Biosafety Manual PDF - Lawrence Berkeley National Laboratory
Biosafety Manual PDF - Lawrence Berkeley National Laboratory
Biosafety Manual PDF - Lawrence Berkeley National Laboratory
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<strong>Biosafety</strong> <strong>Manual</strong><br />
<strong>Lawrence</strong> <strong>Berkeley</strong> <strong>National</strong> <strong>Laboratory</strong><br />
May 18, 2010<br />
This work was supported by the U.S. Department of Energy under Contract No. DE-AC02-<br />
05CH11231.<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.
<strong>Biosafety</strong> <strong>Manual</strong><br />
IBC-approved version (May 18, 2010)<br />
<strong>Biosafety</strong> <strong>Manual</strong><br />
Contents<br />
1.0 Introduction<br />
1.1 Policy<br />
1.2 Purpose<br />
1.3 Scope<br />
1.4 <strong>Manual</strong> Administration and Presentation<br />
1.5 Terms, Acronyms, and Abbreviations<br />
1.6 Roles, Responsibilities, and Whom to Call<br />
2.0 Starting and Conducting Work Safely<br />
3.0 Work and Risk Assessment<br />
3.1 LBNL Assessment and Authorization Processes<br />
3.2 <strong>Biosafety</strong> Risk Assessment Process<br />
3.3 Material or Agent Hazards and Requirements<br />
3.1.1 Risk Group Classification<br />
3.3.2 Pathogenic Agents and Toxins<br />
3.3.2.1 Pathogen and Toxin Information and Guidance<br />
3.3.2.2 Human Pathogens<br />
3.3.2.3 Plant and Animal Pathogens<br />
3.3.2.4 Biological Toxins<br />
3.3.2.5 Select Agents and Toxins<br />
3.3.2.6 Prions<br />
3.3.3 USDA-Regulated Materials, Organisms, and Agents<br />
3.3.4 Bloodborne Pathogens and Human Materials<br />
3.3.5 Recombinant Materials, Organisms, and Agents<br />
3.3.6 Animals<br />
3.4 <strong>Laboratory</strong> Procedure Hazards<br />
3.5 Worker Competence and Health<br />
4.0 <strong>Biosafety</strong> Principles and Levels<br />
4.1 <strong>Laboratory</strong> Practices<br />
4.2 Safety and Personal Protective Equipment<br />
4.3 Facility Design and Construction<br />
4.4 <strong>Biosafety</strong> Containment Levels and Criteria<br />
4.4.1 <strong>Laboratory</strong> Containment Levels<br />
4.4.1.1 <strong>Laboratory</strong> <strong>Biosafety</strong> Level 1<br />
4.4.1.2 <strong>Laboratory</strong> <strong>Biosafety</strong> Level 2<br />
4.4.2 Additional Containment Categories<br />
4.4.2.1 Recombinant Large-Scale Containment Levels<br />
4.4.2.2 Recombinant Plant Containment Levels<br />
4.4.2.3 Vertebrate Animal Containment Levels<br />
4.4.2.4 Arthropod Containment Levels<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.<br />
2
<strong>Biosafety</strong> <strong>Manual</strong><br />
IBC-approved version (May 18, 2010)<br />
5.0 Specific <strong>Biosafety</strong> Controls<br />
5.1 Work Authorizations<br />
5.2 Training, Instruction, and Qualification<br />
5.2.1 Job Hazards Analysis<br />
5.2.2 Training Courses and Tracking<br />
5.2.3 Job-Specific Instruction, Information, and Practices<br />
5.3 Occupational Health and Immunization<br />
5.4 Personal Protective Clothing and Equipment<br />
5.4.1 Body Protection<br />
5.4.2 Eye and Face Protection<br />
5.4.3 Hand Protection<br />
5.4.4 Foot Protection<br />
5.4.5 Respiratory Protection, Respirators, and Face Masks<br />
5.5 Labels and Signs<br />
5.6 Facilities, <strong>Laboratory</strong> Equipment, and Related Practices<br />
5.6.1 Cleanable Surfaces and Furnishings<br />
5.6.2 Doors and Windows<br />
5.6.3 Plumbing Systems and Equipment<br />
5.6.3.1 Sinks and Handwashing<br />
5.6.3.2 Drains and Disposal<br />
5.6.3.3 Water Systems and Backflow Prevention<br />
5.6.3.4 Emergency Eyewashes and Showers<br />
5.6.4 Ventilation and Hoods<br />
5.6.4.1 Room Ventilation<br />
5.6.4.2 Hoods and <strong>Biosafety</strong> Cabinets<br />
5.6.5 Food Facilities and Eating<br />
5.6.6 <strong>Laboratory</strong> Tools and Equipment<br />
5.6.6.1 Sharps<br />
5.6.6.2 Centrifuges<br />
5.6.6.3 Waste Containers<br />
5.6.6.4 Equipment Connected to Building Vacuum Systems<br />
5.7 Decontamination, Waste, and Decommissioning<br />
5.7.1 Decontamination Processes and Antimicrobials<br />
5.7.2 Surface and Equipment Decontamination<br />
5.7.3 Waste Decontamination and Disposal<br />
5.7.4 <strong>Laboratory</strong> and Equipment Decommissioning and Moves<br />
5.8 Access and Security<br />
5.9 Pest Management<br />
5.10 Incident, Accident, and Emergency Response<br />
5.10.1 General Incident Response and Reporting<br />
5.10.2 Worker Exposure, Injury, or Illness<br />
5.10.3 Biological Spills and Cleanup<br />
5.10.4 Additional <strong>Biosafety</strong> Incident Reporting<br />
5.11 Procurement, Transportation, and Transfer<br />
5.11.1 Procurement<br />
5.11.2 Transportation and Shipping<br />
5.11.3 Import, Export, and Transfer Restrictions<br />
6.0 Assessment and Improvement<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.<br />
3
<strong>Biosafety</strong> <strong>Manual</strong><br />
IBC-approved version (May 18, 2010)<br />
7.0 Standards, Policies, References, and Resources<br />
7.1 Standards<br />
7.2 Policies<br />
7.2.1 Health and Safety <strong>Manual</strong> (PUB-3000) Chapters<br />
7.2.2 Other <strong>Biosafety</strong>-related LBNL Publications<br />
7.3 References<br />
7.4 Resources<br />
Appendix A Glossary<br />
Appendices<br />
Appendix B Pathogen and Toxin Lists<br />
B.1 Introduction and Scope<br />
B.2 NIH Guidelines Human Etiologic Agents<br />
B.2.1 Risk Group 1 Agents<br />
B.2.2 Risk Group 2 Agents<br />
B.2.3 Risk Group 3 Agents<br />
B.2.4 Risk Group 4 Agents<br />
B.2.5 Animal Viral Etiologic Agents in Common Use<br />
B.2.6 Murine Retroviral Vectors<br />
B.3 Select Agents and Toxins<br />
B.4 Plant Pathogens<br />
B.4.1 Plant Pathogen Bacteria (by Scientific Name)<br />
B.4.2 Plant Pathogen Fungi (by Scientific Name)<br />
B.4.3 Plant Pathogen Viruses (Regulated by the State of<br />
California)<br />
Appendix C <strong>Laboratory</strong> <strong>Biosafety</strong> Level 1 and 2 Criteria<br />
C.1 Introduction and Scope<br />
C.2 <strong>Laboratory</strong> <strong>Biosafety</strong> Level 1<br />
C.2.1 BL1 Standard Microbiological Practices<br />
C.2.2 BL1 Special Practices<br />
C.2.3 BL1 Safety Equipment (Primary Barriers and Personal<br />
Protective Equipment)<br />
C.2.4 BL1 <strong>Laboratory</strong> Facilities (Secondary Barriers)<br />
C.3 <strong>Laboratory</strong> <strong>Biosafety</strong> Level 2<br />
C.3.1 BL2 Standard Microbiological Practices<br />
C.3.2 BL2 Special Practices<br />
C.3.3 BL2 Safety Equipment (Primary Barriers and Personal<br />
Protective Equipment33)<br />
C.3.4 BL2 <strong>Laboratory</strong> Facilities (Secondary Barriers)<br />
Appendix D Good Microbiological Practice<br />
D.1 Introduction and Scope<br />
D.2 Good Microbiological Practice<br />
D.2.1 Aseptic Technique<br />
D.2.2 Personal Hygiene and Dress<br />
D.2.3 Area Cleanliness and Organization<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.<br />
4
<strong>Biosafety</strong> <strong>Manual</strong><br />
IBC-approved version (May 18, 2010)<br />
D.2.4 <strong>Biosafety</strong> Cabinets and Airborne Contamination<br />
D.2.5 Manipulation Techniques for Minimizing Aerosols<br />
D.2.6 Worker Qualifications<br />
D.2.7 Microbial Contamination Checks<br />
D.3 References<br />
Appendix E<br />
Appendix F<br />
<strong>Biosafety</strong> Cabinets<br />
E.1 Introduction and Scope<br />
E.2 <strong>Biosafety</strong> Cabinet Classifications<br />
E.2.1 Class I <strong>Biosafety</strong> Cabinet<br />
E.2.2 Class II <strong>Biosafety</strong> Cabinet<br />
E.2.3 Class III <strong>Biosafety</strong> Cabinet<br />
E.2.4 Clean Benches (Not BSCs)<br />
E.3 <strong>Biosafety</strong> Cabinet Work Practices and Procedures<br />
E.3.1 Preparing for BSC Work<br />
E.3.2 Material Placement inside the BSC<br />
E.3.3 Operations within a Class II BSC<br />
E.4 <strong>Biosafety</strong> Cabinet Decontamination and Moves<br />
E.4.1 Cabinet Surface Decontamination<br />
E.4.2 Internal Cabinet Gaseous Decontamination<br />
E.5 <strong>Biosafety</strong> Cabinet Installation and Engineering<br />
E.6 <strong>Biosafety</strong> Cabinet Testing and Certification<br />
Decontamination and Antimicrobials<br />
F.1 Introduction and Scope<br />
F.2 Decontamination Principles and Terms<br />
F.2.1 Decontamination Processes and Levels<br />
F.2.1.1 Sterilization<br />
F.2.1.2 Disinfection<br />
F.2.1.3 Sanitization<br />
F.2.1.4 Antisepsis<br />
F.2.2 Antimicrobial Categories<br />
F.2.3 Antimicrobial Selection and Registered Disinfectants<br />
F.3 Chemical Antimicrobials<br />
F.3.1 Surfactants (Soaps and Detergents)<br />
F.3.1.1 Soaps<br />
F.3.1.2 Detergents and Quaternary Ammonium<br />
Compounds<br />
F.3.2 Halogens (Chlorine and Iodine)<br />
F.3.2.1 Chlorine and Sodium Hypochlorite<br />
F.3.2.2 Iodine and Iodophors<br />
F.3.3 Alcohols<br />
F.3.4 Phenol and Phenol Derivatives (Phenolics)<br />
F.3.5 Oxidizing Agents (Hydrogen Peroxide)<br />
F.3.6 Alkylating Agents (Formaldehyde, Glutaraldehyde,<br />
Ethylene Oxide)<br />
F.3.6.1 Formaldehyde<br />
F.3.6.2 Glutaraldehyde<br />
F.3.6.3 Ethylene Oxide<br />
F.4 Physical Antimicrobials<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.<br />
5
<strong>Biosafety</strong> <strong>Manual</strong><br />
IBC-approved version (May 18, 2010)<br />
F.4.1 Heat<br />
F.4.1.1 Dry Heat (Baking and Incineration)<br />
F.4.1.2 Wet Heat (Boiling and Autoclaving)<br />
F.4.2 Ultraviolet (UV) Radiation<br />
F.4.2.1 UV Light Health Effects and Categories<br />
F.4.2.2 <strong>Biosafety</strong> Cabinet UV Light<br />
F.4.3 Ionizing Radiation<br />
F.4.4 Visible Light<br />
F.4.5 Filtration (HEPA Filters)<br />
F.5 Autoclave Sterilization and Safety<br />
F.5.1 Autoclaves and Sterilization<br />
F.5.2 Autoclave Operation and Safety<br />
F.5.2.1 Autoclave Instruction<br />
F.5.2.2 Autoclave Operation Modes<br />
F.5.2.3 Autoclave Container Selection<br />
F.5.2.4 Autoclave Preparation and Loading<br />
F.5.2.5 Autoclave Cycle and Time Selection<br />
F.5.2.6 Removing Autoclave Loads<br />
F.5.2.7 Autoclave Material Staging<br />
F.5.2.8 Burn Emergencies<br />
F.5.3 Autoclave Maintenance and Monitoring<br />
F.6 References<br />
Appendix G Biological Spills and Cleanup<br />
G.1 Introduction and Scope<br />
G.2 Precleanup Considerations<br />
G.3 Biological Spill outside a <strong>Biosafety</strong> Cabinet<br />
G.4 Biohazardous Spill inside a <strong>Biosafety</strong> Cabinet<br />
G.5 Centrifuge Malfunction or Spill<br />
G.6 Radioactive and Biohazardous Spill<br />
G.7 Chemical and Biohazardous Spill<br />
G.8 Cleanup of Small Dead Animals, Nests, or Droppings<br />
Appendix H Transportation and Shipping<br />
H.1 Introduction and Scope<br />
H.2 How to Determine Transportation Mode and Requirements<br />
H.3 Requirements and Processes for Receiving, Transporting, and Shipping<br />
H.3.1 Employee Transportation of Materials<br />
H.3.1.1 Hand-Carry Transport between Laboratories<br />
H.3.1.2 Hand-Carry Transport between Buildings<br />
H.3.1.3 Personal Transportation in a Motor Vehicle<br />
H.3.1.4 Personal Transportation on an LBNL Bus<br />
H.3.2 LBNL Receiving, Transportation, and Shipping<br />
H.3.2.1 LBNL Receiving<br />
H.3.2.2 LBNL Transportation<br />
H.3.2.3 LBNL and Common Carrier Shipping<br />
H.4 Unregulated and Regulated Materials<br />
H.4.1 Unregulated Biological Materials<br />
H.4.2 Regulated Biological Materials<br />
H.5 References and Resources<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.<br />
6
<strong>Biosafety</strong> <strong>Manual</strong><br />
IBC-approved version (May 18, 2010)<br />
Appendix I<br />
Import, Export, and Transfer Restrictions<br />
I.1 Introduction and Scope<br />
I.2 Importing or Transfer into the U.S. and California<br />
I.2.1<br />
I.2.2<br />
CDC and APHIS Select Agent and Toxin Restrictions<br />
APHIS Agricultural Permits<br />
I.2.2.1 APHIS Plant Health Permits<br />
I.2.2.2 APHIS Animal Health Permits<br />
I.2.2.3 APHIS Genetically Engineered Organisms Permits<br />
I.2.3 CDC Agents or Vectors of Human Disease Permits<br />
I.2.4 Food and Drug Administration Import Program<br />
I.2.5 Fish and Wildlife Service Permits<br />
I.3 Exporting or Transfer from the U.S.<br />
I.3.1 Commerce Control List<br />
I.3.2 U.S. Munitions List<br />
I.3.3 Biological Weapons Convention Lists<br />
I.4 References<br />
Tables<br />
Table 1 Guidance for Starting and Conducting Work<br />
Table 2 Risk Group Classification<br />
Table 3 Pathogenic Agent and Toxin Categories<br />
Table 4 Materials Regulated by USDA-APHIS<br />
Table 5 Materials Covered by the OSHA Bloodborne Pathogens Standard<br />
Table 6 Examples of Zoonotic and Other Diseases Related to Animals<br />
Table 7 Equipment Hazard Examples<br />
Table 8 <strong>Laboratory</strong> <strong>Biosafety</strong> Containment Levels<br />
Table 9 Additional Containment Categories<br />
Table 10 <strong>Biosafety</strong>-Related Training Courses<br />
Table 11 Decontamination Processes, Levels, and Antimicrobial Examples<br />
Table B-1 <strong>National</strong> Select Agent Registry Select Agents and Toxins<br />
Table B-2 Additional Information for <strong>National</strong> Select Agent Registry Toxins<br />
Table E-1 Protection Offered by Classes of <strong>Biosafety</strong> Cabinets<br />
Table E-2 Characteristics of <strong>Biosafety</strong> Cabinet Classes<br />
Table F-1 Levels of Chemical Disinfection<br />
Table F-2 Activity Levels of Selected Liquid Germicides<br />
Table F-3 Descending Order of Organism Resistance to Germicidal Chemicals<br />
Table H-1 Transportation Modes and Biological Materials Not Allowed<br />
Table I-1 Wildlife or Plant Specimens That May Require an FWS Permit<br />
Table I-2 Commerce Control List of Biological Agents<br />
Table I-3 BWC Agents Not On the Commerce Control List<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.<br />
7
<strong>Biosafety</strong> <strong>Manual</strong><br />
IBC-approved version (May 18, 2010)<br />
1.0 Introduction<br />
1.1 Policy<br />
Work with or potential exposure to biological materials in the course of performing research or<br />
other work activities at <strong>Lawrence</strong> <strong>Berkeley</strong> <strong>National</strong> <strong>Laboratory</strong> (LBNL) must be conducted in<br />
a safe, ethical, environmentally sound, and compliant manner. Work must be conducted in<br />
accordance with established biosafety standards, the principles and functions of Integrated<br />
Safety Management (ISM), this <strong>Biosafety</strong> <strong>Manual</strong>, Chapter 26 (<strong>Biosafety</strong>) of the Health and<br />
Safety <strong>Manual</strong> (PUB-3000), and applicable standards and LBNL policies.<br />
1.2 Purpose<br />
The purpose of the <strong>Biosafety</strong> Program is to protect workers, the public, agriculture, and the<br />
environment from exposure to biological agents or materials that may cause disease or other<br />
detrimental effects in humans, animals, or plants. This manual provides workers; line<br />
management; Environment, Health, and Safety (EH&S) Division staff; Institutional <strong>Biosafety</strong><br />
Committee (IBC) members; and others with a comprehensive overview of biosafety principles,<br />
requirements from biosafety standards, and measures needed to control biological risks in work<br />
activities and facilities at LBNL.<br />
1.3 Scope<br />
This <strong>Biosafety</strong> <strong>Manual</strong> and the <strong>Biosafety</strong> Program apply to biosafety issues related to worker<br />
safety, public health, agricultural protection, and environmental protection for work activities at<br />
locations where LBNL has Environment, Safety, and Health (ES&H) management<br />
responsibilities. The work must also include:<br />
• Biological materials, agents, and other materials of biological origin (e.g., organisms,<br />
cells, viruses, and toxins) that pose different levels of risk to humans, animals, or plants<br />
when stored or used; or<br />
• Workers who may be exposed to disease-causing biological agents related to<br />
designated job duties (e.g., bloodborne pathogens in health care).<br />
The <strong>Biosafety</strong> <strong>Manual</strong> addresses:<br />
• <strong>Biosafety</strong> risk assessment and containment controls.<br />
• Principles, programmatic elements, and controls required by ISM and the biosafety<br />
standards outlined in PUB-3000, Section 26.5.<br />
1.4 <strong>Manual</strong> Administration and Presentation<br />
The <strong>Biosafety</strong> <strong>Manual</strong> was written by the EH&S Division, and approved by the IBC and the<br />
EH&S Division <strong>Biosafety</strong> Officer. The EH&S Division Director provides management approval.<br />
The <strong>Biosafety</strong> <strong>Manual</strong> is presented in a manner that:<br />
• Further describes requirements outlined in PUB-3000, Chapter 26 (<strong>Biosafety</strong>).<br />
• Integrates similar requirements by topic from different biosafety standards and LBNL<br />
policies, and explains when certain requirements are applicable to specific work.<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.<br />
8
<strong>Biosafety</strong> <strong>Manual</strong><br />
IBC-approved version (May 18, 2010)<br />
• Is similar to the structure of <strong>Biosafety</strong> in Microbiological and Biomedical<br />
Laboratories (BMBL, fifth edition).<br />
• Links to LBNL policies and biosafety regulations, standards, and guidelines available<br />
online.<br />
1.5 Terms, Acronyms, and Abbreviations<br />
Terms, acronyms, and abbreviations used in this manual are defined in the Glossary (Appendix<br />
A).<br />
Statements used in this manual that describe needed conditions commonly use the terms<br />
“must” and “should.” In addition, sections of the manual sometimes begin by stating that the<br />
conditions presented in the section are “guidelines.” These terms are defined below:<br />
• Must means the condition is required. Requirements are derived from LBNL ES&H<br />
standards or LBNL policies.<br />
• Should means there is an expectation that the condition will be met unless there is an<br />
equally forceful reason for not meeting the condition, and an alternative approach that<br />
does not conflict with other requirements and accomplishes the same safety objective.<br />
When the term “should” is used in guidelines, the condition is a best-management<br />
practice, and the condition or safe alternatives will be implemented when needed to<br />
control apparent risk.<br />
• Guidelines are a set of nonmandatory but desirable criteria, conditions, or bestmanagement<br />
practices that should typically be considered when determining controls<br />
needed to mitigate risk.<br />
When determining if a needed condition must or should be implemented, or is recommended,<br />
the reader of this manual should read all parts of the statement or section and use term<br />
definitions (see Appendix A, Glossary) as needed to determine the applicability of the condition<br />
to the work to be conducted.<br />
1.6 Roles, Responsibilities, and Whom to Call<br />
<strong>Biosafety</strong>-related roles and responsibilities are covered in Section 26.4 and Appendix B of PUB-<br />
3000, Chapter 26 (<strong>Biosafety</strong>).<br />
The <strong>Biosafety</strong> Officer in the EH&S Industrial Hygiene Group has primary oversight responsibility<br />
for this manual, but other groups and individuals also provide specific subject matter expertise,<br />
program management, or direction. The following groups or individuals may be contacted for<br />
additional information:<br />
• The Industrial Hygiene Group of the EH&S Division and the <strong>Biosafety</strong> Officer at<br />
(510) 495-2768 or (510) 486-7837<br />
• The Waste Management Group of the EH&S Division and the<br />
Medical/Biohazardous Waste Coordinator at (510) 486-7579<br />
• The Health Services Group of the EH&S Division at (510) 486-6266<br />
• The Training Group of the EH&S Division at (510) 495-2228<br />
• The EH&S Division Web site at (510) 486-5514<br />
• The Security and Emergency Operations Group of the EH&S Division at (510) 486-<br />
6234<br />
• Division Safety Coordinators<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.<br />
9
<strong>Biosafety</strong> <strong>Manual</strong><br />
IBC-approved version (May 18, 2010)<br />
2.0 Starting and Conducting Work Safely<br />
This section and Table 1 provide a simplified list to assist supervisors, work leads, and principal<br />
investigators in getting work with biological materials planned, assessed, authorized, and<br />
conducted. Specific sections of this manual and PUB-3000, Chapter 26 (<strong>Biosafety</strong>) should be<br />
consulted for additional information and requirements:<br />
Table 1<br />
Guidance for Starting and Conducting Work<br />
Guidance<br />
Work Operation Planning, Risk Assessment, Authorization, and<br />
Control<br />
Complete and maintain a <strong>Biosafety</strong> Work Authorization (i.e., Biological Use<br />
Authorization (BUA), Biological Use Registration (BUR), Biological Use<br />
Notification (BUN), or Exposure Control Plan (ECP)) and any other<br />
required authorization. The <strong>Biosafety</strong> Work Authorization documents the<br />
work, risks, and controls. Implement the controls listed in the authorization.<br />
• Complete the Biological Use Application Form for research with biological<br />
materials.<br />
o Submit the application form to the Environment, Health, and Safety<br />
(EH&S) <strong>Biosafety</strong> Office (BWKing@lbl.gov and VMXian@lbl.gov) for<br />
review by the Institutional <strong>Biosafety</strong> Committee (IBC) and/or <strong>Biosafety</strong><br />
Officer.<br />
o Resolve any review comments.<br />
o Work with the <strong>Biosafety</strong> Office to get the completed <strong>Biosafety</strong> Work<br />
Authorization signed and authorized. The <strong>Biosafety</strong> Office will load<br />
the authorization into the <strong>Biosafety</strong> Authorization System (BAS).<br />
o Ensure workers are familiar with the authorization document,<br />
understand the required controls, and are trained.<br />
o Ensure the containment controls noted in the authorization document<br />
are implemented. Also implement applicable controls noted in PUB-<br />
3000, Chapter 26 (<strong>Biosafety</strong>) and the <strong>Biosafety</strong> <strong>Manual</strong>. Standard<br />
<strong>Laboratory</strong> <strong>Biosafety</strong> Level (BL) 1 and BL2 criteria are summarized<br />
in Appendix C.<br />
o Update personnel, biosafety training, and work locations as needed in<br />
the BAS.<br />
o Update and submit for review the authorization document to the<br />
<strong>Biosafety</strong> Office prior to the target renewal date. Ensure authorization<br />
document is re-authorized as needed.<br />
LBNL Policy<br />
Section<br />
<strong>Biosafety</strong><br />
<strong>Manual</strong>,<br />
Section 5.1<br />
PUB-3000,<br />
Section<br />
26.8.2<br />
PUB-3000,<br />
Section<br />
26.8.2<br />
PUB-3000,<br />
Table 26-6<br />
<strong>Biosafety</strong><br />
<strong>Manual</strong>,<br />
Section 5.2<br />
<strong>Biosafety</strong><br />
<strong>Manual</strong>,<br />
Sections 4.0<br />
and 5.0<br />
PUB-3000,<br />
Table 26-6<br />
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10
<strong>Biosafety</strong> <strong>Manual</strong><br />
IBC-approved version (May 18, 2010)<br />
Guidance<br />
• Complete and submit an Exposure Control Plan for nonresearch work<br />
that involves exposure to bloodborne pathogen materials.<br />
• Complete and get approval on any required protocols for research<br />
involving vertebrate animals or human subjects (including human-derived<br />
data or human-derived tissues) via the Animal Welfare and Research<br />
Committee (AWRC) or Human Subjects Committee (HSC),<br />
respectively.<br />
Worker Authorization and Control<br />
Identify the work, hazards, and controls for each worker or subcontractor<br />
and ensure the controls are implemented. Ensure each worker completes:<br />
• A Job Hazards Analysis (JHA) or Subcontractor Job Hazards<br />
Analysis and Work Authorization (SJHAWA).<br />
• Required controls on their JHA or SJHAWA, including required training<br />
courses.<br />
• Applicable job and operation-specific instruction related to biosafety.<br />
• Any required medical surveillance noted on their JHA, SJHAWA, or work<br />
authorization document.<br />
Assessment and Improvement<br />
• Assess and continuously improve the biosafety of the work.<br />
• Conduct periodic biosafety assessments of the operation as specified in<br />
the Division Self-Assessment Program, including assessment of the<br />
safety of tasks being performed, safety of the work area and equipment,<br />
training, and compliance with the <strong>Biosafety</strong> Work Authorization and<br />
standards.<br />
• Participate in periodic <strong>Biosafety</strong> or other Environment, Safety, and Health<br />
(ES&H) Technical Assurance Program (TAP) assessments of the<br />
operation, when scheduled.<br />
• Continuously improve the biosafety of the work, including tracking and<br />
correcting deficiencies when required in the Corrective Action Tracking<br />
System (CATS).<br />
Whom to Call<br />
Refer to the Whom to Call list in Section 1.6.<br />
LBNL Policy<br />
Section<br />
PUB-3000,<br />
Section<br />
26.8.3<br />
PUB-3000,<br />
Chapter 22<br />
<strong>Biosafety</strong><br />
<strong>Manual</strong>,<br />
Section 5.2.1<br />
<strong>Biosafety</strong><br />
<strong>Manual</strong>,<br />
Section 5.2.2<br />
<strong>Biosafety</strong><br />
<strong>Manual</strong>,<br />
Section 5.2.3<br />
<strong>Biosafety</strong><br />
<strong>Manual</strong>,<br />
Section 5.3<br />
PUB-3000,<br />
Section 26.9<br />
PUB-3000,<br />
Section 26.9<br />
PUB-3000,<br />
Section 26.9<br />
PUB-3000,<br />
Section 26.9<br />
<strong>Biosafety</strong><br />
<strong>Manual</strong>,<br />
Section 1.6<br />
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Work with biological materials, like all work at LBNL, must be conducted using the guiding<br />
principles and five core functions of Integrated Safety Management (ISM) (e.g., define scope of<br />
work, analyze hazards, develop and implement controls, perform work within controls, feedback<br />
and continuous improvement) as discussed in PUB-3000, Section 1.4. These core functions are<br />
integrated into the work authorization and control functions summarized above in Table 1.<br />
3.0 Work and Risk Assessment<br />
The work scope must be defined and the hazards and risks must be assessed before work<br />
begins. These work-planning processes are the first two core ISM functions and required by<br />
biosafety standards. Biological work and risks at LBNL are defined using established<br />
institutional assessment and authorization processes, a structured approach as required by the<br />
Department of Energy (DOE), and the standard biosafety risk assessment process defined by<br />
the Centers for Disease Control and Prevention (CDC) and the <strong>National</strong> Institutes of<br />
Health (NIH). It is a primary responsibility of workers, work leads, and supervisors to ensure<br />
these processes are implemented before work begins.<br />
3.1 LBNL Assessment and Authorization Processes<br />
LBNL uses the following institutional assessment and authorization processes and documents<br />
to define work, identify biological hazards and potential exposures, assess biological risks, and<br />
establish biosafety controls:<br />
• A Job Hazards Analysis (JHA) is prepared for each worker (see PUB-3000, Chapter 32).<br />
• A Subcontractor Job Hazards Analysis and Work Authorization (SJHAWA) is prepared<br />
for each subcontractor, vendor, or guest (see Chapter 31).<br />
• <strong>Biosafety</strong> Work Authorizations are prepared for work with biological materials in specific<br />
operations or projects. In the case of research involving biological materials, the<br />
Institutional <strong>Biosafety</strong> Committee (IBC) reviews and approves the definition of work, risk<br />
assessment, and controls as part of the authorization process. See Section 5.1 below<br />
and PUB-3000, Section 26.8, for details.<br />
3.2 <strong>Biosafety</strong> Risk Assessment Process<br />
The institutional assessment and authorization processes and documents noted in Section 3.1<br />
above incorporate the standard biosafety risk assessment process defined and required by<br />
CDC, NIH, and DOE in the <strong>Biosafety</strong> in Microbiological and Biomedical Laboratories (BMBL),<br />
the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines),<br />
and the Worker Health and Safety Program (WSHP).<br />
The standard biosafety risk assessment process starts with considering three primary factors: 1)<br />
the inherent work hazard posed by the biological material or agent, 2) the susceptible hosts (i.e.,<br />
receptors) that may be affected by the material or agent, and 3) the exposure pathways<br />
between the threat hazard and the susceptible host.<br />
In addition, BMBL outlines the following five-step approach for laboratory supervisors and work<br />
leads to assess biological risk and to select controls for laboratory work:<br />
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1. Identify material or agent hazards, and perform an initial risk assessment.<br />
2. Identify laboratory procedure hazards.<br />
3. Make a final determination of the appropriate biosafety containment level, and select<br />
additional controls indicated by the risk assessment.<br />
4. Evaluate a worker’s proficiency in safe work practices, and ensure the integrity of safety<br />
equipment.<br />
5. Review the risk assessment with the biosafety professional, subject matter expert, and<br />
the IBC.<br />
The remaining sections of Section 3.0 below present in greater detail the key factors underlined<br />
above that must be considered when conducting risk assessments and selecting controls.<br />
Primary factors include material or agent hazards (perceived or real) and procedure hazards.<br />
Secondary factors include staff proficiencies and other personal factors.<br />
See Section II of BMBL for more information on biological risk assessment.<br />
3.3 Material or Agent Hazards and Requirements<br />
The material or agent hazard(s) and associated requirements must be considered at the start of<br />
the risk assessment. Terms used to describe biological materials must also be defined and<br />
understood before a risk assessment takes place. This is because these terms often have<br />
specific meanings, associated requirements, and associated lists (see below):<br />
The term biological materials is used in this manual, PUB-3000 (Chapter 26), and the risk<br />
assessment process to describe a broad range of organisms, cells, viruses, and other materials<br />
of biological origin that pose differing levels of risks to plants, animals, or humans.<br />
The term biological agent or agent is used to describe a specific biological organism or<br />
material that is often directly responsible for producing an effect (e.g., disease). Examples of<br />
biological agents include a microorganism (e.g., bacterium, fungus, or parasite), virus, prion, or<br />
biological toxin. For example, humans are composed of tissues that contain blood; the blood<br />
contains fluids and cells; and the blood may contain the viral pathogen hepatitis B. Although the<br />
human body, tissues, blood, cells, fluids, and pathogens are all biological materials, only the<br />
hepatitis B virus is a biological agent.<br />
In addition, the risk assessment should consider the state or treatment of the biological material<br />
that may change or eliminate the hazardous characteristics of the material, and this information<br />
should be included in the <strong>Biosafety</strong> Work Authorization when the information significantly<br />
describes the safety aspects of the work. For example, biohazardous characteristics of a<br />
biological material may not be present if the material is in a nonviable, fixed, inactive, or<br />
decontaminated state. These terms are listed below along with simplified definitions and<br />
examples:<br />
• Nonviable means the material or agent is not capable of living or developing under<br />
favorable conditions. Examples include sections of plant or animal tissue that are often<br />
not capable of propagating, and extracts of biological samples such as DNA or RNA that<br />
cannot replicate without cells. These materials may not pose risks as long as there is no<br />
potential for the presence of pathogens.<br />
• Fixed means the material has been treated so that it has been stabilized and preserved<br />
in place. For example, properly fixing cells with paraformaldehyde or glutaraldehyde<br />
typically kills the cells and most potential pathogens.<br />
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• Inactive means the material is not capable of acting or reacting normally. For example,<br />
infectious proteins (i.e., prions) may be inactivated by chemical destruction.<br />
• Decontaminated means the material has been treated (e.g., sterilized or disinfected) so<br />
that biological contaminants or components have been reduced or inactivated to an<br />
acceptable level to reduce or eliminate the possibility of transmission of pathogens to<br />
undesired hosts. For example, fresh human bones may be decontaminated internally by<br />
radiation.<br />
Biological material and agent hazards are further covered in Section 3.3 as follows:<br />
• The risk group (RG) classification system used to categorize agents and materials<br />
based on the risk of disease in humans (see Section 3.3.1 below)<br />
• Biological risks and concerns related to the following categories of biological materials<br />
and agents:<br />
o Pathogenic agents and toxins (see Section 3.3.2 below)<br />
o U.S. Department of Agriculture (USDA)-regulated materials, organisms, and<br />
agents (see Section 3.3.3 below)<br />
o Bloodborne pathogens and human materials (see Section 3.3.4 below)<br />
o Recombinant materials, organisms, and agents (see Section 3.3.5 below)<br />
o Animals (see Section 3.3.6 below)<br />
3.3.1 Risk Group Classification<br />
The principal hazardous characteristics of the agents that are present, or may be present in the<br />
biological material, must be considered while completing the initial risk assessment. This<br />
consideration includes an assessment of the agent’s capability to infect and cause disease in a<br />
susceptible human or other host, the severity of disease, and the availability of preventive<br />
measures and effective treatments. To facilitate this assessment process, the World Health<br />
Organization (WHO) and NIH established an agent risk group (RG) classification for<br />
laboratories. This RG classification system, which was also adopted by the CDC, describes four<br />
general RGs based on the hazardous characteristics of agents, and the transmission route of<br />
natural disease in humans.<br />
LBNL uses the four RG levels and definitions provided in Appendix B of the U.S.-based NIH<br />
Guidelines (see Table 2 below). As shown in Table 2, a higher RG level indicates a higher risk<br />
for disease in humans. Assignments of RGs to specific agents may be found in various sources,<br />
including:<br />
• Appendix B, Section B.2, of this manual: Provides a list of human pathogens and their<br />
RG designations as excerpted from Appendix B of the NIH Guidelines (Classification of<br />
Human Etiologic Agents on the Basis of Hazard)<br />
• The American Biological Safety Association (ABSA) Risk Group Database<br />
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Table 2<br />
Risk Group Classification<br />
Risk Group<br />
(RG) Level<br />
Risk Group Definition<br />
1 Agents that are not associated with disease in healthy adult humans<br />
2 Agents that are associated with human disease that is rarely serious, and for<br />
which preventive or therapeutic interventions are often available<br />
3 Agents that are associated with serious or lethal human disease for which<br />
preventive or therapeutic interventions may be available (high individual risk<br />
but low community risk)<br />
4 Agents that are likely to cause serious or lethal human disease for which<br />
preventive or therapeutic interventions are not usually available (high<br />
individual risk and high community risk)<br />
Source: Adapted from the NIH Guidelines, Appendix B, Table 1.<br />
As required by LBNL policy, each biological material or agent used for research must be<br />
categorized by RG in the <strong>Biosafety</strong> Work Authorization, and the RG must be based on the<br />
agent’s or material’s potential for causing disease in humans. This categorization should be<br />
based on the following principles:<br />
• Agents must be assigned the RG designated by NIH, unless a risk assessment in the<br />
<strong>Biosafety</strong> Work Authorization indicates an alternate RG is warranted for the specific<br />
agent to be used.<br />
• Agents not classified as RG2, 3, or 4 by NIH are not automatically or implicitly classified<br />
as RG1. A risk assessment must be conducted for unclassified agents based on their<br />
known properties and relationship to agents listed in NIH RGs.<br />
• Some information sources for biological agents (e.g., see Section 3.3.2.1) only state the<br />
recommended biosafety level (BL) to be used for the agent. An agent’s recommended<br />
BL is typically the same as its RG (i.e., RG2 agents are handled at BL2). If an agent has<br />
not been assigned an RG by NIH, the risk assessment process must be used to<br />
determine its BL. See Section 4.4 for information on BLs.<br />
• Bloodborne pathogen materials should be designated RG2. This is because BMBL<br />
specifies BL2 containment practices for bloodborne pathogen materials and compliance<br />
with the OSHA Bloodborne Pathogens Standard (see Section 3.3.4).<br />
3.3.2 Pathogenic Agents and Toxins<br />
The risk assessment includes identification and assessment of the pathogenic agents or toxins<br />
that are involved with the work, or may be present in the biological material. A pathogen is an<br />
infectious microbe (e.g., bacteria, protozoa, fungi, viruses, etc.) or other agent (e.g., prion) that<br />
causes disease in a healthy host organism such as a human, animal, or plant. A toxin is a<br />
poisonous substance produced by a living organism.<br />
Depending on potential hosts and impacts (e.g., humans or livestock), pathogens and toxins<br />
may be regulated by a variety of agencies. Table 3 below and the remainder of this section<br />
identify categories of pathogens and toxins used in biosafety standards and by regulatory<br />
agencies to identify agents, toxins, and associated requirements. Appendix B of this manual<br />
also provides lists of many pathogens and toxins.<br />
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Table 3<br />
Pathogenic Agent and Toxin Categories<br />
Agent or<br />
Toxin<br />
Category<br />
Human<br />
Pathogens<br />
Plant and<br />
Animal<br />
Pathogens<br />
Toxins<br />
Select<br />
Agents and<br />
Toxins<br />
Prions<br />
Agent or Toxin<br />
Subcategory<br />
Human Etiologic<br />
Agents (NIH<br />
Guidelines)<br />
Human<br />
Pathogens<br />
(BMBL)<br />
Biological<br />
Etiologic Agents<br />
(DOE WSHP)<br />
Bloodborne<br />
Pathogens<br />
(Occupational<br />
Safety and<br />
Health<br />
Administration,<br />
OSHA)<br />
Select Agents<br />
(CDC)<br />
General Example or Source<br />
Risk Group 2, 3, or 4 agents such as the bacterial,<br />
fungal, parasitic, viral, and rickettsial agents listed in<br />
Appendix B of the NIH Guidelines<br />
Bacterial, fungal, parasitic, rickettsial, viral, and<br />
arbovirus agents that are included in BMBL agent<br />
summary statements and require BL2 or greater<br />
containment<br />
Human pathogens such as those listed in Appendix B<br />
of the NIH Guidelines<br />
Pathogens such as the human immunodeficiency<br />
virus (HIV), hepatitis B and C viruses (HBV and<br />
HCV).<br />
Pathogens categorized by CDC as select agents<br />
because of their severe threat to humans (e.g.,<br />
biological weapons)<br />
Materials, organisms, or agents regulated by USDA-<br />
APHIS that may harm domestic or native animals or<br />
plants, or natural resources.<br />
Bacterial, fungal, algal, and animal toxins.<br />
Human, animal, and plant pathogens and toxins<br />
categorized by CDC and Animal and Plant Health<br />
Inspection Service (APHIS) as select agents and<br />
toxins because of their potential severe threat to<br />
humans (e.g., biological weapons)<br />
Misfolded proteins and materials potentially containing<br />
other misfolded proteins that cause diseases known as<br />
transmissible spongiform encephalopathies<br />
(TSEs)<br />
3.3.2.1 Pathogen and Toxin Information and Guidance<br />
Documentation of the hazardous characteristics and controls for well-known pathogens and<br />
toxins is usually readily available and should be considered in the risk assessment. Listed in this<br />
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section are agencies and organizations that provide such information, along with links to the<br />
information sources.<br />
Because it may be difficult to find information on lesser-known pathogens or toxins, variants of<br />
pathogens, or opportunistic pathogens, their use may require additional risk assessments. For<br />
example, special technical information might be needed for avirulent or attenuated agents that<br />
have been physiologically modified or genetically altered and therefore several orders of<br />
magnitudes less likely to produce disease in a healthy host organism. In addition, “opportunistic<br />
pathogens” may not be listed as pathogens because they may only infect immunocompromised<br />
hosts.<br />
• BMBL Agent Summary Statements<br />
Section III of BMBL provides summary statements for many agents associated with<br />
laboratory-acquired infections or increased public health concern. Risk assessments must<br />
consider any information from these agent summary statements that apply to specific LBNL<br />
work activities. Categories included in the agent summary statements are listed below:<br />
o bacterial agents<br />
o fungal agents<br />
o parasitic agents<br />
o rickettsial agents<br />
o viral agents<br />
o arboviruses and related zoonotic viruses<br />
o alphabetic listing of 597 arboviruses and hemorrhagic fever viruses<br />
o toxin agents<br />
o prion diseases<br />
• Canadian Material Safety Data Sheets for Infectious Substances<br />
The Public Health Agency of Canada produces and provides material safety data sheets for<br />
infectious substances as a safety resource for Canadian laboratory workers who may be<br />
exposed to these agents in research, public health, teaching, and other laboratories.<br />
• CDC Health Information<br />
The CDC A-Z Index provides information on topics with relevance to a broad cross-section<br />
of CDC audiences. The items are representative of popular topics and frequent inquiries, or<br />
have critical importance to CDC’s public health mission. Topics such as diseases and<br />
vaccinations are covered.<br />
• CDC Travelers’ Health<br />
CDC Travelers' Health offers information to assist travelers and their health care providers in<br />
deciding the vaccines, medications, and other measures necessary to prevent illness and<br />
injury during international travel.<br />
3.3.2.2 Human Pathogens<br />
Human pathogens are infectious microbes (e.g., bacteria, protozoa, fungi, viruses, etc.) or<br />
other agents (e.g., prions) that cause disease in healthy humans. Pathogens are also often<br />
referred to as etiologic agents or infectious agents. Etiologic is an adjective that means<br />
disease-causing. The terms infectious agent and infectious material are also used in<br />
biosafety standards and in this manual. The term infectious agent means human pathogen.<br />
The term infectious material means a biological material that potentially contains human<br />
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pathogens or infectious agents. Listed and linked below are biosafety standards that cover<br />
human pathogens.<br />
• Human Etiologic Agents (NIH Guidelines)<br />
The NIH Guidelines provide a list of human pathogens and their RG2, RG3, and RG4<br />
designations in Appendix B (Classification of Human Etiologic Agents on the Basis of<br />
Hazard) of the NIH Guidelines (also see Appendix B of this manual). Work with human<br />
pathogens at LBNL will be conducted in accordance with the agent-specific RG designations<br />
in Appendix B of the NIH Guidelines and this manual.<br />
• BMBL Human Pathogens<br />
BMBL agent summary statements contain BL-specific containment guidance for specific<br />
human pathogens (see Section 3.3.2.1). Work with human pathogens at LBNL will be<br />
conducted in accordance with the IBC. The IBC will determine the proper containment level<br />
for pathogenic work, and use the recommended BL guidance presented in BMBL agent<br />
summary statements when available and applicable to the work activity. See Section 4.4 of<br />
this manual for additional information on BLs.<br />
• DOE WSHP Biological Etiologic Agents<br />
The DOE WSHP regulation (10 CFR 851, Appendix A, Section 7) has specific requirements<br />
for “biological etiologic agents.” LBNL’s program to comply with 10 CFR 851 defines a<br />
biological etiologic agent as an agent of biological origin (e.g., bacterium, fungus,<br />
parasite, virus, etc.) that causes disease in humans (i.e., pathogenic to humans). See<br />
Appendix B of this manual for the NIH list of human etiologic agents. See PUB-3000,<br />
Chapter 26, Appendix D, for specific LBNL requirements related to biological etiologic<br />
agents under 10 CFR 851.<br />
• OSHA Bloodborne Pathogens<br />
See Section 3.3.3 and Appendix C of this manual for requirements related to human<br />
pathogens that are considered bloodborne pathogens (BBPs) under the OSHA<br />
Bloodborne Pathogens Standard.<br />
• CDC Select Agents<br />
The Health and Human Services (HHS) CDC regulation on select agents and toxins lists<br />
agents that are both select agents and human pathogens. See Section 3.3.2.5 of this<br />
manual for more information.<br />
3.3.2.3 Plant and Animal Pathogens<br />
See Section 3.3.3 (USDA-Regulated Materials, Organisms, and Agents) for information on plant<br />
and animal pathogens and Section 3.3.2.5 for more information on plant and animal pathogens<br />
that are also select agents.<br />
3.3.2.4 Biological Toxins<br />
Biological toxin, biotoxin, or toxin is a poisonous substance produced by a living organism.<br />
The poisonous nature of toxins means that they may cause death or severe incapacitation at<br />
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relatively low exposure levels. Toxins include, for example, bacterial toxins, fungal toxins, algal<br />
toxins, and animal toxins. Examples include microcystins produced by freshwater<br />
cyanobacteria, or venoms produced by snakes or spiders. The word “toxin” without other<br />
descriptors such as “bio” is used in this manual and is a proper technical term to specifically<br />
describe toxins of biological origin. Toxic substances that are not of biological origin are properly<br />
termed “poisons.”<br />
Typical laboratory work with very small quantities of most toxins can be performed with minimal<br />
risk to the worker. Toxins do not replicate, are not infectious, and are difficult to transmit<br />
mechanically or manually from person to person. Other characteristics that further limit the<br />
spread of toxins include the fact that many commonly employed toxins are relatively unstable in<br />
the environment (especially in the case of protein toxins) and have very low volatility.<br />
Toxins must be handled using the general and “particularly hazardous substance” sections of<br />
the LBNL Chemical Hygiene and Safety Plan (CHSP). In addition, safety and security controls<br />
(presented below) based on a risk assessment must be used for each specific laboratory<br />
operation. The main laboratory risks are accidental exposure by direct contamination of the<br />
mouth, eyes, or other mucous membranes; inadvertent aerosol generation; and needlestick or<br />
other accidents that may compromise the normal barrier of the skin.<br />
Requirements and guidelines for storage and work with toxins in the laboratory are covered and<br />
summarized below. See Section 3.3.2.5 for additional information on toxins listed in the <strong>National</strong><br />
Select Agent Registry.<br />
• BMBL Guidelines for Work with Toxins<br />
According to Appendix I of BMBL, toxins of biological origin must be reviewed and should be<br />
incorporated into work with toxins based on a risk assessment approved by the IBC. Key<br />
criteria in the guidelines and LBNL policies:<br />
o A risk assessment should be conducted to develop safe operating procedures and a<br />
specific chemical plan. It is LBNL policy that this toxin assessment and plan should be<br />
documented in the <strong>Biosafety</strong> Work Authorization and should cover applicable topics and<br />
guidelines presented in Appendix I of BMBL. General topics should include: description<br />
of work; safety and security risks, hazards, or concerns; and safety and security controls.<br />
o Each worker must be trained in the theory and practice of toxins, with emphasis on<br />
practical hazards associated with laboratory operations. This training includes how to<br />
handle transfers of toxins or liquids containing toxin, where to place waste solutions and<br />
contaminated materials or equipment, and how to decontaminate work areas after<br />
routine operations as well as after accidental spills.<br />
o An inventory control system should be in place to account for toxin use and disposition.<br />
At LBNL, original primary containers of toxin must have LBNL chemical barcodes and be<br />
entered into the LBNL Chemical Management System.<br />
o<br />
o<br />
o<br />
Access to work areas should be controlled.<br />
Routine operations with dilute toxin solutions should be conducted under BL2<br />
containment with the aid of personal protective equipment, laboratory hoods, biosafety<br />
cabinets, or comparable engineering controls.<br />
Work with dry toxins should be minimized or eliminated.<br />
• BMBL Toxin Agent Summary Statements<br />
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Section VIII-G of BMBL contains information and guidance on specific toxins. When<br />
applicable, this guidance must be reviewed and should be incorporated into the work in<br />
accordance with the IBC-approved risk assessment.<br />
