLEDGMENTS
This Biosafety Guide was compiled based on material from multiple resources, which not have been appropriately attributed in the body of the Guide. Therefore, the GRU Division of EH&S wishes to acknowledge these valuable resources, and thank those who developed them, but in particular:
• Yale University Biosafety Manual http://www.yale.edu/oehs/Documents/Bio/Biosafety%20Manual.pdf
• Andy Glode and David Gillum, Universtity of New Hampshire UNH Shipping of Biological Materials Manual (2006) http://www.unh.edu/ehs/pdf/UNH-Shipping-Biological-Materials.pdf UNH Guide to Shipping with Dry Ice (2007) http://www.unh.edu/ehs/pdf/Guide-to-Shipping-with-Dry-Ice.pdf
• Stanford University Lab Animal Safety Data Sheets http://www.stanford.edu/dept/EHS/prod/asds/
• Iowa State University Center for Food Security and Public Health Disinfectant Tables http://www.cfsph.iastate.edu/BRM/resources/Disinfectants/AntimicrobialSpectr umDisinfectants1207.pdf http://www.cfsph.iastate.edu/BRM/resources/Disinfectants/CharacteristicsSelect edDisinfectants.pdf
• University of Virginia Policies Policy on the Use of Human Cell Lines for Laboratory Personnel https://vprgsecure.web.virginia.edu/bio/uva_human_cell_line_policy.cfm
Vaccinia Virus Use & Immunization Policy for Laboratory Personnel https://vprgsecure.web.virginia.edu/bio/vaccinia.cfm
• Georgia State University Biosafety Manual http://www.ovpr.gsu.edu/biosafety/files/GSU%20Biosafety%20Manual_April04.pdf
• The American Biological Safety Association (ABSA) Principles and Practices Training Materials
Material in this Guide has also been adapted from information derived from the following resources:
Governmental Resources: • The CDC/NIH Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th edition (2007) http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm • The NIH Guidelines for Research with Recombinant DNA Materials (2002) http://www4.od.nih.gov/oba/rac/guidelines/guidelines.html • The NIH Stem Cell Information Resource http://stemcells.nih.gov/
Georgia Regents University Biosafety Guide-June 2008 Acknowledgments • OSHA Blood-borne Pathogens (29 CFR 1910.1030) Standards http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_table=STANDARDS&p_ id=10051 • OSHA Respiratory Protection (29 CFR 1910.134) Standards http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_id=12716&p_table=STA NDARDS • National Institutes of Occupational Safety and Health (NIOSH) Resources on Respirators http://www.cdc.gov/niosh/npptl/topics/respirators/ • National Institutes of Occupational Safety and Health (NIOSH) Resourceses on Latex Allergies: http://www.cdc.gov/Niosh/latexalt.html • Environmental Protection Agency (EPA) Mold References: http://www.epa.gov/mold/
Other GRU Compliance and Service Offices: • Office of Human Research Protection: http://www.gru.edu/research/ohrp/ • Division of Laboratory Animal Services: http://www.gru.edu/research/animal/ • Institutional Animal Care and Use Committee: http://www.gru.edu/research/animal/IACUCHomePage.htm • Office of Institutional Audits and Compliance: http://www.gru.edu/audits/ • Compliance Oversight Council: http://www.gru.edu/audits/coc.htm • Office of Technology Transfer and Economic Development: http://www.gru.edu/research/techtransfer/
Georgia Regents University Biosafety Guide-June 2008 Acknowledgments