3.3.2.5 Select Agents and Toxins<br />
Select agents and toxins are specific pathogenic agents and toxins regulated by<br />
the HHS-CDC and The USDA-APHIS due to their potential threat (e.g., as biological<br />
weapons) to human, animal, and plant health. Specific genetic elements,<br />
recombinant nucleic acids, and recombinant organisms that may pose a similar<br />
threat are also regulated. Appendix B, Section B.2, of this manual provides the list of select<br />
agents and toxins and additional toxin information.<br />
Possession, use, storage, or transfer of select agents and toxins must be conducted in<br />
compliance with the HHS-CDC and USDA-APHIS regulations related to human, plant, and<br />
animal select agents and toxins. Specific controls for select agents are detailed in LBNL’s<br />
<strong>Biosafety</strong>, Security, and Incident Response Plan for Select Agents, a controlled document.<br />
Controls for select agents have also been integrated into the overall biosafety program<br />
described in this manual.<br />
See the <strong>National</strong> Select Agent Registry (NSAR) Program Web site for additional information<br />
on select agents provided by HHS-CDC and USDA-APHIS. The NSAR Program oversees<br />
possession of select agents and toxins for the HHS-CDC Division of Select Agents and Toxins<br />
and the USDA-APHIS Agricultural Select Agent Program.<br />
3.3.2.6 Prions<br />
A prion is an infectious agent composed of protein. All such agents discovered to date<br />
propagate by transmitting a misfolded protein; the protein does not itself self-replicate and the<br />
process is dependent on the presence of the polypeptide in the host organism. The misfolded<br />
form of the prion protein has been implicated in prion diseases known as transmissible<br />
spongiform encephalopathies (TSEs). TSEs are neurodegenerative diseases that affect humans<br />
and a variety of domestic and wild animal species. Examples are Creutzfeldt-Jakob disease<br />
(CJD) in humans and bovine spongiform encephalopathy (BSE), also known as mad cow<br />
disease in cattle. All known prion diseases affect the structure of the brain or other neural tissue,<br />
are currently untreatable, and are always fatal.<br />
Normal and diseased (misfolded) prions.<br />
Source: ScienceBlogs, Basic Concepts: Prions, by Shelley Batts (February 11, 2007).<br />
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Prion diseases are transmissible by inoculation or ingestion of infected tissue or homogenates.<br />
Prion infections usually occur in brain or other central nervous system tissues, and to a lesser<br />
extent in lymphoid tissues including spleen, lymph nodes, gut, bone marrow, and blood.<br />
Section VIII, Appendix H, of BMBL provides an agent summary statement that includes<br />
guidelines for prion diseases. When applicable, this guidance must be used to incorporate<br />
controls based on a risk assessment into the <strong>Biosafety</strong> Work Authorization.<br />
3.3.3 USDA-Regulated Materials, Organisms, and Agents<br />
The USDA-APHIS defends America’s animal and plant resources from agricultural pests and<br />
diseases by regulating materials, organisms, or agents that may harm domestic or native<br />
animals or plants, or natural resources. These materials, organisms, or agents may cause harm<br />
directly (e.g., predator or pathogen) or indirectly (e.g., vector). General examples include<br />
specific animals, plants, genetically engineered organisms, animal pathogens, plant pathogens,<br />
soil that may contain such pathogens, and agents that pose a severe threat.<br />
The transfer, storage, use, and disposal of APHIS-regulated materials at LBNL must be<br />
conducted in accordance with APHIS regulations. Generally, APHIS requires a permit or other<br />
document to import, export, or store regulated materials from or to locations outside the<br />
continental United States (U.S.) or between U.S. states. APHIS permits are issued to individuals<br />
and are not transferrable to others. The APHIS permit and sometimes an accompanying<br />
“compliance agreement” dictate specific controls and limitations when working with regulated<br />
materials. Individuals responsible for the transfer, storage, use, or disposal of such materials will<br />
obtain permits when required, ensure that the materials and permits are covered in the LBNL<br />
<strong>Biosafety</strong> Work Authorization, and ensure that specific requirements in the permit and<br />
compliance agreement are implemented.<br />
Materials, organisms, and agents that threaten animal and plant health are regulated by<br />
branches of the USDA-APHIS and examples are listed below in Table 4. Additional agency<br />
requirements and Web links for more information are detailed in Appendix I, Section I.2.2, of this<br />
manual. See Section 3.3.2.5 above for more information on select agents and toxins.<br />
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Table 4<br />
Materials Regulated by USDA-APHIS<br />
APHIS<br />
Branch<br />
Plant Protection<br />
and Quarantine<br />
(PPQ)<br />
Veterinary Services<br />
(VS)<br />
Biotechnology<br />
Regulatory<br />
Services (BRS)<br />
Agricultural Select<br />
Agent Program<br />
Examples of USDA-APHIS-Regulated Materials, Organisms,<br />
and Agents<br />
Plant pests such as soil, plant pathogens, plants, plant products,<br />
weeds, insects, mollusks, and nematodes<br />
Material, organisms, vectors, animal pathogens, animal products,<br />
cell cultures and their products, live animals, semen, embryos,<br />
and veterinary biologics (e.g., vaccines, antibodies, and<br />
diagnostic kits) that may harm animal health<br />
Certain genetically engineered organisms that may pose a plant<br />
pest risk, including organisms that are plants, insects, or microbes<br />
Animal and plant pathogens that are select agents<br />
3.3.4 Bloodborne Pathogens and Human Materials<br />
The federal OSHA Bloodborne Pathogens Standard has comprehensive requirements for<br />
workers who are or may be exposed to BBPs or designated materials assumed to contain<br />
BBPs. LBNL uses the term “BBP materials” to describe the pathogens and materials covered<br />
by the OSHA standard. These BBP materials are summarized in Table 5 and discussed in the<br />
next paragraph. BMBL guidelines for working with human and mammalian cells and tissues are<br />
also discussed below.<br />
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Table 5<br />
Materials Covered by the OSHA Bloodborne Pathogens Standard*<br />
• Bloodborne pathogens such as human immunodeficiency virus (HIV), hepatitis B virus (HBV),<br />
and hepatitis C virus (HCV)<br />
• Human blood: includes blood, blood components, and products made from human blood<br />
• Other potentially infectious materials (OPIM):<br />
o Unfixed human tissue or organ (other than intact skin) from a living or dead human<br />
o Primary human tissue cultures. These cultures are explants of living human tissue placed in<br />
a medium for tissue culture.<br />
o Primary human cell strains.** These cell strains are propagated in vitro from primary explants<br />
of human tissue or body fluids that have a finite lifetime (i.e., nontransformed) in tissue culture<br />
for 20 to 70 passages.<br />
o Established human cell lines.** These cell lines are immortalized cells that have been<br />
transformed by spontaneous mutation or natural or laboratory infection with an immortalizating<br />
agent, and then propagated or passed many times (e.g., in vitro or in animals such as mice).<br />
o Human body fluids. Fluids that are assumed to be potentially infectious include semen,<br />
vaginal secretions, cerebrospinal fluid, synovial fluid, pleural fluid, pericardial fluid, peritoneal<br />
fluid, amniotic fluid, saliva in dental procedures, any body fluid that is visibly contaminated with<br />
blood, and all body fluids in situations where it is difficult or impossible to differentiate between<br />
body fluids. Some human secretions that do not contain visible blood are not considered OPIM<br />
(e.g., urine, feces, vomit, tears, sweat, sputum, nasal secretions, and saliva).<br />
o HIV or HBV infected materials. HIV-containing cell or tissue cultures, organ cultures, and HIVor<br />
HBV-containing culture medium or other solutions; and blood, organs, or other tissues from<br />
humans or experimental animals infected with HIV or HBV<br />
Table Footnotes:<br />
* Text taken from OSHA Bloodborne Pathogen Standard 29 CFR 1910.1030 and the OSHA Standard<br />
Interpretation on Applicability of 1910.1030 to Establish Human Cell Lines.<br />
** Most primary human cell strains and established human cell lines at LBNL (e.g., American Type<br />
Culture Collection cell lines) are OPIM as required by the OSHA Standard Interpretation on such<br />
cells. If the researcher does not want to consider the cells OPIM, the cells must be “characterized.”<br />
Characterization must include documented screening of the cell lines or strains for viruses specified<br />
as BBPs in the OSHA standard, including human immunodeficiency viruses, hepatitis viruses, and<br />
herpes viruses (e.g., Epstein-Barr virus) if the cells are capable of propagating such viruses.<br />
Documentation that the cell line in culture is free of BBPs must be reviewed and approved by the<br />
<strong>Biosafety</strong> Officer and the Institutional <strong>Biosafety</strong> Committee.<br />
BBPs are infectious agents capable of causing human disease, and are transmitted through<br />
human blood and tissues. Examples include HBV and HIV. According to the OSHA Bloodborne<br />
Pathogens Standard, materials that are regulated based on their potential to contain BBPs<br />
include human blood, human blood components, products made from human blood, and OPIM<br />
listed in Table 5. LBNL uses the term BBP materials to describe all of these materials covered<br />
by the OSHA standard. Dried blood and some human secretions (e.g., urine, feces, vomit, tears,<br />
sweat, sputum, nasal secretions, and saliva) that do not contain visible blood are not considered<br />
OPIM even though they may contain other types of infectious agents or present health<br />
concerns.<br />
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Blood Collection. Source: HHS CDC Office of Health and Safety, <strong>Biosafety</strong> in the <strong>Laboratory</strong><br />
presentation (Web accessed May 2010)<br />
Appendix H of BMBL states that a risk assessment should be conducted for human and primate<br />
cells based on the origin and source of cells or tissues, and such cells should be handled using<br />
BL2 practices and containment (see Section 4.4 for further discussion of BLs). While many<br />
requirements in the BMBL and OSHA Bloodborne Pathogen Standard are similar to each other,<br />
the OSHA standard additionally requires initial and annual BBP training, availability of hepatitis<br />
B vaccination at no cost to employees, and a written Exposure Control Plan (ECP).<br />
Researchers satisfy documentation requirements for a risk assessment, BL2 containment, and<br />
an ECP once they have an approved Biological Use Authorization (BUA). BL2 containment<br />
must be used unless the BUA risk assessment indicates that alternative controls are sufficient.<br />
BUAs are further discussed in Section 5.1 of this manual and PUB-3000.<br />
LBNL work that involves BBP materials will be performed in compliance with the Fed/OSHA<br />
Bloodborne Pathogens Standard and BMBL. LBNL’s program for compliance with these<br />
standards is integrated into the larger LBNL biosafety program that is described in this manual.<br />
3.3.5 Recombinant Materials, Organisms, and Agents<br />
This section defines basic biological terms and processes that are key to<br />
understanding recombinant risks and concerns. Genetic material plays a<br />
fundamental role in determining the structure and nature of cell<br />
substances. It exists in the nucleus, mitochondria, and cytoplasm of a cell<br />
or organism, and is capable of self-propagation and genetic variation.<br />
The genetic material of a cell can be a gene, a part of a gene, a group of genes, a<br />
deoxyribonucleic acid (DNA) molecule, a fragment of DNA, a group of DNA molecules, or the<br />
entire genome of an organism. A nucleic acid is a macromolecule composed of chains of<br />
monomeric nucleotides. In biochemistry, nucleic acids carry genetic information or form<br />
structures within cells. The most common nucleic acids are DNA and ribonucleic acid (RNA).<br />
Nucleic acids are universal in living things, as they are found in all cells and viruses. The term<br />
genetic recombination is used to describe the process by which the strand of genetic material<br />
(usually DNA, but can also be RNA) is broken and then joined to a different DNA molecule to<br />
create recombinant genetic material. The NIH Guidelines defines recombinant DNA<br />
molecules as molecules constructed outside living cells by joining natural or synthetic nucleic<br />
acid segments to nucleic acid molecules that can replicate in a living cell or molecules that<br />
result from the replication of such molecules.<br />
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Vectors are commonly used in genetic engineering to create recombinant materials, organisms,<br />
agents, or cells. In molecular biology, a vector is a DNA molecule used as a vehicle to transfer<br />
foreign genetic material into another cell. Such a vector usually does not cause disease itself,<br />
but may change the properties and risks associated with the host cell. The four major types of<br />
vectors are plasmids, bacteriophages and other viruses, cosmids, and artificial chromosomes.<br />
Two common vectors are plasmids and viral vectors.<br />
• Plasmid vectors are commonly used to multiply or express particular genes. Many<br />
plasmids are commercially available for such uses. Plasmids are DNA segments that are<br />
separate from chromosomal DNA and are capable of replicating independently of the<br />
chromosomal DNA. In many cases, a plasmid is circular and double-stranded. Plasmids<br />
usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms.<br />
Plasmids are considered transferable genetic elements, capable of autonomous<br />
replication within a suitable host. Plasmid host-to-host transfer requires direct,<br />
mechanical transfer by "conjugation" or changes in host gene expression allowing the<br />
intentional uptake of the genetic element by "transformation." Plasmids provide a<br />
mechanism for horizontal gene transfer within a population of microbes and typically<br />
provide a selective advantage under a given environmental state. For example, plasmids<br />
may carry genes that provide resistance to naturally occurring antibiotics in a competitive<br />
environmental niche, or alternatively the proteins produced may act as toxins under<br />
similar circumstances. If these plasmids are inserted into a different host bacterium, the<br />
new host may acquire antibiotic resistance or produce toxic protein.<br />
• Viral vectors are a viral tool commonly used to deliver genetic material into cells. This<br />
process can be performed inside a living organism (in vivo) or in cell culture (in vitro).<br />
Viruses have evolved specialized molecular mechanisms to efficiently transport their<br />
genomes inside the cells they infect. Delivery of genes by a virus is termed transduction,<br />
and the infected cells are described as transduced. Although viral vectors are<br />
occasionally created from pathogenic viruses, they are modified in such a way as to<br />
minimize the risk of handling them. This usually involves the deletion of a part of the viral<br />
genome critical for viral replication. Such a virus can efficiently infect cells, but once the<br />
infection has taken place, it requires a helper virus to produce new virions. Examples of<br />
recombinant viral vectors include:<br />
• retroviral vectors from retroviruses such as the Moloney murine leukemia virus,<br />
• lentiviral vectors from lentiviruses (a subclass of retroviruses) such as HIV,<br />
• adenoviral vectors from adenoviruses, and<br />
• the adeno-associated virus (AAV).<br />
Genetic engineering may also use or create a transgenic organism. A transgenic organism is<br />
an organism whose genome has been altered by the transfer of a gene or genes from another<br />
species or breed. Examples of transgenic organisms include vertebrates such as mice, plants,<br />
and microbes.<br />
Work with or the creation of recombinant organisms or specific recombinant genomic materials<br />
and nucleic acids may create new risks to humans, animals, plants, or the environment. These<br />
potential recombinant risks must be identified and evaluated during the risk assessment<br />
process. Examples of genetic modifications that may increase risk include modifications that<br />
increase an agent’s pathogenicity or susceptibility to effective treatments (e.g., antibiotics), or<br />
increase an organism’s ability to compete in the natural environment.<br />
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Requirements and specific practices for constructing and handling recombinant DNA molecules,<br />
and organisms and viruses containing recombinant DNA molecules, are specified in the NIH<br />
Guidelines. APHIS permits may also be required for the importation, interstate movement, or<br />
environmental release of certain genetically engineered organisms that may be plant pests (see<br />
Appendix I of this manual). Recombinant research requires a risk assessment, establishment of<br />
containment levels and controls, and a <strong>Biosafety</strong> Work Authorization (for more information, see<br />
Sections 2.0 to 5.0 of this manual and PUB-3000, Section 26.8).<br />
3.3.6 Animals<br />
Working with animals in research, caring for animals in animal care facilities, or coming in<br />
contact with animals or vectors in the field may cause zoonotic or other diseases. A zoonosis<br />
or zoonose is an infectious disease that can be transmitted (in some instances, by a vector)<br />
from nonhuman animals, both wild and domestic, to humans, or from humans to nonhuman<br />
animals (the latter is sometimes called reverse zoonosis). Human diseases caused by a<br />
noninfectious, etiological agent derived from animals or their vectors are not considered a<br />
zoonosis (e.g., allergic reactions to animal products such as dander or urine). Work involving<br />
animals may expose workers to etiologic agents in a variety of ways such as wound infections,<br />
inhalation of aerosols (e.g., dust from animal bedding), and animal bites or scratches. See Table<br />
6 for examples of zoonotic diseases and other diseases related to animals.<br />
Worker safety, agricultural, and recombinant risks related to working with animals must be<br />
evaluated during the risk assessment, and proper containment measures must be employed.<br />
See the following sections and standards for additional information:<br />
• Sections 3.3.3 and 3.3.5 of this manual discuss agricultural and recombinant risks,<br />
respectively;<br />
• Section 4.4 of this manual provides an overview of laboratory and animal biosafety level<br />
containment categories and criteria; and<br />
• Section VIII of BMBL provides agent summary statements for zoonotic agents. It also<br />
recommends containment levels for laboratory use of a zoonotic agent and containment<br />
levels for handling animals infected with an agent.<br />
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Table 6<br />
Examples of Zoonotic and Other Diseases Related to Animals<br />
Disease<br />
Reservoir<br />
Vectors<br />
Causative<br />
Agent<br />
Exposure Routes<br />
Inhalation Ingestion Skin Contact<br />
allergies<br />
vertebrate<br />
animals<br />
animal allergens<br />
dander, urine,<br />
or saliva in<br />
dust or<br />
bedding<br />
— —<br />
anthrax<br />
animals<br />
Bacillus<br />
anthracis<br />
contaminated<br />
dust with<br />
spores<br />
contaminated<br />
with spores<br />
contaminated<br />
materials with<br />
spores<br />
hantavirus<br />
pulmonary<br />
syndrome<br />
rodents/deer<br />
mice<br />
sin nombre virus<br />
contaminated<br />
dust from<br />
dried urine,<br />
saliva,<br />
droppings<br />
— —<br />
herpes B<br />
virus<br />
infection<br />
nonhuman<br />
primates,<br />
particularly<br />
endemic in<br />
rhesus and<br />
cynomolgus<br />
members of<br />
the macaque<br />
genus<br />
Herpesvirus<br />
simiae or<br />
B virus<br />
aerosolized<br />
macaque<br />
saliva<br />
mucosal<br />
splashes (e.g.,<br />
monkey fluids<br />
contact the<br />
worker’s eyes or<br />
mouth)<br />
monkey bites,<br />
monkey scratches,<br />
or cage scratches;<br />
direct contamination<br />
of a preexisting<br />
wound with<br />
macaque saliva;<br />
needle-stick injuries<br />
following needle use<br />
in macaques<br />
lyme<br />
disease<br />
rodents/deer<br />
Borrelia<br />
burgdorferi<br />
— — ixodid tick bite<br />
plague rodents/fleas Yersinia pestis — —<br />
flea (Xenopsylla<br />
cheopis, Pulex<br />
irritans) bite<br />
Q fever<br />
sheep, goats,<br />
cattle<br />
Coxiella buretii<br />
barnyard dust<br />
contaminated<br />
by birth<br />
material and<br />
excreta<br />
milk ingestion,<br />
regurgitation,<br />
and perspiration<br />
—<br />
rabies rabid animals rabies virus — —<br />
bites and saliva from<br />
an infected animal<br />
Rocky<br />
Mountain<br />
spotted<br />
fever<br />
ticks<br />
Rickettsia<br />
rickettsii<br />
— —<br />
tick bites or skin<br />
contact with<br />
contaminated<br />
materials<br />
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Disease<br />
Reservoir<br />
Vectors<br />
Causative<br />
Agent<br />
Exposure Routes<br />
Inhalation Ingestion Skin Contact<br />
tetanus<br />
animals<br />
Clostridium<br />
tetani<br />
— —<br />
wounds<br />
contaminated with<br />
dirt or objects<br />
containing animal or<br />
human feces or<br />
saliva<br />
various<br />
diseases<br />
such as<br />
skin<br />
infections<br />
or gastroenteritis<br />
fish aquarium<br />
water<br />
Mycobacterium<br />
marinum,<br />
M. fortuitum,<br />
Aeromonas<br />
hydrophila, other<br />
bacteria, and<br />
Cryptosporidium<br />
spp. protozoa<br />
— —<br />
skin contact with<br />
aquarium water,<br />
especially if skin has<br />
cuts or abrasions<br />
3.4 <strong>Laboratory</strong> Procedure Hazards<br />
The BMBL five-step approach to assessing biological risk and selecting controls for laboratory<br />
work was initially presented in Section 3.2 of this manual. Step 2 (identifying laboratory<br />
procedure hazards) of this approach is discussed in this section.<br />
Historical data on laboratory acquired infections (LAIs) are an indicator of laboratory procedure<br />
hazards that have resulted in disease. Historical data show that past LAIs have occurred from:<br />
• parenteral inoculation by a contaminated sharp or syringe needle,<br />
• spills or splashes of contaminated materials directly onto the skin and mucous<br />
membranes,<br />
• ingestion through mouth pipetting,<br />
• animal bites and scratches, and<br />
• inhalation of infectious aerosol.<br />
See Section II of BMBL for more information regarding LAIs. Prevention of LAIs depends on the<br />
conscientious and proficient use of standard microbiological practices and special practices (see<br />
Section 4.1 of this manual) and the correct use of laboratory equipment. Table 7 below lists<br />
examples of hazards that may be found in laboratories using biological materials.<br />
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Table 7<br />
Equipment Hazard Examples<br />
Equipment<br />
Type<br />
aerosol<br />
generating<br />
cryogenic<br />
temperatures<br />
high<br />
temperatures<br />
high pressure<br />
oxygen<br />
deficiencies<br />
rotational<br />
energies<br />
Hazards<br />
The diameter of aerosols generated from<br />
certain types of equipment will vary from<br />
0.1 to 100 microns.<br />
• Bacterial cells and spores are 0.3 to 10<br />
microns in diameter.<br />
• Viruses are 0.02 to 0.3 micron in<br />
diameter.<br />
• Biological particles generated from<br />
liquid or powder form particles that are<br />
0.5 micron diameter.<br />
Cryogenic temperatures of –80°C are<br />
used to remove moisture from materials<br />
and contain low-temperature refrigerants.<br />
If protective equipment is not used,<br />
exposure to low temperature may cause<br />
cryogenic burns and frostbite.<br />
The use of heat to decontaminate or<br />
sterilize materials is widely used in the<br />
biological research laboratory. Physical<br />
injury from burns may occur from sudden<br />
accidental releases of heat sources or<br />
from the handling of hot items.<br />
Compressed gas cylinders and<br />
pressurized equipment are commonly<br />
used in the laboratory. Injury may occur<br />
from rupture high-pressure lines.<br />
Low-temperature freezers may include a<br />
backup system involving the use of a<br />
cryogenic liquid. Backup systems may<br />
consist of 50–200 liters of liquid nitrogen<br />
or liquid carbon dioxide under pressure.<br />
Liquid helium is also used in nuclear<br />
magnetic resonance (NMR) laboratories.<br />
Sudden release of such rotational<br />
energies can cause serious physical<br />
injury from unbalanced equipment or<br />
flying shrapnel.<br />
Examples<br />
• blender: 2 micron diameter particles<br />
• sonicator: 4.8 micron diameter particles<br />
• dropping bacterial flask: 3.5 micron diameter<br />
particles<br />
• dropping lyophilized culture: 10 micron<br />
diameter particles<br />
• pipette blow out: 4.9 micron diameter<br />
particles<br />
• vortex culture: 4.8 micron diameter particles<br />
• centrifuge: 4 micron diameter particles<br />
• freezers<br />
• lyophilizers (freeze dryers)<br />
• use of dry ice in shipping and receiving<br />
• dry heat temperatures used for sterilization<br />
range from 80°C to 200°C<br />
• wet heat is utilized by autoclaves to sterilize<br />
materials and can range between 80°C to<br />
500°C<br />
• saturated steam operates at 121°C<br />
• autoclaves operate at high pressures of<br />
1,000 kilo Pascal (145 psig)<br />
Oxygen deficiency environment may result<br />
from:<br />
• the displacement of oxygen by expanding<br />
gases (i.e., 700 parts of air to 1 part liquid<br />
nitrogen),<br />
• the linear displacement of oxygen from<br />
carbon dioxide (gas) generated from the use<br />
of dry ice, and<br />
• compressed gas cylinders or tanks.<br />
Tabletop and floor-mounted low, high, and<br />
ultracentrifuges rotate at speeds ranging from<br />
less than 5,000 to more than 100,000 rpm with<br />
rotor masses up to several kilograms.<br />
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Equipment<br />
Type<br />
sharps<br />
ultraviolet (UV)<br />
C radiation<br />
Hazards<br />
Any device having corners, edges, or<br />
projections capable of cutting or piercing<br />
the skin. LBNL's definition of sharps<br />
includes regulated sharps (medical<br />
waste), unregulated biohazardous<br />
sharps, and unregulated uncontaminated<br />
sharps that pose a safety hazard to<br />
custodians and other personnel.<br />
UVC radiation is used for inactivating<br />
microorganisms. Its usefulness,<br />
however, is limited by a variety of factors<br />
(e.g., low penetrating power). The eyes<br />
and skin can be damaged by exposure<br />
to direct or strongly reflected UV<br />
radiation.<br />
Examples<br />
• needles with or without syringes<br />
• needles with vacutainers<br />
• needles with attached tubing<br />
• blades (razors, scalpels, X-ACTO knives)<br />
• broken glass<br />
• glassware with sharp edges or points<br />
• pasteur pipettes and glass slides<br />
UV lights must be evaluated to determine if the<br />
benefits outweigh the potential hazards. UV<br />
radiation is sometimes used in conjunction<br />
with:<br />
• unoccupied tissue culture rooms<br />
• biological safety cabinets<br />
• UV light boxes<br />
3.5 Worker Competence and Health<br />
The BMBL five-step approach to assessing biological risk and selecting controls for laboratory<br />
work was initially presented in Section 3.2 of this manual. Step 4 of this approach (i.e., the<br />
evaluation of a worker’s proficiencies or competence) and the evaluation of a worker’s health<br />
are discussed in this section. Step 4 is an ongoing process where the supervisor or work lead<br />
evaluates a worker’s training, instructions, qualifications, behavior, and health. Worker training<br />
and health requirements are also a component of the <strong>Biosafety</strong> Work Authorization.<br />
Workers are the first line of defense for protecting themselves, others in the laboratory, and the<br />
public from exposure to biohazardous agents. <strong>Laboratory</strong> staff must therefore be properly<br />
trained, instructed, and qualified before conducting work. Supervisors and work leads should<br />
train and evaluate staff to the point where knowledge of the agent and procedure hazards,<br />
aseptic techniques, safety practices, use of safety equipment, caution, and attentiveness<br />
become second nature. Knowledge and experience prior to job assignment may also be<br />
necessary qualifications. See Section 5.2 for more information on training, instruction, and<br />
qualification.<br />
In addition, a worker’s health status may affect his or her susceptibility to an infection or ability<br />
to receive immunizations or prophylactic intervention. Workers who know they have an illness or<br />
medical condition that affects their immune system or their ability to receive vaccines or<br />
medications should seek an evaluation by Health Services in Building 26. See Section 5.4 for<br />
additional information regarding worker health and immunization.<br />
4.0 <strong>Biosafety</strong> Principles and Levels<br />
To determine which controls are required to mitigate hazards and perform work safely,<br />
supervisors and work leads must understand and apply the processes and requirements for<br />
defining work, identifying hazards, and assessing risks, as discussed in Section 3.0 of this<br />
manual. Controls are safeguards employed to contain biological agents or materials and<br />
therefore prevent the exposure of workers, other people, or the environment to agents that may<br />
harm them.<br />
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In biosafety, the term “containment” describes the set of controls, including safe methods,<br />
equipment, and facilities needed to protect workers and the environment from biohazardous<br />
materials or agents. Controls used for containment in laboratories are described in <strong>Biosafety</strong> in<br />
Microbiological and Biomedical Laboratories (BMBL), Section III, as the “Principles of<br />
<strong>Biosafety</strong>.” These containment controls are listed below and summarized in the next sections:<br />
• laboratory practices and techniques,<br />
• safety and personal protective equipment, and<br />
• facility design and construction.<br />
The LBNL <strong>Biosafety</strong> Work Authorization is used to define work, identify hazards, assess risks,<br />
and implement any of the containment controls listed above. See Sections 2.0 and 5.1 of this<br />
manual and PUB-3000, Section 26.8, for additional information on work authorization<br />
documents.<br />
4.1 <strong>Laboratory</strong> Practices<br />
The first and most important element of control for laboratory containment and research product<br />
protection is strict adherence to laboratory biosafety containment practices and good<br />
microbiological practice (GMP). <strong>Biosafety</strong> containment practices include standard<br />
microbiological practices and special practices specified by the Centers for Disease Control and<br />
Prevention (CDC) and the <strong>National</strong> Institutes of Health (NIH). GMP is based on widely accepted<br />
aseptic practices.<br />
Standard microbiological practices and special practices are administrative controls listed<br />
as biosafety level (BL) containment criteria in BMBL and the NIH Guidelines to protect workers<br />
and the environment. (See Section 4.4 of this manual for additional information on BL<br />
containment categories and criteria.) These practices, along with requirements from other<br />
biosafety standards, are used for the safe performance of work documented in the LBNL<br />
<strong>Biosafety</strong> Work Authorization. Standard microbiological practices or special practices for<br />
laboratories apply to most LBNL work with biological materials. Standard practices for BL1 and<br />
BL2 laboratories address the following topics (see Appendix C and Section 5.0 for more<br />
information):<br />
• access control<br />
• hand hygiene<br />
• food and eating<br />
• pipetting<br />
• sharps control<br />
• spill, splash, and aerosol control<br />
• decontamination of work surfaces, equipment, materials, and spills<br />
• signage and hazard communication<br />
• pest management<br />
• worker training and proficiency<br />
• occupational health, immunization, and personal health<br />
• incident reporting, evaluation, and worker treatment<br />
• biosafety manuals or documents<br />
Good Microbiological Practice (GMP) is also typically needed for containment and good<br />
research. GMP is based on aseptic techniques and other good microbiological practices<br />
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necessary to prevent contamination of the laboratory with the agents being handled and<br />
contamination of the work with agents from the environment. See Appendix D for common<br />
GMP. Supervisors and work leads are responsible for selecting and instructing workers on the<br />
specific GMP needed to conduct the work, or additional safety practices needed for specific<br />
agents or procedures.<br />
4.2 Safety and Personal Protective Equipment<br />
Worker exposure to infectious agents may be prevented by the use of standard and activityspecific<br />
safety and personal protective equipment (PPE) as primary barriers or controls. The<br />
need for additional activity-specific safety equipment or PPE must be determined during risk<br />
assessment, and any equipment needed for safety should be included in the <strong>Biosafety</strong> Work<br />
Authorization.<br />
Standard safety equipment and PPE are equipment controls listed as BL containment criteria<br />
in BMBL and the NIH Guidelines. They provide primary barriers that prevent worker exposure to<br />
infectious agents. See Section 4.4 of this manual for additional information on BL containment<br />
categories and criteria. These standard equipment requirements, along with requirements from<br />
other biosafety standards, are used and customized for the work to be conducted. They are also<br />
summarized in the LBNL <strong>Biosafety</strong> Work Authorization. Standard safety equipment and PPE are<br />
applicable to most work with biological materials at LBNL. The following types of standard<br />
equipment and PPE are further discussed in Appendix C and Section 5.0 of this manual:<br />
• biosafety cabinets<br />
• PPE<br />
• other physical containment devices such as centrifuge safety cups<br />
4.3 Facility Design and Construction<br />
Facility design and equipment provide secondary barriers that protect laboratory workers,<br />
persons outside the laboratory, the public, and the environment from potentially hazardous<br />
materials or agents that may be accidentally released from the laboratory.<br />
Standard facilites provide secondary barriers.<br />
Source: HHS CDC Office of Health and Safety.<br />
LBNL designs and operates its facilities where work with biological materials is conducted in<br />
accordance with applicable standard facilities criteria. Standard facilities are design features,<br />
materials, and equipment incorporated into the laboratory or facility in accordance with BL<br />
containment criteria stated in BMBL and the NIH Guidelines (see Section 4.4 for more<br />
information).<br />
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Standard BL1 and BL2 laboratory facility barriers are sufficient to control most work at LBNL.<br />
This is because risks related to most work are associated with direct contact with materials or<br />
agents in standard laboratories. Examples of standard facility barriers and equipment are listed<br />
below. See Appendix C for standard laboratory facilities criteria that summarize how these<br />
barriers must be employed. See Section 5.0 for additional information on each of these topics.<br />
• doors<br />
• sinks<br />
• cleanable surfaces and furnishings<br />
• window screens<br />
• ventilation and biosafety cabinets<br />
• vacuum line filters and traps<br />
• eyewashes<br />
• autoclaves<br />
If the risk assessment indicates that there is a risk of exposure to an infectious aerosol, then<br />
higher levels of safety equipment and PPE (primary barriers) or multiple secondary facilities<br />
barriers are necessary. Multiple secondary facilities barriers are not typically needed at LBNL.<br />
Some standard facility barriers are summarized in the <strong>Biosafety</strong> Work Authorization. Any<br />
additional special facility barriers that are required should also be included in the authorization.<br />
4.4 <strong>Biosafety</strong> Containment Levels and Criteria<br />
LBNL requires researchers who work with biological materials to implement containment<br />
controls in accordance with an established biosafety level (BL). BL is a standard combination of<br />
practices and techniques, safety equipment, and facilities to safely contain biohazardous<br />
materials or agents to be used in the work, as specified by BMBL or the NIH Guidelines.<br />
Work at LBNL requires routine application of BLs developed for biological laboratories, and<br />
occasional application of BLs developed for other types of work such as large-scale<br />
recombinant operations. BLs for laboratories are presented in the next section. BLs for largescale,<br />
plant, and animal uses are presented in Section 4.4.2 this manual.<br />
The appropriate BL must be selected once the risk assessment has been completed. The final<br />
BL determination should consider all aspects of the work, hazards, and controls. The principal<br />
investigator (PI) or supervisor should propose the appropriate BL(s) when submitting the<br />
authorization for review. The final BL(s) are determined by the Institutional <strong>Biosafety</strong> Committee<br />
(IBC).<br />
4.4.1 <strong>Laboratory</strong> Containment Levels<br />
Containment controls for laboratory biosafety are categorized into four BLs. Definitions of each<br />
laboratory biosafety level (BL) are provided in Table 8. Work at LBNL is commonly conducted<br />
at BL1 or BL2, while work at BL3 or BL4 is not currently conducted. <strong>Laboratory</strong> work at LBNL<br />
must be conducted in accordance with the standard and special work practices, safety<br />
equipment, and facility requirements noted in the laboratory BL1 and BL2 criteria listed in<br />
Appendix C.<br />
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Present<br />
at<br />
LBNL<br />
<strong>Biosafety</strong><br />
Level (BL)<br />
Table 8<br />
<strong>Laboratory</strong> <strong>Biosafety</strong> Containment Levels<br />
<strong>Biosafety</strong> Level Definition<br />
yes 1 BL1 is suitable for work involving agents of unknown or minimal<br />
potential hazard to laboratory personnel and the environment, 1 or<br />
work with defined and characterized strains of viable microorganisms<br />
not known to consistently cause disease in healthy adult humans. 2<br />
2 BL2 is suitable for laboratory work involving agents of moderate<br />
potential hazard to personnel and the environment. 1 Primary hazards<br />
to personnel working with these agents relate to accidental<br />
percutaneous or mucous membrane exposures, or ingestion of<br />
infectious materials. 2<br />
no 3 BL3 is applicable to facilities in which work is conducted with<br />
indigenous or exotic agents that may cause serious or potentially<br />
lethal disease as a result of exposure by the inhalation route. 3<br />
4 BL4 is required for work with dangerous and exotic agents that pose<br />
a high individual risk of life-threatening disease, aerosol transmission,<br />
or related agents having an unknown transmission risk. 3<br />
Footnotes:<br />
1 NIH Guidelines<br />
2 BMBL, fifth edition, Section III<br />
3 BMBL, fifth edition, Section IV<br />
When developing the <strong>Biosafety</strong> Work Authorization, the appropriate laboratory BL must be<br />
selected after conducting the risk assessment. Typical BLs used for various materials and<br />
agents are listed in Sections 4.4.1.1 and 4.4.1.2 of this manual. The final BL(s) are determined<br />
by the IBC.<br />
4.4.1.1 <strong>Laboratory</strong> <strong>Biosafety</strong> Level 1<br />
BL1 laboratories are not necessarily separated from the general traffic patterns in the building.<br />
Work is typically conducted on open benchtops using standard microbiological practices.<br />
Special containment equipment or facility design is not required, but may be used as<br />
determined by a risk assessment. <strong>Laboratory</strong> personnel must have specific training in the<br />
procedures conducted in the laboratory and must be supervised by a scientist trained in<br />
microbiology or a related science.<br />
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<strong>Laboratory</strong> <strong>Biosafety</strong> Level 1 work with open benchtops and standard microbiological<br />
practices. Source: LBNL EH&S.<br />
The BL will be determined as part of the risk assessment. BL1 containment is typically required<br />
for laboratory work involving:<br />
• biological agents that meet the definition of Risk Group (RG) 1 (i.e., agents not<br />
associated with disease in healthy adult humans);<br />
• biological materials not suspected of containing RG2 or higher agents in a quantity or<br />
form that may cause human disease (e.g., many soils and nonprimate animal cells);<br />
• biological agents or materials not characterized by the supplier as RG2 or higher;<br />
• transgenic or wild-type laboratory animals that have size or growth requirements<br />
allowing the use of containment for laboratory animals (e.g., rodents) and are<br />
o free of zoonotic diseases, and<br />
o not infected with, implanted with, or containing RG2 or higher agents or materials;<br />
• laboratory growth of nongreenhouse transgenic plants (see Section 4.4.3 of this<br />
manual);<br />
• biological agents, materials, or animals not typically categorized as RG2 or BL2 (or<br />
higher) as detailed Section 4.4.1.2 of this manual.<br />
4.4.1.2 <strong>Laboratory</strong> <strong>Biosafety</strong> Level 2<br />
BL2 laboratories follow BL1 requirements and additional BL2 requirements such as:<br />
• <strong>Laboratory</strong> personnel have specific training in handling any pathogenic agents and are<br />
supervised by scientists competent in handling infectious agents and associated<br />
procedures.<br />
• Access to the laboratory is restricted when work is being conducted.<br />
• All procedures in which infectious aerosols or splashes may be created are conducted in<br />
biological safety cabinets (BSCs) or other physical containment equipment.<br />
The BL will be determined as part of the risk assessment. <strong>Laboratory</strong> BL2 containment is<br />
typically required for laboratory work involving:<br />
• biological agents categorized as RG2 in the NIH Guidelines or by the supplier;<br />
• uses of biological agents described as BL2 in BMBL agent summary statements or other<br />
BMBL;<br />
• biological materials that may contain RG2 agents (e.g., sewage);<br />
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• Bloodborne pathogen (BBP) materials (e.g., human blood, human tissues, or human<br />
cells);<br />
• nonhuman primate tissues or cells;<br />
• viral vectors that are replication defective but still infectious to human cells;<br />
• laboratory animals (e.g., rodents) infected with, implanted with, or containing RG2<br />
agents or materials (e.g., infected with a human pathogen or containing a xenotransplant<br />
of human cells); and<br />
• tissues or cells potentially containing an RG2 agent (e.g., cells transformed with a RG2<br />
virus).<br />
4.4.2 Additional Containment Categories<br />
Additional types of containment specified in the NIH Guidelines or BMBL may also be applicable<br />
to work with biological materials at LBNL. Table 9 below lists these additional containment<br />
categories and relevant NIH Guidelines or BMBL section. When selecting a containment level<br />
for a type of work listed in Table 9, the supervisor, work lead, and IBC should carefully review<br />
this table to determine if the containment category and criteria apply to the planned LBNL work.<br />
<strong>Laboratory</strong>- or operation-specific authorizations, biosafety manuals, or other documents may be<br />
used to document the containment requirements related to the work. If the containment<br />
categories or criteria presented in Table 9 are not applicable to the work, the laboratory BLs<br />
presented in Section 4.4.1 of this manual are applied.<br />
4.4.2.1 Recombinant Large-Scale Containment Levels<br />
Physical containment guidelines from Appendix K of the NIH Guidelines must be used for largescale<br />
research or production activities involving viable organisms containing recombinant DNA<br />
molecules. Large scale (BL–Large Scale) is a term used in the NIH Guidelines and the LBNL<br />
biosafety policy to describe uses of and containment levels for organisms containing<br />
recombinant DNA molecules involving a quantity of culture greater than 10 liters. Note that this<br />
quantity category typically means the quantity of a material in a single batch of liquid culture;<br />
however, this batch quantity is not defined by NIH and should be used as a guideline to<br />
determine the applicability of large-scale containment criteria. Criteria for large-scale<br />
containment address the biological hazard associated with organisms containing recombinant<br />
DNA only. Large-scale containment criteria must be selected based on the findings of the risk<br />
assessment, and then documented in the <strong>Biosafety</strong> Work Authorization.<br />
4.4.2.2 Recombinant Plant Containment Levels<br />
<strong>Biosafety</strong> containment levels and criteria for recombinant research with plants must be selected<br />
based on the findings of the risk assessment, and then documented in the <strong>Biosafety</strong> Work<br />
Authorization. <strong>Laboratory</strong> or plant biosafety containment levels must be applied to the work as<br />
follows:<br />
• <strong>Laboratory</strong> BLs and criteria discussed in Section 4.4.1 of this manual and Appendix G of<br />
the NIH Guidelines should be used when the research plants are of a size, number, or<br />
have growth requirements that allow good containment when using laboratory BLs.<br />
• Plant BLs (BL-P) must be used when the research plants are of a size, number, or have<br />
growth requirements that preclude the use of laboratory BLs. For plant BLs and criteria,<br />
see Appendix P (Physical and Biological Containment for Recombinant DNA Research<br />
Involving Plants) of the NIH Guidelines.<br />
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Table 9<br />
Additional Containment Categories<br />
Containment<br />
Category<br />
large-scale uses<br />
of organisms<br />
containing<br />
recombinant DNA<br />
molecules<br />
recombinant DNA<br />
research involving<br />
plants<br />
recombinant DNA<br />
research involving<br />
animals<br />
vertebrate<br />
animal BL criteria<br />
for indoor<br />
research facilities<br />
(e.g., vivariums)<br />
arthropod<br />
containment<br />
guidelines<br />
Standard<br />
and<br />
Section<br />
NIH<br />
Guidelines,<br />
Appendix K<br />
NIH<br />
Guidelines,<br />
Appendix P<br />
NIH<br />
Guidelines,<br />
Appendix Q<br />
BMBL,<br />
Section V<br />
BMBL,<br />
Appendix E<br />
Focused Scope of Containment Criteria<br />
Physical containment guidelines for large-scale (greater<br />
than 10 liters of culture) research or production activities<br />
involving viable organisms containing recombinant DNA<br />
molecules<br />
Physical and biological containment conditions and<br />
practices suitable to greenhouse operations that conduct<br />
experiments involving plants, plant-associated<br />
microorganisms, and small animals (e.g., arthropods or<br />
nematodes)<br />
Containment and confinement practices for research<br />
involving whole animals when the animals are of a size or<br />
have growth requirements that preclude the use of<br />
containment for laboratory animals (i.e., including but not<br />
limited to nonhuman primates, cattle, swine, sheep, goats,<br />
horses, and poultry) and:<br />
• The animals’ genomes have been altered by the<br />
introduction of recombinant DNA or DNA derived<br />
therefrom into the germ line (transgenic animals), or<br />
• Experiments involving viable recombinant DNAmodified<br />
microorganisms have been tested on whole<br />
animals.<br />
Use of experimentally infected animals housed in indoor<br />
research facilities (e.g., vivariums), and the maintenance<br />
of laboratory animals that may naturally harbor zoonotic<br />
infectious agents<br />
Risk assessment and containment for arthropods of public<br />
health importance including those that transmit pathogens.<br />
Arthropods that only bite, sting, or cause myiasis and<br />
infestation are not included. Myiasis is an infestation of<br />
tissue by fly larvae, or a disease resulting from such<br />
infestation.<br />
The plant BLs listed in Appendix P of the NIH Guidelines specify physical and biological<br />
containment conditions and practices suitable for conducting greenhouse experiments involving<br />
recombinant DNA-containing plants, plant-associated microorganisms, and small animals.<br />
Acronyms for plant BLs are BL1-P through BL4-P. The following bullets further clarify terms and<br />
applicability of the plant biosafety levels:<br />
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• The term greenhouse refers to a structure with walls, a roof, and a floor designed and<br />
used principally for growing plants in a controlled and protected environment. The walls<br />
and roof are usually constructed of transparent or translucent material to allow the<br />
passage of sunlight for plant growth. The term greenhouse facility includes the actual<br />
greenhouse rooms or compartments for growing plants, including all immediately<br />
contiguous hallways and head-house areas, and is considered part of the confinement<br />
area.<br />
• The plants covered in Appendix P of the NIH Guidelines include but are not limited to<br />
mosses, liverworts, macroscopic algae, and vascular plants including terrestrial crops,<br />
forest, and ornamental species.<br />
• Plant-associated microorganisms include viroids, virusoids, viruses, bacteria, fungi,<br />
protozoans, certain small algae, and microorganisms that have a benign or beneficial<br />
association with plants, such as certain Rhizobium species and microorganisms known<br />
to cause plant diseases. Microorganisms being modified to foster an association with<br />
plants are also included.<br />
• Plant-associated small animals include those arthropods that have an obligate<br />
association with plants, are plant pests or plant pollinators, or transmit plant disease<br />
agents. They also include other small animals such as nematodes that require the use of<br />
plants to test their biological properties. Microorganisms associated with such small<br />
animals (e.g., pathogens or symbionts) are also included.<br />
A Practical Guide to Containment developed by Virginia Polytechnic and State University is a<br />
good example of how to apply plant BLs in research with greenhouse transgenic plants and<br />
microbes.<br />
4.4.2.3 Vertebrate Animal Containment Levels<br />
<strong>Biosafety</strong> containment levels and criteria for the use or care of vertebrate animals must be<br />
selected or developed following the risk assessment and covered in the <strong>Biosafety</strong> Work<br />
Authorization. <strong>Laboratory</strong> or animal biosafety containment levels must be applied to the work as<br />
follows:<br />
• <strong>Laboratory</strong> biosafety level criteria should be used for laboratory animals<br />
such as rodents whose size or growth requirements allow the use of<br />
laboratory containment levels specified by the NIH Guidelines. <strong>Laboratory</strong><br />
BLs and criteria are discussed in Section 4.4.1 of this manual.<br />
• Animal biosafety levels must be applied when 1) recombinant research<br />
involves larger animals (e.g., nonhuman primates), 2) animals are infected<br />
with human pathogens, or 3) animals may harbor zoonotic agents (see<br />
Table 9 for more information). Acronyms for animal biosafety levels are<br />
BL1-N through BL4-N. In some cases, animal use, animal care, and<br />
hazards at LBNL may not be directly applicable to these established animal biosafety<br />
levels and criteria. In these cases, specific criteria that may be applicable may be<br />
selected, customized, and incorporated into the <strong>Biosafety</strong> Work Authorization.<br />
Agent summary statements for zoonotic agents in Section VIII of BMBL also recommend<br />
containment levels for laboratory use of zoonotic agents, and for handling animals infected with<br />
the agent.<br />
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4.4.2.4 Arthropod Containment Levels<br />
<strong>Biosafety</strong> containment levels and criteria for the use of arthropods must be selected or<br />
developed after the risk assessment. <strong>Laboratory</strong> or arthropod biosafety containment levels must<br />
be applied to the work as follows:<br />
• <strong>Laboratory</strong> biosafety level criteria should be used for arthropods that do not present risks<br />
to humans, plants, or animals (e.g., most research uses of Drosophila spp.).<br />
• Arthropod containment guidelines discussed in Appendix E of BMBL must be used for<br />
arthropods of public health importance including those that transmit pathogens.<br />
Arthropods that only bite, sting, or cause myiasis and infestation are not included. Most<br />
uses of Drosophila spp. are also excluded from these arthropod containment guidelines.<br />
The Appendix E of BMBL references the Arthropod Containment Guidelines published<br />
by the American Society of Tropical Medicine and Hygiene.<br />
5.0 Specific <strong>Biosafety</strong> Controls<br />
This section further describes biosafety controls, including safe methods, equipment, and<br />
facilities that were generally introduced in Section 4.0 of this manual.<br />
5.1 Work Authorizations<br />
LBNL Job Hazards Analyses (JHAs), Subcontractor Job Hazards Analysis and Work<br />
Authorizations (SJHAWAs), and <strong>Biosafety</strong> Work Authorizations document the definition of work,<br />
identification of hazards, risk assessments, and controls. <strong>Biosafety</strong> Work Authorizations include:<br />
• Biological Use Authorizations (BUAs),<br />
• Biological Use Registrations (BURs),<br />
• Biological Use Notifications (BUNs), and<br />
• Exposure Control Plans (ECPs).<br />
PIs, work leads, workers, Division Safety Coordinators, and Environment, Health, and Safety<br />
(EH&S) Division <strong>Biosafety</strong> Program personnel have access to their BUAs, BURs, and BUNs<br />
through the <strong>Biosafety</strong> Authorization System (BAS) so that controls may be implemented and<br />
authorizations updated. Documentation, review, and authorization of new work should be<br />
initiated by filling out the Biological Use Application Form. See Section 2.0 of this manual and<br />
PUB-3000, Section 26.8, for additional information on these work authorizations.<br />
These work authorizations consolidate and document a wide variety of biosafety requirements<br />
and controls to meet various biosafety standards (see standards in PUB-3000, Sections 26.4<br />
and 26.10). For example the BUA is also regarded as:<br />
• the registration document that must be submitted to the LBNL Institutional <strong>Biosafety</strong><br />
Committee (IBC) as required by the NIH Guidelines for recombinant work (BURs also<br />
document such recombinant work),<br />
• the laboratory-specific biosafety manual required by <strong>Biosafety</strong> in Microbiological and<br />
Biomedical Laboratories (BMBL) when <strong>Biosafety</strong> Level (BL) 2 work is performed with<br />
Risk Group (RG) 2 agents or materials, and<br />
• the ECP required by the Occupational Safety and Health Administration (OSHA)<br />
Bloodborne Pathogens (BBP) Standard when BBP agents or materials are used.<br />
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5.2 Training, Instruction, and Qualification<br />
This section describes the requirements and administrative systems for institutional and<br />
operation-specific training, information, and instruction based on the biosafety-related standards<br />
and LBNL policies summarized in PUB-3000, Section 26.7.8.1.<br />
Work leads, supervisors, and principal investigators are responsible for ensuring their workers<br />
have sufficient skills, knowledge, and ability to perform their work safely. This includes<br />
understanding of the work, hazards, and controls through technical competence, training,<br />
instruction, and a commitment to safety in Integrated Safety Management (ISM) terms. Each<br />
worker’s competence must be commensurate with his or her responsibilities. This competence<br />
is a major component of biosafety containment and includes both required LBNL courses and<br />
sufficient operation-specific information and instruction. These courses, information, and<br />
instruction provide workers with awareness of the potential hazards, required training, and<br />
proficiency in the practices and techniques required for handling biological materials safely and<br />
in accordance with laboratory standard microbiological practices and special practices<br />
discussed in Section 4.1.<br />
Work leads must provide or arrange for appropriate training and instruction for each person,<br />
including but not limited to the:<br />
• completion of required LBNL courses specified on all work authorization documents, and<br />
• job- and operation-specific instruction and information.<br />
5.2.1 Job Hazards Analysis<br />
Supervisors, work leads, and staff must use the JHA or Subcontractor Job Hazards Analysis<br />
(SJHA) to define work with biological materials, determine the potential for exposure to<br />
biological hazards, and establish biosafety controls for each worker or subcontractor (for more<br />
information, see PUB-3000, Chapter 32 and Chapter 31, respectively). The <strong>Laboratory</strong>-wide<br />
JHA identifies workers who work with or have potential exposure to biological materials (e.g.,<br />
BBP materials). The JHA process is based on each individual’s work and activities. It also lists<br />
general controls including any required EH&S courses and <strong>Biosafety</strong> Work Authorizations for<br />
work or activities in which the worker participates.<br />
5.2.2 Training Courses and Tracking<br />
Specific biosafety, biohazardous waste, and occupational health courses are developed and<br />
maintained by the EH&S Division to meet requirements that can be fulfilled at an institutional<br />
level. LBNL course requirements are presented below and summarized in Table 10. See the<br />
EH&S Training Web site for additional course information, to register for a course, or to take an<br />
online course.<br />
• Anyone who works with biological material of any risk level (e.g., microorganisms, cells,<br />
cell lines, tissue cultures, recombinant nucleic acids, blood, body fluids or tissues, or<br />
animals) must complete EHS0739 (General <strong>Biosafety</strong> Training) online or in a classroom.<br />
In addition, the online course EHS0730 (Medical/Biohazardous Waste Training) is<br />
recommended for anyone who works with biological material, and required for anyone<br />
who works with medical or biohazardous waste.<br />
• Anyone who works with or may be exposed to human blood or blood products or to<br />
human materials (e.g., cells, tissues, or fluids) defined by the OSHA Bloodborne<br />
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Pathogens Standard as other potentially infectious materials (OPIM) (see Section 3.3.4<br />
of this manual for more information) must also complete EHS0735 (Bloodborne<br />
Pathogen Training) and EHS0745 (Hepatitis B Medical Surveillance). EHS0735 must be<br />
renewed annually through EHS0738. Courses EHS0739 and EHS0738 are also<br />
available as Web-based challenge exams.<br />
Table 10<br />
<strong>Biosafety</strong>-Related Training Courses<br />
Work or<br />
Exposure<br />
EHS0739<br />
General<br />
<strong>Biosafety</strong><br />
Training<br />
EHS0730<br />
Medical/<br />
Biohazardous<br />
Waste<br />
EHS0735<br />
Bloodborne<br />
Pathogen<br />
Training<br />
(Initial)<br />
EHS0738<br />
Bloodborne<br />
Pathogen<br />
Retraining<br />
(Annual)<br />
EHS0745<br />
Hepatitis B<br />
Medical<br />
Surveillance<br />
Use of biological<br />
materials of any<br />
risk level, or<br />
generation of<br />
medical/biohazard<br />
ous waste<br />
X<br />
X<br />
Use or exposure<br />
to bloodborne<br />
pathogen<br />
materials<br />
X X X X X<br />
<strong>Biosafety</strong> training course requirements for each worker are identified through each worker’s<br />
JHA, and inclusion in the Personnel and Training sections of the <strong>Biosafety</strong> Work Authorization in<br />
the BAS. Each worker’s course requirements and training status are then displayed in the<br />
worker’s Training Profile, JHA Profile, and the <strong>Biosafety</strong> Work Authorization.<br />
5.2.3 Job-Specific Instruction, Information, and Practices<br />
As discussed above in Section 5.2, supervisors and work leads are responsible for ensuring that<br />
workers receive job- and operation-specific instructions. These instructions should include:<br />
• Individual JHA job duties and controls<br />
• Hazards and controls in authorization documents including BUAs, BURs, BUNs, and<br />
ECPs. Controls in these documents include, for example, standard microbiological<br />
practices and special practices customized as needed for the work (see Appendix C of<br />
this manual). These authorizations must be available and accessible to each worker so<br />
that they can understand the work, hazards, and required controls. Each worker has<br />
access to their authorization, registration, and notification through the BAS.<br />
• Good microbiological practices as needed to perform the work safely (see Section 4.1<br />
and Appendix D of this manual)<br />
• Incident, accident, and emergency response procedures (e.g., LBNL Emergency<br />
Response Guide)<br />
• Any operation-specific safety procedure<br />
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Additional instruction or demonstration of proficiency may be needed for work that involves<br />
higher hazards. For example, workers must demonstrate their proficiency in standard<br />
microbiological practices and special practices before working with RG2 agents (for more<br />
information, see Section 4.1 of this manual).<br />
Labels and signs must also be used to advise workers of hazards and controls (see Section 5.5<br />
of this manual).<br />
5.3 Occupational Health and Immunization<br />
LBNL occupational health and immunization policies, programs, and services are provided by<br />
the Health Services Group of the EH&S Division under the direction of the Site Occupational<br />
Medical Director. These policies, programs, and services are described in the Health Services<br />
Web site, policies (e.g., immunization and serum banking), and Chapter 3 of PUB-3000. The<br />
occupational health program related to biosafety is designed to proactively identify and prepare<br />
workers who may be exposed to certain biological materials or agents, and provide procedures<br />
for the treatment and management of workers who have been injured or may have been<br />
exposed. Employees who are aware of personal illnesses that may affect their ability to combat<br />
infection or receive medications or vaccines should visit Health Services for an evaluation of<br />
how this may affect their individual risk for work with biological agents.<br />
Potential exposures to biological agents or materials that generate health concerns or may<br />
cause disease are assessed as part of the work review and authorization process discussed<br />
above in Sections 3.0 and 5.1. This assessment includes an evaluation and determination of the<br />
need for employee medical evaluations, immunizations, serum banking, or other occupational<br />
health controls. For research projects, this assessment is conducted by IBC members including<br />
the <strong>Biosafety</strong> Officer and the SOMD. The IBC review includes SOMD recommendations and is<br />
the basis of required or recommended occupational health controls for potentially exposed<br />
employees. These controls are then documented in the <strong>Biosafety</strong> Work Authorization.<br />
Employee consultation with LBNL Health Services. Source: LBNL EH&S.<br />
Requirements or recommendations for occupational health controls (e.g., vaccinations) for<br />
specific agents or materials are discussed in BMBL, Section VIII (Agent Summary Statements).<br />
Requirements for BBP materials are provided by the OSHA Bloodborne Pathogens Standard.<br />
OSHA requirements and LBNL programmatic policies and systems for implementing these<br />
requirements are summarized below:<br />
• Hepatitis B Vaccination: The OSHA Bloodborne Pathogens Standard requires that the<br />
hepatitis B vaccination series must be made available and offered to all LBNL<br />
employees who have occupational exposure to BBPs or materials that are regulated<br />
based on their potential to contain BBPs (e.g., human blood, tissues, and cells). This<br />
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requirement is managed at LBNL by ensuring that workers who are potentially exposed<br />
to BBP materials are identified in their BUA or ECP and are required to complete<br />
EHS0745 (Hepatitis B Medical Surveillance). Workers fulfill hepatitis B surveillance<br />
requirements by 1) completing the online EHS0745 course and 2) indicating on the<br />
integral surveillance form that they wish to be vaccinated or wish to decline the<br />
vaccination.<br />
• Post-Exposure Evaluation and Follow-up: The OSHA Bloodborne Pathogens Standard<br />
requires that postexposure evaluations and follow-ups must be made available to all<br />
employees who have had an exposure incident. Employees who have had an exposure<br />
incident must report their exposure to their supervisor and Health Services in<br />
accordance with LBNL’s policies on “Incident Review and Reporting” (see PUB-3000,<br />
Sections 5.1 and 26.7.12).<br />
• Sharps Injury Log: The OSHA Reporting and Recording Occupational Injuries and<br />
Illnesses Standard requires a Sharps Injury Log for the recording of percutaneous<br />
injuries from sharps contaminated with BBP material (see 29 CFR1904.8). The<br />
information in the Sharps Injury Log must be recorded and maintained in such a manner<br />
as to protect the confidentiality of the injured employee. The Sharps Injury Log must<br />
contain the type and brand of device involved in the incident, the department or work<br />
area where the exposure incident occurred, and an explanation of how the incident<br />
occurred. This log may be maintained on the OSHA 300 Form, provided that the type<br />
and brand of the device causing the sharps injury is recorded, and sharps injury records<br />
may be easily separated from other types of work-related injuries and illnesses. LBNL<br />
maintains sharps injury information on the OSHA 300 Form in accordance with OSHA<br />
regulations. In addition, the LBNL Health Services Group maintains a separate,<br />
confidential log containing sharps injury information required by OSHA.<br />
Consult the LBNL Health Services Group ((510) 486-6266), Health Services Web site, and<br />
PUB-3000 (i.e., Chapter 3 (Health Services)) for additional information. See Section 5.10.2 of<br />
this manual for additional information on worker exposure, injury, and illness reporting.<br />
5.4 Personal Protective Clothing and Equipment<br />
Use of safety equipment including personal protective equipment (PPE) is<br />
another element of BL1 and BL2 containment. PPE is clothing or equipment worn<br />
by workers to protect the body from injury by hazardous agents or materials. PPE<br />
may include foot, hand, eye, face, body, and respiratory protection.<br />
PPE must be used, maintained, and disposed of in accordance with federal regulations,<br />
biosafety standards, and LBNL-specific PPE policies to prevent the spread of contamination and<br />
accidental infection. LBNL policies related to PPE when working with biological materials are<br />
described in this section and the following policy documents:<br />
• PUB-3000, Chapter 19 (Personal Protective Equipment)<br />
• PUB-3000, Chapter 4, Section 4.13, and the LBNL Respiratory Protection Program<br />
• Medical and Biohazardous Waste Generator’s Guide (PUB-3095)<br />
The PPE section of the Chemical Safety Hygiene Plan (CHSP) should also be consulted<br />
regarding PPE requirements and guidelines related to work with chemicals.<br />
The following PPE requirements are related to biosafety:<br />
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• Area-specific PPE requirements must be established for all Technical Areas (e.g.,<br />
laboratories) and must be posted on the LBNL entrance placard. Minimum PPE for<br />
laboratories where biological materials are stored or handled includes safety protective<br />
eyewear, long pants, and closed-toe shoes. Area PPE requirements apply to the entire<br />
Technical Area unless an exception is granted in accordance with the procedure<br />
described in PUB-3000, Chapter 19, Appendix A.<br />
• Activity- or operation-specific PPE requirements are assessed and defined in the<br />
<strong>Biosafety</strong> Work Authorization, which covers what PPE must be used (e.g., gloves,<br />
laboratory coats, and safety glasses) and any maintenance (e.g., laundering) or disposal<br />
requirements.<br />
General requirements and conditions for use of PPE related to biosafety include:<br />
• The supervisor or work lead is responsible for:<br />
o Determining what PPE is required to prevent occupational exposure<br />
o Providing at no cost to an employee the PPE required by this section or specified in<br />
the <strong>Biosafety</strong> Work Authorization. This PPE must be readily available in appropriate<br />
sizes.<br />
o Ensuring that employees and visitors properly use and store required PPE<br />
• The EH&S Division is available to assist supervisors or work leads in evaluating work<br />
activities and selecting appropriate PPE.<br />
• Employees and visitors are responsible for using PPE when required and whenever the<br />
work poses a reasonable probability of eye injury or exposure.<br />
• In general, removed PPE must be:<br />
o Decontaminated when needed, or<br />
o Disposed of in accordance with LBNL medical/biohazardous, hazardous, and<br />
radiological waste management requirements.<br />
• PPE that protects against exposure to BBP materials is considered appropriate if it does<br />
not permit BBP material (e.g., human blood or cell culture solution) to pass through the<br />
employee's work clothes, street clothes or undergarments, skin, eyes, or other mucous<br />
membranes under normal conditions of use and for the duration of time in which the<br />
PPE will be worn.<br />
5.4.1 Body Protection<br />
Protective laboratory clothing is a garment such as a lab coat, gown, smock,<br />
or uniform designed to keep personal clothing, forearms, or other exposed<br />
bodily surfaces protected from contamination by biological materials or<br />
exposure to other hazards. The term "protective laboratory clothing” typically<br />
applies to garments worn in the laboratory, but may also apply to garments<br />
worn in nonlaboratory work (e.g., health care).<br />
The following biosafety criteria are applicable to wearing protective laboratory clothing:<br />
• Protective laboratory clothing should be worn to prevent contamination of personal<br />
clothing when working at BL1.<br />
• Protective laboratory clothing must be worn when working at BL2 or when working with<br />
RG2 or other hazardous materials. This clothing must be removed and left in the<br />
laboratory before leaving for nonlaboratory areas (e.g., cafeteria, library, administrative<br />
offices).<br />
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Protective laboratory clothing removed after use at BL2 or with biohazardous materials must be<br />
handled in one of the following ways:<br />
• Placed in a laundry bag or container for cleaning by a qualified laundry service<br />
• Disposed of in accordance with LBNL medical/biohazardous, hazardous, and<br />
radiological waste management requirements<br />
• Stored for reuse if not contaminated. Such clothing stored for reuse should be stored in<br />
a manner that would not contaminate other items in case the protective clothing has<br />
unknown contamination (e.g., separate coat hook).<br />
Protective laboratory clothing and other laundry contaminated with RG2 materials should be<br />
handled as noted below, and laundry contaminated with BBP materials must be handled as<br />
follows:<br />
• Handled as little as possible with a minimum of agitation<br />
• Bagged or containerized at the location where it was used but not sorted or<br />
rinsed in the location of use<br />
• Placed in bags or containers that have biohazard labels, are red in color, or<br />
are identified by an alternative laundry labeling or color-coding system that<br />
uses universal precautions<br />
• Placed and transported in bags or containers that prevent soak-through or<br />
leakage of fluids to the exterior if the laundry is sufficiently wet<br />
Place protective clothing in properly identified laundry bags.<br />
5.4.2 Eye and Face Protection<br />
Eye protection is a safety device such as safety glasses with side shields or<br />
goggles worn over the eyes to prevent injury to the eye or exposure to<br />
biological agents. Face protection is a safety device such as a face mask,<br />
face shield, or other splatter guard worn over all or part of the face to protect<br />
the face from injury or exposure to biological agents. Face masks or<br />
respirators that are occasionally used for face protection are discussed in<br />
Section 5.4.5 of this manual.<br />
Eye and face protection is used by laboratory and other workers to protect the eyes and face<br />
from splashes, splatters, or flying debris and hand-eye contact with biological materials. Contact<br />
by these means may result in injuries to the eyes and face or accidental inoculation via the<br />
eyes, nose, or mouth and subsequent infection and disease.<br />
The risks noted above are prevented by using eye and face protection in accordance with the<br />
following requirements:<br />
• As a minimum requirement, safety glasses with side shields must be worn at all times<br />
when in a Technical Area such as a laboratory. Area PPE requirements apply to the<br />
entire Technical Area unless an exception is granted in accordance with the procedure<br />
described in PUB-3000, Chapter 19, Appendix A. Additional eye or face protection may<br />
be necessary when handling chemicals or biological materials (e.g., goggles, face<br />
shield).<br />
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• Eye protection must be worn when conducting procedures that have the potential to<br />
create splashes of biological agents, biohazardous materials, or other hazardous<br />
materials.<br />
• Eye and face protection (e.g., goggles, face mask, face shield, or other splatter guard)<br />
must be used when it is anticipated that splashes, sprays, splatters, or droplets of<br />
infectious or other hazardous materials may be generated and could contaminate the<br />
eyes, nose, or mouth (e.g., when RG2 microorganisms must be handled outside the<br />
biosafety cabinet or containment device). This eye and face protection must be included<br />
in the <strong>Biosafety</strong> Work Authorization risk assessment and disposed of with other<br />
contaminated laboratory waste or decontaminated before reuse.<br />
Eye protection using safety glasses. Source: LBNL EH&S.<br />
LBNL provides prescription safety glasses when needed via the EH&S Health Services Group<br />
and a staff optometrist. Personnel who need consultation or require prescription safety glasses<br />
should schedule an appointment with the optometrist by calling the Health Services Group at<br />
(510) 486-6266.<br />
5.4.3 Hand Protection<br />
Hand protection is a glove or other safety device used on the hand to prevent<br />
injury to the hand or direct skin contact with biological materials. Hand<br />
protection is used by laboratory and other workers to protect the hands from<br />
harmful physical, chemical, biological, radiological, or other agents or hazards.<br />
These agents or hazards may cut, lacerate, abrade, or burn the skin; absorb<br />
through the skin; pass through breaks in the skin; or be spread as<br />
contamination. Although there are relatively few microbes that can penetrate unbroken skin,<br />
there are many circumstances that may cause a break in the skin, such as a cut or puncture<br />
from a sharp (see sharps in Section 5.6.6.1). In the case of biological materials, gloves prevent<br />
the worker’s hands, fingers, and nails from being contaminated. Spread of biological<br />
contamination from the worker’s exposed hands or contaminated gloves to the worker’s mucous<br />
membranes or other surfaces may also cause infection and disease in the worker or other<br />
people.<br />
Glove selection may need to consider protection of the worker from different hazards or serve<br />
multiple purposes. For example, gloves used for handling chemical and biological materials may<br />
need to be resistant to the chemicals being handled, liquid permeation, and physical damage<br />
(see the PPE section of the CHSP). But the remainder of this section is focused only on glove<br />
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criteria that are important for biosafety and gloves that provide protection from biological<br />
materials or liquids.<br />
The following criteria are applicable to glove selection, use, and disposal:<br />
• In general, gloves should be worn to protect the hands from exposure to biological<br />
materials or organisms that may present a biological risk. Gloves must be worn to<br />
protect hands from exposure to hazardous materials, including: organisms containing<br />
recombinant DNA, recombinant experimental animals, RG2 materials, BBP materials or<br />
surfaces and items contaminated with BBP materials, when touching mucous<br />
membranes and nonintact skin of patients, and when performing vascular access<br />
procedures such as phlebotomies.<br />
• Glove selection should be based on an appropriate risk assessment. Use of standard<br />
nitrile or latex examination gloves is considered adequate for handling most biological<br />
materials, and is assumed in the <strong>Biosafety</strong> Work Authorization. The need for gloves with<br />
any additional safety features to handle biological materials should be documented in<br />
the <strong>Biosafety</strong> Work Authorization. The JHA process should be used to assess other<br />
hand hazards and glove requirements.<br />
• Alternatives to latex gloves should be available because some workers are known to<br />
develop allergic reactions to latex. Exposures to latex may result in skin rashes; hives;<br />
flushing; itching; nasal, eye, or sinus symptoms; asthma; and (rarely) shock.<br />
• When working at BL1 and BL2, workers should remember the following:<br />
o Change gloves when contaminated, when their integrity has been compromised, or<br />
when otherwise necessary. When working at BL2, wear two pairs of gloves when<br />
appropriate.<br />
o Remove gloves and wash hands when work with hazardous materials has been<br />
completed and before leaving the laboratory. Gloves that were used in BL1 or BL2<br />
work must not be worn outside the laboratory.<br />
o Do not wash or reuse disposable gloves. Dispose of used gloves with other<br />
contaminated laboratory waste. Hand washing protocols must be rigorously followed.<br />
5.4.4 Foot Protection<br />
Footwear appropriate to the worker’s work activities and conditions must be worn<br />
at all times. In a Technical Area such as a laboratory, or areas where chemical or<br />
biological materials are stored or handled, closed-toe shoes must be worn at all<br />
times, and open-toe shoes and sandals are not permitted. In some cases, LBNL<br />
requires workers to wear safety shoes for other hazards such as falling heavy<br />
objects. See PUB-3000, Section 19.3 (Foot Protection), for additional<br />
information.<br />
5.4.5 Respiratory Protection, Respirators, and Face Masks<br />
Workers who conduct procedures that may generate aerosols containing harmful<br />
levels of infectious agents must use controls such as biosafety cabinets (BSCs),<br />
enclosed containment systems, or respirators to avoid inhaling the agents. In<br />
general, a BSC should be used as the principal device in laboratories to contain<br />
infectious splashes or aerosols generated by numerous microbiological<br />
procedures (see Section 5.6.4.2 and Appendix E of this manual for additional<br />
BSC information). Other engineered containment devices such as safety<br />
centrifuge cups should also be used. When engineering controls are not feasible<br />
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or appropriate for the work, respirators may be needed to provide respiratory protection (e.g.,<br />
potential exposure to airborne transmissible disease agents during patient care).<br />
Respirators or face masks are occasionally worn by workers while conducting work with<br />
biological materials. There are important differences in design, purpose, and requirements<br />
between types of respirators and face masks that may be used for biological materials:<br />
• A respirator is a device designed and certified to protect the wearer from the inhalation<br />
of harmful atmospheres. A respirator may be a required respiratory control or worn<br />
voluntarily by the worker. A respirator might also provide face or product protection.<br />
Types and examples of some respirators:<br />
o A negative-pressure, air-purifying respirator is a tight-fitting respirator in which<br />
the air pressure inside the facepiece is negative during inhalation with respect to the<br />
ambient air pressure outside the respirator, and an air-purifying filter or cartridge<br />
removes specific air contaminants. Examples include the following types of cartridge<br />
and filtering facepiece respirators:<br />
‣ A negative-pressure, air-purifying, cartridge respirator is a respirator that<br />
uses a filter, sorbent, or catalyst housed inside a cartridge to remove<br />
contaminants from the air. Examples are respirators using an N95 or P100<br />
cartridge particulate filter that is 95% and 100% efficient, respectively.<br />
‣ A filtering facepiece respirator is a negative pressure, air-purifying respirator<br />
with a particulate filter as an integral part of the facepiece or with the entire<br />
facepiece composed of the filtering medium. A filtering facepiece respirator is<br />
sometimes incorrectly referred to as a dust mask or an N95 respirator. The<br />
term “dust mask” is an inaccurate term because a filtering facepiece respirator is<br />
a respirator, not a face mask. In addition, filtering facepiece respirators are not to<br />
be confused with N95 respirators, because only cartridge-type respirators use<br />
N95 filters.<br />
o A positive-pressure respirator is a respirator designed to maintain positive<br />
pressure inside the facepiece during exhalation and inhalation. Examples include a<br />
powered air-purifying respirator or a supplied-air respirator, which are not normally<br />
used at LBNL for biosafety purposes.<br />
• A face mask is a loose-fitting, disposable device that covers the worker’s nose and<br />
mouth and is not a respirator. Examples of face masks include products labeled as<br />
surgical, medical, dental, or isolation masks. A face mask might be worn in combination<br />
with eye protection to protect the nose and mouth from splatters or sprays, or the face<br />
mask might prevent the wearer from contaminating a product, patient, lab animal, or<br />
surface from particles (e.g., droplets) expelled from the nose or mouth. Face masks are<br />
not intended to protect the wearer from inhalation of airborne agents and must not be<br />
used for respiratory protection.<br />
Filtering facepiece respirator.<br />
Face mask<br />
Source: CDC courtesy of Moldex Metric Inc. (April 2009). Source: CDC (April 2009)<br />
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The following requirements are applicable to respirator uses, regardless of why the respirator is<br />
worn:<br />
• The respirator must be issued and worn in accordance with PUB-3000, Section 4.13,<br />
which includes the LBNL Respiratory Protection Program document. See these polices<br />
for additional information and consult your EH&S Industrial Hygienist. Voluntary use of a<br />
filtering facepiece that is not a required respiratory control requires a hazard evaluation<br />
and training before use, but unlike other required respirator uses, does not require a<br />
medical evaluation or fit-testing.<br />
• A risk assessment for the respirator must be documented in the <strong>Biosafety</strong> Work<br />
Authorization if the respirator use is related to the handling of biological materials.<br />
5.5 Labels and Signs<br />
Biological materials, agents, waste, potentially contaminated items, and laboratory rooms must<br />
be properly identified with labels, signs, or colors. Identification is needed so that<br />
responsibilities, material identities, hazards, or controls are communicated to workers, visitors,<br />
and others. These labels, signs, and colors must be displayed in accordance with LBNL policies<br />
and applicable requirements in the biosafety standards as summarized in this section.<br />
A biohazard label or red color is typically required to provide warning when a biohazardous<br />
condition may be present. A biohazard label is a sign that is predominantly fluorescent orange<br />
or orange-red. It also contains a biohazard symbol and the word “Biohazard” in a contrasting<br />
color. The label shown below displays the required biohazard legend:<br />
Biohazard label. Source: 29 CFR 1910.1030(g)(1)<br />
The following are biosafety criteria for labels, signs, and colors:<br />
• Information or labels should be visible on containers of biological materials or agents so<br />
that their content can be identified.<br />
• A biohazard label should be posted as a best management practice on primary<br />
equipment that uses, stores, or may be contaminated with RG2 agents or materials.<br />
• Work with BBP materials requires:<br />
o Biohazard labels, red containers, or red bags for waste containers, refrigerators,<br />
freezers, or other containers used to store, transport, or ship BBP materials<br />
o Biohazard labels, red containers, or red bags for containers or bags used for laundry<br />
that may be contaminated with BBP materials<br />
o Biohazard labels used to indicate which equipment parts remain contaminated with<br />
BBP materials<br />
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• Caution placards and other information must be posted at laboratory entrances,<br />
including a biohazard label for BL2 work areas. See additional details in the next<br />
paragraph.<br />
• Consult the Medical and Biohazardous Waste Generator’s Guide (PUB-3095) for details<br />
on labels and colors for sharps containers, waste containers, and waste bags in<br />
designated red-bag or clear-bag areas. A determination must be made in the <strong>Biosafety</strong><br />
Work Authorization as to whether the work will generate either regulated medical waste<br />
(i.e., red-bag waste that is regulated by the California Department of Health Services) or<br />
nonregulated biohazardous waste not (i.e., clear-bag waste).<br />
The following criteria must be implemented when posting an entrance to BL1 or BL2 laboratory<br />
area:<br />
• A Caution placard must be posted at the entrance to a Technical Area as specified in the<br />
LBNL CHSP.<br />
• Area PPE requirements must be included on the Caution Placard as specified Chapter<br />
19 of PUB-3000 (PPE).<br />
• A biohazard label must be posted (typically on a placard) at the entrance to each BL2<br />
work area to advise entering personnel of potential biological hazards.<br />
• When infectious agents (i.e., human pathogens) are present or there are organisms that<br />
require special provisions for entry (e.g., vaccination), additional biological hazard<br />
warning signage is required at the entrance to the laboratory. This signage must<br />
incorporate the universal biohazard symbol and include: the laboratory’s biosafety level;<br />
the identity of the agent(s) or the words “Infectious Agent(s)”; the name and telephone<br />
number of the supervisor, work lead, prinicipal investigator (PI), or other responsible<br />
personnel; and any special requirements or procedures for entering and exiting the<br />
laboratory. The CHSP Caution Placard will be used to accomplish these additional<br />
signage requirements. Any requirements for posting identities of agents or posting<br />
special entry and exit procedures will be specified in the BUA.<br />
• Other LBNL requirements for signage (e.g., radiological) may also apply to the entrance<br />
to be posted.<br />
5.6 Facilities, <strong>Laboratory</strong> Equipment, and Related Practices<br />
This section describes in a topical manner biosafety engineering and work practice controls<br />
related to standard facility design and laboratory equipment. Properly designed and used<br />
facilities, facility equipment, laboratory equipment, and lab tools provide protection for laboratory<br />
workers, persons outside the laboratory, the public, and the environment.<br />
See Sections 4.1, 4.2, and 4.3 of this manual for a general discussion of the principles of<br />
standard laboratory practices, equipment, and facilities. See Appendix C of this manual for a<br />
summary of standard laboratory practices, equipment, and facilities categorized as BL1 and<br />
BL2. See PUB-3000, Section 26.7.6, for a discussion of the facility design process at LBNL<br />
related to biosafety.<br />
5.6.1 Cleanable Surfaces and Furnishings<br />
In general, laboratory facilities and furnishings should be designed and maintained so that they<br />
are durable, will not trap contamination, and can be easily cleaned. The following BL1 and BL2<br />
laboratory criteria specified by BMBL and apply to this objective:<br />
• The laboratory should be designed so that it can be easily cleaned or decontaminated.<br />
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• Carpets and rugs in laboratories are not permitted.<br />
• <strong>Laboratory</strong> furniture must be capable of supporting anticipated loads and uses.<br />
• Spaces between benches, cabinets, and equipment should be accessible for cleaning.<br />
• Benchtops must be impervious to water and resistant to heat, organic solvents, acids,<br />
alkalis, and other chemicals.<br />
• Chairs used in laboratory work must be covered with a nonporous material that can be<br />
easily cleaned and decontaminated with an appropriate disinfectant.<br />
BMBL, fifth edition, added the new requirement noted above for chairs used in BL1 laboratory<br />
work. This new requirement for BL1 work involves significant costs to replace or modify chairs<br />
covered with porous material (e.g., cloth or mesh cushions). LBNL divisions may develop and<br />
document a corrective action that specifies a phase-in period to replace chairs with porous<br />
cushions used in BL1 laboratory work. During any phase-in period, new chairs used in BL1<br />
laboratory work must meet the requirement to be covered with a nonporous material.<br />
5.6.2 Doors and Windows<br />
<strong>Laboratory</strong> doors and windows provide a means to control personnel access to the laboratory,<br />
control vectors such as insects and rodents, and maintain laboratory air-flow balance. These<br />
controls are elements of standard BL1 or BL2 laboratory practices or facilities.<br />
The following biosafety criteria from BMBL and Appendix C are applicable to laboratory doors<br />
and windows:<br />
• BL1 and BL2 laboratories should have doors for access control. BL2 laboratory doors<br />
should be self-closing and have locks in accordance with LBNL standards. When the<br />
laboratory is unoccupied during nonbusiness hours, access to the laboratory should be<br />
controlled (e.g., by locking doors to the laboratory areas and/or doors to the building<br />
entrance).<br />
• BL1 laboratory windows that open to the exterior should be fitted with screens. BL2<br />
laboratory windows that open to the exterior are not recommended. However, if a BL2<br />
laboratory has windows that open to the exterior, they must be fitted with screens.<br />
5.6.3 Plumbing Systems and Equipment<br />
Plumbing-related systems and equipment that have requirements related to biosafety include<br />
handwashing sinks, sanitary sewer drains, water systems and backflow protection, emergency<br />
eyewash and shower units, and pipes. These systems provide needed utilities and containment<br />
when used properly. When used incorrectly, these systems may provide a route of exposure to<br />
personnel or the environment.<br />
5.6.3.1 Sinks and Handwashing<br />
BL1 and BL2 laboratories must have a sink with running water for handwashing. In BL2<br />
laboratories, the sink should be located near the exit door and may be manually, hands-free, or<br />
automatically operated. Handwashing sinks should be provided with a soap dispenser and<br />
paper towel dispenser as a best management practice. When working with BBP materials, the<br />
sink facility is called a handwashing facility. A handwashing facility must have an adequate<br />
supply of potable running water, soap, and single-use towels or hot-air drying machines.<br />
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Personnel working at BL1 or BL2 laboratories must wash their hands a) after working with<br />
potentially hazardous materials, recombinant materials, and animals; b) after removing gloves;<br />
and c) before leaving the laboratory.<br />
Handwashing with soap and water. Source: LBNL EH&S.<br />
When work involves potential exposure to BBP materials outside of the laboratory (e.g., health<br />
care) and handwashing facilities (e.g., potable water and a sink) are not feasible, an appropriate<br />
antiseptic hand cleanser in conjunction with clean cloth/paper towels or antiseptic towelettes<br />
may be provided. When antiseptic hand cleansers or towelettes are used, hands must be<br />
washed with soap and running water as soon as possible.<br />
5.6.3.2 Drains and Disposal<br />
<strong>Laboratory</strong> sinks must typically be drained into the sanitary sewer system. In general and as a<br />
best management practice, liquids that contain biological material that is potentially viable or<br />
biologically active and not contaminated with other hazardous or radioactive material should be<br />
properly decontaminated with a disinfectant before disposal into the sanitary sewer system (see<br />
Section 5.7 (Decontamination, Waste, and Decommissioning) below. All biological liquid<br />
material considered medical/biohazardous waste must be decontaminated before disposal (see<br />
the Medical and Biohazardous Waste Generator’s Guide (PUB-3095)).<br />
5.6.3.3 Water Systems and Backflow Prevention<br />
Backflow-prevention devices are required in building water systems or<br />
connection points to prevent contaminated liquid or water from being<br />
inadvertently sucked into the potable water system of the building. For<br />
example, a backflow-prevention device called a vacuum breaker is often<br />
integrated into the gooseneck of the laboratory sink faucet. This device<br />
prevents liquids from being drawn up into the faucet’s water system in case<br />
a laboratory worker connects tubing to the faucet’s serrated hose end.<br />
<strong>Laboratory</strong> faucet with backflow prevention device. Source: Grainger (May 2010).<br />
Potable water is typically supplied to each laboratory building. This water supply is separated<br />
through backflow-prevention devices in the building’s plumbing system into potable and<br />
industrial water systems or sources. Plumbing fixtures that must be supplied with potable water<br />
include emergency eyewashes and showers and fixtures used in restrooms, in kitchens, or as<br />
drinking sources (e.g., toilets, sinks, or drinking faucets). Water connected to other fixtures or<br />
equipment in the laboratory or building must be separated from the fixtures that require potable<br />
water by proper backflow-prevention devices. When the water system is correctly designed and<br />
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labeled, water pipes labeled as industrial water are separated from the potable water system by<br />
a backflow-prevention device(s) in the building’s water system. Connection of laboratory sinks,<br />
laboratory equipment, or industrial equipment to pipes that also provide water to potable fixtures<br />
requires proper installation of a backflow-prevention device. Contact your building’s facilities<br />
service provider (e.g., LBNL Facilities) for proper plumbing advice and hardware.<br />
5.6.3.4 Emergency Eyewashes and Showers<br />
Emergency eyewash is a plumbing unit designed to properly flush chemical,<br />
biological, or other hazardous agents off the face and out of mucous<br />
membranes such as the eyes. Use of an eyewash prevents injury to the eye or<br />
exposed body surfaces. It also prevents an agent from penetrating into the<br />
body. An emergency eyewash must be readily available to BL2 work areas.<br />
Ready access to a sink and emergency eyewash without strict distance-to-use requirements is<br />
normally sufficient for washing biological contamination from the body, because:<br />
• Intact skin is considered a good barrier to most biological agents;<br />
• Biological agents do not cause immediate tissue damage to skin or eyes; and<br />
• An eyewash unit works well to flush the face (e.g., eyes, nose, and mouth areas).<br />
However, in areas where there is also a splash hazard to certain chemicals (e.g., corrosives,<br />
eye irritants, chemicals that are toxic via skin or eye contact), the CHSP specifies that a<br />
combination emergency eyewash and shower unit must be reachable within 10 seconds via<br />
an unobstructed path. When combination eyewash and shower units are provided for potential<br />
chemical exposures, the number and placement of units is often sufficient to also meet the<br />
biosafety requirement for an emergency eyewash being readily available in BL2 work areas.<br />
Installation, maintenance, and use of all emergency eyewash and shower units must comply<br />
with the eyewash and shower requirements in the CHSP.<br />
5.6.4 Ventilation and Hoods<br />
Room ventilation and hoods provide for control of potential biological aerosols, other harmful<br />
atmospheres, odors, and smoke caused by fires by providing general room air dilution,<br />
directional air flow, and enclosure to contain and exhaust airborne agents. Room ventilation and<br />
hoods must be designed and maintained to established standards, guidelines, and LBNL<br />
policies.<br />
5.6.4.1 Room Ventilation<br />
The volume and balance of laboratory room ventilation are important safety controls. There may<br />
be specific ventilation requirements for specific laboratory uses, but the following design<br />
requirements generally apply to laboratory rooms that use biological and hazardous materials:<br />
• <strong>Laboratory</strong> rooms must be negative in pressure relative to any fire exit corridor.<br />
• <strong>Laboratory</strong> rooms should be negative in pressure relative to nonlaboratory rooms (e.g.,<br />
offices).<br />
• BL2 rooms should be negative in pressure relative to other areas. If researchers indicate<br />
that a BL2 area should be positive in pressure for research purposes (e.g.,<br />
contamination control), a negative-pressure anteroom leading to the BL2 area may be<br />
required, or the risk assessment process may indicate that it is acceptable for air to flow<br />
from the BL2 area into another laboratory area.<br />
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• Air exhausted from laboratories should not be recirculated to rooms outside the<br />
laboratory.<br />
• <strong>Laboratory</strong> exhaust ventilation flow rates must meet minimum requirements (e.g., 1<br />
cubic foot per minute of exhaust air per square foot of laboratory space).<br />
5.6.4.2 Hoods and <strong>Biosafety</strong> Cabinets<br />
Hoods are enclosures or shaped inlets designed to conduct contaminated air into an exhaust<br />
duct system, or a filter that safely captures the contaminant. This section discusses hoods<br />
designed to provide for the safety of the worker or the environment such as biosafety cabinets,<br />
laboratory fume hoods, exhausted equipment enclosures, gloveboxes, and other local exhaust<br />
points. This section does not cover ventilated enclosures such as laminar flow clean benches<br />
that are not designed to protect the worker or the environment from contaminated air.<br />
Hoods used for safety must be designed, installed, tested, and surveyed in accordance with<br />
LBNL Environment, Safety, and Health (ES&H) standards and policies for all hoods and highefficiency<br />
particulate air (HEPA) filters (see PUB-3000, Section 4.6). The EH&S Industrial<br />
Hygiene Group manages the ventilation safety program and records hood locations, surveys,<br />
and testing in the Ventilation Database. Supervisors or work leads should ensure that hood<br />
safety survey stickers or labels indicate the hood has been surveyed or tested and determined<br />
to be safe for use.<br />
<strong>Laboratory</strong> hood. Hood survey label and monitor. BSC certification label.<br />
Source: LBNL EH&S Source: LBNL EH&S Source: LBNL EH&S<br />
5.6.4.2 (a) Biological Safety Cabinets and Other HEPA-filtered Containment<br />
Biological safety cabinets or biosafety cabinets (BSCs) are hoods with HEPA<br />
filters that provide personnel, environmental, and product protection when<br />
appropriate practices and procedures are followed. Appendix E of this manual<br />
summarizes BSC types and provides additional BSC information. Various types<br />
of BSCs and similar hoods are used at LBNL. Listed below are more common<br />
types:<br />
• Typical BSCs used at LBNL are Class II, Type A2 BSCs built by BSC manufacturers.<br />
These BSCs discharge exhaust directly though a HEPA filter and into the laboratory.<br />
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• Class II, Type B1 BSCs are used less commonly than Class II, Type A2 BSCs. These<br />
BSCs discharge exhaust air through a HEPA filter, but exhaust air is then ducted to the<br />
roof so that toxic chemicals that cannot be filtered by the BSC’s HEPA filter are not<br />
exhausted back into the laboratory.<br />
• Researchers sometimes acquire or build equipment such as cell sorters or robotic<br />
enclosures that cannot be categorized as a BSC. These specialized pieces of equipment<br />
should be tested and managed using many of the same BSC ventilation, testing, and<br />
management principles.<br />
BSCs or other safety equipment, PPE, or other physical containment devices (e.g., safety<br />
centrifuge cups) must be used whenever procedures with a potential to create infectious<br />
aerosols or splashes are conducted, or whenever high concentrations or large volumes of<br />
infectious agents are used. Examples of such procedures include pipetting, centrifuging,<br />
grinding, blending, shaking, mixing, vortexing, sonicating, opening containers with pressure<br />
differentials, or harvesting infected tissues. The BSC is the principal BL2 device used to provide<br />
containment of infectious splashes or aerosols generated by many microbiological procedures.<br />
<strong>Biosafety</strong> cabinet. Source: LBNL EH&S<br />
Aerosols and aerosol generation.<br />
Source: unidentified.<br />
BSCs must be:<br />
• Designed, installed, tested, and surveyed in accordance with LBNL ES&H standards and<br />
policies for all hoods and HEPA filters (i.e., PUB-3000, Section 4.6).<br />
• Designed, constructed, installed, operated, used, decontaminated, and tested in<br />
accordance with BSC guidelines in Appendix A of BMBL and summarized in Appendix E<br />
of this manual.<br />
• Managed in accordance with the following list of BSC policies:<br />
o The <strong>Biosafety</strong> Work Authorization must include specific BSC uses and information,<br />
and an assessment of procedures for RG2 materials that have the potential to<br />
produce aerosols or splashes.<br />
o The EH&S Industrial Hygiene Group is responsible for maintaining records of BSC<br />
locations, surveys, and testing in the Ventilation Database, and managing surveys,<br />
tests, and gaseous decontaminations of BSCs. BSC testing, certification, and<br />
gaseous decontaminations are performed by a subcontractor. The EH&S Division<br />
normally pays for each BSC’s annual safety test and certification when the BSC is<br />
used for safety.<br />
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o<br />
o<br />
o<br />
Line management owners of BSCs have primary responsibility for paying costs and<br />
ensuring the proper purchase, use, maintenance, testing, and decontamination of<br />
BSCs.<br />
BSCs used for BL1, BL2, or other safety levels must be tested and certified before<br />
initial use, after being moved, and on a nominal one-year cycle.<br />
BSCs and their filters must be decontaminated with a gaseous decontaminant before<br />
being moved or repaired internally, unless an alternative procedure is approved by<br />
the <strong>Biosafety</strong> Officer.<br />
o BSCs must be installed and operated according to the manufacturer’s<br />
recommendations.<br />
When a new BSC is needed or a BSC needs to be moved, contact the EH&S Industrial Hygiene<br />
Group or <strong>Biosafety</strong> Office for assistance with selecting, testing, and decontaminating BSCs.<br />
5.6.4.2 (b) <strong>Laboratory</strong> and Other Hoods<br />
Other hoods that are not exhausted through HEPA filters are typically used for most<br />
nonbiological laboratory airborne hazards or concerns. These hoods are generally used for<br />
control of chemical hazards, gas hazards, process emissions, odors, and heat. Examples of<br />
such hoods include laboratory-type (“fume”) hoods, gas chromatograph local exhaust points,<br />
and autoclave canopy hoods. These hoods can be used for chemicals including biological<br />
toxins, but are not adequate for control of potential infectious biological aerosols or toxic<br />
particulate.<br />
5.6.5 Food Facilities and Eating<br />
Eating, drinking, smoking, handling contact lenses, applying cosmetics, and<br />
storing food for human consumption are not permitted in BL1 and BL2<br />
laboratory areas. Food must be stored outside the laboratory area in cabinets<br />
or refrigerators designated and used for this purpose.<br />
5.6.6 <strong>Laboratory</strong> Tools and Equipment<br />
5.6.6.1 Sharps<br />
This section describes types and hazards of sharps, states requirements from biosafety<br />
standards, and outlines LBNL’s policies on sharps related to biosafety. A sharp is an object that<br />
can penetrate the skin. A sharp is often a tool, device, or material that typically has a sharp<br />
edge or point such as a needle, scalpel, razor, blade, broken glass piece, broken capillary tube,<br />
or an exposed wire end.<br />
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Sharps examples. Source: unidentified.<br />
Sharps may cause cut or puncture wounds. In addition, sharps contaminated with a biological<br />
material may result in the parenteral inoculation of a worker with an infectious or recombinant<br />
agent that may cause a laboratory-acquired infection or another disease. Parenteral is an<br />
adjective that refers to a route of administration that involves piercing the mucous membranes<br />
or skin barrier through events such as punctures, lacerations, abrasions, and bites.<br />
Sharp tools are often designed with a built-in safety feature or mechanism that effectively<br />
reduces the risk of accidental skin penetration and a biological exposure incident. These tools<br />
are called safety-engineered sharps or safety-engineered needles. Examples include<br />
devices that blunt, sheath, or withdraw the sharp when the sharp edge or point has been used<br />
or is not in use. The OSHA Bloodborne Pathogens Standard has specific definitions and<br />
requirements for the use of safety-engineered sharps that are discussed in the next section.<br />
5.6.6.1 (a) Sharps Risk Assessment and Documentation<br />
Use of sharps should be assessed as part of the risk assessment for work with biological<br />
materials. The following general process should be followed:<br />
• The use of sharps is assessed and controls are defined in each:<br />
o Worker’s JHA for use of sharp tools<br />
o BUA and ECP for all sharps involved with RG2 and BBP materials<br />
• The sharps risk assessment that is conducted when developing the BUA or ECP should:<br />
o Evaluate what sharps may be needed or might be present<br />
o Evaluate if a safer alternative to the sharp can be used to accomplish the work. For<br />
example: Plasticware should be substituted for glassware whenever possible at BL2.<br />
o Evaluate available sharp tools and pick the safest device that will accomplish the<br />
work. For example:<br />
– Safety-engineered needles rather than needles that cannot be sheathed after<br />
use<br />
– Scalpels with longer handles that are often more controllable than razor<br />
blades<br />
– Razor blade holders rather than unprotected blades<br />
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o<br />
Evaluate the machine’s point-of-operation guarding if there is an exposed sharp<br />
edge or point on a machine. For example:<br />
– A cutting blade on a microtome<br />
– Needles on a colony picking robotic machine<br />
Use of sharps with RG2 materials should be documented in the BUA. In addition, use of sharps<br />
with BBP materials must be documented in the BUA or ECP, and the annual review and update<br />
of these plans must reflect changes in technology that eliminate or reduce exposure to BBPs<br />
(e.g., newly available devices designed to reduce exposure).<br />
In addition, when the BUA or ECP covers medical procedures or devices that involve exposure<br />
to BBP material:<br />
• Sharps with engineered sharps injury protection (ESIP) must be specified and used<br />
with a few exceptions. OSHA defines sharps with ESIP as a non-needle sharp or a<br />
needle device used for withdrawing body fluids, accessing a vein or artery, or<br />
administering medications or other fluids, with a built-in safety feature or mechanism that<br />
effectively reduces the risk of an exposure incident. See the OSHA fact sheet on safety<br />
needles and needleless systems for additional information. When a needle must be used<br />
as described above, a needle device with ESIP must be used unless one of the following<br />
four OSHA exceptions is documented in the BUA or ECP:<br />
o No needleless systems or sharps devices with ESIP are available in the marketplace<br />
for the procedure.<br />
o A licensed health care professional directly involved with a patient’s care determines<br />
that available needleless systems or sharps devices with ESIP would compromise<br />
the patient’s care or safety.<br />
o Available needleless systems and sharps devices with ESIP are not more effective in<br />
preventing exposure to BBPs than the alternative being used.<br />
o Sufficient information is not available on the safety performance of needleless<br />
systems or sharps devices with ESIP available in the marketplace, and the<br />
supervisor or work lead is actively evaluating such devices.<br />
• The BUA or ECP’s annual review and update must:<br />
o Reflect changes in technology that eliminate or reduce exposure to BBPs (e.g.,<br />
newly available medical devices designed to reduce needlesticks).<br />
o Document consideration and implementation of appropriate, commercially available,<br />
effective, and safer medical devices.<br />
o Document how input was solicited from nonmanagerial employees responsible for<br />
direct patient care who are potentially exposed to injuries from contaminated sharps<br />
in the identification, evaluation, and selection of effective engineering and work<br />
practice controls.<br />
5.6.6.1 (b) Sharps Use and Disposal<br />
Sharps must be used and disposed of in accordance with:<br />
• <strong>Laboratory</strong> BL1 and BL2 criteria in Appendix C of this manual<br />
• The Medical and Biohazardous Waste Generator’s Guide (PUB-3095)<br />
When applying force to a handheld sharps tool, the sharp end of the tool should be pointed<br />
away from the worker’s body.<br />
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Syringe with capped needle, needle disposal without recapping in sharps container, and glass<br />
sharps container. Sources: unidentified.<br />
5.6.6.1 (c) Sharps Injury Reporting and Log<br />
See Section 5.10 of this manual for requirements and procedures related to injury and accident<br />
reporting, and Section 5.3 for requirements and responsibilities related to logging sharps<br />
injuries.<br />
5.6.6.2 Centrifuges<br />
Rotational energies involved with most centrifuges can generate two serious hazards:<br />
mechanical failure, and dispersion of aerosols or droplets. This section describes general<br />
classes of centrifuges, and general operation and maintenance guidelines to minimize<br />
centrifuge hazards. Elements of these guidelines may or may not be applicable to specific<br />
centrifuge operations. Information in this section was adapted from the University of Minnesota’s<br />
“Bio Basics Fact Sheet: Centrifuge Safety.”<br />
There are three general classes of centrifuges:<br />
• Low-speed centrifuges that do not exceed 5,000 rpm are commonly made<br />
for benchtop use.<br />
• High-speed centrifuges that do not exceed 25,000 rpm may include<br />
benchtop or floor models.<br />
• Ultracentrifuges that may exceed 100,000 rpm are often found in core<br />
equipment areas. These centrifuges are the most expensive and<br />
potentially the most dangerous.<br />
5.6.6.2 (a) Centrifugation Operation Guidelines<br />
Before centrifugation:<br />
o Each operator should review or be instructed on proper operating procedures and<br />
necessary information from the user manual.<br />
o Use only rotors compatible with the centrifuge. Check the expiration date for<br />
ultracentrifuge rotors.<br />
o Check tubes, bottles, and rotors for cracks and deformities before each use.<br />
o Make sure that the rotor, tubes, and spindle are dry and clean.<br />
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o<br />
o<br />
o<br />
o<br />
o<br />
o<br />
Examine O-rings. Replace if worn, cracked, or missing.<br />
Never overfill centrifuge tubes (do not exceed three-fourths full).<br />
Cap tubes before centrifugation.<br />
Balance buckets, tubes, and rotors properly.<br />
Check that the rotor is seated on the drive correctly, put lid on rotor, close the lid on the<br />
centrifuge, and secure it.<br />
When using swinging bucket rotors, make sure that all buckets are hooked correctly and<br />
move freely.<br />
During centrifugation:<br />
o Keep the lid closed at all times during operation. Never open a centrifuge until the rotor<br />
has stopped.<br />
o Do not exceed safe rotor speed.<br />
o The operator should not leave the centrifuge until full operating speed is attained and the<br />
machine appears to be running safely without vibration.<br />
o Stop the centrifuge immediately if an unusual condition (e.g., noise or vibration) begins,<br />
and rebalance the load if needed. If a loud noise indicates significant mechanical failure<br />
such as rotor or container breakage, follow guidelines in Appendix G, Section G.5<br />
(Centrifuge Malfunction or Spills) of this manual. Report other unusual conditions to the<br />
work lead. Evaluation by a manufacturer’s representative may be needed.<br />
After centrifugation:<br />
o Allow the centrifuge to come to a complete stop before opening.<br />
o Wear gloves to remove rotor and samples.<br />
o Check inside of centrifuge for possible spills and leaks. Disinfect centrifuge and rotor<br />
thoroughly if necessary.<br />
o Wash hands after removing gloves.<br />
Centrifuging RG2 materials:<br />
Follow the safety procedures noted above plus:<br />
o Place a biohazard label on the centrifuge.<br />
o Wear gloves when handling tubes or rotors.<br />
o Avoid the use of celluloid tubes with biohazards. If celluloid tubes must be used, an<br />
appropriate chemical disinfectant must be used to decontaminate them.<br />
o Use sealed safety cups, safety buckets, or sealed rotors with O-ring as secondary<br />
containment.<br />
o Fill centrifuge tubes, load into rotors, remove from rotors, and open tubes within a<br />
biological safety cabinet.<br />
o Wipe exterior of tubes or bottles with disinfectant prior to loading into rotor or bucket.<br />
Seal rotor or bucket, remove outer gloves, and transport to the centrifuge.<br />
o Wait at least 10 minutes after the run to allow aerosols to settle before opening the<br />
centrifuge. Check for possible spills or leaks.<br />
o Decontaminate centrifuge interior, safety cups or buckets, and rotors if spills or tube<br />
breakage occurs. Follow guidelines in Appendix G, Section G.5 (Centrifuge Malfunction<br />
or Spills).<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.<br />
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5.6.6.2 (b) Centrifuge Maintenance Guidelines<br />
Moisture, chemicals, strong cleaning agents, and other substances can promote corrosion of<br />
centrifuge parts and cause centrifuge failure. Long-term centrifuge use may also cause<br />
centrifuge failure. The following are general maintenance recommendations:<br />
o<br />
o<br />
o<br />
o<br />
o<br />
o<br />
o<br />
o<br />
o<br />
Follow manufacturer instructions for maintenance and cleaning.<br />
Keep the centrifuge clean and dry.<br />
Clean up all nonhazardous spills immediately. Follow guidelines in Appendix G, Section<br />
G.5 (Centrifuge Malfunction or Spills), for biohazardous spills.<br />
Decontaminate rotors used with biological or radioactive materials (e.g., use 10% bleach<br />
for 30 minutes followed by 70% ethanol; let air dry to clean rotors and cups).<br />
Never clean rotors and associated parts with abrasive wire brushes.<br />
Store the rotor upside down in a dry place, with lids or plugs removed, to prevent<br />
condensation.<br />
Remove adapters after use. Inspect them for corrosion.<br />
Inspect rotors regularly. Remove rotors from use if they show any signs of defects.<br />
Report the defective rotors to a manufacturer's representative for inspection.<br />
To avoid rotor failure, record the length of time and speed for each high-speed rotor in a<br />
log book. Track and discard rotors according to the manufacturer's recommended<br />
schedule.<br />
5.6.6.3 Waste Containers<br />
Containers used to hold medical/biohazardous waste, sharps waste, or pathological waste must<br />
be placed in biohazardous waste containers and bags in accordance with the Medical and<br />
Biohazardous Waste Generator’s Guide (PUB-3095).<br />
5.6.6.4 Equipment Connected to Building Vacuum Systems<br />
House vacuum systems used to evacuate air from containers, enclosures, or lines that contain<br />
biological materials should be equipped with a HEPA filter (or equivalent filter) to prevent<br />
biological materials or aerosols from being sucked inadvertently into the vacuum line. This is a<br />
general guideline for all biological materials, but the BMBL criteria for BL2 laboratory facilities<br />
specifically states that vacuum lines should be protected with a HEPA filter or equivalent, the<br />
filter must be replaced as needed, and liquid disinfectant traps may be required. Liquid<br />
disinfectant traps typically used in conjunction with tissue culture work inside a BSC are further<br />
detailed in Appendix E, Section E.3.3, of this manual.<br />
5.7 Decontamination, Waste, and Decommissioning<br />
Work surfaces, work areas, furniture, equipment, materials, and wastes involved in most work<br />
with biological materials must be routinely decontaminated during the work, and prior to transfer<br />
or disposal. This section 1) discusses principles of decontamination, 2) provides examples of<br />
antimicrobials used to decontaminate, and 3) summarizes or references requirements from the<br />
standards related to antimicrobials and decontamination of surfaces, equipment, and wastes.<br />
See the following policy sections and standards for additional information:<br />
• Appendix F of this manual for more detailed information on decontamination processes<br />
and antimicrobials.<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.<br />
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• PUB-3000, Section 26.5.7, for a summary of decontamination and waste standards and<br />
LBNL policies.<br />
• BMBL, Appendix B, for BMBL guidance on strategies for decontaminating laboratory<br />
surfaces, items, and areas.<br />
5.7.1 Decontamination Processes and Antimicrobials<br />
Decontamination is a process that uses an antimicrobial to reduce or inactivate<br />
biological contaminants or components to an acceptable level so as to reduce or<br />
eliminate the possibility of transmitting pathogens to undesired hosts. An<br />
antimicrobial is a chemical or physical agent that is used in a decontamination<br />
process to prevent microbial growth. Prevention of microbial growth and pathogen<br />
transmission is needed to control contamination of the work, and to prevent disease in hosts<br />
such as laboratory workers, the general public, and other organisms in the environment. The<br />
decontamination process, level, antimicrobial, frequency, and specific method should be based<br />
on the work activity, agents that need inactivation, and decontamination objectives or<br />
requirements. Definitions of decontamination processes and levels, along with common<br />
examples of antimicrobials and processes, are listed in Table 11 below. Refer to Appendix F of<br />
this manual for additional information on decontamination and antimicrobials.<br />
When using a chemical or physical antimicrobial to ensure decontamination is accomplished for<br />
biosafety purposes (i.e., protection of workers, public, agriculture, or environment):<br />
• There should be information indicating that the selected antimicrobial will be effective<br />
when used in a certain manner for the biological materials or agents and equipment or<br />
surfaces that need to be decontaminated; and<br />
• The antimicrobial should be used in accordance with its antimicrobial activity capabilities<br />
and conditions of use.<br />
Antimicrobial information in Appendix F of this manual, information provided by manufacturers<br />
(e.g., labels or technical specifications), and other information may be used for selecting and<br />
using the appropriate antimicrobial. Effective decontamination can also be ensured by using an<br />
Environmental Protection Agency (EPA)–registered or Food and Drug Administration<br />
(FDA)–cleared antimicrobial product within its manufacturer-specified limits. See Appendix F,<br />
Section F.2.3, of this manual for additional information on commercial disinfectants and<br />
sterilants registered or cleared by the EPA and FDA.<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.<br />
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General<br />
Decontamination<br />
Process and Level<br />
Sterilization is the<br />
process of completely<br />
destroying all living<br />
microorganisms and<br />
viruses.<br />
Disinfection is the<br />
process of generally<br />
eliminating nearly all<br />
recognized pathogenic<br />
microorganisms but<br />
not necessarily all<br />
microbial forms (e.g.,<br />
bacterial spores) on<br />
inanimate objects.<br />
Sanitization is the<br />
process of generally<br />
reducing microbes by<br />
the use of general<br />
cleaning agents.<br />
Antisepsis is the<br />
application of a liquid<br />
antimicrobial chemical<br />
to human or animal<br />
living tissue.<br />
Table 11<br />
Decontamination Processes, Levels, and Antimicrobial Examples*<br />
Antimicrobial<br />
Example<br />
Wet heat-steam<br />
Dry heat<br />
Wet or dry heat<br />
Chlorine in<br />
sodium<br />
hypochlorite<br />
Ethyl or isopropyl<br />
alcohol<br />
Iodine<br />
Formaldehyde<br />
Ultraviolet (UV)<br />
light<br />
Soap and water,<br />
quaternary<br />
ammonium<br />
compounds, or<br />
disinfectants<br />
Iodine<br />
Example Decontamination Process<br />
Autoclave at 121°C (250°F) for 15 minutes or<br />
more.<br />
Bake at 171°C for at least 1 hour, or Incinerate.<br />
Place solid waste in a biohazardous waste<br />
container for autoclaving or incineration by a<br />
licensed LBNL subcontractor.<br />
Wipe clean hard work surfaces and equipment<br />
with a 1% solution of fresh household bleach, and<br />
allow to air dry for intermediate-level disinfection.<br />
Add household bleach to liquid biohazardous<br />
spills or liquid waste until a 10% concentration of<br />
household bleach is achieved for 20 minutes for<br />
high-level disinfection.<br />
Wipe clean hard work surfaces with a 70%<br />
solution of alcohol for low-level disinfection.<br />
Submerge precleaned items in 70% alcohol for 10<br />
minutes for intermediate-level disinfection.<br />
Wipe clean hard work surfaces with an idophor<br />
such as Wescodyne ® for intermediate-level<br />
disinfection.<br />
Use formaldehyde in water (i.e., formalin) or in<br />
alcohol at 1% to 8% for low- to high-level<br />
disinfection, respectively.<br />
UV light inside biosafety cabinet. Not recommended<br />
as a biosafety control because<br />
disinfection is limited, and light damages human<br />
tissue.<br />
Launder clothing or generally clean laboratory,<br />
restroom, room, and equipment surfaces.<br />
Wash hands with Betadine ® skin cleanser<br />
containing povidone-iodine (PVP-I), or apply<br />
10% PVP-I solution in water to the injection site<br />
on a research animal.<br />
* See Appendix F of this manual for additional information and specific conditions.<br />
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The OSHA Bloodborne Pathogens Standard requires that work surfaces contaminated with BBP<br />
material (as defined in Section 3.3.4 of this manual) must be cleaned with an “appropriate<br />
disinfectant.” Appropriate disinfectants include household bleach diluted to concentrations<br />
ranging from 1% (i.e., 1:100) to 10% (i.e., 1:10) in water and certain disinfectants registered by<br />
the EPA or FDA. Household bleach at these concentrations is one of the most common and<br />
effective disinfectants used in the laboratory. Household bleach is a water-based solution of<br />
sodium hypochlorite (NaOCl) with a typical concentration of 5.25% by weight of the active<br />
sodium hypochlorite ingredient. In the U.S., Clorox ® bleach is the best-known brand. See<br />
Appendix F, Section F.3.2.1, of this manual for additional details on the properties and use of<br />
bleach.<br />
Steam heat used in autoclaves is also a common laboratory antimicrobial. An autoclave is a<br />
piece of equipment with a chamber used to sterilize items by applying wet heat (i.e., highpressure<br />
steam) at temperatures above the normal boiling point of water and pressures above<br />
normal atmospheric pressure. Autoclaves are used to sterilize laboratory equipment or materials<br />
such as glassware, media, reagents, or waste. See Appendix F, Section F.5, of this manual for<br />
general information and guidelines on autoclave principles, operation, and maintenance typically<br />
needed to sterilize equipment and ensure operator safety.<br />
5.7.2 Surface and Equipment Decontamination<br />
In general, surface and equipment decontamination guidelines for BL1 and BL2 areas include:<br />
• The work area should be cleaned and maintained in a sanitary condition.<br />
• Surfaces or equipment where work with biological materials is conducted should be<br />
routinely decontaminated.<br />
• Surfaces, furniture, or equipment contaminated with biohazardous materials should be<br />
decontaminated after spills and before repair, maintenance, or removal from the<br />
laboratory.<br />
<strong>Biosafety</strong> cabinet surface decontamination. Source: unidentified.<br />
<strong>Laboratory</strong> standard microbiological practices from BMBL and NIH Guidelines (see Appendix C<br />
of this manual) specifically require the following surface and equipment decontamination<br />
practices:<br />
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• At BL1 and BL2, work surfaces must be decontaminated with an appropriate disinfectant<br />
after completion of work and after any spill or splash of a potentially infectious or viable<br />
recombinant material.<br />
• At BL2, laboratory equipment should be decontaminated on a routine basis and after<br />
spills, splashes, or other potential contamination.<br />
o Spills involving infectious materials must be contained, decontaminated, and cleaned<br />
by staff properly trained and equipped to work with infectious material.<br />
o Equipment must be decontaminated before repair, maintenance, or removal from the<br />
laboratory.<br />
5.7.3 Waste Decontamination and Disposal<br />
<strong>Laboratory</strong> standard microbiological practices and special practices from BMBL and NIH<br />
Guidelines (see Appendix C of this manual) for BL1 and BL2 specifically require that all cultures,<br />
stocks, and other potentially infectious or viable recombinant materials must be decontaminated<br />
before disposal using an effective method. Effective decontamination methods are covered in<br />
Section 5.7.1 and Appendix F of this manual. Responsibility for decontamination starts with the<br />
waste generator. In some cases, the waste generator performs the actual decontamination. In<br />
other cases, the generator selects the decontamination system and then prepares the waste<br />
materials for treatment by others.<br />
LBNL uses the term medical/biohazardous waste to describe wastes that are biological<br />
materials, or that may be contaminated with biological materials and require inactivation (i.e.,<br />
decontamination) in an approved manner prior to final disposal. See the Medical and<br />
Biohazardous Waste Generator’s Guide (PUB-3095) for LBNL definitions and requirements for<br />
disposal of medical/biohazardous waste. Decontamination, collection, and disposal of<br />
medical/biohazardous waste will be conducted in accordance with PUB-3095, methods<br />
approved or known to inactivate the materials, and any requirements specified in regulatory<br />
permits (e.g., U.S. Department of Agrigulture (USDA)) issued to individuals.<br />
Contaminated items considered<br />
medical/biohazardous waste.<br />
Source: Michigan State University, Office<br />
of Radiation, Chemical, and Biological<br />
Safety (May 2010).<br />
Labeled biohazardous container lined with a red<br />
biohazard bag. Transfer of closed biohazard bag to<br />
waste pickup container. Source: LBNL EH&S.<br />
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The EH&S Division Waste Management Group manages the disposal of LBNL<br />
medical/biohazardous waste. It also manages the contract with a licensed subcontractor that<br />
transports, treats, and disposes of LBNL’s solid waste as regulated medical waste. Examples of<br />
such solid waste include materials that are placed in lined and labeled biohazardous waste<br />
containers, biologically contaminated sharps in sharps containers, and pathological materials<br />
such as carcasses.<br />
Although Waste Management Group manages the waste component of the <strong>Biosafety</strong> Program,<br />
it is the responsibility of the waste generator to ensure that medical/biohazardous waste is<br />
properly:<br />
• Inactivated before disposal (e.g., treatment of liquid culture with bleach prior to sanitary<br />
sewer disposal), or<br />
• Contained in durable leakproof containers, labeled, and documented in the work area<br />
before further handling by the EH&S Division or the licensed LBNL subcontractor.<br />
The Joint Genome Institute (JGI) is the only LBNL site that does not use a licensed<br />
subcontractor to dispose of solid biological waste as regulated medical waste. Instead, JGI uses<br />
autoclaves to sterilize solid, recombinant, biohazardous waste prior to disposal as detailed in<br />
PUB-3095.<br />
5.7.4 <strong>Laboratory</strong> and Equipment Decommissioning and Moves<br />
All surfaces and equipment should be cleaned and put into a safe condition prior to vacating<br />
laboratory spaces or relocating equipment. The <strong>Laboratory</strong>’s Space Management Policy in the<br />
Regulations and Procedure <strong>Manual</strong> (§1.20) requires that laboratory and shop spaces be cleared<br />
of debris and contamination prior to transfer of ownership. The decommissioning section of the<br />
CHSP provides a good general description of requirements and resources for decommissioning<br />
laboratories and equipment.<br />
Decommissioning should include decontamination and waste disposal methods appropriate for<br />
the biological materials that may be present and the materials or equipment to be<br />
decontaminated. Decommissioning may include:<br />
• <strong>Laboratory</strong> surfaces and equipment should be decontaminated. Household bleach is<br />
commonly used in the concentrations and manners discussed in Appendix F, Section<br />
F.3.2.1, of this manual. Appendix F also provides other decontamination methods.<br />
• Biohazard labels should be posted on any equipment or containers that still contain or<br />
may be contaminated with RG2 agents or materials as discussed in Section 5.5.<br />
• Dispose of medical/biohazardous waste as described in Section 5.7.4.<br />
• BSCs and their filters must be decontaminated with a gaseous decontaminant prior to<br />
being moved, unless approved by the <strong>Biosafety</strong> Officer (see Section 5.6.4.2 (a)).<br />
• Custodians of equipment that will be moved by the LBNL Transportation Department<br />
must verify that the equipment is free of biological, chemical, and radiological hazards.<br />
This verification is accomplished when the equipment custodian places a completed<br />
LBNL Transportation Authorization Form on each piece of equipment to be transported<br />
(see PUB-3000, Section 5.8.13.1). Transportation Authorization Forms are issued to<br />
equipment custodians when they request an equipment move through the Work Request<br />
Center.<br />
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5.8 Access and Security<br />
<strong>Laboratory</strong> supervisors and work leads conducting work at BL1 or BL2 must enforce LBNL<br />
Institutional policies that control access to the site and to laboratory facilities as described in the<br />
LBNL Site Security Plan. Policies and practices include, for example, the hosting of visitors and<br />
the issuance of gate passes, badges, and/or keys to control access to the site, building, and/or<br />
room based on each individual’s business needs. In addition, laboratory areas should have<br />
doors for access control. Consult the Safeguards and Security Web site for security policies and<br />
additional information.<br />
In addition to the above access requirements, the following additional controls are applicable<br />
when working at BL2:<br />
• <strong>Laboratory</strong> doors should be self-closing and have locks designed in accordance with<br />
LBNL standards. When the laboratory is unoccupied during nonbusiness hours, access<br />
to the laboratory should be controlled (e.g., by locking doors to the laboratory areas<br />
and/or doors to the building entrance).<br />
• All persons entering the laboratory must be advised of the potential hazards and meet<br />
any specific entry/exit requirements as communicated through laboratory door postings<br />
specified in Section 5.5 of this manual. Minimum biosafety hazard advisories include a<br />
required biohazard symbol posted at the entrance to the BL2 laboratory. Any additional<br />
biosafety requirements necessary for advising and protecting personnel entering and<br />
exiting the area will be specified in the BUA based on a risk assessment.<br />
Additional security assessments and security measures should be considered when<br />
select agents, other agents of high public health or agricultural concern, or agents of<br />
high commercial value are introduced into the laboratory. In this case, advisory<br />
recommendations of Section VI (Principles of <strong>Laboratory</strong> Biosecurity) of BMBL<br />
should be considered. In addition, when a security risk assessment has determined that<br />
additional physical security measures are needed to mitigate specific vulnerabilities, the<br />
laboratory or facility may be designated a property protection area. Lastly, when the agents are<br />
select agents or toxins (see Section 3.3.2.5), then the security requirements of the select agent<br />
regulations must be implemented as outlined in a specific security plan for the laboratory or<br />
building. The term biosecurity is often used to describe the administrative and physical security<br />
measures used to protect higher-consequence microbial agents or toxins from loss, theft,<br />
diversion, or intentional misuse.<br />
5.9 Pest Management<br />
<strong>Biosafety</strong> level (e.g., BL1 and BL2) criteria in BMBL and the NIH Guidelines require a program<br />
to control pests such as insects and rodents. Pests such as flies, cockroaches, ants, or mice<br />
can mechanically transmit biological materials and pathogens.<br />
Appendix G of BMBL provides guidance and requirements for Integrated Pest Management<br />
(IPM). IPM is a comprehensive program approach that integrates housekeeping, maintenance,<br />
and pest control services. The primary goal of IPM is to prevent pest problems by managing the<br />
facility environment to make it less conducive to pest infestation. Along with limited applications<br />
of pesticides, pest control is achieved through proactive operational and administrative<br />
intervention strategies to correct conditions that foster pest problems. Research supervisors,<br />
work leads, and LBNL Facilities are each responsible for elements of IPM for each operation.<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.<br />
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The LBNL Facilities Division is responsible for the general construction and maintenance of<br />
facilities including the design of laboratory buildings, periodic floor cleaning, disposal of general<br />
trash, and pest management. Pest management includes maintenance of a contract with a<br />
licensed California State/County applicator to provide insect and rodent control services. The<br />
licensed applicator conducts preventative services (e.g., periodically spraying the foundation of<br />
a building) and controls reported infestations. The Facilities Division also maintains the Facilities<br />
Work Request Center (510-486-6274) to track and respond to requests to repair and clean<br />
facilities and control infestations.<br />
Research supervisors and work leads must ensure implementation of the following IPM<br />
elements:<br />
• Program area surfaces and equipment can be easily cleaned (see Section 5.6.1) and<br />
are routinely cleaned and decontaminated (see Section 5.7.3).<br />
• Medical/biohazardous wastes are routinely placed in designated waste collection barrels<br />
(see Section 5.7.4).<br />
• The Facilities Work Request Center is contacted if additional services are needed from<br />
the Facilities Division to repair or clean the facility, or to control a pest infestation.<br />
The following general guidelines may be used to prevent or control rodent infestations:<br />
• Use rodent-proof containers with tight-fitting lids for storing food, washed utensils, and<br />
garbage so that rodents are not attracted to the building. Dispose of trash as soon as<br />
possible.<br />
• Seal, screen, and cover all building openings greater than a quarter of an inch.<br />
• Place sheds, wood piles, or other structures and debris away (e.g., 100 feet) from<br />
buildings. Cut grass, brush, and dense shrubbery.<br />
• If a building has been abandoned or closed for long periods, open doors and windows<br />
to help ventilate the building, and then wait for at least 30 minutes before entering.<br />
Use mechanical ventilation if needed.<br />
• Use spring-loaded traps or appropriate EPA-approved rodenticides to control the<br />
rodent population.<br />
Note Appendix G of this manual for guidelines on the cleanup of small dead animals, nests,<br />
or droppings.<br />
5.10 Incident, Accident, and Emergency Response<br />
This section outlines policy-related incident response and reporting. <strong>Biosafety</strong>-related incidents<br />
may include worker exposure to biological material, injuries or illnesses involving or resulting<br />
from exposure to biological material, spillage of biological material, or release of biological<br />
material outside of biosafety secondary containment. Such incidents may require reporting,<br />
medical evaluation and treatment, emergency response, incident review and documentation,<br />
and/or corrective actions.<br />
Response to biosafety-related incidents will be managed in accordance with this section and the<br />
following guidelines, policies, and authorizations:<br />
• LBNL Emergency Response Guide<br />
• PUB-3000, Section 5.1 (Incident Reviewing and Reporting)<br />
• PUB-3000, Chapter 9 (Emergency Management)<br />
• PUB-533, Master Emergency Program Plan for LBNL<br />
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• PUB-3000, Chapter 14 (Lessons Learned)<br />
• PUB-2488, Occurrence Reporting and Processing System (ORPS)<br />
• Safeguards and Security Program Planning and Management, DOE <strong>Manual</strong> 470.4-1,<br />
Section N (Incidents of Security Concern)<br />
• applicable <strong>Biosafety</strong> Work Authorizations (see Section 5.1 of this manual)<br />
5.10.1 General Incident Response and Reporting<br />
Worker instructions for reporting incidents and general emergency<br />
response are covered in the LBNL Emergency Response Guide.<br />
This guide provides response guidelines for a variety of common<br />
emergencies including biological spills and personal injury. It also<br />
provides both emergency and nonemergency telephone numbers.<br />
The guide is available on the EH&S Emergency Services Web site<br />
and as a wall-mountable flip chart. The Emergency Response Guide<br />
must be posted in areas wherever work with biological materials is<br />
conducted, and emergency response guidelines should be employed<br />
when responding to incidents.<br />
Chapter 5, Section 5.1, of PUB-3000 also provides general<br />
requirements for incident reviewing and reporting such, as<br />
responding to emergencies, and reporting and reviewing incidents<br />
and occupational injuries or illnesses.<br />
Division Directors and their designees are also responsible for reporting certain adverse or<br />
abnormal occurrences in accordance with the Occurrence Reporting and Processing System<br />
(ORPS) polices and system. In addition to ORPS reporting, incidents of security concern must<br />
be reported to LBNL Security.<br />
5.10.2 Worker Exposure, Injury, or Illness<br />
Workers are responsible for immediately reporting all occupational injuries, illnesses, and<br />
exposures to biological materials of concern to their supervisor and Health Services. The<br />
<strong>Biosafety</strong> Officer must also be notified of exposures to biological materials of concern or any<br />
related illness. Health Services will manage the occupational health case and initiate a<br />
Supervisor Accident Analysis Report (SAAR). In addition, an incident review team will be<br />
assigned to review the case and determine the causes and any needed actions. See PUB-3000,<br />
Chapter 5, Section 5.1, for additional information.<br />
Biological materials of concern related to exposures include materials or animals that may<br />
contain agents or properties that have known, potential, or unknown health risks. Examples of<br />
materials include all recombinant genomic materials, viable biological microbes in research, or<br />
Risk Group 2 or higher agents or materials. Examples of worker exposures to such biological<br />
materials of concern include:<br />
• Biological materials in contact with mucous membranes such as eyes, nose, or mouth.<br />
• Biological materials in contact with an open area of skin (e.g., cut or abrasion).<br />
• Cuts or punctures with sharp objects that may be contaminated with biological materials.<br />
• Exposures to humans or animals in research in a manner that is known to transmit<br />
disease.<br />
• Exposure to the blood of other people.<br />
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Additional information on biosafety-related accidents, response, and reporting is contained in<br />
the applicable BUA or ECP (see Section 5.1).<br />
5.10.3 Biological Spills and Cleanup<br />
Supervisors, work leads, and PIs are responsible for ensuring that spill response procedures<br />
and materials needed to safely respond to potential biological spills are maintained in operations<br />
where biological materials are used.<br />
The Emergency Response Guide, which must be posted in work areas, provides guidance and<br />
materials needed to safely respond to and clean up most biological spills at LBNL. Additional<br />
guidance regarding a variety of biohazardous spills inside and outside of biosafety cabinets is<br />
provided in Appendix G. Any additional guidance or materials needed to safely respond to or<br />
clean up biological spills must be included in the operation’s <strong>Biosafety</strong> Work Authorization (see<br />
Section 5.1).<br />
Response to biological spills should be conducted in accordance with applicable guidelines or<br />
requirements contained in the “Biological Spill” section of the Emergency Response Guide,<br />
Appendix G of this manual, and the operation’s <strong>Biosafety</strong> Work Authorization.<br />
5.10.4 Additional <strong>Biosafety</strong> Incident Reporting<br />
Line management, the <strong>Biosafety</strong> Officer, the Responsible Official, Waste Management, the IBC,<br />
and other LBNL employees have various internal and regulatory responsibilities for reporting<br />
biosafety-related incidents. The following incidents must be reported to the <strong>Biosafety</strong> Officer in<br />
the EH&S Division:<br />
• Worker exposure to biological materials of concern (see Section 5.10.2).<br />
• Injuries or illnesses involving or resulting from exposure to biological materials (see<br />
Section 5.10.2).<br />
• Release occurring outside of secondary biosafety containment of medical/biohazardous<br />
waste, biohazardous materials, recombinant genomic materials, or other regulated<br />
biological materials that have not been inactivated.<br />
• Incidents related to select agents or toxins (see definitions in Section 3.3.2.5).<br />
• <strong>Biosafety</strong>-related regulatory inspections or findings.<br />
Release outside of secondary containment includes, for example:<br />
• Spill of a material outside of its laboratory facility and outside of its primary and<br />
secondary containers.<br />
• Medical/biohazardous waste that has not been decontaminated but is disposed of in a<br />
sanitary sewer or in trash outside the laboratory where the work is conducted.<br />
• Environmental release of a viable agent, animal, plant, or pest material that is regulated<br />
against release or may cause damage to humans, plants, animals, or the environment.<br />
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5.11 Procurement, Transportation, and Transfer<br />
5.11.1 Procurement<br />
Procurement of biological agents, biological toxins, and other selected laboratory equipment or<br />
supplies are controlled at LBNL using a graded approach through the procurement process.<br />
These controls are designed to screen for biosafety and other hazards. They also provide a<br />
means for EH&S to assist requestors in implementing biosafety controls or complying with<br />
regulations. The following LBNL procurement controls are related to biosafety:<br />
• Expenditures for goods and services must be performed in accordance with LBNL<br />
procurement policies and through the Procurement and Property Management<br />
Department.<br />
• Only personnel authorized by the Chief Financial Officer or the Procurement and<br />
Property Manager may commit the <strong>Laboratory</strong> to goods or services. These authorized<br />
personnel categorize items to be procured so that assigned EH&S personnel will be<br />
notified of the procurement.<br />
• EH&S personnel notification or pre-approval for EH&S-related items that are on the<br />
restricted items list. EH&S personnel are notified of items such as, but not limited to,<br />
biological agents, biosafety cabinets, hoods, HEPA filters, chemicals, gases,<br />
eyewashes, safety showers, respirators, dust masks, and laboratory refrigerators. Items<br />
specific to biosafety that are on the restricted items list include:<br />
o Biological agents – The LBNL <strong>Biosafety</strong> Officer is sent a notification e-mail that<br />
procurement of a biological agent has been initiated, and the <strong>Biosafety</strong> Officer<br />
contacts the requestor if needed.<br />
o Select agents and toxins – Only individuals in LBNL Procurement may purchase<br />
select agents or toxins (see Section 3.3.2.5 and Appendix B, Section B.2, of this<br />
manual) with approval from the <strong>Biosafety</strong> Officer.<br />
o <strong>Biosafety</strong> cabinets – Selected EH&S Industrial Hygienists are e-mailed a notification<br />
that procurement of a BSC has been initiated, and the hygienist contacts the<br />
requestor as needed.<br />
5.11.2 Transportation and Shipping<br />
Employees who wish to transport or ship a biological material must ensure the material is<br />
moved safely and in accordance with LBNL biosafety transportation and shipping policy detailed<br />
in Appendix H of this manual. Appendix H should be used to assess if the material is a<br />
regulated biological material and how it should be moved. LBNL’s policy for workers handling<br />
materials at LBNL is based on biosafety requirements and U.S. and international transportation<br />
and shipping regulations. A number of biological materials may be transported directly by LBNL<br />
researchers in accordance with LBNL requirements, but all biological materials shipped by a<br />
contracted shipping company (e.g., a common carrier such as FedEx or UPS must be moved<br />
through LBNL Receiving, Transportation, and Shipping. See Appendix H for additional<br />
information.<br />
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5.11.3 Import, Export, and Transfer Restrictions Borders<br />
Materials being transferred (i.e., imported, exported, or transferred) from one location or person<br />
to another may be subject to regulatory restrictions or permit requirements. U.S., state, and<br />
foreign government agencies restrict and permit the movement of certain biological materials<br />
across borders to prevent threats to public health, agriculture, environment, and national<br />
security.<br />
The supervisor, work lead, person transferring the biological material, person requesting<br />
transfer of the biological material, and permit holder all have LBNL or legal responsibilities for<br />
complying with transfer requirements, obtaining any required permits, and following the<br />
conditions of the permit. Regulatory requirements, permits, and permit conditions related to the<br />
transfer of biological materials should also be included in the <strong>Biosafety</strong> Work Authorization. The<br />
LBNL <strong>Biosafety</strong> Office and IBC will review the researcher’s assessment and documentation of<br />
transfer requirements during the work authorization review process.<br />
Appendix I of this manual provides an outline of U.S.-based regulatory restrictions, permits, and<br />
lists related to the transfer (i.e., import, export, or transfer) of biological and related materials.<br />
Appendix I may be used by LBNL personnel as a starting point for determining whether<br />
biological materials are potentially regulated by U.S. agencies. It may also be used to determine<br />
whether there are restrictions or permits applicable to transfer of the material. Contact the LBNL<br />
<strong>Biosafety</strong> Office for additional advice.<br />
General controls for exporting from LBNL are outlined in the <strong>Berkeley</strong> Lab Export Control<br />
<strong>Manual</strong>. Export controls are based on government rules and regulations that govern the transfer<br />
of the following items to non-U.S. entities or individuals, regardless of where or how the transfer<br />
takes place:<br />
• Goods (systems, components, equipment, or materials)<br />
• Technologies (technical data, information, or assistance)<br />
• Software/codes (commercial or custom)<br />
6.0 Assessment and Improvement<br />
The fifth core function of Integrated Safety Management (ISM) requires that feedback and<br />
continuous improvement are incorporated into the work cycle for activities that involve work with<br />
biological materials or exposure to biological materials. This function is accomplished when<br />
supervisors, work leads, principal investigators (PIs), line management, Environment, Health,<br />
and Safety (EH&S), and others assess and continuously improve the biosafety of work<br />
conducted at LBNL.<br />
See PUB-3000, Chapter 26, Section 26.9, for a description of how LBNL assessment and<br />
improvement processes are incorporated into work with biological materials and the <strong>Biosafety</strong><br />
Program. The bulleted paragraphs below provide an overview of assessment and improvement<br />
processes and resources for supervisors, work leads, and PIs.<br />
Supervisors, work leads, and PIs must:<br />
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• Conduct periodic Environment, Safety, and Health (ES&H) assessments of their<br />
operation as specified in the Division Self-Assessment Program, including assessment<br />
of the safety of tasks being performed, safety of the work area and equipment, training,<br />
and compliance with the <strong>Biosafety</strong> Work Authorization and standards.<br />
• Participate in periodic biosafety assessments or other ES&H Technical Assurance<br />
Program (TAP) assessments of their operation when scheduled by EH&S.<br />
• Continuously improve the biosafety of their work, including correcting deficiencies and<br />
tracking actions in the Corrective Action Tracking System (CATS) when required.<br />
• Update their <strong>Biosafety</strong> Work Authorization with changes in personnel, training<br />
requirements, locations, and significant changes in the work.<br />
Supervisors, work leads, and PIs may use the following key resources to assess the biosafety<br />
and compliance of their operations:<br />
• The <strong>Biosafety</strong> Work Authorization for the operation.<br />
• The training requirements and tracking feature for personnel listed on the work<br />
authorization in the <strong>Biosafety</strong> Authorization System (BAS).<br />
• <strong>Laboratory</strong> <strong>Biosafety</strong> Level (BL) 1 and BL2 criteria listed in Appendix C of this manual.<br />
7.0 Standards, Policies, References, and Resources<br />
7.1 Standards<br />
• 7 CFR 331 and 9 CFR 121, Possession, Use, and Transfer of Biological Agents and<br />
Toxins, U.S. Department of Agriculture (USDA), Animal and Plant Health Inspection<br />
Service (APHIS)<br />
• 7 CFR 330, Plant Pest Regulations; General; Plant Pests; Soil, Stone, and Quarry<br />
Products; Garbage. Importation of Plant Pests, USDA/APHIS<br />
• 9 CFR Parts 92, 94, 95 96, 122 and 130 (note especially Part 122, Organisms and<br />
Vectors). Importation of Etiologic Agents of Livestock, Poultry, and Other Animal<br />
Diseases; USDA/APHIS<br />
• 10 CFR 851, Worker Safety and Health Program, Department of Energy (DOE)<br />
• 29 CFR 1904.8, Recording criteria for needle stick and sharps injuries, Occupational<br />
Safety and Health Administration (OSHA)<br />
• 29 CFR 1910.1030, Bloodborne Pathogens, OSHA<br />
• 42 CFR 71, Foreign Quarantine, Part 71.54 Etiologic agents, hosts, and vectors;<br />
Importation of Etiological Agents of Human Disease and Other Materials That May<br />
Contain These Agents; United States Public Health Service (PHS)<br />
• 42 CFR 73, Select Agents and Toxins, Department of Health and Human Services<br />
(HHS)<br />
• 49 CFR 171.8 (Definitions), 173.134 (Infectious Substances), and 173.6 (Materials of<br />
Trade), Hazardous Material Regulations (HMR), U.S. Department of Transportation<br />
(DOT)<br />
• <strong>Biosafety</strong> in Microbiological and Biomedical Laboratories, fifth edition, Centers for<br />
Disease Control (CDC) and <strong>National</strong> Institutes of Health (NIH)<br />
• California Health and Safety Code, Sections 117600 - 118360, California Medical Waste<br />
Management Act<br />
• Guidelines for Research Involving Recombinant DNA Molecules, <strong>National</strong> Institutes of<br />
Health (NIH), Federal Register (current version)<br />
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• <strong>Laboratory</strong> <strong>Biosafety</strong> <strong>Manual</strong>, 2nd ed. (revised), Interim Guidelines, World Health<br />
Organization (WHO), Geneva 2003, as applicable to biological etiologic agents<br />
• OSHA Standard Interpretation on Applicability of 1910.1030 to Establish Human Cell<br />
Lines<br />
7.2 Policies<br />
7.2.1 Health and Safety <strong>Manual</strong> (PUB-3000) Chapters<br />
• General Policy and Responsibilities (Chapter 1)<br />
• Health Services (Chapter 3)<br />
• Industrial Hygiene (Chapter 4)<br />
• Transportation (Chapter 5)<br />
• Safe Work Authorizations (Chapter 6)<br />
• Emergency Management (Chapter 9)<br />
• Personal Protective Equipment (Chapter 19)<br />
• Hazardous Waste Disposal (Chapter 20)<br />
• Research with Human and Animal Subjects (Chapter 22)<br />
• Environment, Health, and Safety (EH&S) Training (Chapter 24)<br />
• <strong>Biosafety</strong> (Chapter 26)<br />
7.2.2 Other <strong>Biosafety</strong>-related LBNL Publications<br />
• <strong>Berkeley</strong> Lab Export Control <strong>Manual</strong><br />
• <strong>Biosafety</strong>, Security, and Incident Response Plan for Select Agents, LBNL, latest version.<br />
• PUB-5341, Chemical Hygiene and Safety Plan, LBNL, latest version<br />
• PUB-533, Master Emergency Program Plan for <strong>Lawrence</strong> <strong>Berkeley</strong> <strong>National</strong> <strong>Laboratory</strong>,<br />
LBNL, latest version<br />
• PUB-3140, Integrated Environment, Health & Safety Management Plan, LBNL, latest<br />
version<br />
• PUB-3095, Medical and Biohazardous Waste Generator Guidelines, LBNL, latest<br />
revision<br />
• Site Safeguards and Security Plan, LBNL, latest version (a controlled document)<br />
• Site Security Plan for the <strong>Lawrence</strong> <strong>Berkeley</strong> <strong>National</strong> <strong>Laboratory</strong><br />
7.3 References<br />
• Emergency Response Guide (wall posting) and EH&S Emergency Preparedness Web<br />
page, LBNL<br />
• Facility Safety Plan Requirements, United States Army Medical Research and Materiel<br />
Command (USAMRMC) Web site<br />
• How to Import Foreign Soil and How to Move Soil within the United States, Circular Q-<br />
330.300-1 Soil (10/2006), USDA/APHIS, Plant Protection and Quarantine (PPQ)<br />
• <strong>National</strong> Sanitation Foundation (NSF)/ American <strong>National</strong> Standard (ANSI) Standard 49:<br />
Class II (laminar flow) biosafety cabinetry, March 19, 2002<br />
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7.4 Resources<br />
• LBNL EH&S groups, contact information, and Web links that may assist with biosafetyrelated<br />
matters are listed in Section 1.6 of this manual.<br />
• American Biological Safety Association (ABSA)<br />
• Canadian Fact Sheets: The Health Protection Branch of the <strong>Laboratory</strong> Centre for<br />
Disease Control in Ottawa, Canada, has developed fact sheets for many<br />
microorganisms that are similar to a chemical material safety data sheet.<br />
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Appendix A<br />
Glossary<br />
Terms, acronyms, and abbreviations used in this manual are defined in this appendix.<br />
Adeno-associated virus (AAV) is a virus that infects humans and some other primate species.<br />
AAV is a very attractive candidate for creating viral vectors because it is not known to cause<br />
disease in humans, can infect both dividing and nondividing cells, and may incorporate its<br />
genome into that of the host cell.<br />
American Biological Safety Association (ABSA) is a professional association that promotes<br />
biosafety as a scientific discipline and serves the growing needs of biosafety professionals<br />
throughout the world.<br />
Animal and Plant Health Inspection Service (APHIS) is an agency of the U.S. Department of<br />
Agriculture (USDA) that is responsible for protecting and promoting U.S. agricultural health,<br />
administering the Animal Welfare Act, and carrying out wildlife damage management activities.<br />
Animal <strong>Biosafety</strong> Level (BL-N) is standard containment and confinement practice for research<br />
involving whole animals when 1) recombinant research involves larger animals (e.g., nonhuman<br />
primates), 2) animals are infected with human pathogens, or 3) animals may harbor zoonotic<br />
agents (see this manual for details).<br />
Animal Welfare and Research Committee (AWRC) is an LBNL committee that reviews and<br />
approves proposed LBNL research for animal welfare concerns. Federal law uses the term<br />
Institutional Animal Care and Use Committee (IACUC).<br />
Antimicrobial is a chemical or physical agent that is used in the decontamination process to<br />
prevent microbial growth.<br />
Antisepsis is the application of a liquid antimicrobial chemical to human or animal living tissue<br />
to prevent sepsis.<br />
Antiseptic is a disinfecting chemical agent that is applied to living tissue and used to prevent<br />
sepsis.<br />
Australia Group (AG) is an informal forum of countries that, through the harmonization of<br />
export controls, seeks to ensure that exports do not contribute to the development of chemical<br />
or biological weapons.<br />
Autoclave is a piece of equipment with a chamber that is used to sterilize items by applying wet<br />
heat (i.e., high-pressure steam) at temperatures above the normal boiling point of water and<br />
pressures above normal atmospheric pressure.<br />
Biohazard is a biological material or condition that presents potential detrimental risk to the<br />
health of humans or other organisms, either directly through infection or indirectly through<br />
damage to the environment.<br />
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Biohazard label is a sign that is predominately fluorescent orange or orange-red and contains a<br />
biohazard symbol and the word “Biohazard” in a contrasting color.<br />
Biohazardous is an adjective used to describe biological materials that present potential<br />
detrimental risk to the health of humans or other organisms, either directly through infection or<br />
indirectly through damage to the environment.<br />
Biohazardous waste is waste that requires inactivation (i.e., decontamination) in an approved<br />
manner prior to disposal, but is not regulated by the California Department of Health Services as<br />
regulated medical waste. See PUB-3095, Medical and Biohazardous Waste Generator<br />
Guidelines, for additional information.<br />
Biological agent or agent is a very specific biological organism or material that is often directly<br />
responsible for producing an effect (e.g., disease). Agent examples include a microorganism<br />
(e.g., bacterium, fungus, or parasite), virus, prion, or biological toxin.<br />
Biological etiologic agent is an agent of biological origin (e.g., bacterium, fungus, parasite,<br />
virus, etc.) that causes disease in humans (i.e., pathogenic to humans).<br />
Biological materials are a broad range of organisms, cells, viruses, and other materials of<br />
biological origin that pose differing levels of risks to plants, animals, or humans.<br />
Biological products are materials that are regulated by Department of Transportation (DOT)<br />
and International Air Transport Association (IATA) for shipping that are derived from living<br />
organisms and manufactured for use in the prevention, diagnosis, treatment, or cure of disease<br />
in humans or animals and are certified by the USDA, Food and Drug Administration (FDA), or<br />
other national authority. Examples of biological products include certain viruses, therapeutic<br />
serums, toxins, antitoxins, vaccines, blood, and blood products.<br />
Biological toxin, biotoxin, or toxin. See toxin.<br />
Biological Use Application is the form completed by a prinicipal investigator (PI) or supervisor<br />
and submitted to the Environment, Health, and Safety (EH&S) biosafety office for review,<br />
approval, and authorization by a <strong>Biosafety</strong> Officer, the Institutional <strong>Biosafety</strong> Committee (IBC),<br />
or line management. Authorized applications result in a Biological Use Authorization (BUA),<br />
Biological Use Registration (BUR), or Biological Use Notification (BUN).<br />
Biological Use Authorization (BUA) is a type of LBNL formal biosafety authorization for work<br />
involving Risk Group (RG) 2 or higher biological materials, <strong>Biosafety</strong> Level (BL) 2 used for<br />
safety, or a regulatory permit or registration.<br />
Biological Use Notification (BUN) is a type of LBNL biosafety authorization for work involving<br />
RG1 biological materials, including work with <strong>National</strong> Institutes of Health (NIH)-exempt<br />
recombinant DNA molecules.<br />
Biological Use Registration (BUR) is a type of LBNL biosafety authorization for work involving<br />
RG1 work with recombinant DNA molecules and organisms or viruses containing recombinant<br />
DNA molecules.<br />
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Biological Weapons Convention (BWC) is a multilateral disarmament treaty that prohibits the<br />
development, production, acquisition, transfer, retention, stockpiling, and use of biological and<br />
toxin weapons and is a key element in the international community’s efforts to address the<br />
proliferation of weapons of mass destruction.<br />
<strong>Biosafety</strong> or biological safety is the general administrative and physical safety measures and<br />
efforts employed in a certain environment (e.g., LBNL) to protect workers, the public,<br />
agriculture, and the environment from exposure to biological agents or materials that may cause<br />
disease or other detrimental effects in humans, plants, or animals.<br />
<strong>Biosafety</strong> Authorization System (BAS) is the LBNL online system used to manage and<br />
provide BUNs, BURs, BUAs, and related information.<br />
<strong>Biosafety</strong> cabinet or biological safety cabinet (BSC) is a hood with high-efficiency particulate<br />
air (HEPA) filters that provides personnel, environmental, and/or product protection when<br />
appropriate practices and procedures are followed.<br />
<strong>Biosafety</strong> in Microbiological and Biomedical Laboratories (BMBL) is the title of an NIH-<br />
Centers for Disease Control and Prevention (CDC) national code of practice and LBNL standard<br />
for biosafety that outlines and defines biosafety risk assessment and control.<br />
<strong>Biosafety</strong> Level (BL) is a standard combination of practices and techniques, safety equipment,<br />
and facilities to safely contain biohazardous materials or agents to be used in work, as specified<br />
by BMBL and the NIH Guidelines. The NIH Guidelines uses the acronym BL, and BMBL uses<br />
the acronym BSL. The term biosafety level and acronym BL may be used generally to apply to<br />
any work with biological materials, but the acronym BL when used without additional letters or<br />
words technically applies only to laboratory BLs. When other letters or words are added to the<br />
BL acronym, other containment categories are indicated (e.g., BL-Large Scale, BL-P for plants,<br />
and BL-N for animals).<br />
<strong>Biosafety</strong> <strong>Manual</strong> is a comprehensive LBNL policy and tool developed that covers fundamental<br />
principles of biosafety, integrates requirements from the biosafety standards, and provides<br />
direction on identifying biological risks and required controls.<br />
<strong>Biosafety</strong> Officer is a person in the EH&S Division that oversees the development and<br />
maintenance of the primary structure and function of the <strong>Biosafety</strong> Program in accordance with<br />
the biosafety standards.<br />
<strong>Biosafety</strong> Work Authorization is a BUA, BUR, BUN, or Exposure Control Plan (ECP).<br />
Biosecurity is the administrative and physical security measures used to protect higherconsequence<br />
microbial agents or toxins and related information from loss, theft, diversion, or<br />
intentional misuse.<br />
Biotechnology Regulatory Services (BRS) is a branch of APHIS that regulates the<br />
introduction (importation, interstate movement, or environmental release) of certain genetically<br />
engineered organisms that may pose a plant pest risk, including organisms that are plants,<br />
insects, or microbes.<br />
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Blood as used in the Occupational Safety and Health Administration (OSHA) Bloodborne<br />
Pathogens Standard means human blood, human blood components, and products made from<br />
human blood.<br />
Bloodborne pathogen (BBP) material is a term used at LBNL to describe biological agents or<br />
materials that are covered by the OSHA Bloodborne Pathogens Standard including, for<br />
example, bloodborne pathogens, human blood, human blood components, products made from<br />
human blood, and other potentially infectious materials (OPIM).<br />
Bloodborne pathogens (BBPs) are infectious agents such as the human immunodeficiency<br />
virus (HIV) and the hepatitis B virus (HBV) that are capable of causing human disease and are<br />
transmitted through human blood.<br />
Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disease in cattle<br />
caused by a prion infection. BSE causes the animal’s brain and spinal cord to degenerate, and<br />
is characterized by the spongy appearance of infected brain tissue. BSE—also known as mad<br />
cow disease—is a type of transmissible spongiform encephalopathy. Humans who ingest brain<br />
or spinal cord tissue from infected cattle carcasses may develop a TSE known as new variant or<br />
variant Creutzfeldt-Jakob disease (nvCJD or vCJD).<br />
Bureau of Industry and Security (BIS) is an agency of the U.S. Department of Commerce that<br />
deals with issues involving national security and high technology. The BIS is responsible for<br />
implementing and enforcing the Export Administration Regulations (EAR) and has a principal<br />
goal of stopping proliferation of weapons of mass destruction, while furthering the growth of U.S.<br />
exports.<br />
California Department of Food and Agriculture (CDFA) is an agency in the California state<br />
government that is responsible for ensuring the state’s food safety, the protection of the state’s<br />
agriculture from invasive species, and promoting the state’s agricultural industry.<br />
Category A Infectious Substances see Infectious Substances, Category A.<br />
Category B Infectious Substances see Infectious Substances, Category B.<br />
Center for Veterinary Biologics (CVB) is a group within APHIS Veterinary Services (VS) that<br />
regulates veterinary biologics including vaccines, antibodies, diagnostic kits, and certain<br />
immunomodulators, including those developed using genetically engineered organisms.<br />
Centers for Disease Control and Prevention (CDC) is one of the 13 major operating<br />
components of the U.S. Department of Health and Human Services.<br />
Chemical Safety Hygiene Plan (CHSP) is a comprehensive LBNL policy and tool that provides<br />
requirements and guidance to employees on the safe handling, use, and storage of hazardous<br />
materials such as chemicals and engineered nanomaterials in laboratory, shop, and office<br />
settings.<br />
Commerce Control List (CCL) is a section of the EAR that lists specific goods, technologies,<br />
and software and the countries to which those items may or may not be exported, along with<br />
any special restrictions or exceptions that may apply.<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.<br />
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Common carrier is a person or company that transports goods or people for any person or<br />
company and that is responsible for any possible loss of the goods during transport (e.g., FedEx<br />
or UPS).<br />
Containment is a set of controls including the safe methods, equipment, and facilities needed<br />
to protect workers and the environment from biohazardous materials or agents.<br />
Contaminated means the potential presence of biohazardous material on an item or surface.<br />
The OSHA Bloodborne Pathogens Standard defines contaminated as the presence or the<br />
reasonably anticipated presence of blood or other potentially infectious materials on an item or<br />
surface.<br />
Corrective Action Tracking System (CATS) is an online LBNL database tool used to identify,<br />
track, and resolve issues and their associated corrective actions as well as determine the<br />
effectiveness of those corrective actions.<br />
Creutzfeldt-Jakob disease (CJD) is an incurable neurodegenerative and fatal human disease<br />
caused by a prion infection. CJD causes brain nerve cells to degenerate and is characterized by<br />
the spongy appearance of infected brain tissue. Although CJD is rare, it is the most common<br />
type of transmissible spongiform encephalopathy in humans. Three major categories of CJD are<br />
sporadic CJD, hereditary CJD, and acquired CJD.<br />
Customs and Border Protection (CBP) or United States Customs and Border Protection is a<br />
federal law enforcement agency of the U.S. Department of Homeland Security charged with<br />
regulating and facilitating international trade, collecting import duties, and enforcing U.S.<br />
regulations including trade (e.g., import and export), drug, and immigration.<br />
Dangerous Goods Regulations (DGR) is a manual published by IATA to provide procedures<br />
for shippers and operators by which articles and substances with hazardous properties can be<br />
safely and efficiently transported by air on all commercial air transport. The manual provides<br />
lists and classifications of articles and substances (e.g., infectious substances) and<br />
requirements for training, packing, labeling, documentation, handling, and reporting.<br />
Decontamination is the process of reducing or inactivating biological contaminants or<br />
components to an acceptable level to reduce or eliminate the possibility of transmission of<br />
pathogens to undesired hosts such as laboratory workers, the general public, and other<br />
organisms in the environment.<br />
Deoxyribonucleic acid (DNA) is a nucleic acid that contains the genetic instructions used in<br />
the development and functioning of all known living organisms and some viruses.<br />
Department of Energy (DOE) is a Cabinet-level department of the U.S. government concerned<br />
with the U.S. policies regarding energy and safety in handling nuclear material. DOE also<br />
sponsors basic and applied scientific research mostly through its system of U.S. DOE national<br />
laboratories such as LBNL.<br />
Department of Transportation (DOT) is a federal Cabinet-level department of the U.S.<br />
government that is concerned with interstate transportation to keep the traveling public safe and<br />
secure, increase their mobility, and have a transportation system that contributes to the nation's<br />
economic growth.<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.<br />
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Detergent is a synthetic surfactant.<br />
Disease is any deviation from or interruption of the normal structure or function of any body<br />
part, organ, or system that is manifested by a characteristic set of symptoms and signs and<br />
whose etiology, pathology, and prognosis may be known or unknown.<br />
Disinfectant is a chemical germicide or physical agent that is applied to inanimate objects to kill<br />
microbes, but is not capable of killing endospores, some viruses, or mycobacterium.<br />
Disinfectants are typically chemical germicides.<br />
Disinfection is the process of generally eliminating nearly all recognized pathogenic<br />
microorganisms but not necessarily all microbial forms (e.g., bacterial spores) from inanimate<br />
objects (e.g., work surfaces, equipment). Common disinfectants include diluted household<br />
bleach or 70% isopropanol.<br />
Dust mask is a common, but inaccurate name for a filtering facepiece respirator.<br />
Emergency eyewash is a plumbing unit designed to properly flush chemical, biological, or<br />
other hazardous agents off the face, and out of mucous membranes such as the eyes, so as to<br />
prevent injury to the eye and exposed body surfaces or penetration of an agent into the body.<br />
Emergency eyewash and shower is a combined plumbing unit(s) designed to properly flush<br />
chemical, biological, or other hazardous agents off of the skin or the face, and out of mucous<br />
membranes such as the eyes, so as to prevent injury to the exposed body surfaces or<br />
penetration of an agent into the body.<br />
Emergency Response Guide is an online LBNL guide and wall-mountable flip chart that<br />
covers worker instructions and telephone numbers for reporting incidents and general<br />
emergency response for a variety of common emergencies including biological spills and<br />
personal injury.<br />
Environment, Health, and Safety (EH&S) Division at LBNL manages environment, safety, and<br />
health programs to ensure LBNL fulfills their requirements.<br />
Environment, Safety, and Health (ES&H) is a term used to describe subjects (e.g., policies,<br />
responsibilities, and functions) related to protecting the safety and health of workers, the public,<br />
and the environment.<br />
Environmental Protection Agency (EPA) is an agency of the U.S. government charged to<br />
protect human health and the environment and has primary responsibility for setting and<br />
enforcing national standards under a variety of environmental laws. The EPA also conducts<br />
environmental assessment, research, and education and works with industry and government in<br />
voluntary pollution prevention and energy conservation efforts.<br />
ES&H Technical Assurance Program (TAP) is one component of the LBNL Self-Assessment<br />
Program that is managed by the EH&S Division. The ES&H <strong>Biosafety</strong> Program TAP reviews<br />
biosafety programs and processes <strong>Laboratory</strong>-wide to ensure they are compliant with guiding<br />
regulations, effective, and properly implemented by <strong>Laboratory</strong> divisions.<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.<br />
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Etiologic is an adjective that means disease-causing.<br />
Export Administration Regulations (EAR) are regulations that contain the CCL, are issued by<br />
the U.S. Department of Commerce BIS under laws relating to the control of certain exports,<br />
reexports, and activities, and contain the CCL.<br />
Exposure Control Plan (ECP) is an LBNL authorization document that defines work, hazards,<br />
and controls in accordance with the requirements of the OSHA Bloodborne Pathogens Standard<br />
for work with or potential exposure to BBP materials. The BUA is the ECP for work that pertains<br />
to research.<br />
Eye protection is a safety device such as safety glasses or goggles worn over the eyes to<br />
prevent injury to the eye or exposure to biological agents.<br />
Face mask is a loose-fitting, disposable device that covers the worker’s nose and mouth and is<br />
not a respirator (e.g., products labeled as surgical, medical, dental, or isolation masks).<br />
Face protection is a safety device such as a face mask, face shield, or other splatter guard<br />
worn over all or part of the face to protect the face from injury or exposure to biological agents.<br />
Filtering facepiece respirator is a negative-pressure, air-purifying respirator with a particulate<br />
filter as an integral part of the facepiece or with the entire facepiece composed of the filtering<br />
medium. A filtering facepiece respirator is sometimes poorly referred to as a “dust mask” or<br />
improperly called a “N95 respirator.”<br />
Fixed means the biological material has been treated so that it has been stabilized and<br />
preserved in place. Fixing cells with some fixatives (e.g., paraformaldehyde or glutaraldehyde)<br />
kills the cells and most potential pathogens.<br />
Food and Drug Administration (FDA) is an agency of the U.S. Health and Human Services<br />
Department responsible for protecting and promoting public health through the regulation of<br />
food safety, tobacco products, dietary supplements, medications, vaccines, biopharmaceuticals,<br />
blood transfusions, medical devices, electromagnetic radiation emitting devices, veterinary<br />
products, cosmetics, and other concerns.<br />
Foot protection is an enclosed shoe or safety shoe worn on the foot to protect the foot from<br />
injury or exposure to biological agents.<br />
Genetic material is material found in the nucleus, mitochondria, and cytoplasm of a cell or<br />
organism. It plays a fundamental role in determining the structure and nature of cell substances<br />
and is capable of self-propagating and variation. The genetic material of a cell can be a gene, a<br />
part of a gene, a group of genes, a DNA molecule, a fragment of DNA, a group of DNA<br />
molecules, or the entire genome of an organism.<br />
Genetic recombination is the process by which the strand of genetic material (usually DNA,<br />
but can also be RNA) is broken and then joined to a different DNA molecule to create<br />
recombinant genetic material.<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.<br />
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Genetically Modified Organisms (GMO) or microorganisms (GMMO) are organisms and<br />
microorganisms that are regulated by DOT and IATA for shipping in which genetic material has<br />
been purposely altered through genetic engineering in a way that does not occur naturally.<br />
Germicide is an antimicrobial substance or physical agent that kills microbes.<br />
Good Microbiological Practice (GMP) refers to aseptic techniques and other good<br />
microbiological practices that are necessary to prevent contamination of the laboratory with the<br />
agents being handled and contamination of the work with agents from the environment.<br />
Greenhouse is a structure with walls, a roof, and a floor designed and used principally for<br />
growing plants in a controlled and protected environment.<br />
Greenhouse facility includes the actual greenhouse rooms or compartments for growing plants<br />
and all immediately contiguous hallways and head-house (i.e., work) areas, and is considered<br />
part of the confinement area.<br />
Guidelines are a set nonmandatory but desirable criteria, conditions, or best management<br />
practices that should typically be considered when determining controls needed to mitigate risk.<br />
Hand protection is a glove or other safety device used on the hand to prevent injury to the<br />
hand or direct skin contact with biological materials.<br />
Handwashing facility is a facility that is required when work with BBP materials is conducted. It<br />
has an adequate supply of running potable water, soap, and single-use towels or hot-air-drying<br />
machines.<br />
Handwashing sink is basin with running water and a drain that is designed for washing of<br />
hands and that should be provided with a soap dispenser and paper towel dispenser as a best<br />
management practice.<br />
Hazardous Material Regulations (HMR) are DOT regulations that govern the movement of<br />
hazardous materials (e.g., infectious substances) in vehicles, airplanes, railcars, or vessels via<br />
public right-of-ways such as roadways, airways, railways, and sea lanes that are accessible to<br />
the public.<br />
Health and Human Services (HHS) is a Cabinet department of the U.S. government that<br />
contains the U.S. Public Health Service and has the goal of protecting the health of all<br />
Americans and providing essential human services.<br />
Hepatitis B virus (HBV) is a pathogen that causes contagious liver disease (i.e., hepatitis B) in<br />
humans. HBV is a common BBP.<br />
Hepatitis C virus (HCV) is a pathogen that causes contagiouis liver disease (i.e., hepatitis C) in<br />
humans. HCV is a common BBP.<br />
High-efficiency particulate air (HEPA) filter is a device composed of fibrous materials<br />
capable of trapping and retaining at least 99.97% of airborne monodispersed particles<br />
0.3 micrometers (µm) in diameter.<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.<br />
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Hood is an enclosure or shaped inlet designed to conduct contaminated air into an exhaust duct<br />
system or a filter that safely captures the contaminant.<br />
Household bleach is a water-based solution of sodium hypochlorite with a typical concentration<br />
of 5.25% by weight of the active sodium hypochlorite ingredient.<br />
Human immunodeficiency virus (HIV) is a lentivirus (a member of the retrovirus family) that<br />
causes acquired immunodeficiency syndrome (AIDS), a condition in humans in which the<br />
immune system begins to fail, leading to life-threatening opportunistic infections. HIV is a<br />
common BBP.<br />
Human pathogen or infectious agent is an infectious microbe (e.g., bacteria, protozoa, fungi,<br />
viruses, etc.) or other agent (e.g., prions) that causes disease in healthy humans.<br />
Human Subjects Committee (HSC) is an LBNL committee that reviews proposed research<br />
projects involving human subjects, human-derived data, or human-derived tissues, for ethical<br />
concerns in accordance with HHS regulations and DOE Orders.<br />
Inactive means the biological material is not capable of acting or reacting normally.<br />
Infectious agent or human pathogen is an infectious microbial (e.g., bacteria, protozoa, fungi,<br />
viruses, etc.) or other agent (e.g., prions) that causes disease in healthy humans.<br />
Infectious substances are materials regulated by DOT and IATA for shipping that are known<br />
to be, or are reasonably suspected to contain, an animal or human pathogen. A pathogen is a<br />
virus, microorganism (including bacteria, plasmids, or other genetic elements), proteinaceous<br />
infectious particle (prion), or recombinant microorganism (hybrid or mutant) that is known or<br />
reasonably expected to cause disease in humans or animals.<br />
Infectious substances, Category A, are materials regulated for shipping by DOT and IATA<br />
that are capable of causing permanent disability, or life threatening or fatal disease in humans<br />
or animals when exposure to them occurs.<br />
Infectious substances, Category B are materials regulated for shipping by DOT and IATA that<br />
are infectious, but do not meet the standard for inclusion in Category A.<br />
Institutional <strong>Biosafety</strong> Committee (IBC) is an LBNL committee that provides oversight,<br />
administration, and review of LBNL policies and projects involving research with biological<br />
materials that may pose safety, health, or environmental risks.<br />
Institutional Review Board (IRB) is an HHS-mandated committee that requires the use of<br />
established principles and requirements during the ethical review of proposed research projects<br />
involving human subjects, human-derived data, or human-derived tissues. The IRB for LBNL is<br />
the HSC.<br />
Integrated Pest Management (IPM) is a term used in the BMBL and LBNL biosafety policy to<br />
describe a comprehensive program approach that integrates housekeeping, maintenance, and<br />
pest control services to prevent pest problems by managing the facility environment to make it<br />
less conducive to pest infestation.<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.<br />
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Integrated Safety Management (ISM) is the safety management system used by LBNL and<br />
the U.S. Department of Energy to systematically integrate safety into management and work<br />
practices at all levels so that missions are accomplished while protecting the public, the worker,<br />
and the environment.<br />
International Air Transport Association (IATA) is an international industry trade group of<br />
airlines that represents, leads, and serves the airline industry and publishes the DGR used for<br />
airlines’ shipping of articles and substances with hazardous properties including infectious<br />
substances.<br />
International Traffic in Arms Regulations (ITAR) is a set of U.S. Department of State<br />
regulations that control the export and import of defense-related articles and services on the<br />
United States Munitions List (USML).<br />
Iodophor is a preparation containing iodine complexed with a solubilizing agent, such as a<br />
surfactant or povidone (a type of water soluble polyvinyl polymer).<br />
Ionizing radiation is radiation of sufficiently high energy to cause ionization in the medium<br />
through which it passes.<br />
Job Hazards Analysis (JHA) is the LBNL process that results in a worker hazard and control<br />
description (Hazards Profile) and Work Authorization prepared for a specific worker according to<br />
the requirements of PUB-3000, Chapter 32.<br />
<strong>Laboratory</strong> acquired infections (LAIs) are all infections acquired through laboratory or<br />
laboratory-related activities regardless of whether they are symptomatic or asymptomatic in<br />
nature.<br />
<strong>Laboratory</strong> <strong>Biosafety</strong> Level (BL) is a standard combination of practices and techniques, safety<br />
equipment, and facilities to safely contain biohazardous materials or agents used in laboratory<br />
work.<br />
Large Scale (BL–Large Scale) is a term used in the NIH Guidelines and LBNL biosafety policy<br />
to describe uses of and containment levels for organisms containing recombinant DNA<br />
molecules involving a quantity of culture greater than 10 liters.<br />
<strong>Lawrence</strong> <strong>Berkeley</strong> <strong>National</strong> <strong>Laboratory</strong> (LBNL), which is also called <strong>Berkeley</strong> Lab, is a DOE<br />
national laboratory that conducts unclassified, interdisciplinary scientific research.<br />
Medical waste is waste generated or produced as a result of the following: diagnosis,<br />
treatment, or immunization of human beings or animals; research pertaining to the diagnosis,<br />
treatment, or immunization of human beings or animals; or the production or testing of<br />
biologicals. See PUB-3095, Medical and Biohazardous Waste Generator Guidelines, for<br />
additional information.<br />
Medical/biohazardous waste is a term used to describe wastes that are biological materials or<br />
contaminated with biological materials and require inactivation (i.e., decontamination) in an<br />
approved manner prior to final disposal.<br />
Must means the condition is required.<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.<br />
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N95 respirator is a term sometimes improperly used to describe a filtering facepiece respirator<br />
that has a 95% efficient filter built into the facepiece.<br />
<strong>National</strong> Center for Import Export (NCIE) is a group within APHIS VS that regulates the<br />
import, export, and interstate movement of all animals and animal products (e.g., tissues, blood,<br />
and semen), including those that are genetically engineered.<br />
<strong>National</strong> Institutes of Health (NIH) is one of eight health agencies that are components of the<br />
Public Health Service (PHS).<br />
<strong>National</strong> Select Agent Registry (NSAR) is a cooperative program between the USDA-APHIS<br />
Agricultural Select Agent Program and the CDC Division of Select Agents and Toxins to<br />
oversee activities involving the possession of biological agents and toxins that have the<br />
potential to pose a severe threat to public health, animal or plant health, or to animal or plant<br />
products.<br />
Negative-pressure, air-purifying, cartridge respirator is a respirator that uses a filter,<br />
sorbent, or catalyst housed inside a cartridge to remove contaminants from the air (e.g.,<br />
respirators using a N95 or P100 cartridge particulate filter that is 95% or 100% efficient,<br />
respectively).<br />
Negative-pressure, air-purifying respirator is a tight-fitting respirator in which the air pressure<br />
inside the facepiece is negative during inhalation with respect to the ambient air pressure<br />
outside the respirator and an air-purifying filter or cartridge removes specific air contaminants<br />
(e.g., filtering facepiece and some cartridge respirators).<br />
NIH Guidelines is an abbreviated title used by NIH for the document titled NIH Guidelines for<br />
Research Involving Recombinant DNA Molecules.<br />
Nonviable means the biological material or agent is not capable of living or developing under<br />
favorable conditions.<br />
Nucleic acid is a macromolecule composed of chains of monomeric nucleotides. In<br />
biochemistry, nucleic acids carry genetic information or form structures within cells. The most<br />
common nucleic acids are DNA and RNA.<br />
Occupational Safety and Health Administration (OSHA) is an agency of the U.S.<br />
government that ensures the safety and health of U.S. workers (e.g., by setting and enforcing<br />
standards).<br />
Occurrence Reporting and Processing System (ORPS) is an LBNL system that is used to<br />
notify and keep <strong>Laboratory</strong> management and applicable elements of the U.S. Department of<br />
Energy (DOE) informed of abnormal occurrences that could adversely affect 1) the health and<br />
safety of employees, guests, visitors, and the general public; 2) the environment; 3) the<br />
intended purpose of LBNL facilities; or 4) the credibility of DOE and/or LBNL.<br />
Office of <strong>Laboratory</strong> Animal Welfare (OLAW) is an office of NIH that oversees compliance<br />
with the PHS Policy on Humane Care and Use of <strong>Laboratory</strong> Animals.<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.<br />
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Organism is any living system (such as animal, plant, fungus, or microorganism). In at least<br />
some form, all organisms are capable of response to stimuli, reproduction, growth and<br />
development, and maintenance of homeostasis as a stable whole. An organism may either be<br />
unicellular (single-celled) or composed of, as in humans, many billions of cells grouped into<br />
specialized tissues and organs. The term multicellular (many-celled) describes any organism<br />
made up of more than one cell.<br />
Other Potentially Infectious Materials (OPIM) are materials other than blood and bloodborne<br />
pathogens that are regulated by the OSHA Bloodborne Pathogens Standard based on their<br />
potential to contain BBPs. See Table 5 of this manual and definitions for blood and bloodborne<br />
pathogens.<br />
Parenteral is an adjective that refers to a route of administration that involves piercing the<br />
mucous membranes or skin barrier through events such as punctures, lacerations, abrasions,<br />
and bites.<br />
Pathogen is an infectious microbe (e.g., bacteria, protozoa, fungi, viruses, etc.) or other agent<br />
that causes disease in healthy host organisms such as humans, animals, or plants.<br />
Patient specimens or diagnostic specimens are any human or animal materials including but<br />
not limited to excreta, secreta, blood, blood components, tissue, and tissue fluids being shipped<br />
for the purpose of diagnosis and regulated by DOT and IATA.<br />
Personal Protective Equipment (PPE) is clothing or equipment worn by workers to protect the<br />
body from injury by hazardous agents or materials. Examples of PPE include foot, hand, eye,<br />
face, body, and respiratory protection. PPE is one element of biosafety containment.<br />
Plant <strong>Biosafety</strong> Level (BL-P) is standard physical and biological containment conditions and<br />
practices suitable to greenhouse operations that conduct experiments involving plants, plantassociated<br />
microorganisms, and small animals (e.g., arthropods or nematodes).<br />
Plant Protection and Quarantine (PPQ) is a branch of APHIS that safeguards agriculture and<br />
natural resources from the risks associated with the entry, establishment, or spread of animal<br />
and plant pests and noxious weeds to ensure an abundant, high-quality, and varied food supply.<br />
Plasmids are DNA segments that are separate from chromosomal DNA and are capable of<br />
replicating independently of the chromosomal DNA. In many cases, a plasmid is circular and<br />
double-stranded. Plasmids usually occur naturally in bacteria, but are sometimes found in<br />
eukaryotic organisms<br />
Positive pressure respirator is a respirator that is designed to maintain positive pressure<br />
inside the facepiece during exhalation and inhalation (e.g., a powered air-purifying respirator or<br />
PAPR).<br />
Potable water or drinking water is water which is satisfactory for drinking, culinary, and<br />
domestic purposes and meets the requirements of the regulatory health authority having<br />
jurisdiction. In laboratory and other industrial water uses, the building’s water supply is<br />
separated through backflow prevention devices in the building’s plumbing system into potable<br />
and industrial water systems or sources.<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.<br />
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Povidone-iodine (PVP-I) is an iodophor antimicrobial composed of a stable chemical complex<br />
of polyvinylpyrrolidone (povidone or PVP) and elemental iodine (ranging from 9.0% to 12.0%<br />
available iodine, calculated on a dry basis).<br />
Principal Investigator (PI) is the individual assigned authority and responsibility to direct a<br />
research experiment, project, or program that is typically funded by a grant.<br />
Prion is an infectious agent that is composed of protein that typically propagates by transmitting<br />
a misfolded protein state.<br />
Protective laboratory clothing is a garment such as a lab coat, gown, smock, or uniform<br />
designed to keep personal clothing, forearms, or other exposed bodily surfaces protected from<br />
contamination by biological materials or exposure to other hazards.<br />
PUB-3000 is the LBNL Health and Safety <strong>Manual</strong>.<br />
Public Health Service (PHS) is an umbrella organization in the U.S. federal government<br />
consisting of eight HHS health agencies, the Office of Public Health and Science, and the<br />
Commissioned Corps (a uniformed service of health professionals). NIH and CDC are agencies<br />
within the PHS.<br />
Quaternary ammonium compound or quat is a cationic detergent compound derived from<br />
ammonia by replacing the hydrogen atoms with organic radicals, and the compound is<br />
especially important as surface-active agents, disinfectants, or in drugs.<br />
Recombinant DNA molecules are defined by the NIH Guidelines as molecules that are<br />
constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic<br />
acid molecules that can replicate in a living cell or molecules that result from the replication of<br />
such molecules.<br />
Recombinant genetic (or genomic) materials are genetic materials that have undergone<br />
genetic recombination. See definitions for genetic materials and genetic recombination.<br />
Respirator is a device such as a filtering facepiece or negative-pressure cartridge respirator<br />
that is designed and certified to protect the wearer from the inhalation of harmful atmospheres.<br />
Respiratory protection is a control such as a biosafety cabinet, enclosed containment system,<br />
or respirator that prevents worker inhalation of an agent to harmful levels.<br />
Responsible Official (RO) is an LBNL person that has the authority and responsibility to<br />
ensure compliance with CDC and USDA regulations for possession, use, or transfer of select<br />
agents and toxins, as specified in the regulations and on behalf of LBNL.<br />
Ribonucleic acid (RNA) is a biologically important type of molecule that consists of a long<br />
chain of nucleotide units. Each nucleotide consists of a nitrogenous base, a ribose sugar, and a<br />
phosphate. RNA is very similar to DNA, but differs in a few important structural details. For<br />
example, in the cell, RNA is usually single-stranded, while DNA is usually double-stranded.<br />
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Risk Group (RG) is a system adopted by the CDC and NIH for classifying biological agents by<br />
the degree of human hazard. There are four risk groups, and a higher RG number indicates a<br />
higher level of hazard.<br />
Safety-engineered sharps or safety-engineered needles are sharp tools with a built-in safety<br />
feature or mechanism that effectively reduces the risk of accidental skin penetration and a<br />
biological exposure incident. Examples include devices that blunt, sheath, or withdraw the sharp<br />
when the sharp edge or point has been used or is not in use. Also see below Sharps with<br />
ESIP.<br />
Sanitization is the process of generally reducing the number of microorganisms by the use of<br />
general cleaning agents.<br />
Select agents and toxins are (a) specific pathogenic agents and toxins listed and strictly<br />
regulated by the CDC and USDA (i.e., under 7 CFR 331, 9 CFR 121, and 42 CFR 73) because<br />
they may be used as agents of mass destruction or pose a severe threat to human, animal, and<br />
plant health, and (b) specific genetic elements, recombinant nucleic acids, and recombinant<br />
organisms that are related to the list of select agents and toxins as described in the regulations.<br />
Sepsis is the presence of infectious organisms in the blood or other tissue of the body.<br />
Sharp is an object that can penetrate the skin. A sharp is often a tool, device, or material that<br />
typically has a sharp edge or point such as a needle, scalpel, razor, blade, broken glass piece,<br />
broken capillary tube, or an exposed wire end.<br />
Sharps with engineered sharps injury protection (ESIP) are defined in the OSHA<br />
Bloodborne Pathogen Standard as a nonneedle sharp or a needle device used for withdrawing<br />
body fluids, accessing a vein or artery, or administering medications or other fluids, with a builtin<br />
safety feature or mechanism that effectively reduces the risk of an exposure incident.<br />
Should means there is an expectation that the condition will be met unless there is a compelling<br />
and countervailing reason for not meeting the condition and the alternative provides a sufficient<br />
level of safety that does not conflict with other requirements. When the term should is used in a<br />
section identified as guidelines, the condition is desirable or is a best management practice, and<br />
the condition or other alternatives should be implemented when needed to control apparent risk.<br />
Soap is sodium or potassium salt of fatty acids.<br />
Soil is defined by the USDA PPQ as a mixture of inorganic and organic materials, when the<br />
organic materials are unidentifiable plant and/or animal parts. The PPQ Soil Circular defines<br />
what is and is not soil.<br />
Standard facilities are design features, materials, and equipment incorporated into the<br />
laboratory or facility in accordance with BL containment criteria stated in BMBL and the NIH<br />
Guidelines.<br />
Standard microbiological practices and special practices are administrative controls listed<br />
as BL containment criteria in BMBL and the NIH Guidelines to protect workers and the<br />
environment.<br />
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Standard safety equipment and PPE are equipment controls listed as BL containment criteria<br />
in BMBL and the NIH Guidelines that provide primary barriers to prevent worker exposure to<br />
infectious agents.<br />
Standards are the external rules established by government, contract, and funding regulations<br />
and nonregulatory standards that form the requirements of the LBNL <strong>Biosafety</strong> Program.<br />
Sterilant is an antimicrobial chemical or physical agent that is capable of killing all microbes<br />
including their spores. It fulfills the sterility assurance level.<br />
Sterile is an adjective that means completely free of all living microorganisms and viruses.<br />
Sterility assurance level is the degree of killing efficacy in a sterilization process equal to the<br />
probability of a microorganism or virus surviving on the item of less than one in one million.<br />
Sterilization is the process of completely destroying all living microorganisms and viruses on an<br />
object. Common sterilization methods include autoclaving and incineration.<br />
Sterilization procedure is a treatment process to which an item is subjected after which the<br />
probability of a microorganism or virus (including a high number of bacterial endospores)<br />
surviving on the item is less than one in one million. This level of killing efficacy is referred to as<br />
the sterility assurance level.<br />
Subcontractor Job Hazards Analysis and Work Authorization (SJHAWA) is the LBNL work<br />
authorization document that identifies work hazards and controls for subcontractors, vendors,<br />
and guests.<br />
Supervisor Accident Analysis Report (SAAR) is the LBNL report that the supervisor must<br />
complete to document the nature, cause, and necessary actions related to an employee injury.<br />
Surfactant is a surface active agent that is usually an organic compound that possesses both<br />
hydrophilic (water-loving) and lipophilic (fat-liking) properties that make the compound soluble in<br />
water and lipids.<br />
Technical Assurance Program (TAP). See ES&H Technical Assurance Program above.<br />
Toxin, biological toxin, or biotoxin is a poisonous substance produced by a living organism.<br />
The term “toxin” is used in this manual.<br />
Transgenic organism is an organism whose genome has been altered by the transfer of a<br />
gene or genes from another species or breed.<br />
Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases such<br />
as Creutzfeldt-Jakob disease (CJD) in humans and bovine spongiform encephalopathy (BSE or<br />
"mad cow disease") that affect humans and a variety of domestic and wild animal species.<br />
Transportation Authorization Form (TAF) is an LBNL form that is 1) generated when LBNL<br />
Transportation is asked via the Facilities Division Work Request Center to move an item, and<br />
then 2) completed by the requester and affixed to the item prior to movement to indicate that the<br />
item is safe and ready for movement.<br />
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Ultraviolet (UV) radiation or UV light is electromagnetic radiation with a wavelength shorter<br />
than that of visible light, but longer than X-rays, in the range of 10 nanometers (nm) to 400 nm,<br />
and energies from 3 electron volts (eV) to 124 eV.<br />
United States Department of Agriculture (USDA) is an agency of the U.S. government with<br />
the following types of mission areas: farm and foreign agriculture, food, food safety, nutrition,<br />
natural resources, environment, research, education, economics, and rural development.<br />
United States Munitions List (USML) is a list of articles, services, and related technology<br />
designated as defense-related that are defined in ITAR and fall under the export and temporary<br />
import jurisdiction of the Department of State.<br />
Veterinary Services (VS) is a branch of APHIS that protects and improves the health, quality,<br />
and marketability of our nation's animals, animal products, and veterinary biologics by<br />
preventing, controlling and/or eliminating animal diseases, and monitoring and promoting animal<br />
health and productivity.<br />
Viral vector is a viral tool commonly used to deliver genetic material into cells.<br />
Virus is a small infectious agent that can only replicate inside the cells of another organism.<br />
Worker Safety and Health Program (WSHP) is a DOE rule (10 CFR 851) that establishes the<br />
framework for DOE’s nonradiological worker safety and health programs just as the<br />
Occupational Safety and Health Administration (OSHA) does for the private industry.<br />
World Health Organization (WHO) is an agency of the United Nations that specializes in the<br />
attainment by all peoples of the highest possible level of health.<br />
Zoonosis or zoonose is an infectious disease that can be transmitted (in some instances, by a<br />
vector) from nonhuman animals, both wild and domestic, to humans or from humans to<br />
nonhuman animals (the latter is sometimes called reverse zoonosis). Zoonotic is an adjective<br />
that pertains to zoonosis.<br />
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Appendix B<br />
Pathogen and Toxin Lists<br />
B.1 Introduction and Scope<br />
Pathogens and toxins are discussed in detail in Section 3.3.2 of this manual. This appendix<br />
provides the following lists of biological agents and toxins presented in Section 3.3.2:<br />
• Human etiologic agents (pathogens) from Appendix B of the NIH Guidelines<br />
• Select agents and toxins from the <strong>National</strong> Select Agent Registry (NSAR)<br />
• Plant pathogens previously identified by U.S. Department of Agriculture (USDA)<br />
These lists are provided for convenience in this manual, but may not reflect the actual regulatory<br />
list or applicable agents or materials at any given time. Regulatory sources, standards, and Web<br />
links noted in this appendix and Section 3.3.2 should be consulted to confirm applicable agents<br />
or toxins.<br />
B.2 NIH Guidelines Human Etiologic Agents<br />
This section provides a list of human pathogens and their Risk Group (RG) 2, RG3, and RG4<br />
designations as excerpted from Appendix B (Classification of Human Etiologic Agents on the<br />
Basis of Hazard) of the NIH Guidelines, Amendment Effective September 22, 2009.<br />
B.2.1 Risk Group 1 Agents<br />
RG1 agents are not associated with disease in healthy adult humans. Examples of RG1 agents<br />
include asporogenic Bacillus subtilis or Bacillus licheniformis (see Appendix C-IV-A, Bacillus<br />
subtilis or Bacillus licheniformis Host-Vector Systems, Exceptions); adeno-associated virus<br />
(AAV) Types 1 through 4; and recombinant AAV constructs, in which the transgene does not<br />
encode either a potentially tumorigenic gene product or a toxin molecule and which are<br />
produced in the absence of a helper virus. A strain of Escherichia coli (see Appendix C-II-A,<br />
Escherichia coli K-12 Host Vector Systems, Exceptions) is an RG1 agent if it 1) does not<br />
possess a complete lipopolysaccharide (i.e., lacks the O antigen); and 2) does not carry any<br />
active virulence factor (e.g., toxins) or colonization factors and does not carry any genes<br />
encoding these factors.<br />
Those agents not listed in RGs 2, 3, and 4 are not automatically or implicitly classified in RG1; a<br />
risk assessment must be conducted based on the known and potential properties of the agents<br />
and their relationship to agents that are listed.<br />
B.2.2 Risk Group 2 Agents<br />
RG2 agents are associated with human disease that is rarely serious and for which preventive<br />
or therapeutic interventions are often available.<br />
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Risk Group 2 Bacterial Agents Including Chlamydia<br />
• Acinetobacter baumannii (formerly Acinetobacter calcoaceticus)<br />
• Actinobacillus<br />
• Actinomyces pyogenes (formerly Corynebacterium pyogenes)<br />
• Aeromonas hydrophila<br />
• Amycolata autotrophica<br />
• Archanobacterium haemolyticum (formerly Corynebacterium haemolyticum)<br />
• Arizona hinshawii - all serotypes<br />
• Bacillus anthracis<br />
• Bartonella henselae, B. quintana, B. vinsonii<br />
• Bordetella including B. pertussis<br />
• Borrelia recurrentis, B. burgdorferi<br />
• Burkholderia (formerly Pseudomonas species) (except those listed in Appendix B-III-A (RG3))<br />
• Campylobacter coli, C. fetus, C. jejuni<br />
• Chlamydia psittaci, C. trachomatis, C. pneumoniae<br />
• Clostridium botulinum, Cl. chauvoei, Cl. haemolyticum, Cl. histolyticum, Cl. novyi, Cl. septicum, Cl.<br />
tetani<br />
• Corynebacterium diphtheriae, C. pseudotuberculosis, C. renale<br />
• Dermatophilus congolensis<br />
• Edwardsiella tarda<br />
• Erysipelothrix rhusiopathiae<br />
• Escherichia coli - all enteropathogenic, enterotoxigenic, enteroinvasive and strains bearing K1<br />
• antigen, including E. coli O157:H7<br />
• Haemophilus ducreyi, H. influenzae<br />
• Helicobacter pylori<br />
• Klebsiella: All species except K. oxytoca (RG1)<br />
• Legionella including L. pneumophila<br />
• Leptospira interrogans: All serotypes<br />
• Listeria<br />
• Moraxella<br />
• Mycobacterium (except those listed in Appendix B-III-A (RG3)) including M. avium complex, M.<br />
asiaticum, M.bovis BCG vaccine strain, M. chelonei, M. fortuitum, M. kansasii, M. leprae, M.<br />
malmoense, M. marinum, M.paratuberculosis, M. scrofulaceum, M. simiae, M. szulgai, M. ulcerans, M.<br />
xenopi<br />
• Mycoplasma, except M. mycoides and M. agalactiae, which are restricted animal pathogens<br />
• Neisseria gonorrhoeae, N. meningitidis<br />
• Nocardia asteroides, N. brasiliensis, N. otitidiscaviarum, N. transvalensis<br />
• Rhodococcus equi<br />
• Salmonella including S. arizonae, S. cholerasuis, S. enteritidis, S. gallinarum-pullorum, S. meleagridis,<br />
S.paratyphi, A, B, C, S. typhi, S. typhimurium<br />
• Shigella including S. boydii, S. dysenteriae, type 1, S. flexneri, S. sonnei<br />
• Sphaerophorus necrophorus<br />
• Staphylococcus aureus<br />
• Streptobacillus moniliformis<br />
• Streptococcus including S. pneumoniae, S. pyogenes<br />
• Treponema pallidum, T. carateum<br />
• Vibrio cholerae, V. parahemolyticus, V. vulnificus<br />
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• Yersinia enterocolitica<br />
Risk Group 2 Fungal Agents<br />
• Blastomyces dermatitidis<br />
• Cladosporium bantianum, C. (Xylohypha) trichoides<br />
• Cryptococcus neoformans<br />
• Dactylaria galopava (Ochroconis gallopavum)<br />
• Epidermophyton<br />
• Exophiala (Wangiella) dermatitidis<br />
• Fonsecaea pedrosoi<br />
• Microsporum<br />
• Paracoccidioides braziliensis<br />
• Penicillium marneffei<br />
• Sporothrix schenckii<br />
• Trichophyton<br />
Risk Group 2 Parasitic Agents<br />
• Ancylostoma human hookworms including A. duodenale, A. ceylanicum<br />
• Ascaris including Ascaris lumbricoides suum<br />
• Babesia including B. divergens, B. microti<br />
• Brugia filaria worms including B. malayi, B. timori<br />
• Coccidia<br />
• Cryptosporidium including C. parvum<br />
• Cysticercus cellulosae (hydatid cyst, larva of T. solium)<br />
• Echinococcus including E. granulosis, E. multilocularis, E. vogeli<br />
• Entamoeba histolytica<br />
• Enterobius<br />
• Fasciola including F. gigantica, F. hepatica<br />
• Giardia including G. lamblia<br />
• Heterophyes<br />
• Hymenolepis including H. diminuta, H. nana<br />
• Isospora<br />
• Leishmania including L. braziliensis, L. donovani, L. ethiopia, L. major, L. mexicana, L. peruvania, L. tropica<br />
• Loa loa filaria worms<br />
• Microsporidium<br />
• Naegleria fowleri<br />
• Necator human hookworms including N. americanus<br />
• Onchocerca filaria worms including, O. volvulus<br />
• Plasmodium including simian species, P. cynomologi, P. falciparum, P. malariae, P. ovale, P. vivax<br />
• Sarcocystis including S. sui hominis<br />
• Schistosoma including S. haematobium, S. intercalatum, S. japonicum, S. mansoni, S. mekongi<br />
• Strongyloides including S. stercoralis<br />
• Taenia solium<br />
• Toxocara including T. canis<br />
• Toxoplasma including T. gondii<br />
• Trichinella spiralis<br />
• Trypanosoma including T. brucei brucei, T. brucei gambiense, T. brucei rhodesiense, T. cruzi<br />
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• Wuchereria bancrofti filaria worms<br />
Risk Group 2 Viruses<br />
Adenoviruses, human: All types<br />
Alphaviruses (togaviruses), group A arboviruses:<br />
• Eastern equine encephalomyelitis virus<br />
• Venezuelan equine encephalomyelitis vaccine strain TC-83<br />
• Western equine encephalomyelitis virus<br />
Arenaviruses:<br />
• Lymphocytic choriomeningitis virus (nonneurotropic strains)<br />
• Tacaribe virus complex<br />
• Other viruses as listed in BMBL<br />
Bunyaviruses:<br />
• Bunyamwera virus<br />
• Rift Valley fever virus vaccine strain MP-12<br />
• Other viruses as listed in BMBL<br />
Calciviruses<br />
Coronaviruses<br />
Flaviviruses (togaviruses), group B arboviruses:<br />
• Dengue virus, serotypes 1, 2, 3, and 4<br />
• Yellow fever virus vaccine strain 17D<br />
• Other viruses as listed in BMBL<br />
Hepatitis A, B, C, D, and E viruses<br />
Herpesviruses, except Herpesvirus simiae (monkey B virus) (see Appendix B-IV-D, Risk Group 4 (RG4) –<br />
Viral Agents):<br />
• Cytomegalovirus<br />
• Epstein Barr virus<br />
• Herpes simplex, types 1 and 2<br />
• Herpes zoster<br />
• Human herpesvirus, types 6 and 7<br />
Orthomyxoviruses:<br />
• Influenza viruses, types A, B, and C<br />
• Tick-borne orthomyxoviruses<br />
Papovaviruses: All human papilloma viruses<br />
Paramyxoviruses:<br />
• Newcastle disease virus<br />
• Measles virus<br />
• Mumps virus<br />
• Parainfluenza viruses, types 1, 2, 3, and 4<br />
• Respiratory syncytial virus<br />
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Parvoviruses: Human parvovirus (B19)<br />
Picornaviruses:<br />
• Coxsackie viruses, types A and B<br />
• Echoviruses, all types<br />
• Polioviruses, all types, wild and attenuated<br />
• Rhinoviruses, all types<br />
Poxviruses: All types except monkeypox virus (see Appendix B-III-D, Risk Group 3 (RG3)—Viruses and<br />
Prions) and restricted poxviruses including alastrim, smallpox, and whitepox (see BMBL Section V-L)<br />
Reoviruses: All types including coltivirus, human rotavirus, and orbivirus (Colorado tick fever virus)<br />
Rhabdoviruses:<br />
• Rabies virus, all strains<br />
• Vesicular stomatitis virus (<strong>Laboratory</strong>-adapted strains including VSV-Indiana, San Juan, and Glasgow)<br />
Togaviruses (see alphaviruses and flaviviruses): Rubivirus (rubella)<br />
B.2.3 Risk Group 3 Agents<br />
RG3 agents are associated with serious or lethal human disease for which preventive or therapeutic<br />
interventions may be available.<br />
Risk Group 3 Bacterial Agents Including Rickettsia<br />
• Bartonella<br />
• Brucella including B. abortus, B. canis, B. suis<br />
• Burkholderia (Pseudomonas) mallei, B. pseudomallei<br />
• Coxiella burnetii<br />
• Francisella tularensis<br />
• Mycobacterium bovis (except BCG strain, see Appendix B-II-A, Risk Group 2 (RG2) - Bacterial Agents<br />
Including Chlamydia), M. tuberculosis<br />
• Pasteurella multocida type B: "Buffalo" and other virulent strains<br />
• Rickettsia akari, R. australis, R. canada, R. conorii, R. prowazekii, R. rickettsii, R, siberica, R.<br />
tsutsugamushi, R. typhi (R. mooseri)<br />
• Yersinia pestis<br />
Risk Group 3 Fungal Agents<br />
• Coccidioides immitis (sporulating cultures; contaminated soil)<br />
• Histoplasma capsulatum, H. capsulatum var. duboisii<br />
Risk Group 3 Parasitic Agents<br />
None<br />
Risk Group 3 Viruses and Prions<br />
Alphaviruses (Togaviruses), group A arboviruses:<br />
• Semliki Forest virus<br />
• St. Louis encephalitis virus<br />
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• Venezuelan equine encephalomyelitis virus (except the vaccine strain TC-83, see Appendix B-II-D<br />
(RG2))<br />
• Other viruses as listed in BMBL<br />
Arenaviruses:<br />
• Flexal<br />
• Lymphocytic choriomeningitis virus (LCM) (neurotropic strains)<br />
Bunyaviruses:<br />
• Hantaviruses including Hantaan virus<br />
• Rift Valley fever virus<br />
Flaviviruses (togaviruses), group B arboviruses:<br />
• Japanese encephalitis virus<br />
• Yellow fever virus<br />
• Other viruses as listed in BMBL<br />
Orthomyxoviruses: Influenza viruses 1918–1919 H1N1 (1918 H1N1), human H2N2 (1957–1968), and<br />
highly pathogenic avian influenza H5N1 strains within the Goose/Guangdong/96-like H5 lineage (HPAI<br />
H5N1)<br />
Poxviruses: Monkeypox virus<br />
Prions: Transmissible spongioform encephalopathy (TME) agents (Creutzfeldt-Jacob disease and kuru<br />
agents) (see BMBL, for containment instruction)<br />
Retroviruses<br />
• Human immunodeficiency virus (HIV) types 1 and 2<br />
• Human T cell lymphotropic virus (HTLV) types 1 and 2<br />
• Simian immunodeficiency virus (SIV)<br />
Rhabdoviruses: Vesicular stomatitis virus<br />
B.2.4 Risk Group 4 Agents<br />
RG4 agents are likely to cause serious or lethal human disease for which preventive or<br />
therapeutic interventions are not usually available.<br />
Risk Group 4 Bacterial Agents<br />
None<br />
Risk Group 4 Fungal Agents<br />
None<br />
Risk Group 4 Parasitic Agents<br />
None<br />
Risk Group 4 Viral Agents<br />
Arenaviruses<br />
• Guanarito virus<br />
• Lassa virus<br />
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• Junin virus<br />
• Machupo virus<br />
• Sabia<br />
Bunyaviruses (Nairovirus): Crimean-Congo hemorrhagic fever virus<br />
Filoviruses<br />
• Ebola virus<br />
• Marburg virus<br />
• Flaviruses (Togaviruses), group B arboviruses: Tick-borne encephalitis virus complex including<br />
Absetterov, Central European encephalitis, Hanzalova, Hypr, Kumlinge, Kyasanur Forest disease,<br />
Omsk hemorrhagic fever, and Russian spring-summer encephalitis viruses<br />
Herpesviruses (alpha): Herpesvirus simiae (herpes B or monkey B virus)<br />
Paramyxoviruses: Equine morbillivirus<br />
Hemorrhagic fever agents and viruses as yet undefined<br />
B.2.5 Animal Viral Etiologic Agents in Common Use<br />
The following list of animal etiologic agents is appended to the list of human etiologic agents.<br />
None of these agents are associated with disease in healthy adult humans; they are commonly<br />
used in laboratory experimental work. A containment level appropriate for RG1 human agents is<br />
recommended for their use. For agents that are infectious to human cells, e.g., amphotropic and<br />
xenotropic strains of murine leukemia virus, a containment level appropriate for RG2 human<br />
agents is recommended.<br />
Baculoviruses:<br />
• Herpesviruses<br />
• Herpesvirus ateles<br />
• Herpesvirus saimiri<br />
• Marek's disease virus<br />
• Murine cytomegalovirus<br />
Papovaviruses:<br />
• Bovine papilloma virus<br />
• Polyoma virus<br />
• Shope papilloma virus<br />
• Simian virus 40 (SV40)<br />
Retroviruses:<br />
• Avian leukosis virus<br />
• Avian sarcoma virus<br />
• Bovine leukemia virus<br />
• Feline leukemia virus<br />
• Feline sarcoma virus<br />
• Gibbon leukemia virus<br />
• Mason-Pfizer monkey virus<br />
• Mouse mammary tumor virus<br />
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• Murine leukemia virus<br />
• Murine sarcoma virus<br />
• Rat leukemia virus<br />
B.2.6 Murine Retroviral Vectors<br />
Murine retroviral vectors to be used for human transfer experiments (less than 10 liters) that<br />
contain less than 50% of their respective parental viral genome and that have been<br />
demonstrated to be free of detectable replication competent retrovirus can be maintained,<br />
handled, and administered under <strong>Biosafety</strong> Level (BL) 1 containment.<br />
B.3 Select Agents and Toxins<br />
Table B-1 provides the list of select agents and toxins on the <strong>National</strong> Select<br />
Agent Registry (NSAR) established by the Department of Health and Human<br />
Services (HHS) Centers for Disease Control and Prevention (CDC) and United<br />
States Department of Agriculture (USDA). The most-recent online list may be<br />
found at http://www.selectagents.gov/index.html. Listed select agents and toxins<br />
are categorized as follows:<br />
• Agents and toxins that cause disease in humans are listed by HHS CDC as:<br />
o HHS select agents and toxins that affect humans<br />
o OVERLAP select agents and toxins that affect both (or OVERLAP with) humans and<br />
animals<br />
• Agents and toxins that cause disease in agricultural animals or plants are listed by USDA<br />
as:<br />
o OVERLAP select agents and toxins that affect humans and animals<br />
o USDA select agents and toxins that affect animals<br />
o USDA Plant Protection and Quarantine (PPQ) select agents and toxins that affect<br />
plants<br />
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Table B-1<br />
<strong>National</strong> Select Agent Registry Select Agents and Toxins<br />
HHS and USDA Select Agents and Toxins<br />
7 CFR Part 331, 9 CFR Part 121, and 42 CFR Part 73<br />
HHS Select Agents and Toxins<br />
Abrin<br />
Botulinum neurotoxins<br />
Botulinum neurotoxin producing species of<br />
Clostridium<br />
Cercopithecine herpesvirus 1 (Herpes B<br />
virus)<br />
Clostridium perfringens epsilon toxin<br />
Coccidioides posadasii/Coccidioides<br />
immitis<br />
Conotoxins<br />
Coxiella burnetii<br />
Crimean-Congo hemorrhagic fever virus<br />
Diacetoxyscirpenol<br />
Eastern Equine Encephalitis virus<br />
Ebola virus<br />
Francisella tularensis<br />
Lassa fever virus<br />
Marburg virus<br />
Monkeypox virus<br />
Reconstructed replication competent forms<br />
of the 1918 pandemic influenza virus<br />
containing any portion of the coding<br />
regions of all eight gene segments<br />
(Reconstructed1918 Influenza virus)<br />
Ricin<br />
Rickettsia prowazekii<br />
Rickettsia rickettsii<br />
Saxitoxin<br />
Shiga-like ribosome inactivating proteins<br />
Shigatoxin<br />
South American hemorrhagic fever viruses<br />
(Flexal, Guanarito, Junin, Machupo, Sabia)<br />
Staphylococcal enterotoxins<br />
T-2 toxin<br />
Tetrodotoxin<br />
Tick-borne encephalitis complex (flavi)<br />
OVERLAP Select Agents and Toxins<br />
Bacillus anthracis<br />
Brucella abortus<br />
Brucella melitensis<br />
Brucella suis<br />
Burkholderia mallei (formerly Pseudomonas<br />
mallei)<br />
Burkholderia pseudomallei (formerly<br />
Pseudomonas pseudomallei)<br />
Hendra virus<br />
Nipah virus<br />
Rift Valley fever virus<br />
Venezuelan equine encephalitis virus<br />
USDA Select Agents and Toxins<br />
African horse sickness virus<br />
African swine fever virus<br />
Akabane virus<br />
Avian influenza virus (highly pathogenic)<br />
Bluetongue virus (exotic)<br />
Bovine spongiform encephalopathy agent<br />
Camel pox virus<br />
Classical swine fever virus<br />
Ehrlichia ruminantium (Heartwater)<br />
Foot-and-mouth disease virus<br />
Goat pox virus<br />
Japanese encephalitis virus<br />
Lumpy skin disease virus<br />
Malignant catarrhal fever virus<br />
(Alcelaphine herpesvirus, Type 1)<br />
Menangle virus<br />
Mycoplasma capricolum subspecies<br />
capripneumoniae<br />
(contagious caprine pleuropneumonia)<br />
Mycoplasma mycoides subspecies mycoides<br />
small colony (Mmm SC) (contagious bovine<br />
pleuropneumonia)<br />
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HHS and USDA Select Agents and Toxins<br />
7 CFR Part 331, 9 CFR Part 121, and 42 CFR Part 73<br />
viruses (Central European tick-borne<br />
encephalitis, Far Eastern tick-borne<br />
encephalitis, Kyasanur Forest disease,<br />
Omsk hemorrhagic fever, Russian Spring<br />
and Summer encephalitis)<br />
Variola major virus (Smallpox virus)<br />
Variola minor virus (Alastrim)<br />
Yersinia pestis<br />
Peste des petits ruminants virus<br />
Rinderpest virus<br />
Sheep pox virus<br />
Swine vesicular disease virus<br />
Vesicular stomatitis virus (exotic): Indiana<br />
subtypes VSV-IN2 and VSV-IN3<br />
Virulent Newcastle disease virus 1<br />
USDA Plant Protection and Quarantine<br />
(PPQ) Select Agents and Toxins<br />
Peronosclerospora philippinensis<br />
(Peronosclerospora sacchari)<br />
Phoma glycinicola (formerly Pyrenochaeta<br />
glycines)<br />
Ralstonia solanacearum race 3, biovar 2<br />
Rathayibacter toxicus<br />
Sclerophthora rayssiae var zeae<br />
Synchytrium endobioticum<br />
Xanthomonas oryzae<br />
Xylella fastidiosa (citrus variegated chlorosis<br />
strain)<br />
Source: NSAR list updated 11/17/2008<br />
1 A virulent Newcastle disease virus (avian paramyxovirus serotype 1) has an intracerebral<br />
pathogenicity index in day-old chicks (Gallus gallus) of 0.7 or greater or has an amino acid sequence<br />
at the fusion (F) protein cleavage site that is consistent with virulent strains of Newcastle disease<br />
virus. A failure to detect a cleavage site that is consistent with virulent strains does not confirm the<br />
absence of a virulent virus.<br />
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Table B-2 provides additional information, permissible toxin amounts, and synonyms for biological<br />
toxins that are listed on the NSAR of select agents and toxins. A permissible toxin amount is the<br />
maximum quantity of biological toxin that can be under the control of a principal investigator at<br />
any time without regulation under the CDC and USDA select agent and toxin regulations.<br />
Table B-2<br />
Additional Information for <strong>National</strong> Select Agent Registry Toxins<br />
Name<br />
(Permissible<br />
Toxin Amount)<br />
Abrin<br />
(100 mg)<br />
Botulinum<br />
neurotoxin<br />
(0.5 mg)<br />
Clostridium<br />
perfringens<br />
epsilon toxin<br />
(100 mg)<br />
Conotoxins<br />
(100 mg)<br />
Diacetoxyscirpenol<br />
(1,000 mg)<br />
Ricin<br />
(100 mg)<br />
Synonyms/Types (Strains)/ Key<br />
Words<br />
Abrina, Abrin B, Abrin C, Abrin D,<br />
Abrin reconstituted (A+B mix),<br />
Abrin agglutinin, Toxalbumin<br />
Botulinum neurotoxin, Types A, B,<br />
C, C1, C2, D, E, F, and G (7<br />
serotypes with a few subtypes).<br />
Clostridium botulinum toxin,<br />
botulinum toxin, botulinus toxin,<br />
botulin toxin<br />
Clostridium perfringens Type B<br />
epsilon toxin; Clostridium<br />
perfringens, Type D epsilon toxin<br />
Conotoxins GI, GIA, GII, GIV,<br />
GIIIA, GIIIB, GIIIC, GIVA, GVIB,<br />
GVIC, Im1, MI,MVIIA,MVIIB,<br />
MVIIC,MVIIIv, MVIIDSIA, SVIB<br />
(plus more). Conus geographus<br />
venom, Conus magus venom,<br />
Conus straiatus venom<br />
Diacetoxyscirpenol; Anguidin;<br />
Auguidine; Insariotoxin; DAS; 4,15-<br />
Diacetoxyscirpen-3-OL;<br />
Scirpenetriol 4,15-diacetate; 4<br />
beta, 15-diacetoxy-3-alphahydroxy-12,<br />
13-epoxytrichotech-9-<br />
ene<br />
Ricinotoxin, Ricinus toxin, Ricin A,<br />
Ricin B, Ricin C, Ricin D, Ricin<br />
Toxin-Con A, Concanvalin A, Ricin<br />
nitrogen, Ricine, Ricin total<br />
CAS Numbers<br />
1393-62-0 (Abrin)<br />
53597-23-2 (Abrin A)<br />
53597-24-3 (Abrin C)<br />
Description<br />
A powerful phytotoxin<br />
present in the seeds of<br />
Abrus precatorius<br />
(common names include<br />
precatory bean, rosemary<br />
pea, and jequirity).<br />
93384-43-1 (Type A) Produced by the soil<br />
bacterium Clostridium<br />
botulinum under anaerobic<br />
conditions. The most<br />
potent toxin known but<br />
heat labile and neutralized<br />
by specific antibodies.<br />
None found<br />
81133-24-6 (IV)<br />
76862-65-2 (GI)<br />
156467-85-5 (Im 1)<br />
106375-28-4 (GVIA)<br />
107452-89-1 (MVIIA)<br />
147794-23-8 (MVIIC)<br />
150433-82-2 (SVIB)<br />
2270-40-8<br />
4297-61-4 (3-Aacetyldiacetoxyscir-penol).<br />
9009-86-3 (Ricin),<br />
63099-95-6<br />
9040-12-4 (Ricin D)<br />
72514-84-2 (Ricin D<br />
One of 12 protein toxins<br />
produced by the bacterium<br />
Clostridium perfringens. Of<br />
the 5 types of Clostridium.<br />
perfringens, only 2 (Types<br />
B and D) make the epsilon<br />
neurotoxin.<br />
Small peptide venoms<br />
produced by cone shells<br />
(Conidia) and marine<br />
snails (carnivorous<br />
gastropod “cone”<br />
mollusks). Venoms vary<br />
between species. Act on<br />
neuronal communications<br />
but each (alpha-mu-, and<br />
omega-conotoxins) target<br />
a different aspect of the<br />
process.<br />
Trichothecene compound<br />
toxins (mycotoxins)<br />
produced by various<br />
fungus Fusarium, which<br />
grow on barley, corn, rye,<br />
wheat, etc.<br />
A powerful phytotoxin<br />
present in the seeds of the<br />
castor bean oil plant<br />
(Ricinus communis).<br />
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Name<br />
(Permissible<br />
Toxin Amount)<br />
Saxitoxin<br />
(100 mg)<br />
Shigatoxin<br />
(100 mg)<br />
Shiga-likeribosome<br />
inactivating<br />
proteins<br />
(100 mg)<br />
Staphylococcal<br />
enterotoxin<br />
(5.0 mg)<br />
Tetrotoxin<br />
(100 mg)<br />
T-2 Toxin<br />
(1000 mg)<br />
Synonyms/Types (Strains)/ Key<br />
Words<br />
hydrolysate, Ricinus lectin, Ricnus<br />
agglutinin<br />
Mytilotoxin; Saxitoxin hydrate;<br />
Saxitoxin hydrochloride; Saxitoxin<br />
dihydrochloride; STX<br />
dihydrochloride; neo-Saxitoxin<br />
(neo-STX); Saxitoxin p-bromo<br />
benzenesulfonate; Mytilus<br />
californianus poison/toxin;<br />
Saxidomas giganteus poison/<br />
toxin; Gonyaulas catenella poison/<br />
toxin; Saxitonin diacetate salt<br />
Shigella dysenteria neurotoxin;<br />
shigella diysenteriae exotoxin Type<br />
I; Verocytotoxin; Verotoxin<br />
A-chain portion of Shigella<br />
dysenteria Shigatoxin;<br />
Enterohemorrhagic Escherichia<br />
coli toxin SLT-1 and SLT-2;<br />
Escherichia coli 0157; H7 toxin<br />
Staphylococcus enterotoxins types<br />
A,B,F. Enterotoxin F is the Toxic<br />
Shock Syndrome “Toxin-1.”<br />
Fugu poison; fugtoxin;<br />
Anhydroepiterodotoxin;<br />
Deoxytetrodotoxin; 4-<br />
Deoxytetrodotoxin;<br />
Deoxyterttoxin;Diateylanhydrotetrd<br />
otoxin; Diacetate 4,9-<br />
anhydrotetrodotoxin;<br />
Ethoxytetrodotoxin; Maculotoxin;<br />
Ethyl tetrodotoxin; 4-Deoxy<br />
tetrodotoxin; Spheroidine;<br />
Tarichatoxin; 4-amino-4-deoxy,<br />
4,9-Anhydrotetrodotoxin; 8,8-<br />
Diacetate 4,9-anhydrotetrodotoxin;<br />
tetrodotoxin citrate; TTX; (4-alpha)-<br />
4-amino-4-deoxy-tetrodotoxin<br />
Toxin T-2; T-2 mycotoxin; T-2<br />
hemisuccinate; T-2 tetraol; T-2<br />
Toxin d3; T-2 Triol; 2,4,5-T-2<br />
ethylhexyl ester; 2,4,5-T-2<br />
methylpropyl ester; Insariotoxin;<br />
12,13-tricothecene; Fusariotoxine<br />
T-2; Scirpenol<br />
Source: LBNL EH&S Group (July 2003).<br />
CAS Numbers<br />
ananine chain)<br />
66419-04-03 (Ricin D isoleucine<br />
chain reduced)<br />
35523-89-8<br />
35554-08-6<br />
80450-05-01<br />
64296-20-4 (neo-STX from<br />
dinoflagellates)<br />
220355-66-8 (Saxitoxin<br />
doacetate salt)<br />
Description<br />
Toxin produced by<br />
bacterium that grow in<br />
other organisms; e.g.,<br />
poisonous mussels<br />
(Mytilus), clams<br />
(Saxidomas, and Plankton<br />
(Gonyaulax).<br />
7575-64-1 Protein exotoxin produced<br />
by the bacterium Shigella<br />
dysenteriae that affects<br />
both the gut and the<br />
central nervous system.<br />
None found<br />
Group of structurallyrelated<br />
toxins similar to<br />
shigatoxin that block cell<br />
protein synthesis.<br />
11100-45-1 (Enterotoxin B) Toxin produced by a strain<br />
of Staphylococcus aureus.<br />
Acts on receptors in gut.<br />
4368-28-9 (tetrodotoxin)<br />
13072-89-4 (4,9-<br />
anhydrotetrodotoxin)<br />
13285-84-2 (8,8-diacetate<br />
4,9-anhydro tetrodotoxin)<br />
7724-38-1 [(4 alpha)-4-<br />
amino-4-deoxytetrodotoxin]<br />
7724-41-6 (4-deoxytetrodotoxin)<br />
18660-81-6 (Tetrodotoxin<br />
citrate salt)<br />
7724-39-2<br />
[O(sup 4)- methyl<br />
tetrodotoxin]<br />
7724-40-5 [O(sup 5)-ethyl<br />
tetrodotoxin]<br />
21259-20-1 (T-2 Toxin)<br />
34114-99-3 (T-2 tetraol)<br />
120467-83-6 (T-2 Toxind3)<br />
34114-98-2 (T-2 triol)<br />
1928-47-8 (2,4,5-T2<br />
ethylhexyl ester)<br />
4938-72-1 (2,4,5-T-2-<br />
methylproply ester)<br />
Highly lethal neurotoxin<br />
present in numerous<br />
species of puffer fish<br />
(Tetraodontoidea) and<br />
newts (Tarika).<br />
Trichothecene compound<br />
toxins (mycotoxins)<br />
produced by various<br />
species of fungus<br />
Fusarium, which grows on<br />
barley, corn, rye, wheat.<br />
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B.4 Plant Pathogens<br />
This appendix of the <strong>Biosafety</strong> <strong>Manual</strong> provides lists of bacterial, fungal, and viral plant<br />
pathogens that may be used to identify agents that might be considered plant pathogens.<br />
Current USDA Web sites and the USDA permit process may be needed to determine if the<br />
USDA considers agents in specific locations (e.g., California) to be plant pathogens.<br />
B.4.1 Plant Pathogen Bacteria (by Scientific Name)<br />
Agrobacterium radiobacter, Agrobacterium rubi, Agrobacterium tumefaciens, Agrobacterium<br />
vitis, Burkholderia andropogonis, Burkholderia caryophylli, Burkholderia cepacia, Burkholderia<br />
cichorii, Burkholderia corrugata, Burkholderia gladioli pv. gladioli, Clavibacter michiganensis<br />
subsp. insidiosus, Clavibacter michiganensis subsp. michiganensis, Clavibacter michiganensis<br />
subsp. sepedonicus, Curtobacterium flaccumfaciens pv. flaccumfaciens, Erwinia amylovora,<br />
Erwinia carotovora subsp. atroseptica, Erwinia carotovora subsp. carotovora, Erwinia<br />
chrysanthemi, Erwinia chrysanthemi pv. chrysanthemi, Erwinia chrysanthemi pv.<br />
dieffenbachiae, Erwinia chrysanthemi pv. zeae, Erwinia tracheiphila, Pantoea stewartii subsp.<br />
stewartii, Pseudomonas syringae pv. apii, Pseudomonas syringae pv. atrofaciens,<br />
Pseudomonas syringae pv. coronafaciens, Pseudomonas syringae pv. glycinea, Pseudomonas<br />
syringae pv. lachrymans, Pseudomonas syringae pv. mori, Pseudomonas syringae pv.<br />
papulans, Pseudomonas syringae pv. phaseolicola, Pseudomonas syringae pv. pisi,<br />
Pseudomonas syringae pv. syringae, Pseudomonas syringae pv. tabaci, Pseudomonas<br />
syringae pv. tomato1, Ralstonia solanacearum2, Rhodococcus fascians, Spiroplasma citri,<br />
Streptomyces scabies, Xanthomonas campestris pv. armoraciae, Xanthomonas campestris pv.<br />
campestris, Xanthomonas campestris pv. carotae, Xanthomonas campestris pv. cucurbitae,<br />
Xanthomonas campestris pv. hederae, Xanthomonas campestris pv. juglandis, Xanthomonas<br />
campestris pv. papavericola, Xanthomonas campestris pv. pelargonii, Xanthomonas campestris<br />
pv. pruni, Xanthomonas campestris pv. raphani, Xanthomonas campestris pv. vitians,<br />
Xanthomonas campestris pv. zinniae, Xanthomonas fragariae, Xanthomonas phaseoli pv.<br />
alfalfae, Xanthomonas phaseoli pv. begoniae, Xanthomonas phaseoli pv. glycines,<br />
Xanthomonas phaseoli pv. phaseoli, Xanthomonas translucens pv. translucens, Xanthomonas<br />
vesicatoria.<br />
B.4.2 Plant Pathogen Fungi (by Scientific Name)<br />
CHYTRIDIOMYCETES<br />
Physoderma maydis<br />
OOMYCETES<br />
Albugo candida, Peronospora sojae, Peronospora trifoliorum, Peronospora viticola,<br />
Phytophthora cactorum, Phytophthora capsici, Phytophthora cinnamomi, Phytophthora citricola,<br />
Phytophthora fragariae, Phytophthora infestans, Phytophthora megasperma, Phytophthora<br />
megasperma f.sp. medicaginis, Phytophthora rubi s.sp. fragariae, Phytophthora sojae,<br />
Plasmodiophora brassicae, Pythium aphanidermatum, Pythium arrhenomanes, Pythium<br />
graminicola, Pythium irregulare, Pythium ultimum, Sclerophthora macrospora.<br />
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ASCOMYCETES<br />
Apiosporina morbosa (black knot), Botryosphaeria obtusa, Botryosphaeria ribis (B. dothidea, B.<br />
berengeriana), Claviceps purpurea, Cymadothea trifolii (sooty blotch), Diaporthe phaseolorum,<br />
Gaeumannomyces graminis, Gibberella zeae, Glomerella cingulata, Leptosphaerulina trifolii,<br />
Monilinia fructicola (Sclerotinia fructicola), Nectria cinnabarina, Ophiostoma ulmi (Ceratocystis<br />
ulmi), Pseudopeziza medicaginis, Pseudopeziza trifolii, Sclerotinia sclerotiorum (Whetzelinia<br />
sclerotiorum), Sclerotinia trifoliorum, Valsa ambiens, Venturia inaequalis (apple scab), Xylaria<br />
polymorpha.<br />
Powdery Mildews<br />
Erysiphe graminis, Microsphaera vaccinii (on Ericaceae), Podosphaera clandestina (on<br />
Rosaceae), Sphaerotheca Asteraceae, Cucurbitaceae, Scrophulariaceae), Sphaerotheca<br />
macularis (on hops and strawberry), Unicinula viticola.<br />
Coelomycetes<br />
Colletotrichum acutatum, Colletotrichum coccodes, Colletotrichum destructivum, Colletotrichum<br />
fragariae, Colletotrichum gloeosporioides, Colletotrichum graminicola, Colletotrichum trifolii,<br />
Macrophomina phaseolina (Macrophoma phaseolina, M. phaseoli, Botryodiplodia phaseoli),<br />
Phoma medicaginis, Phomopsis juniperovora, Phomopsis sojae, Phomopsis viticola, Septoria<br />
rubi, Septoria tritici, Sphaeropsis sapinea (Diplodia pinea), Stagonospora nodorum (Septoria<br />
nodorum), Stenocarpelia maydis (Diplodia zeae, D. zeae-maydis).<br />
Hyphomycetes<br />
Alternaria alternata, Alternaria solani, Bipolaris maydis (Heminthosporium maydis, Drechslera<br />
maydis), Bipolaris sorokiniana (Helminthosporium sorokiniana, Drechslera sorokiniana),<br />
Bipolaris victoriae (Helminthosporium victoriae, Drechslera victoriae), Botrytis cinerea.<br />
Cercospora medicaginis, Cercospora zeae-maydis, Cladosporium herbarum, Drechslera<br />
avenae (on oats, other grasses), Drechslera graminea (on barley, other grasses), Drechslera<br />
poae (on grasses), Drechslera teres (on barley, other grasses), Drechslera tritici-repentis (on<br />
cereals, other grasses), Exserohilum turcicum (Helminthosporium turcicum, Bipolaris turcicum),<br />
Fusarium acuminatum, Fusarium avenaceum, Fusarium culmorum, Fusarium equiseti,<br />
Fusarium graminearum, Fusarium moniliforme, Fusarium oxysporum, Fusarium oxysporum,<br />
Fusarium roseum, Fusarium solani, Penicillium expansum, Rhynchosporium secalis,<br />
Thielaviopsis basicola, Verticillium albo-atrum, Verticillium dahliae.<br />
HEMIASCOMYCETES<br />
Taphrina caerulescens (leaf blister on oak, Ostrya, Rhus), Taphrina communis (plum pocket on<br />
Prunus), Taphrina deformans (peach leaf curl).<br />
BASIDIOMYCETES<br />
Wood Rotters and Root-Collar Rotters<br />
Armillaria mellea, Ceratobasidium cerealea, Daedaleopsis confragosa (Daedalea confragosa),<br />
Ganoderma applanatum (Fomes applanatus), Ganoderma lucidum, Hirschioporus pargamenus<br />
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(Trichaptum biformis, Polyporus pargamenus), Laetiporus sulphureus (Polyporus sulphureus),<br />
Phellinus gilius, Phellinus robiniae, Schizophyllum commune, Stereum ostrea, Trametes<br />
versicolor (Polyporus versicolor, Coriolus versicolor).<br />
Rusts<br />
Gymnosporangium clavipes (cedar-quince rust), Gymnosporangium globosum (cedar-hawthorn<br />
rust), Gymnosporangium juniperi-virginianae (cedar-apple rust), Puccinia coronata (on<br />
Rhamnaceae, Eleganaceae/Poaceae), Puccinia graminis (on Berberis/Poaceae), Puccinia<br />
recondita (on Ranunculaceae/Poaceae), Pucciniastrum americanum (late leaf rust on<br />
raspberry).<br />
Smuts<br />
Tilletia caries (Tilletia tritici), Tilletia laevis (Tilletia foetida), Ustilago avenae, Ustilago hordei,<br />
Ustilago tritici, Ustilago zeae.<br />
Other Basidiomycetes<br />
Rhizoctonia solani (Thanatephorus cucumeris), Sclerotium rolfsii.<br />
B.4.3 Plant Pathogen Viruses (Regulated by the State of California)<br />
Alfalfa mosaic, barley yellow dwarf, bean common mosaic, bean yellow mosaic, beet curly top,<br />
beet mosaic, cactus virus X, camellia yellow mottle, carnation mottle, cauliflower mosaic,<br />
chrysanthemum mosaic, chrysanthemum virus B, cucumber mosaic, cymbidium mosaic,<br />
dasheen mosaic, fig mosaic, impatiens necrotic spot, lettuce big vein, lettuce mosaic, lily<br />
symptomless, maize dwarf mosaic, odontoglossum ringspot, papaya ringspot, pepper mottle,<br />
plum line pattern, potato leafroll, potato virus S, potato virus X, potato virus Y, prune dwarf,<br />
prunus necrotic ringspot, squash mosaic, sugarcane mosaic, tobacco etch, tomato mosaic,<br />
tomato spotted wilt, turnip mosaic, watermelon mosaic virus 2, zucchini yellow mosaic.<br />
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Appendix C<br />
<strong>Laboratory</strong> <strong>Biosafety</strong> Level 1 and 2 Criteria<br />
C.1 Introduction and Scope<br />
This appendix describes criteria for laboratory <strong>Biosafety</strong> Level 1 (BL1) and BL2 in the same<br />
manner and level of detail presented in <strong>Biosafety</strong> in Microbiological and Biomedical Laboratories<br />
(BMBL), fifth edition. Requirements from the NIH Guidelines and Occupational Safety and<br />
Health Administration (OSHA) Bloodborne Pathogens (BBP) Standard were also added by<br />
LBNL to each BMBL criteria statement as needed to integrate requirements from all of these<br />
standards. LBNL requirements were also succinctly added when needed to clarify important<br />
requirements or implementation policy specifically related to BMBL criteria statements.<br />
See Section 4.0 of this manual for additional information on biosafety principles and levels, and<br />
Section 4.4.1 for additional information on laboratory biosafety levels. See Section 5.0 of this<br />
manual for additional information and requirements on controls described in specific criteria<br />
statements.<br />
C.2 <strong>Laboratory</strong> <strong>Biosafety</strong> Level 1<br />
<strong>Biosafety</strong> Level 1 is suitable for work involving well-characterized agents not<br />
known to consistently cause disease in immunocompetent adult humans, and<br />
present minimal potential hazard to laboratory personnel and the environment. BL1 laboratories<br />
are not necessarily separated from the general traffic patterns in the building. Work is typically<br />
conducted on open benchtops using standard microbiological practices. Special containment<br />
equipment or facility design is not required but may be used as determined by appropriate risk<br />
assessment. <strong>Laboratory</strong> personnel must have specific training in the procedures conducted in<br />
the laboratory and must be supervised by a scientist with training in microbiology or a related<br />
science.<br />
The following standard practices, safety equipment, and facility requirements apply to<br />
BL1:<br />
C.2.1 BL1 Standard Microbiological Practices<br />
1. The laboratory supervisor and work lead must enforce LBNL institutional policies that control<br />
access to the site and laboratory facilities as described in the LBNL Site Security Plan.<br />
Policies and practices include, for example, the hosting of visitors and the issuance of gate<br />
passes, badges, and/or keys to control access to the site, building, and/or room based on<br />
each individual’s business need and experiments in progress. In addition, laboratory areas<br />
should have doors for access control.<br />
2. Persons must wash their hands: (a) after working with potentially hazardous materials,<br />
recombinant materials, and animals; (b) after removing gloves; and (c) before leaving the<br />
laboratory.<br />
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3. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for<br />
human consumption are not permitted in laboratory areas. Food must be stored outside the<br />
laboratory area in cabinets or refrigerators designated and used for this purpose.<br />
4. Mouth pipetting is prohibited; mechanical pipetting devices must be used.<br />
5. Policies for the safe handling of sharps, such as needles, scalpels, pipettes, and broken<br />
glassware, must be developed and implemented. Whenever practical, laboratory<br />
supervisors should adopt improved engineering and work practice controls that reduce the<br />
risk of sharps injuries.<br />
Precautions, including those listed below, must always be taken with sharp items. These<br />
include:<br />
a. Careful management of needles and other sharps are of primary importance. Needles<br />
must not be bent, sheared, broken, recapped, removed from disposable syringes, or<br />
otherwise manipulated by hand before disposal.<br />
b. Used disposable sharps must be carefully placed in conveniently located punctureresistant<br />
containers used for sharps disposal.<br />
c. Nondisposable sharps must be placed in a hard-walled container for transport to a<br />
processing area for decontamination, preferably by autoclaving.<br />
d. Broken glassware must not be handled directly. Instead, it must be removed using a<br />
brush and dustpan, tongs, or forceps. Plasticware should be substituted for glassware<br />
whenever possible.<br />
6. Perform all procedures to minimize splashes and/or aerosols.<br />
7. Decontaminate work surfaces after completion of work and after any spill or splash of<br />
potentially infectious or viable recombinant material with appropriate disinfectant.<br />
8. Decontaminate all cultures, stocks, and other potentially infectious or recombinant materials<br />
before disposal using an effective method. Depending on where the decontamination will be<br />
performed, the following methods should be used prior to transport:<br />
a. Materials to be decontaminated outside of the immediate laboratory must be placed in a<br />
durable leak-proof container and secured for transport.<br />
b. Materials to be removed from the facility for decontamination must be packed in<br />
accordance with applicable local, state, and federal regulations.<br />
9. An effective integrated pest management program is required.<br />
10. The laboratory supervisor must ensure that laboratory personnel receive appropriate training<br />
regarding their duties, the necessary precautions to prevent exposures, and exposure<br />
evaluation procedures. Personnel must receive annual updates or additional training when<br />
procedural or policy changes occur. Personal health status may impact an individual’s<br />
susceptibility to infection or ability to receive immunizations or prophylactic interventions.<br />
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Therefore, all laboratory personnel and particularly women of childbearing age should be<br />
provided with information regarding immune competence and conditions that may<br />
predispose them to infection. Individuals who have these conditions should be encouraged<br />
to identify themselves to the institution’s health care provider for appropriate counseling and<br />
guidance.<br />
C.2.2 BL1 Special Practices<br />
None required.<br />
C.2.3 BL1 Safety Equipment (Primary Barriers and Personal Protective Equipment)<br />
1. Special containment devices or equipment, such as biosafety cabinets (BSCs), are not<br />
generally required.<br />
2. Protective laboratory clothing (e.g., coats, gowns, or uniforms) should be worn to prevent<br />
contamination of personal clothing.<br />
3. Eye protection must be worn in the laboratory and when conducting procedures that have<br />
the potential to create splashes of biological materials or other hazardous materials.<br />
4. Gloves must be worn to protect hands from exposure to hazardous materials. Glove<br />
selection should be based on an appropriate risk assessment. Alternatives to latex gloves<br />
should be available. Wash hands prior to leaving the laboratory. In addition, BL1 workers<br />
should:<br />
a. Change gloves when contaminated, when their integrity has been compromised, or<br />
when otherwise necessary.<br />
b. Remove gloves and wash hands when work with hazardous materials has been<br />
completed and before leaving the laboratory.<br />
c. Do not wash or reuse disposable gloves. Dispose of used gloves with other<br />
contaminated laboratory waste. Hand washing protocols must be rigorously followed.<br />
C.2.4 BL1 <strong>Laboratory</strong> Facilities (Secondary Barriers)<br />
1. Laboratories should have doors for access control.<br />
2. Laboratories must have a sink for hand washing.<br />
3. The laboratory should be designed so that it can be easily cleaned. Carpets and rugs in<br />
laboratories are not appropriate.<br />
4. <strong>Laboratory</strong> furniture must be capable of supporting anticipated loads and uses. Spaces<br />
between benches, cabinets, and equipment should be accessible for cleaning.<br />
a. Benchtops must be impervious to water and resistant to heat, organic solvents, acids,<br />
alkalis, and other chemicals.<br />
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b. Chairs used in laboratory work must be covered with a nonporous material that can be<br />
easily cleaned and decontaminated with appropriate disinfectant.<br />
5. <strong>Laboratory</strong> windows that open to the exterior should be fitted with screens.<br />
C.3 <strong>Laboratory</strong> <strong>Biosafety</strong> Level 2<br />
<strong>Biosafety</strong> Level 2 builds upon BL1. BL2 is suitable for work involving<br />
agents that pose moderate hazards to personnel and the environment.<br />
It differs from BL1 in that 1) laboratory personnel have specific training in handling pathogenic<br />
agents and are supervised by scientists competent in handling infectious agents and associated<br />
procedures; 2) access to the laboratory is restricted when work is being conducted; and 3) all<br />
procedures in which infectious aerosols or splashes may be created are conducted in BSCs or<br />
other physical containment equipment.<br />
The following standard and special practices, safety equipment, and facility requirements apply<br />
to BL2:<br />
C.3.1 BL2 Standard Microbiological Practices<br />
1. The laboratory supervisor and work lead must enforce the institutional policies that control<br />
access to the site and laboratory facilities as described in the LBNL Site Security Plan.<br />
Policies and practices include, for example, the hosting of visitors and the issuance of gate<br />
passes, badges, and/or keys to control access to the site, building, and/or room based on<br />
each individual’s business need and experiments in progress. Access to the laboratory<br />
should be controlled when the laboratory is unoccupied during nonbusiness hours, (e.g., by<br />
locking doors to the laboratory areas and/or doors to the building entrance).<br />
2. Persons must wash their hands (a) after working with potentially hazardous materials,<br />
recombinant materials, and animals; (b) after removing gloves; and (c) before leaving the<br />
laboratory.<br />
3. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for<br />
human consumption are not permitted in laboratory areas. Food must be stored outside the<br />
laboratory area in cabinets or refrigerators designated and used for this purpose.<br />
4. Mouth pipetting is prohibited; mechanical pipetting devices must be used.<br />
5. Policies for the safe handling of sharps, such as needles, scalpels, pipettes, and broken<br />
glassware, must be developed and implemented. Whenever practical, the laboratory<br />
supervisor and work lead should adopt improved engineering and work practice controls that<br />
reduce risk of sharps injuries. Use of sharps with Risk Group (RG) 2 materials should be<br />
restricted and included in the Biological Use Authorization (BUA) as part of the risk<br />
assessment.<br />
Precautions, including those listed below, must always be taken with sharp items:<br />
a. Careful management of needles and other sharps are of primary importance. Needles<br />
must not be bent, sheared, broken, recapped, removed from disposable syringes, or<br />
otherwise manipulated by hand before disposal.<br />
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b. Used disposable sharps must be carefully placed in conveniently located, properly<br />
labeled, leakproof, puncture-resistant, and closable containers used for sharps disposal.<br />
Contaminated disposable sharps are disposed of immediately after use in containers<br />
that are not overfilled. These containers are closed immediately when full.<br />
c. Nondisposable sharps must be placed in a properly labeled, leakproof, punctureresistant,<br />
hard-walled container for transport to a processing area for decontamination,<br />
preferably by autoclaving. In addition, these sharps must not be stored or processed in a<br />
manner that requires workers to reach by hand into the containers where these sharps<br />
have been placed.<br />
d. Broken glassware must not be handled directly. Instead, it must be removed using a<br />
brush and dustpan, tongs, or forceps. Plasticware should be substituted for glassware<br />
whenever possible.<br />
6. Perform all procedures to minimize the creation of splashes and/or aerosols.<br />
7. Decontaminate work surfaces after completion of work and after any spill or splash of<br />
potentially infectious or viable recombinant material with appropriate disinfectant.<br />
8. Decontaminate all cultures, stocks, and other potentially infectious or recombinant materials<br />
before disposal, using an effective method. Depending on where the decontamination will be<br />
performed, the following methods should be used prior to transport:<br />
a. Materials to be decontaminated outside of the immediate laboratory must be placed in a<br />
durable, leak-proof container and secured for transport.<br />
b. Materials to be removed from the facility for decontamination must be packed in<br />
accordance with applicable local, state, and federal regulations.<br />
9. When infectious agents (i.e., human pathogens) are present or there are organisms that<br />
require special provisions for entry (e.g., vaccination), additional biological hazard warning<br />
signage is required at the laboratory entrance. This signage must incorporate the universal<br />
biohazard symbol and include the laboratory’s biosafety level; the identity of the agent(s) or<br />
the words Infectious Agent(s); the name and telephone number of the supervisor, work lead,<br />
PI, or other responsible personnel; and any special requirements or procedures for entering<br />
and exiting the laboratory. The Chemical Safety Hygiene Plan (CHSP) Caution Placard will<br />
be used to accomplish these additional signage requirements. Any requirements for posting<br />
identities of agents or posting special entry and exit procedures will be specified in the BUA.<br />
10. An effective integrated pest management program is required.<br />
11. The laboratory supervisor must ensure that laboratory personnel receive appropriate training<br />
regarding their duties, the necessary precautions to prevent exposures, and exposure<br />
evaluation procedures. Personnel must receive annual updates or additional training when<br />
procedural or policy changes occur. Personal health status may impact an individual’s<br />
susceptibility to infection, or ability to receive immunizations or prophylactic interventions.<br />
Therefore, all laboratory personnel and particularly women of childbearing age should be<br />
provided with information regarding immune competence and conditions that may<br />
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predispose them to infection. Individuals who have these conditions should be encouraged<br />
to identify themselves to the institution’s health care provider for appropriate counseling and<br />
guidance.<br />
C.3.2 BL2 Special Practices<br />
1. All persons entering the laboratory must be advised of the potential hazards and meet any<br />
specific entry/exit requirements as communicated through laboratory door postings or other<br />
means. Minimum biosafety hazard advisories include a required biohazard symbol posted at<br />
the entrance to the BL2 laboratory. Any additional biosafety requirements necessary for<br />
advising and protecting personnel entering and exiting the area will be specified in the BUA<br />
based on a risk assessment.<br />
2. <strong>Laboratory</strong> personnel must be provided with medical surveillance and offered appropriate<br />
immunizations for agents handled or potentially present in the laboratory.<br />
3. When appropriate, a baseline serum sample should be stored.<br />
4. A laboratory-specific biosafety manual must be prepared and adopted as policy, and must<br />
be available and accessible.<br />
5. The laboratory supervisor must ensure that laboratory personnel demonstrate proficiency in<br />
standard and special microbiological practices before working with BL2 agents.<br />
6. Potentially infectious materials must be placed in a durable, leak-proof container during<br />
collection, handling, processing, storage, or transport within a facility.<br />
7. <strong>Laboratory</strong> equipment should be decontaminated on a routine basis and after spills,<br />
splashes, or other potential contamination.<br />
a. Spills involving infectious materials must be contained, decontaminated, and cleaned by<br />
staff properly trained and equipped to work with infectious material.<br />
b. Equipment must be decontaminated before repair, maintenance, or removal from the<br />
laboratory.<br />
8. Incidents that may result in exposure to infectious materials must be immediately evaluated<br />
and treated according to procedures described in the laboratory biosafety safety manual. All<br />
such incidents must be reported to the laboratory supervisor. Medical evaluation,<br />
surveillance, and treatment should be provided. Appropriate records should be maintained.<br />
9. Animals and plants not associated with the work being performed must not be permitted in<br />
the laboratory.<br />
10. All procedures involving the manipulation of infectious materials that may generate an<br />
aerosol should be conducted within a BSC or other physical containment device.<br />
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C.3.3 BL2 Safety Equipment (Primary Barriers and Personal Protective Equipment)<br />
1. Properly maintained BSCs (preferably Class II), other appropriate personal protective<br />
equipment, or other physical containment devices must be used whenever:<br />
a. Procedures with a potential for creating infectious aerosols or splashes are conducted.<br />
These may include pipetting, centrifuging, grinding, blending, shaking, mixing,<br />
sonicating, opening containers of infectious materials, inoculating animals intranasally,<br />
and harvesting infected tissues from animals or eggs.<br />
b. High concentrations or large volumes of infectious agents or organisms containing<br />
recombinant DNA are used. Such materials may be centrifuged in the open laboratory<br />
using sealed rotor heads or centrifuge safety cups. In this case, the rotor heads and<br />
centrifuge cups must be opened inside a BSC.<br />
2. Protective laboratory clothing (e.g., coats, gowns, smocks, or uniforms) designated for<br />
laboratory use should be worn to prevent contamination of personal clothing and must be<br />
worn when working at BL2 or when working with RG2 or other hazardous materials.<br />
Remove protective clothing before leaving for nonlaboratory areas (e.g., cafeteria, library,<br />
administrative offices). Dispose of protective clothing appropriately, or deposit it for<br />
laundering services provided by an LBNL subcontractor. <strong>Laboratory</strong> clothing must not be<br />
taken home.<br />
3. Eye protection must be worn in the laboratory. Eye and face protection (goggles, mask, face<br />
shield, or other splatter guard) must be used when it is anticipated that splashes, sprays,<br />
splatters, or droplets of infectious or other hazardous materials may be generated and could<br />
contaminate the eyes, nose, or mouth (e.g., when RG2 microorganisms must be handled<br />
outside the BSC or containment device). This eye and face protection must be disposed of<br />
with other contaminated laboratory waste or decontaminated before reuse.<br />
4. Gloves must be worn to protect hands from exposure to hazardous materials. Glove<br />
selection should be based on an appropriate risk assessment. Alternatives to latex gloves<br />
should be available. Gloves that were used in BL1 and BL2 work must not be worn outside<br />
the laboratory. In addition, BL2 laboratory workers should:<br />
a. Change gloves when contaminated, when their integrity has been compromised, or<br />
when otherwise necessary. Wear two pairs of gloves when appropriate.<br />
b. Remove gloves and wash hands when work with hazardous materials has been<br />
completed and before leaving the laboratory.<br />
c. Do not wash or reuse disposable gloves. Dispose of used gloves with other<br />
contaminated laboratory waste. Hand washing protocols must be rigorously followed.<br />
5. Eye, face, and respiratory protection should be used in rooms containing infected animals,<br />
as determined by the risk assessment.<br />
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C.3.4 BL2 <strong>Laboratory</strong> Facilities (Secondary Barriers)<br />
1. <strong>Laboratory</strong> areas should have doors for access and ventilation control, and the doors should<br />
be self-closing and have locks designed in accordance with LBNL standards.<br />
2. Laboratories must have a sink for hand washing. The sink may be manually, hands-free, or<br />
automatically operated. The sink should be located near the exit door.<br />
3. The laboratory should be designed so that it can be easily cleaned and decontaminated.<br />
Carpets and rugs in laboratories are not permitted.<br />
4. <strong>Laboratory</strong> furniture must be capable of supporting anticipated loads and uses. Spaces<br />
between benches, cabinets, and equipment should be accessible for cleaning.<br />
a. Benchtops must be impervious to water and resistant to heat, organic solvents, acids,<br />
alkalis, and other chemicals.<br />
b. Chairs used in laboratory work must be covered with a nonporous material that can be<br />
easily cleaned and decontaminated with appropriate disinfectant.<br />
5. <strong>Laboratory</strong> windows that open to the exterior are not recommended. However, if a<br />
laboratory does have windows that open to the exterior, they must be fitted with screens.<br />
6. BSCs must be installed so that fluctuations of the room air supply and exhaust do not<br />
interfere with proper operations. BSCs should be located away from doors, windows that<br />
can be opened, heavily traveled laboratory areas, and other possible airflow disruptions.<br />
7. Vacuum lines should be protected with high-efficiency particulate air (HEPA) filters or their<br />
equivalent. Filters must be replaced as needed. Liquid disinfectant traps may be required.<br />
8. An eyewash station must be readily available.<br />
9. There are no specific requirements on ventilation systems. However, planning of new<br />
facilities should consider mechanical ventilation systems that provide an inward flow of air<br />
without recirculation to spaces outside of the laboratory.<br />
10. HEPA filtered exhaust air from a Class II BSC can be safely recirculated back into the<br />
laboratory environment if the cabinet is tested and certified at least annually and operated<br />
according to manufacturer’s recommendations. BSCs can also be connected to the<br />
laboratory exhaust system by either a thimble (canopy) connection or a direct (hard)<br />
connection. Provisions to ensure proper safety cabinet performance and air system<br />
operation must be verified.<br />
11. A method for decontaminating all laboratory wastes should be available in the facility (e.g.,<br />
autoclave, chemical disinfection, incineration, or other validated decontamination method).<br />
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Appendix D<br />
Good Microbiological Practice<br />
D.1 Introduction and Scope<br />
This appendix describes 1) customary principles of good microbiological practice (GMP), and 2)<br />
explains the differences between GMP and laboratory biosafety practices defined by the<br />
Centers for Disease Control and Prevention (CDC) and the <strong>National</strong> Institutes of Health (NIH)<br />
and provided in Appendix C (<strong>Laboratory</strong> <strong>Biosafety</strong> Level 1 and 2 Criteria) of this manual. These<br />
GMP principles are guidelines that may be used to control the biosafety and research quality<br />
aspects of laboratory work. These guidelines are not biosafety requirements unless other<br />
sections of this manual describe them as biosafety requirements.<br />
The first and most important element of control for research product protection and laboratory<br />
containment is strict adherence to 1) GMP, and 2) standard microbiological practices and<br />
special practices. These sets of practices have different main objectives, but include many<br />
overlapping practices and secondary objectives. Both sets of practices should be used when<br />
conducting work.<br />
• Good Microbiological Practice (GMP) is aseptic techniques and other good<br />
microbiological practices that are not uniformly defined but are necessary to prevent<br />
contamination of the laboratory with the agents being handled and contamination of the<br />
work with agents from the environment. GMP is used to keep the agents being handled<br />
inside their primary containers without any other organisms getting in and contaminating<br />
the research materials. The main objective of GMP is to ensure that contamination does<br />
not affect the research results.<br />
• Standard microbiological practices and special practices are defined by the CDC<br />
and NIH, discussed in Section 4.1, and listed in detail in Appendix C of this manual.<br />
Standard microbiological practices and special practices are used much like GMP to<br />
keep agents inside their primary containers. However, the main objective of these<br />
practices is to provide safety controls needed to protect workers and the environment<br />
from contamination in the event that the agents are accidentally released from their<br />
primary container.<br />
D.2 Good Microbiological Practice<br />
GMP involves the use of aseptic techniques and other good microbiological practices. These<br />
practices and techniques achieve two objectives:<br />
• Prevent handled organisms from contaminating the laboratory, and<br />
• Prevent organisms in a laboratory environment from contaminating the work.<br />
Both objectives are important, but the first objective is primarily important for the safety of the<br />
worker, while the second objective is mostly important for the quality of the research.<br />
The principles of GMP should generally be applied to all types of work involving microorganisms<br />
and tissue cultures, regardless of containment level.<br />
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D.2.1 Aseptic Technique<br />
An aseptic technique is a procedure used to grow a microorganism or culture of interest in a<br />
clean micro-environment isolated from the outside world. This micro-environment is usually<br />
some sort of culture or holding container such as a flask, bottle, or petri dish. The organisms or<br />
cells can either be on a solid agar medium or be suspended in a broth, diluent, or other fluid<br />
medium.<br />
Examples of aseptic techniques include ensuring all components of the system are sterile prior<br />
to use (e.g., container interior, growth medium, and any items used in manipulation) and using<br />
special care and techniques to avoid cross-contamination during the inoculation, incubation, and<br />
processing steps. They also include:<br />
• Keeping the container closed except for the minimum time required to introduce or<br />
remove materials.<br />
• Holding open containers at an angle whenever possible to prevent contaminants from<br />
entering the container.<br />
• Protecting sterile containers from contamination, and working with these containers<br />
inside a biosafety cabinet (BSC). When working outside a BSC, a Bunsen burner may<br />
be used to flame the opening of the container whenever tops are removed (i.e., passing<br />
the opening quickly through the Bunsen flame). The upwards current of hot air created<br />
by the Bunsen burner prevents contaminated air or particles from dropping into the<br />
culture container when the lid is open.<br />
• Using manipulation techniques that minimize the possibility of cross-contamination (e.g.,<br />
opening lids with the little finger so that tops are not put down on the work surface).<br />
• Ensuring that all tools (e.g., pipette tips or loops) or other items that may come in contact<br />
with the culture are sterile and not contaminated by casual contact with the bench,<br />
fingers, or outside of the bottle. Also ensuring that these tools are disposed of or<br />
decontaminated immediately after use.<br />
In addition to aseptic technique, GMP includes a wide range of other working methods that<br />
minimize the cross-contamination of the work and workplace. Examples of these methods are<br />
provided in the remaining sections of this appendix.<br />
D.2.2 Personal Hygiene and Dress<br />
• Wash hands prior to and following manipulations of organisms or cultures and whenever<br />
contamination is suspected.<br />
• Wear personal protective equipment (PPE) to protect the worker and to prevent research<br />
materials from contamination. Change gloves when contaminated. Routinely clean lab<br />
coats or throw away disposable coats.<br />
• Tie back or confine loose or long hair.<br />
• Do not touch the skin, face, or unclean or nonsterile surfaces.<br />
• Keep fingernail tips at a length of one-quarter inch or shorter.<br />
D.2.3 Area Cleanliness and Organization<br />
Keep the laboratory and work area clean and organized, such as in the following examples:<br />
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• Keep only items necessary for the task in progress on the bench or in the BSC. This<br />
practice avoids unnecessary clutter that may collect contaminants, prevent surface<br />
disinfection and spill cleanup, and increase the possibility of things getting knocked over.<br />
• Plan and lay out work so that everything needed for a procedure is ready to be handled<br />
in a logical order. This practice should allow the worker to sit at the BSC or bench and<br />
handle the items efficiently using aseptic techniques.<br />
• Use appropriate chemical antimicrobials (e.g., disinfectants) and decontamination<br />
procedures. See Appendix F of this manual.<br />
• Wash hands and disinfect work surfaces before and after work.<br />
• Immediately clean spills, and then disinfect the work surface and wash hands.<br />
• Organize the work area when work is complete.<br />
• Avoid putting items on the floor. This practice allows the cleanliness of the floor to be<br />
viewed, allows all parts of the floor to be cleaned routinely, eases spill cleanup, and<br />
prevents tripping hazards.<br />
• Routinely clean water baths to minimize microbial contamination of the water.<br />
• Routinely clean laboratory surfaces such as open shelving, benchtops, windowsills, and<br />
items on them to prevent accumulation of dust and debris. Store infrequently used items<br />
in cabinets and drawers.<br />
• Routinely clean floors and difficult-to-access areas to prevent buildup of dust and debris.<br />
• Routinely clean sink faucets and basins.<br />
• Identify areas and systems in the laboratory and support areas (e.g., wash and<br />
autoclave area) for storage and staging of dirty, contaminated, clean, and sterilized<br />
items that are being stored, used, or processed for eventual reuse. Ensure everyone<br />
understands and follows the system.<br />
• Periodically review items stored in refrigerators and freezers and on shelves and<br />
benches. Dispose of items that are no longer needed.<br />
D.2.4 <strong>Biosafety</strong> Cabinets and Airborne Contamination<br />
• Use a BSC when needed to protect biological research materials and when procedures<br />
may generate biohazardous aerosols. See Appendix E, Section E.3 (<strong>Biosafety</strong> Cabinet<br />
Work Practices and Procedures) for additional GMP and containment work practices<br />
related to work in a BSC.<br />
• Minimize personnel traffic and unnecessary movements around the work area or BSC.<br />
Such movements cause area air turbulence that may transport contaminants into the<br />
work area and onto the biological materials that need protection. Such movements also<br />
disturb clean laminar airflows inside BSCs responsible for containing aerosols and<br />
protecting biological materials.<br />
D.2.5 Manipulation Techniques for Minimizing Aerosols<br />
Manipulation techniques should be used that minimize the possibility of producing aerosols.<br />
Examples include:<br />
• Mixing by gentle rolling and swirling rather than vigorous shaking (to avoid frothing).<br />
• Pipetting by putting the tip into a liquid or onto a surface prior to gently ejecting the<br />
pipette contents (to avoid bubbling and splashing).<br />
• Placing containers in very close proximity to each other when transferring liquids<br />
between them (to avoid drops that fall and splash).<br />
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• Allowing loops to cool down after incineration or flaming before using the loop (to avoid<br />
sizzling).<br />
• Not overfilling centrifuge tubes (to avoid leakage into centrifuge).<br />
• Slowly removing tube caps or stoppers.<br />
• Not popping caps off of tubes.<br />
• Carrying and storing cultures (e.g., bottles and plates) in racks and spill-proof containers<br />
(to prevent dropping and breakage).<br />
D.2.6 Worker Qualifications<br />
Workers who handle microorganisms and cultures should have sufficient technical competence,<br />
training, and experience in GMP and containment practices. In addition, workers should use<br />
GMP and biosafety containment in anticipation of unexpected hazards when handling<br />
microorganisms and cultures (including Risk Group 1). Workers should conservatively approach<br />
their safety by assuming, for example, that an unexpected pathogen may be present or<br />
contaminate the culture; a pathogen may be unintentionally cultured; the disease potential of the<br />
agent may be altered under laboratory conditions; or exposure to an RG1 agent may cause an<br />
opportunistic infection.<br />
D.2.7 Microbial Contamination Checks<br />
Routine microbial contamination checks should be incorporated into protocols and undertaken<br />
at various stages of experiments. Examples of contamination checks include:<br />
• Taking a loopful of fluid from the vessel and plating (or streaking) it out onto a<br />
nonselective solid nutrient medium to look for single colonies.<br />
• Incubating culture samples at a suitable temperature (usually 30°C) to allow growth of<br />
contaminants originating from the general environment and human sources.<br />
• After incubation, examining plates for evidence of any contamination as indicated by<br />
colony types.<br />
The purity of a liquid culture can also be obtained by microscopic examination. This is done by<br />
placing a loopful of the culture on a microscope slide. The slide is then examined wet either by<br />
phase contrast microscopy, or by fixed or Gram staining. Contaminant organisms should be<br />
instantly and clearly visible.<br />
Contamination checks are particularly useful in evaluating GMP competence. Workers with poor<br />
aseptic techniques will have frequent contamination problems, while workers skilled in GMP will<br />
have problems only occasionally. It is important to recognize that poor practices not only result<br />
in contaminated cultures, but may also result in spreading biological materials and<br />
contamination to work surfaces and workers in the laboratory.<br />
D.3 References<br />
University of Edinburgh, Health and Safety, “Good Microbiological Practice and Containment,”<br />
Web page information from the Health and Safety Department, August 2003.<br />
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Appendix E<br />
<strong>Biosafety</strong> Cabinets<br />
E.1 Introduction and Scope<br />
Biological safety cabinets or biosafety cabinets (BSCs) are hoods with high-efficiency<br />
particulate air (HEPA) filters that provide personnel, environmental, and product protection when<br />
appropriate practices and procedures are followed. Key BSC information and requirements are<br />
summarized in Section 5.6.4.2. This appendix provides the following information and<br />
requirements on BSCs:<br />
• Classifications<br />
• Work practices and procedures<br />
• Decontamination<br />
• Installation and engineering<br />
• Testing and certification<br />
Information in this appendix primarily contains information that was excerpted and adapted from<br />
Appendix A (Primary Containment for Biohazards: Selection, Installation, and Use of Biological<br />
Safety Cabinets) of <strong>Biosafety</strong> in Microbiological and Biomedical Laboratories (BMBL), fifth<br />
edition, and minimally reiterates LBNL policies presented in Section 5.6.4.2 (Hoods and<br />
<strong>Biosafety</strong> Cabinets) of this manual.<br />
E.2 <strong>Biosafety</strong> Cabinet Classifications<br />
Three primary types of BSCs have been developed to meet varying research and clinical needs.<br />
These primary BSC types are designated as Class I, II, and III. Class II BSCs are also further<br />
subdivided into different Class II types. Tables E-1 and E-2 summarize the similarities and<br />
differences in the types of protection and physical characteristics of different classes of BSCs.<br />
The sections following these tables summarize and illustrate the characteristics of BSC classes<br />
used at LBNL. This information should be used in BSC selection and risk assessment.<br />
Table E-1<br />
Protection Offered by Classes of <strong>Biosafety</strong> Cabinets<br />
Biological<br />
Protection Provided<br />
Risk Assessed Personnel Product Environmental BSC Class<br />
<strong>Biosafety</strong> Level Yes No Yes I<br />
(BL) 1 to 3<br />
BL1 to 3 Yes Yes Yes II (A1, A2, B1, B2)<br />
BL4 Yes Yes Yes III<br />
II - when used in room<br />
with suit<br />
Source: adapted from BMBL, fifth edition, Appendix A, Table 1.<br />
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Table E-2<br />
Characteristics of <strong>Biosafety</strong> Cabinet Classes<br />
BSC<br />
Class,<br />
Type<br />
Face<br />
Velocity<br />
(fpm)<br />
Airflow Pattern<br />
Nonvolatile<br />
Toxic<br />
Chemicals and<br />
Radionuclides<br />
Applications<br />
Volatile Toxic<br />
Chemicals<br />
and<br />
Radionuclides<br />
I 75 In at front through HEPA to the<br />
outside or into the room<br />
through HEPA (Figure 1)<br />
II, A1 75 70% recirculated to the cabinet<br />
work area through HEPA; 30%<br />
balance can be exhausted<br />
through HEPA back into the<br />
room or to outside through a<br />
canopy unit<br />
II, A2 100 Similar to II, A1, but has 100<br />
linear fpm intake air velocity<br />
and plenums are under<br />
negative pressure to room<br />
(Figure 2); exhaust air can be<br />
ducted to outside through a<br />
canopy unit (Figure 3)<br />
II, B1 100 30% recirculated, 70%<br />
exhausted. Exhaust cabinet air<br />
must pass through a dedicated<br />
duct to the outside through a<br />
HEPA filter (Figure 4)<br />
II, B2 100 No recirculation; total exhaust<br />
to the outside through a HEPA<br />
filter<br />
III N/A Supply air is HEPA filtered.<br />
Exhaust air passes through<br />
two HEPA filters in series and<br />
is exhausted to the<br />
outside via a hard connection<br />
Yes<br />
Yes<br />
(minute<br />
amounts)<br />
Yes<br />
Yes<br />
Yes<br />
Yes<br />
When<br />
exhausted<br />
outdoors 1,2<br />
No<br />
When<br />
exhausted<br />
outdoors<br />
(formerly "B3")<br />
(minute<br />
amounts) 1,2<br />
Yes<br />
(minute<br />
amounts) 1,2<br />
Yes. (small<br />
amounts) 1,2<br />
Yes<br />
(small<br />
amounts) 1,2<br />
(Figure 5)<br />
Footnotes:<br />
1. Installation may require a special duct to the outside, an in-line charcoal filter,<br />
and a spark-proof (explosion-proof) motor and other electrical components in the<br />
cabinet. Discharge of a Class I or Class II Type A2 cabinet into a room should<br />
not occur if volatile chemicals are used.<br />
2. In no instance should the chemical concentration approach the lower explosion<br />
limits of the compounds.<br />
Source: adapted from BMBL, fifth edition, Appendix A, Table 2.<br />
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E.2.1 Class I <strong>Biosafety</strong> Cabinet<br />
The Class I BSC provides personnel and environmental protection, but no product protection. It<br />
is similar in air movement to a chemical fume hood, but has a HEPA filter in the exhaust system<br />
to protect the environment. Figure 1 shows a diagram of a Class I BSC.<br />
Figure 1. Class I BSC. (A) front opening, (B) sash, (C) exhaust HEPA filter, and (D) exhaust<br />
plenum. Note: The cabinet needs to be hard connected to the building exhaust system if toxic<br />
vapors are to be used. Source: BMBL, fifth edition, Appendix A.<br />
E.2.2 Class II <strong>Biosafety</strong> Cabinet<br />
Class II BSCs (Types A1, A2, B1 and B2) provide personnel, environmental, and product<br />
protection. Airflow is drawn into the front grille of the cabinet, providing personnel protection. In<br />
addition, the downward laminar flow of HEPA-filtered air provides product protection by<br />
minimizing the chance of cross-contamination across the work surface of the cabinet. Because<br />
cabinet exhaust air is passed through a certified HEPA filter, the exhaust air is particulate-free<br />
(environmental protection), and may be recirculated to the laboratory (i.e., Type A1 and A2<br />
BSCs only) or discharged from the building via the exhaust duct system and a canopy<br />
connection. Exhaust air from Types B1 and B2 BSCs must be discharged to the outdoors via a<br />
hard duct connection. Figure 2 shows a diagram of a Class II Type A2 BSC, which is the most<br />
common type of BSC at LBNL. Figure 3 shows a diagram of a canopy (or thimble) unit that is<br />
normally required when connecting a Class II Type A1 or A2 BSC to an exhaust duct system.<br />
Figure 4 shows a Class II, Type B1 BSC, which is also used at LBNL. Installation of a Class II,<br />
Type B1 BSC typically requires a hard duct connection to the exhaust system without a canopy<br />
or thimble unit connection.<br />
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HEPA filters are effective at trapping particulates and thus infectious agents but do not capture<br />
volatile chemicals or gases. Only Type A2 exhausted or Types B1and B2 BSCs exhausting to<br />
the outside should be used when working with volatile toxic chemicals, but amounts must be<br />
limited. See Table 2 for additional information.<br />
Figure 2. Class II, Type A2 BSC. Tabletop model. (A) front opening, (B) sash, (C)<br />
exhaust HEPA filter, (D) supply HEPA filter, (E) positive-pressure common plenum, (F)<br />
negative-pressure plenum. Unless it is connected to the building exhaust system, the<br />
Class II, Type A2 BSC is not equivalent to what was formerly called a Class II, Type B3<br />
BSC. Note: The Class II, Type A2 BSC should be canopy connected to the exhaust<br />
system. Source: adapted from BMBL, fifth edition, Appendix A.<br />
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Figure 3. Canopy (Thimble) Unit. Canopy (thimble) units for connecting a Class II,<br />
Type A1 or A2 BSC to the exhaust duct system. (A) balancing damper, (B) flexible<br />
connector to exhaust system, (C) cabinet exhaust HEPA filter housing, (D) canopy unit,<br />
(E) BSC. Note: There is a one-inch gap between (D) the canopy unit and (E) the exhaust<br />
filter housing through which room air is exhausted. Source: adapted from BMBL, fifth<br />
edition, Appendix A.<br />
A<br />
Figure 4. Class II, Type B1 BSC (classic design). (A) Front opening, (B) sash, (C) exhaust<br />
HEPA filter, (D) supply HEPA filter, (E) negative-pressure dedicated exhaust plenum, (F)<br />
blower, (G) additional HEPA filter for supply air. Note: The cabinet exhaust needs to be hard<br />
connected to the building exhaust system. Source: BMBL, fifth edition, Appendix A.<br />
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E.2.3 Class III <strong>Biosafety</strong> Cabinet<br />
A standard Class III BSC (Figure 5) is designed for working with highly infectious microbiological<br />
agents and conducting hazardous operations. It is a gas-tight enclosure with a nonopening view<br />
window that provides maximum protection for the environment and the worker. Access for<br />
passage of materials into the cabinet is through a chemical dunk tank accessible through the<br />
cabinet floor, or a double-door pass-through box (e.g., an autoclave) that can be<br />
decontaminated between uses. Reversing that process allows materials to be removed from the<br />
Class III BSC safely. Both supply and exhaust air pass through a HEPA filter on a Class III<br />
cabinet. Exhaust air must pass through two HEPA filters, or a HEPA filter and an air incinerator,<br />
before discharge to the outdoors. Airflow is maintained by a dedicated, independent exhaust<br />
system exterior to the cabinet, which keeps the cabinet under negative pressure (minimum<br />
pressure of 0.5 inches of water gauge). Some Class III BSCs may not have all of these controls,<br />
based on the risk assessment conducted (e.g., types of materials and manner of work).<br />
Long, heavy-duty rubber gloves are attached in a gas-tight manner to ports in the cabinet and<br />
allow direct manipulation of the materials isolated inside and prevent the user's direct contact<br />
with the hazardous materials. Depending on the design of the cabinet, the supply HEPA filter<br />
provides particulate-free, albeit somewhat turbulent, airflow within the work environment.<br />
Laminar airflow is not a characteristic of a Class III cabinet.<br />
Figure 5. Class III BSC. (A) Glove ports with O-ring for attaching arm-length gloves to<br />
cabinet, (B) sash, (C) exhaust HEPA filter, (D) supply HEPA filter, (E) double-ended<br />
autoclave or pass-through box. The cabinet exhaust needs to be hard connected to an<br />
independent dedicated exhaust system. The exhaust air must be double HEPA filtered or<br />
HEPA filtered and incinerated. Source: adapted from BMBL, fifth edition, Appendix A.<br />
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E.2.4 Clean Benches (Not BSCs)<br />
Horizontal and vertical laminar flow “clean benches” are shown in Figures 6 and 7. These units<br />
may provide protection for the product, but are not considered safety hoods or BSCs and must<br />
not be used for infectious or toxic materials or when a hood or BSC is needed to protect the<br />
worker.<br />
Figure 6. Horizontal Laminar Flow “Clean Bench.” (A) Front opening, (B) grille, (C)<br />
supply HEPA filter, (D) plenum, (E) blower, (F) grille. Source: BMBL, fifth edition, Appendix<br />
A.<br />
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Figure 7. Vertical Laminar Flow “Clean Bench.” (A) Front opening, (B) sash, (C) supply<br />
HEPA filter, (D) blower. Source: BMBL, fifth edition, Appendix A.<br />
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E.3 <strong>Biosafety</strong> Cabinet Work Practices and Procedures<br />
This section discusses in detail standard work practices and procedures for investigators<br />
working in a Class II BSC. In general, these practices and procedures are important for<br />
protection of the worker or the product, but the importance of each practice or procedure for the<br />
safety of the worker often depends on the nature of biological materials and the work being<br />
conducted. A shorter list of key BSC work practices and procedures is provided in Appendix D.<br />
E.3.1 Preparing for BSC Work<br />
This section discusses preparing for work within a Class II BSC.<br />
Air Current Disruptions. Preparing a written checklist of materials necessary for a particular<br />
activity and placing necessary materials in the BSC before beginning work minimizes the<br />
number and extent of air curtain disruptions compromising the fragile air barrier of the cabinet.<br />
The rapid movement of a worker's arms in a sweeping motion into and out of the cabinet will<br />
disrupt the air curtain and compromise the partial containment barrier provided by the BSC.<br />
Moving arms slowly in and out and perpendicular to the face while opening the cabinet will<br />
reduce this risk. Other personnel activities in the room (e.g., rapid movements near the face of<br />
the cabinet, walking traffic, room fans, open/closing room doors, etc.) may also disrupt the<br />
cabinet air barrier.<br />
Personal Protective Equipment (PPE). Eye protection and laboratory coats buttoned over street<br />
clothing must be worn. Latex, vinyl, nitrile, or other suitable gloves must be worn to provide<br />
hand protection. Higher levels of PPE can be included as determined by an individual risk<br />
assessment. For example, a solid front, back-closing laboratory gown provides better protection<br />
of personal clothing than a traditional laboratory coat and is a recommended practice at BL3.<br />
Body and Material Positioning. Before beginning work, the BSC user should adjust the stool<br />
height so that his/her face is above the front opening. Manipulation of materials should be<br />
delayed for approximately one minute after placing the hands/arms inside the cabinet. This<br />
allows the cabinet to stabilize, the user to "air sweep" his or her hands and arms, and to allow<br />
time for turbulence reduction. When the user's arms rest flatly across the front grille, the arms<br />
may occlude the grille opening, and room air laden with particles may flow directly into the work<br />
area rather than being drawn down through the front grille. Raising the arms slightly will alleviate<br />
this problem. The front grille must not be blocked by towels, research notes, discarded plastic<br />
wrappers, pipetting devices, etc. All operations should be performed on the work surface at<br />
least four inches from the front grille. If there is a drain valve under the work surface, it should<br />
be closed prior to beginning work in the BSC.<br />
Materials or equipment placed inside the cabinet may cause disruption of the airflow, resulting in<br />
turbulence, possible cross-contamination and/or breach of containment. Extra supplies (e.g.,<br />
additional gloves, culture plates or flasks, culture media) should be stored outside the cabinet.<br />
Only the materials and equipment required for immediate work should be placed in the BSC.<br />
Purge and Decontamination. If the cabinet has been shut down, the blowers should be operated<br />
at least four minutes before beginning work to allow the cabinet to "purge." This purge will<br />
remove any suspended particulates in the cabinet. The work surface, the interior walls (except<br />
the supply filter diffuser), and the interior surface of the window should be wiped with 70%<br />
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ethanol (EtOH), a 1:100 dilution of household bleach (i.e., 0.05% sodium hypochlorite), or other<br />
disinfectant as determined by the investigator to meet the requirements of the particular activity.<br />
When bleach is used, a second wiping with sterile water is needed to remove the residual<br />
chlorine, which may eventually corrode stainless steel surfaces. Wiping with nonsterile water<br />
may recontaminate cabinet surfaces, a critical issue when sterility is essential (e.g.,<br />
maintenance of cell cultures).<br />
Similarly, the surfaces of all materials and containers placed into the cabinet should be wiped<br />
with 70% EtOH to reduce the introduction of contaminants to the cabinet environment. This<br />
simple step will reduce the introduction of mold spores and thereby minimize contamination of<br />
cultures. The further reduction of microbial load on materials to be placed or used in BSCs may<br />
be achieved by periodic decontamination of incubators and refrigerators.<br />
E.3.2 Material Placement inside the BSC<br />
This section covers placement of materials inside the BSC.<br />
Surface Towels. Plastic-backed absorbent towels can be placed on the work surface but not on<br />
the front or rear grille openings. The use of towels facilitates routine cleanup and reduces<br />
splatter and aerosol generation during an overt spill. It can be folded and placed in a biohazard<br />
bag or other appropriate receptacle when work is completed.<br />
Inside Materials and Sash. All materials should be placed as far back in the cabinet as practical,<br />
toward the rear edge of the work surface and away from the front grille of the cabinet (Figure 8).<br />
Similarly, aerosol generating equipment (e.g., vortex mixers, tabletop centrifuges) should be<br />
placed toward the rear of the cabinet to take advantage of the split in downward laminar air flow<br />
to the front and rear grilles as the air approaches the work surface. Bulky items such as<br />
biohazard bags, discard pipette trays, and vacuum collection flasks should be placed to one<br />
side of the interior of the cabinet. If placing those items in the cabinet requires opening the sash,<br />
make sure that the sash is returned to its original position before work is initiated. The correct<br />
sash position (usually 8 or 10 inches above the base of the opening) should be indicated on the<br />
front of the cabinet. On most BSCs, an audible alarm will sound if the sash is in the wrong<br />
position while the fan is operating.<br />
Practices Do and Do Not Interfere with BSC Operation. Certain common practices interfere with<br />
the operation of the BSC. The biohazard collection bag should not be taped to the outside of the<br />
cabinet. Upright pipette collection containers should not be used in BSCs nor placed on the floor<br />
outside the cabinet. The frequent inward/outward movement needed to place objects in these<br />
containers is disruptive to the integrity of the cabinet air barrier and can compromise both<br />
personnel and product protection. Only horizontal pipette discard trays containing an<br />
appropriate chemical disinfectant should be used within the cabinet. Furthermore, potentially<br />
contaminated materials should not be brought out of the cabinet until they have been surface<br />
decontaminated. Alternatively, contaminated materials can be placed into a closable container<br />
for transfer to an incubator, autoclave, or another part of the laboratory.<br />
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E.3.3 Operations within a Class II BSC<br />
Splatters and Aerosols. Many procedures conducted in BSCs may create splatter or aerosols.<br />
Good microbiological techniques should always be used when working in a BSC. For example,<br />
techniques used to reduce splatter and aerosol generation will also minimize the potential for<br />
personnel exposure to infectious materials manipulated within the cabinet. Class II cabinets are<br />
designed so that horizontally nebulized spores introduced into the cabinet will be captured by<br />
the downward flowing cabinet air within 14 inches of travel. Therefore, as a general rule of<br />
thumb, keeping clean materials at least one foot away from aerosol-generating activities will<br />
minimize the potential for cross-contamination.<br />
Work Flow. The work flow should be from "clean to dirty" (see Figure 8). Materials and supplies<br />
should be placed in the cabinet in such a way as to limit the movement of "dirty" items over<br />
"clean" ones. Several measures can be taken to reduce the chance for cross-contamination of<br />
materials when working in a BSC. Opened tubes or bottles should not be held in a vertical<br />
position. Investigators working with petri dishes and tissue culture plates should hold the lid<br />
above the open sterile surface to minimize direct impaction of downward air. Bottle or tube caps<br />
should not be placed on the towels. Items should be recapped or covered as soon as possible.<br />
Figure 8. Typical Work Layout Inside a BSC. Shown above is a typical layout for working<br />
“clean to dirty” within a Class II BSC. Clean cultures (left) can be inoculated (center);<br />
contaminated pipettes can be discarded in the shallow pan, and other contaminated<br />
materials can be placed in the biohazard bag (right). This arrangement is reversed for lefthanded<br />
persons. Source: adapted from BMBL, fifth edition, Appendix A.<br />
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Burners and Open Flames. Open flames are not required in the near-microbe-free environment<br />
of a BSC. On an open bench, flaming the neck of a culture vessel will create an upward air<br />
current, which prevents microorganisms from falling into the tube or flask. An open flame in a<br />
BSC, however, creates turbulence that disrupts the pattern of HEPA-filtered air being supplied<br />
to the work surface and may cause fires. When deemed absolutely necessary, touch-plate<br />
microburners equipped with a pilot light to provide a flame on demand should be used. These<br />
burners will minimize internal cabinet air disturbance, heat buildup, and fire risk. The burner<br />
must be turned off when work is completed. Small electric "furnaces" are also available for<br />
decontaminating bacteriological loops and needles, and are preferable to an open flame inside<br />
the BSC. Disposable or recyclable sterile loops should be used whenever possible.<br />
A fire inside a BSC occurred when the gas rubber<br />
hose connected to a Touch-O-Matic Bunsen burner<br />
melted and gas in the hose ignited. Brookhaven<br />
<strong>National</strong> <strong>Laboratory</strong>, Lessons Learned 2002-CHBNL-<br />
MED-0003 (July 23, 2007).<br />
BSC fire. Source: Stanford University,<br />
Use of open flames in Cabinets/Tissue<br />
Culture Hoods (May 29, 2003).<br />
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The following are examples of burners and heaters that could be used in a biosafety cabinet if<br />
other sterile techniques are not feasible:<br />
• Burner: Touch-O-Matic Bunsen Burner<br />
Simply depress ON/OFF platform with side of hand to release gas stream, and continuous<br />
pinpoint pilot light ignites gas to produce full flame. Release platform and flame goes out to<br />
conserve gas. To produce continuous flame, depress platform, then turn it slightly; reverse<br />
process to turn off flame.<br />
• Heater: Bacti-Cinerator<br />
Infrared heat chamber sterilizes loops, needles, and culture tubes in 5 to 7 seconds.<br />
Suitable for anaerobic procedures in chambers and hoods. Electric heat source eliminates<br />
hazards from gas and open flames. Within 6 minutes of activation, the interior of the ceramic<br />
cone reaches an optimum sterilizing temperature of 815°C (1,500°F). A prominent light<br />
indicates when the unit is in operation. Weighted cast aluminum stand includes handy<br />
spaces for storage of six inoculating loop handles. Electrical: 120V, 50/60Hz. UL listed. Unit<br />
is not intended for use with scalpels, forceps, or sharp instruments.<br />
Touch-O-Matic Bunsen Burner.<br />
Bacti-Cinerator.<br />
Source: Fisher Scientific (May 2010). Source: VWR (May 2010).<br />
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Aspirator Bottles or Suction Flasks. Aspirator bottles or suction flasks should be connected to an<br />
overflow collection flask containing appropriate disinfectant, and to an in-line HEPA or<br />
equivalent filter (see Figure 9). This combination will provide protection to the central building<br />
vacuum system or vacuum pump, as well as to the personnel who service this equipment.<br />
Inactivation of aspirated materials can be accomplished by placing sufficient chemical<br />
decontamination solution into the flask to inactivate the microorganisms as they are collected.<br />
Once inactivation occurs, liquid materials can be disposed of as noninfectious waste.<br />
Aspirator bottles that collect Risk Group (RG) 1 or RG2 biological materials that do not contain<br />
RG2 infectious agents may be placed outside the BSC as long as the aspirator bottles are<br />
placed inside a secondary spill tray.<br />
E<br />
Figure 9. Aspiration and House Vacuum System Protection. Shown below is one<br />
method to protect a house vacuum system during aspiration of infectious fluids. The left<br />
suction flask (A) is used to collect the contaminated fluids into a suitable decontamination<br />
solution; the right flask (B) serves as a fluid overflow collection vessel. An in-line HEPA filter<br />
(C) is used to protect the vacuum system (D) from aerosolized microorganisms. A spill tray<br />
(E) should be used when the flasks are outside the BSC. Source: adapted from BMBL, fifth<br />
edition, Appendix A.<br />
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E.4 <strong>Biosafety</strong> Cabinet Decontamination and Moves<br />
E.4.1 Cabinet Surface Decontamination<br />
Cabinet Surfaces. With the cabinet blower running, all containers and equipment should be<br />
surface decontaminated and removed from the cabinet when work is completed. At the end of<br />
the workday, the final surface decontamination of the cabinet should include a wipe-down of the<br />
work surface, the cabinet's sides and back, and the interior of the glass. If necessary, the<br />
cabinet should also be monitored for radioactivity and decontaminated when necessary.<br />
Investigators should remove their gloves and gowns in a manner to prevent contamination of<br />
unprotected skin and aerosol generation, and wash their hands as the final step in safe<br />
microbiological practices. The cabinet blower may be turned off after these operations are<br />
completed, or it may be left on.<br />
Small Spills. Small spills within the operating BSC can be handled immediately by removing the<br />
contaminated absorbent paper towel and placing it into the biohazard bag or receptacle. Any<br />
splatter onto items within the cabinet, as well as the cabinet interior, should be immediately<br />
cleaned up with a towel dampened with an appropriate decontaminating solution. Gloves should<br />
be changed after the work surface is decontaminated and before placing clean absorbent towel<br />
in the cabinet. Hands should be washed whenever gloves are changed or removed.<br />
Large Spills. Spills large enough to result in liquids flowing through the front or rear grilles<br />
require more extensive decontamination. All items within the cabinet should be surface<br />
decontaminated and removed. After ensuring that the drain valve is closed, decontaminating<br />
solution can be poured onto the work surface and through the grille(s) into the drain pan.<br />
Decontamination Time and Cleanup. Twenty to 30 minutes is generally considered an<br />
appropriate contact time for decontamination, but this varies with the disinfectant and the<br />
microbiological agent. Manufacturer's directions should be followed. The spilled fluid and<br />
disinfectant solution on the work surface should be absorbed with paper towels and discarded<br />
into a biohazard bag. The drain pan should be emptied into a collection vessel containing<br />
disinfectant. A hose barb and flexible tube should be attached to the drain valve and be of<br />
sufficient length to allow the open end to be submerged in the disinfectant within the collection<br />
vessel. This procedure serves to minimize aerosol generation. The drain pan should be flushed<br />
with water and the drain tube removed.<br />
Radioactive Materials. Should the spilled liquid contain radioactive material, a similar procedure<br />
can be followed. Radiation safety personnel should be contacted for specific instructions.<br />
Work Surface, Grille, and Drain Pan Cleaning. Periodic removal of the cabinet work surface<br />
and/or grilles after the completion of drain pan decontamination may be justified because of dirty<br />
drain pan surfaces and grilles, which ultimately could occlude the drain valve or block airflow.<br />
However, extreme caution should be observed while wiping these surfaces to avoid injury from<br />
broken glass and sharp metal edges. Always use disposable paper towels and avoid applying<br />
harsh force. Wipe dirty surfaces gently. Never leave paper towels on the drain pan because the<br />
paper could block the drain valve or the air passages in the cabinet.<br />
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E.4.2 Internal Cabinet Gaseous Decontamination<br />
BSCs that have been used for work involving infectious materials must be decontaminated<br />
before HEPA filters are changed or internal repair work is done. Before a BSC is relocated, a<br />
risk assessment considering the agents manipulated within the BSC must be performed to<br />
determine the need and method for decontamination. LBNL policy requires that BSCs and their<br />
filters be decontaminated with a gaseous decontaminant prior to being moved or internal repair<br />
work is conducted, unless approved by the <strong>Biosafety</strong> Officer. The most common<br />
decontamination method uses formaldehyde gas, although more recently, hydrogen peroxide<br />
vapor and chlorine dioxide gas have been used successfully.<br />
E.5 <strong>Biosafety</strong> Cabinet Installation and Engineering<br />
Room Ventilation and Secondary Barriers. Whereas BSCs are considered to be the primary<br />
safety barrier for manipulation of infectious materials, the laboratory room itself is considered to<br />
be the secondary safety barrier. Inward directional airflow is established by exhausting a greater<br />
volume of air than is supplied to a given laboratory and by drawing makeup air from the<br />
adjacent space. This directional air flow into the room should generally be accomplished at BL2<br />
(see Section 5.6.4.1 of this manual). The air balance for the entire facility should be established<br />
and maintained to ensure that air flows from areas of least to greatest potential contamination.<br />
The room exhaust system should be sized to handle both the room and all containment devices<br />
vented through the system. Adequate supply air must be provided to ensure appropriate<br />
function of the exhaust system. The facility engineer must be consulted before locating a new<br />
cabinet requiring connection to the building exhaust system. Right angle bends, long horizontal<br />
runs, and transitional connections within the systems will add to the demand on the exhaust fan.<br />
The building exhaust air should be discharged away from supply air intakes to prevent<br />
reentrainment of laboratory exhaust air into the building air supply system. Refer to recognized<br />
design guides for locating the exhaust terminus relative to nearby air intakes.<br />
Utility Services. Utility services needed within a BSC must be planned carefully. Protection of<br />
vacuum systems must be addressed (Figure 9). Electrical outlets inside the cabinet must be<br />
protected by ground fault circuit interrupters and should be supplied by an independent circuit.<br />
When propane or natural gas is provided, a clearly marked emergency gas shutoff valve must<br />
be installed outside the cabinet for fire safety. All nonelectrical utility services should have<br />
exposed, accessible shutoff valves. The use of compressed air within a BSC must be carefully<br />
considered and controlled to prevent aerosol production and reduce the potential for vessel<br />
pressurization.<br />
Ultraviolet (UV) Lamps. UV lamps are not required in BSCs nor are they necessary. If installed,<br />
UV lamps must be cleaned weekly to remove any dust and dirt that may block the germicidal<br />
effectiveness of the ultraviolet light. The lamps should be checked weekly with a UV meter to<br />
ensure that the appropriate intensity of UV light is being emitted. UV lamps must be turned off<br />
when the room is occupied to protect eyes and skin from UV exposure, which can burn the<br />
cornea and cause skin cancer. If the cabinet has a sliding sash, close the sash when operating<br />
the UV lamp.<br />
BSC Placement. BSCs were developed as workstations to provide personnel, environmental,<br />
and product protection during the manipulation of infectious microorganisms. Certain<br />
considerations must be met to ensure maximum effectiveness of these primary barriers.<br />
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Whenever possible, adequate clearance should be provided behind and on each side of the<br />
cabinet to allow easy access for maintenance and to ensure that the cabinet air recirculated to<br />
the laboratory is not hindered. A 12- to 14-inch clearance above the cabinet may be required to<br />
provide for accurate air velocity measurement across the exhaust filter surface and for exhaust<br />
filter changes. When the BSC is hard ducted or connected by a canopy unit to the ventilation<br />
system, adequate space must be provided so that the configuration of the duct work will not<br />
interfere with airflow. The canopy unit must provide adequate access to the exhaust HEPA filter<br />
for testing.<br />
The ideal location for the biological safety cabinet is away from the entry (i.e., the rear of the<br />
laboratory away from traffic), since people walking parallel to the face of a BSC can disrupt the<br />
air curtain. The air curtain created at the front of the cabinet is quite fragile, amounting to a<br />
nominal inward and downward velocity of 1 mph. Open windows, air supply registers, portable<br />
fans, or laboratory equipment that creates air movement (e.g., centrifuges, vacuum pumps)<br />
should not be located near the BSC. Similarly, chemical fume hoods must not be located close<br />
to BSCs.<br />
E.6 <strong>Biosafety</strong> Cabinet Testing and Certification<br />
Class II BSCs are the primary containment devices that protect the worker, product, and<br />
environment from exposure to microbiological agents. BSCs used for BL1, BL2, or other safety<br />
levels must be tested and certified before initial use, after being moved, and on a nominal oneyear<br />
cycle. This testing must verify that BSC operation is in accordance with the <strong>National</strong><br />
Sanitation Foundation (NSF)/ American <strong>National</strong> Standard (ANSI) 49 Standard (Class II<br />
Laminar Flow Biohazard Cabinetry) and be performed by experienced and qualified personnel.<br />
This testing ensures the balance of inflow and exhaust air, distribution of air onto the work<br />
surface, integrity of the cabinet and the filters, and other BSC features. The LBNL Environment,<br />
Health, and Safety (EH&S) Industrial Hygiene Group manages surveys and tests of BSCs<br />
through the LBNL ventilation safety program and qualified vendors contracted to test BSCs (see<br />
Section 5.6.4.2 of this manual).<br />
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Appendix F<br />
Decontamination and Antimicrobials<br />
F.1 Introduction and Scope<br />
This appendix primarily provides information and guidance on decontamination<br />
principles, decontamination terms, and the variety of chemical and physical agents<br />
used to decontaminate. In a few cases, requirements are stated using the words<br />
should or must. See Section 5.7 of this manual for requirements and additional information<br />
regarding decontamination, waste, and decommissioning. Information used to develop this<br />
appendix was taken from a wide variety of Web pages and documents. Primary sources are<br />
listed in the reference section at the end of this appendix.<br />
F.2 Decontamination Principles and Terms<br />
Decontamination is a process that uses an antimicrobial to reduce or inactivate biological<br />
contaminants or components to an acceptable level so as to reduce or eliminate the possibility<br />
of transmitting pathogens to undesired hosts. An antimicrobial is the chemical or physical<br />
agent that is used in a decontamination process to prevent microbial growth. Prevention of<br />
microbial growth and pathogen transmission is needed to control contamination of the work and<br />
prevent disease in hosts such as laboratory workers, the general public, and other organisms in<br />
the environment. The decontamination process, level, antimicrobial, frequency, and specific<br />
method are based on the work activity, agents that need inactivation, and decontamination<br />
objective or requirements.<br />
Sterilization, disinfection, sanitization, and antisepsis are decontamination processes that result<br />
in different levels of decontamination or decontamination of different types of objects. These<br />
processes are discussed in Section F.2.1 below. A variety of terms are also used to describe<br />
the antimicrobials that are used in sterilization, disinfection, sanitization, and antisepsis. These<br />
antimicrobial terms are discussed in Section F.2.2 below.<br />
F.2.1 Decontamination Processes and Levels<br />
F.2.1.1 Sterilization<br />
Sterilization is the process of completely destroying all living microorganisms and viruses on an<br />
object. Any item, device, or solution is considered to be sterile when it is completely free of all<br />
living microorganisms and viruses. Sterility is an absolute term (an item is either sterile or it is<br />
not), but sterilization procedures must be defined to achieve sterility. A sterilization procedure<br />
is a treatment process to which an item is subjected after which the probability of a<br />
microorganism or virus (including a high number of bacterial endospores) surviving on the item<br />
is less than 1 in 1 million. This level of killing efficacy is referred to as the sterility assurance<br />
level.<br />
Sterilization can be accomplished by heat (e.g., autoclave or incineration), ethylene oxide gas,<br />
hydrogen peroxide gas, plasma, ozone, and radiation. Solid biohazardous waste is typically<br />
sterilized prior to disposal.<br />
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F.2.1.2 Disinfection<br />
Disinfection is generally a less lethal process than sterilization. Disinfection is the process of<br />
generally eliminating nearly all recognized pathogenic microorganisms but not necessarily all<br />
microbial forms (e.g., bacterial spores) on inanimate objects (e.g., work surfaces, equipment).<br />
Disinfection does not ensure “overkill'' and therefore lacks the margin of safety achieved by<br />
sterilization procedures. Longer disinfection times or higher concentrations of disinfectant may<br />
be needed if the effectiveness of a disinfection procedure is reduced significantly by a number<br />
of factors such as:<br />
1. More resistant microorganisms (especially bacterial spores)<br />
2. Higher microbial concentrations<br />
3. Presence of more organic matter (e.g., soil, feces, or blood)<br />
4. Rougher surfaces or more porous equipment or material<br />
5. Lower temperatures<br />
Disinfection may involve chemical or physical agents, but the term disinfection more commonly<br />
implies the use of chemical germicides or disinfectants on inanimate objects. See Section F.2.2<br />
below for additional explanation of germicides and disinfectants.<br />
Disinfection is a process that reduces the level of microbial contamination, but there is a broad<br />
range of activity that extends from sterility at one extreme to a minimal reduction in the number<br />
of microbial contaminants at the other. By definition, chemical disinfection and in particular,<br />
high-level disinfection differs from chemical sterilization by its lack of sporicidal power. This is an<br />
oversimplification of the actual situation because a few chemical germicides used as<br />
disinfectants do, in fact, kill large numbers of spores even though high concentrations and<br />
several hours of exposure may be required. Nonsporicidal disinfectants may differ in their<br />
capacity to accomplish disinfection or decontamination. Some germicides rapidly kill only the<br />
ordinary vegetative forms of bacteria such as staphylococci and streptococci, some forms of<br />
fungi, and lipid-containing viruses, whereas others are effective against such relatively resistant<br />
organisms as Mycobacterium tuberculosis var. bovis, nonlipid viruses, and most forms of fungi.<br />
Levels of chemical disinfection and activity levels for chemical disinfectants (or germicides) on<br />
inanimate surfaces may be used to assist in categorizing and selecting disinfection methods<br />
and disinfectants. Levels of chemical disinfection are categorized in Table F-1, and activity<br />
levels of selected disinfectants are shown in Table F-2.<br />
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Level<br />
High<br />
Intermediate<br />
Low<br />
Table F-1<br />
Levels of Chemical Disinfection<br />
Level Definition and Description<br />
High-level disinfection kills vegetative microorganisms and inactivates viruses, but not<br />
necessarily high numbers of bacterial spores. Such disinfectants are capable of<br />
sterilization when the contact time is relatively long (e.g., 6 to 10 hours). As high-level<br />
disinfectants, they are used for relatively short periods of time (e.g., 10 to 30 minutes).<br />
These chemical germicides are potent sporicides and, in the United States, are classified<br />
by the Food and Drug Administration (FDA) as sterilant/disinfectants. They are formulated<br />
for use on medical devices, but not on environmental surfaces such as laboratory benches<br />
or floors.<br />
Intermediate-level disinfection kills vegetative microorganisms, including Mycobacterium<br />
tuberculosis, all fungi, and inactivates most viruses. Chemical germicides used in this<br />
procedure often correspond to Environmental Protection Agency (EPA)-approved "hospital<br />
disinfectants" that are also "tuberculocidal." They are used commonly in laboratories for<br />
disinfection of laboratory benches and as part of detergent germicides used for<br />
housekeeping purposes.<br />
Low-level disinfection kills most vegetative bacteria except M. tuberculosis, some fungi,<br />
and inactivates some viruses. The EPA approves chemical germicides used in this<br />
procedure in the U.S. as "hospital disinfectants" or "sanitizers."<br />
Source: adapted from <strong>Biosafety</strong> in Microbiological and Biomedical Laboratories (BMBL), fifth edition,<br />
Appendix B.<br />
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Table F-2<br />
Activity Levels of Selected Liquid Germicides a<br />
Procedure/Product Aqueous Concentration Disinfection Activity Level<br />
Sterilization<br />
glutaraldehyde variable N/A<br />
hydrogen peroxide 6–30% N/A<br />
formaldehyde 6–8% b N/A<br />
chlorine dioxide variable N/A<br />
peracetic acid variable N/A<br />
Disinfection<br />
glutaraldehyde variable high to intermediate<br />
ortho-phthalaldehyde 0.5% high<br />
hydrogen peroxide 3 to 6% high to intermediate<br />
formaldehyde 1 to 8% high to low<br />
chlorine dioxide variable high<br />
peracetic acid variable high<br />
chlorine compounds c<br />
500 to 5,000 mg/L available intermediate<br />
chlorine (or 1 to 10% household<br />
beach in water)<br />
alcohols(ethyl,isopropyl) d 70% intermediate<br />
phenolic compounds 0.5 to 3% intermediate to low<br />
iodophor compounds e<br />
30 to 50 mg/L free iodine up to intermediate to low<br />
10,000 mg/L available iodine<br />
0.1 to 0.2%<br />
quaternary ammonium<br />
low<br />
compounds<br />
Source: adapted from BMBL, fifth edition, Appendix B.<br />
Footnotes:<br />
a This list of chemical germicides centers on generic formulations. A large number of commercial<br />
products based on these generic components can be considered for use. Users should ensure that<br />
commercial formulations are registered with the EPA or by the FDA.<br />
b Because of the ongoing controversy of the role of formaldehyde as a potential occupational<br />
carcinogen, the use of formaldehyde is limited to certain specific circumstances under carefully<br />
controlled conditions, e.g., for the disinfection of certain hemodialysis equipment. There are no FDAcleared<br />
liquid chemical sterilant/disinfectants that contain formaldehyde.<br />
c Generic disinfectants containing chlorine are available in liquid or solid form (e.g., sodium or calcium<br />
hypochlorite). Although the indicated concentrations are rapid acting and broad spectrum<br />
(tuberculocidal, bactericidal, fungicidal, and virucidal), no proprietary hypochlorite formulations are<br />
formally registered with EPA or cleared by FDA. Common household bleach is an excellent and<br />
inexpensive source of sodium hypochlorite. Concentrations between 500 and 1,000 mg/L (or ppm)<br />
chlorine are appropriate for the vast majority of uses requiring an intermediate level of germicidal<br />
activity. Higher concentrations are extremely corrosive as well as irritating to personnel, and their use<br />
should be limited to situations where there is an excessive amount of organic material or unusually<br />
high concentrations of microorganisms (e.g., spills of cultured material in the laboratory).<br />
d The effectiveness of alcohols as intermediate-level germicides is limited because they evaporate<br />
rapidly, resulting in short contact times, and also lack the ability to penetrate residual organic material.<br />
They are rapidly tuberculocidal, bactericidal, and fungicidal, but may vary in spectrum of virucidal<br />
activity (see text). Items to be disinfected with alcohols should be carefully precleaned and then<br />
completely submerged for an appropriate exposure time (e.g., 10 minutes).<br />
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e<br />
Only those iodophors registered with EPA as hard-surface disinfectants should be used, closely<br />
following the manufacturer's instructions regarding proper dilution and product stability. Antiseptic<br />
iodophors are not suitable for disinfecting devices, environmental surfaces, or medical instruments.<br />
An understanding of the resistance of organisms to chemical germicides should also be<br />
considered when selecting the disinfection methods and disinfectants. Table F-3 shows the<br />
resistance of selected organisms to decontamination, from most to least resistant.<br />
Table F-3<br />
Descending Order of Organism Resistance to Germicidal Chemicals<br />
BACTERIAL SPORES<br />
Bacillus subtilis, Clostridium sporogenes<br />
⇓<br />
MYCOBACTERIA<br />
Mycobacterium tuberculosis var. bovis, nontuberculous mycobacteria<br />
⇓<br />
NONLIPID OR SMALL VIRUSES<br />
Poliovirus, Coxsackievirus, Rhinovirus<br />
⇓<br />
FUNGI<br />
Trichophyton spp., Cryptococcus spp., Candida spp.<br />
⇓<br />
VEGETATIVE BACTERIA<br />
Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella choleraesuis, Enterococci<br />
⇓<br />
LIPID OR MEDIUM-SIZE VIRUSES<br />
Herpes simplex virus, cytomegalovirus, respiratory syncytial virus, hepatitis B virus (HBV), hepatitis C<br />
virus (HCV), human immunodeficiency virus (HIV), Hantavirus, Ebola virus<br />
Source: adapted from BMBL, fifth edition, Appendix B<br />
Note: There are exceptions to this list. Pseudomonas spp. are sensitive to high-level disinfectants, but<br />
if they grow in water and form biofilms on surfaces, the protected cells can approach the resistance of<br />
bacterial spores to the same disinfectant. The same is true for resistance to glutaraldehyde by some<br />
nontuberculous mycobacteria, some fungal ascospores of Microascus cinereus and Cheatomium<br />
globosum, and the pink-pigmented Methylobacteria. Prions are also resistant to most liquid chemical<br />
germicides and are discussed in the last part of this section.<br />
F.2.1.3 Sanitization<br />
Sanitization is the process of generally reducing microorganisms by the use of general<br />
cleaning agents. Sanitization is less effective than disinfection at reducing the number of<br />
microorganisms. General cleaning of laundry or laboratory, restroom, room, and equipment<br />
surfaces with soap and water or another cleaning agent are examples of sanitization. A<br />
particular cleaning method might use a chemical germicide or disinfectant, but the cleaning<br />
process is considered sanitization if the process only generally reduces the number of<br />
microorganisms. See Section F.2.2 below for additional explanation of germicides and<br />
disinfectants.<br />
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In the food industry, the term sanitization has a more specific meaning. According to the<br />
California Retail Food Code (CRFC), sanitization means the application of cumulative heat or<br />
chemicals on cleaned food-contact surfaces that, when evaluated for efficacy, is sufficient to<br />
yield a reduction of five logs, which is equal to a 99.999% reduction, of representative disease<br />
microorganisms of public health importance.<br />
F.2.1.4 Antisepsis<br />
Antisepsis is the application of a liquid antimicrobial chemical to human or animal living tissue.<br />
The purpose of antisepsis is to prevent sepsis by destroying potentially infectious organisms or<br />
by inhibiting their growth and multiplication. Sepsis is the presence of infectious organisms in<br />
the blood or other tissue of the body. No sporicidal activity is implied. Examples of antisepsis<br />
include application of a germicide to the injection site on a research animal, and handwashing<br />
with germicidal solution. With handwashing, the objective includes preventing the spread of<br />
infectious or contaminating agents for safety and quality control.<br />
F.2.2 Antimicrobial Categories<br />
Chemical or physical agents or substances that can decontaminate under ideal conditions have<br />
specific terms with specific meanings. The broadest term for such agents is the term<br />
antimicrobial. Antimicrobial is a chemical or physical agent that can prevent microbial growth<br />
either by some static action or by the direct killing of microbes. Categories of antimicrobials<br />
include:<br />
• Sterilant. An antimicrobial chemical or physical agent that is capable of killing all<br />
microbes including their spores to the sterility assurance level.<br />
• Germicide. An antimicrobial substance or physical agent that kills microbes. Germicides<br />
are a broader category of antimicrobials than disinfectants, since some germicides are<br />
active against endospores and viruses. Germicides, which are also known for the<br />
specific microorganisms they kill, end with the suffix –cidal (e.g., bacteriocide, sporicide,<br />
fungicide, virucide).<br />
• Disinfectant. A chemical germicide or physical agent that is applied to inanimate objects<br />
to kill microbes, but is not capable of killing endospores, some viruses, or<br />
mycobacterium. Disinfectants are typically chemical germicides.<br />
• Antiseptic. A disinfecting chemical agent applied to living tissue and used to prevent<br />
sepsis. Antiseptics are a subset of disinfecting chemical agents. A few agents are<br />
suitable as both disinfectants and antiseptics, although most disinfectants are too harsh<br />
for use on delicate skin.<br />
F.2.3 Antimicrobial Selection and Registered Disinfectants<br />
When using a chemical or physical antimicrobial to ensure decontamination is accomplished for<br />
biosafety purposes (i.e., protection of workers, public, agriculture, or environment):<br />
• There should be information indicating that the selected antimicrobial will be effective<br />
when used in a certain manner for the biological materials or agents and equipment or<br />
surfaces that need to be decontaminated; and<br />
• The antimicrobial should be used in accordance with its antimicrobial activity capabilities<br />
and conditions of use.<br />
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Antimicrobial information in this appendix, information provided by manufacturers (e.g., labels or<br />
technical specifications), and other information may be used for selecting and using the<br />
appropriate antimicrobial. Selecting a commercially available chemical antimicrobial product<br />
registered with the EPA or cleared by the FDA and using the product within its manufacturerspecified<br />
limits also ensure effective decontamination. The following lists of antimicrobials<br />
registered with EPA and FDA are available online:<br />
• Selected EPA-registered Disinfectants including sterilizers, tuberculocides, and<br />
antimicrobial products against certain human public health bacteria and viruses<br />
• FDA-Cleared Sterilants and High-Level Disinfectants with General Claims for Processing<br />
Reusable Medical and Dental Devices<br />
The Occupational Safety and Health Administration (OSHA) Bloodborne Pathogens (BBPs)<br />
Standard requires that work surfaces that are contaminated with BBP material (as defined in<br />
Section 3.3.4 of this manual) must be cleaned with an “appropriate disinfectant.” Appropriate<br />
disinfectants include:<br />
• Household bleach (i.e., approximately 5.25% sodium hypochlorite) diluted to<br />
concentrations ranging from 1% (1:100) to 10% (1:10) in water.<br />
• EPA-registered products as sterilants (List A)<br />
• EPA-registered products as tuberculocides (List B)<br />
• EPA-registered products effective against HIV/HBV (List D), or<br />
• FDA-cleared sterilants and high-level disinfectants<br />
Any of the above products are considered effective when used according to the manufacturer's<br />
instructions, provided the surfaces have not become contaminated with agents, or volumes or<br />
concentrations of agents for which higher level disinfection is recommended. Also note that the<br />
EPA lists contain the primary registrants' products only. The same formulation is frequently<br />
repackaged and renamed and distributed by other companies. These renamed products will not<br />
appear on the list, but their EPA Registration Number must appear on the label. Products<br />
cleared solely by the FDA will not have an EPA Number.<br />
F.3 Chemical Antimicrobials<br />
This section summarizes basic types and characteristics of antimicrobials that are chemical<br />
agents. Section F.4 below summarizes antimicrobials that are physical agents.<br />
All chemical antimicrobials harm microorganisms in some manner, but different chemical<br />
antimicrobials have different mechanisms of action. Mechanisms of harm include protein<br />
denaturation, membrane disruption, nucleic acid damage, and inhibition of metabolism.<br />
Chemical antimicrobials that are summarized in this section include surfactants, halogencontaining<br />
compounds, alcohols, phenol and phenol derivatives, oxidizing agents, and alkylating<br />
agents.<br />
F.3.1 Surfactants (Soaps and Detergents)<br />
A surfactant is a surface active agent that is usually an organic compound that possesses<br />
both hydrophilic (water-loving) and lipophilic (fat-liking) properties that make the compound<br />
soluble in water and lipids. Surfactants therefore increase the solubility of lipids in water<br />
solutions and increase the ability of water solutions to wet (i.e., move across or penetrate) lipid<br />
surfaces. Soaps and detergents are examples of surfactants.<br />
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F.3.1.1 Soaps<br />
Soap is sodium or potassium salts of fatty acids. Soaps are therefore alkaline (pH greater than<br />
7). Soaps either harm bacteria that are sensitive to high pH, or remove pathogens from surfaces<br />
by cleaning the surface.<br />
F.3.1.2 Detergents and Quaternary Ammonium Compounds<br />
Detergent is a synthetic surfactant. A detergent may be cationic (positively charged) or anionic<br />
(negatively charged). Cationic detergents are better at inactivating bacteria than anionic<br />
detergents.<br />
One commonly used type of cationic detergent disinfectant is a quaternary ammonium<br />
compound. Quaternary ammonium compound or quat is a cationic detergent compound<br />
derived from ammonia by replacing the hydrogen atoms with organic radicals, and the<br />
compound is especially important as surface-active agents or disinfectants, or in drugs. Quats<br />
have strong surface activity and can be used for general cleaning and low-level disinfection.<br />
Additional properties of quaternary ammonium compounds include the following:<br />
• Active against Gram-positive bacteria and lipid-containing viruses. They are less active<br />
against Gram-negative bacteria and are not active against nonlipid-containing viruses<br />
and bacterial spores.<br />
• Less effective or inactivated by organic materials, soaps or anionic detergents, or salts<br />
of metals found in water. Quats are often mixed with another agent to overcome some of<br />
these problems.<br />
• Built-in cleaning properties and relatively nontoxic (e.g., can be used for general<br />
cleaning and food equipment).<br />
• Has no odor but acts as a deodorizer.<br />
• Effective at temperatures up to 212°F.<br />
• More effective in alkaline than in acid solutions.<br />
• Typically nonirritating to the skin when used in proper dilution, but prolonged skin or eye<br />
contact should be avoided.<br />
• Stable in storage.<br />
F.3.2 Halogens (Chlorine and Iodine)<br />
Halogens are a group of elements on the periodic table. Chlorine and iodine are two halogens<br />
that are routinely used as antimicrobials.<br />
F.3.2.1 Chlorine and Sodium Hypochlorite<br />
Chlorine-containing solutions are commonly used disinfectants, and sodium hypochlorite in the<br />
form of household bleach is the most common solution used for chlorine disinfection. These<br />
solutions have broad-spectrum antimicrobial activity, but their decay rates and corrosive nature<br />
limit their use. The following bullets provide additional information:<br />
• Concentrations and Effectiveness: Chlorine-containing solutions have broad spectrum<br />
activity, but the concentration of the chlorine-active ingredient in the solution at time of<br />
use affects germicidal activity. Low concentrations of available chlorine (2 to 500 ppm)<br />
are active against vegetative bacteria, fungi, and most viruses. Effectiveness increases<br />
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with concentration of available chlorine. Rapid sporicidal action can be obtained at about<br />
2,500 ppm.<br />
• Active Ingredient Decay: The chlorine-active ingredient typically decays or is consumed.<br />
Decay or decomposition typically occurs over time and is accelerated by unfavorable<br />
storage conditions. Chlorine is also consumed by excess organic materials. Use of<br />
sufficient concentrations and quantity of chlorine, along with precleaning items to be<br />
disinfected, ensures sufficient chlorine is available for disinfection.<br />
• Corrosiveness: Chlorine-containing solutions are strong oxidizers and are very corrosive<br />
to personnel and some surfaces. Personnel handling these solutions must wear required<br />
hand, eye, and body protection (see Section 5.4 of this manual). Surfaces such as<br />
stainless steel may be corroded and should be wiped or rinsed with water following<br />
disinfection.<br />
One of the most common and effective disinfectants used in the laboratory is<br />
sodium hypochlorite (NaOCl) in water or “bleach.” Household bleach is a<br />
water-based solution of sodium hypochlorite with a typical concentration of<br />
5.25% by weight (or 52,500 ppm) of the active sodium hypochlorite<br />
ingredient. Commercial supplies are also available in the 12 to 15% dilution<br />
range, but household bleach is typically sufficient for laboratory use. Many<br />
brands and formulations of bleach are registered with the EPA as a<br />
disinfectant that is effective against bloodborne and other common human<br />
pathogens (see Section F.2.3 above). Clorox ® is the best-known brand of<br />
bleach in the U.S.<br />
Common applications and mixtures of household bleach are listed below.<br />
• Work Surfaces and Equipment: Hard work surfaces and equipment may be disinfected<br />
with 1% solution of fresh household bleach (or 500 ppm sodium hypochlorite). A 1%<br />
household bleach solution can be made by mixing 1 part household bleach with 99 parts<br />
water, or 1/8 to 1/4 cup household bleach with water in a gallon container, or 10 ml of<br />
household bleach with water in a 1 L container. Contact time for bleach is generally<br />
considered to be the time it takes the product to air dry.<br />
• Spills and Liquid Waste: Biohazardous spills and liquid waste may be decontaminated<br />
by adding household bleach to water or the liquid to be decontaminated until a 10%<br />
concentration of household bleach is achieved (or 5,000 ppm sodium hypochlorite). A<br />
10% household bleach solution can be made by mixing one part household bleach with<br />
9 parts water, or 1.5 cups household bleach with water in a gallon container, or 100 ml of<br />
household bleach with water in a 1 L container. The bleach should remain in contact with<br />
the spill or waste material for approximately 20 minutes to ensure adequate germicidal<br />
action. See Appendix G of this manual for additional information on spill cleanup.<br />
Sodium hypochlorite solutions are not very stable, and the antimicrobial activity of the chlorine<br />
typically decays over time. This decay is accelerated by unfavorable storage conditions and<br />
must be compensated by mixing fresh solutions. Favorable storage conditions include:<br />
temperature below 70°F, plastic container (not metal or glass), opaque container (to minimize<br />
exposure to light), and closed container (to minimize exposure to air). It is common to measure<br />
50% decay within one month under favorable storage conditions. Since bleach antimicrobial<br />
activity decays over time, bleach solutions must be sufficiently fresh so that the solution to be<br />
used for decontamination has sufficient antimicrobial activity. Fresh solutions of diluted<br />
household bleach made up daily are recommended for disinfection of work surfaces.<br />
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F.3.2.2 Iodine and Iodophors<br />
Iodine is another halogen that is routinely used as an antimicrobial (at 70 to 150 ppm total<br />
iodine), and iodine has properties similar to chlorine. Iodophor is a preparation containing<br />
iodine complexed with a solubilizing agent, such as a surfactant or povidone (a type of water<br />
soluble polyvinyl polymer). The resulting iodophor is a water-soluble material that increases<br />
penetration (as a surfactant) and slows the release of free iodine over long periods (as a<br />
disinfectant) when in solution. Iodophors are prepared by mixing iodine with the solubilizing<br />
agent. Wescodyne ® is a common laboratory disinfectant iodophor.<br />
Additional properties of iodophors include:<br />
• Rapid germicidal action. Effective against vegetative bacteria, Gram-positive bacteria,<br />
Gram-negative bacteria, fungi, viruses, and tubercle bacilli. Poor activity against<br />
bacterial spores.<br />
• Most effective in acid solutions.<br />
• Should not be used in hot water, since iodine is vaporized at 120 to 125°F. For optimal<br />
germicidal activity, dilute with warm acidic water. Resulting solutions are less stable but<br />
have a higher germicidal activity.<br />
• Effectiveness reduced by organic matter (but not as much as hypochlorites).<br />
• Stable in storage if kept cool and tightly covered.<br />
• Relatively harmless and nontoxic to humans.<br />
• The solution has germicidal activity if the color is brown or yellow.<br />
• Solutions of sodium thiosulfate can be used to inactivate iodophors and remove<br />
iodophor stains.<br />
Iodophors may also be used as antiseptics. Betadine and isodine are examples of antiseptic<br />
iodophors. Iodine may also be used in an alcohol solution (i.e., or tincture) as an antiseptic.<br />
F.3.3 Alcohols<br />
Ethyl or isopropyl (rubbing) alcohol concentrations of 70 to 90% in water are good general-use<br />
disinfectants with some limitations. Alcohol-water mixtures are more penetrating than pure<br />
alcohols, and therefore provide better disinfection. Alcohol concentrations above 90% are less<br />
effective than 70 to 90% concentrations.<br />
Alcohols have some positive and negative characteristics, including:<br />
• Alcohols are effective against a broad spectrum of bacterial species and many viruses,<br />
but they are less active against nonlipid viruses and ineffective against bacterial spores.<br />
• Alcohols evaporate quickly and leave no residue. These characteristics often make<br />
alcohols convenient and efficient, but provide limited penetration and disinfection time.<br />
F.3.4 Phenol and Phenol Derivatives (Phenolics)<br />
Phenol and phenol derivatives (or phenolics) come in various concentrations ranging mostly<br />
from 5 to 10% phenol-based compounds. These disinfectants are especially useful for<br />
disinfecting materials contaminated with organic materials and contaminated surfaces. Lysol ® is<br />
an example of a phenol-based disinfectant.<br />
Additional properties of phenol and phenol derivatives include the following:<br />
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• Effective at killing Gram-negative and Gram-positive bacteria including Mycobacterium<br />
tuberculosis, fungi, and lipid-containing viruses. Not active against spores or most<br />
nonlipid viruses.<br />
• Low solubility in water unless combined with detergent.<br />
• Stable in storage.<br />
• Less adversely affected by organic matter than other common disinfectants.<br />
• Effective over a relatively large pH range.<br />
• Prolonged contact deteriorates rubber.<br />
• Can cause skin and eye irritation.<br />
• Not for use on food contact surfaces.<br />
• Some phenolics are mild enough for use as antiseptics whereas others are too harsh or<br />
otherwise dangerous to be employed on living tissue.<br />
F.3.5 Oxidizing Agents (Hydrogen Peroxide)<br />
Hydrogen peroxide is an oxidizing agent and may be used as a liquid or vapor antimicrobial.<br />
Hydrogen peroxide vapor may be used for decontamination of equipment such as biosafety<br />
cabinets or high-containment (<strong>Biosafety</strong> Level 3) rooms that may be sealed during the<br />
decontamination process.<br />
F.3.6 Alkylating Agents (Formaldehyde, Glutaraldehyde, Ethylene Oxide)<br />
Formaldehyde, glutaraldehyde, and ethylene oxides are alkylating agents. These agents add<br />
carbon-containing functional groups to biological molecules.<br />
F.3.6.1 Formaldehyde<br />
Formaldehyde may be used as a liquid or gaseous antimicrobial. When used as a liquid,<br />
formaldehyde may be mixed with water as formalin or mixed with alcohol. Formaldehyde is also<br />
a human carcinogen, creates respiratory problems, and has a very low occupational exposure<br />
ceiling and short-term exposure limits that are approximately equal to the odor threshold.<br />
Additional information on formaldehyde antimicrobials are listed below:<br />
• Formalin is 37% solution of formaldehyde in water. Dilution of formalin to 5% results in<br />
an effective disinfectant. A concentration of 8% formaldehyde exhibits good activity<br />
against vegetative bacteria, spores, and viruses.<br />
• Formaldehyde and alcohol solutions (8% formaldehyde in 70% alcohol) are considered<br />
very good disinfectants because of their effectiveness against vegetative bacteria, fungi,<br />
spores, and viruses. This is the disinfectant of choice for many applications.<br />
• Formaldehyde gas may be generated by heat-accelerated depolymerization of flake<br />
paraformaldehyde. The resulting gas may be used to decontaminate equipment such as<br />
biosafety cabinets that may be sealed prior to decontamination.<br />
F.3.6.2 Glutaraldehyde<br />
Gluteraldehyde may be used for cold sterilization of equipment (e.g., medical) that cannot be<br />
steam sterilized, but sterilization often requires many hours of exposure. Two percent solutions<br />
exhibit good activity against vegetative bacteria, spores, and viruses. Its use, however, must be<br />
limited and controlled due to its toxic properties and ability to damage the eyes.<br />
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Glutaraldehyde is slightly acidic in aqueous solution and typically used at ambient temperature.<br />
When these solutions are adjusted by sodium bicarbonate (or other buffers) to a pH of 7.5 to<br />
8.5, glutaraldehyde is considered to be activated and the antimicrobial activity enhanced.<br />
Activated glutaraldehyde has limited stability after activation.<br />
F.3.6.3 Ethylene Oxide<br />
Ethylene oxide is a gaseous chemical antimicrobial used to sterilize laboratory, medical, and<br />
pharmaceutical products and equipment that would be damaged by high-temperature steam<br />
sterilization (e.g., prepackaged plastic petri dishes). This gas is especially useful because it<br />
penetrates very well into small crevices.<br />
F.4 Physical Antimicrobials<br />
This section summarizes basic types and characteristics of antimicrobials that are physical<br />
agents. Physical antimicrobials summarized in this section include dry heat, wet heat, ultraviolet<br />
radiation, ionizing radiation, visible light, and filtration.<br />
F.4.1 Heat<br />
Dry heat (e.g., oven) and moist heat (e.g., autoclave) may be used to sterilize materials and<br />
equipment. The following principles and comparisons generally apply to sterilization with dry<br />
and moist heat:<br />
• Moist heat is more effective than dry heat at a given temperature or length of exposure.<br />
• Moist heat is more penetrating than dry heat.<br />
• Temperature and length of exposure are inversely related, and penetration is critical.<br />
• Temperature and length of exposure needed to achieve sterilization are inversely related<br />
(i.e., lower temperatures require longer exposure times).<br />
• Time to achieve sterilization does not start until heat has penetrated into the item and<br />
the required temperature in the item has been achieved.<br />
F.4.1.1 Dry Heat (Baking and Incineration)<br />
Dry heat sterilization may include baking or incineration.<br />
• Baking in an oven to achieve sterilization typically requires 171°C for at least 1 hour,<br />
160°C for at least 2 hours, or 121°C for at least 16 hours.<br />
• Incineration may also be used to achieve dry heat sterilization. Examples include off-site<br />
incineration of biohazardous or pathological waste by an LBNL subcontractor or heating<br />
an inoculating loop in an infrared heat chamber at 815°C (1,500°F).<br />
Specific times and temperatures must be determined for each type of material being sterilized.<br />
Generous safety factors are usually added to allow for variables that can influence the efficiency<br />
of dry heat sterilization, such as:<br />
• The moisture of the sterilization environment as well as the moisture history of<br />
organisms prior to heat exposure.<br />
• The heat transfer properties and the spatial configuration or arrangement of articles in<br />
the load.<br />
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F.4.1.2 Wet Heat (Boiling and Autoclaving)<br />
Use of wet heat may include boiling an item in water or processing the item in an autoclave.<br />
Boiling water is a common means of applying moist heat, but boiling does not kill endospores<br />
and all viruses. Boiling water is 100°C (212°F) at standard atmospheric pressure. Higher wetheat<br />
temperatures and sterilization efficacy may be achieved with a pressurized autoclave.<br />
Autoclaves are commonly used to sterilize laboratory equipment or materials such as<br />
glassware, media, reagents, or waste. See Section F.5 below for general information and<br />
guidelines on autoclave principles, operation, and maintenance.<br />
F.4.2 Ultraviolet (UV) Radiation<br />
UV radiation or UV light is electromagnetic radiation with a wavelength shorter than that of<br />
visible light but longer than X-rays. They are in the range of 10 nanometers (nm) to 400 nm, and<br />
energies from 3 electron volts (eV) to 124 eV. UV radiation is so named because the spectrum<br />
consists of electromagnetic waves with frequencies higher than those that humans identify as<br />
the color violet.<br />
F.4.2.1 UV Light Health Effects and Categories<br />
UV radiation may affect or damage the skin and eyes depending on the wavelength, intensity,<br />
and duration of exposure. Other organs are typically not affected because UV light does not<br />
penetrate deeply into tissue. Acute effects to the skin and eyes are generally not permanent but<br />
can be quite painful.<br />
The UV spectrum is divided into three wavelength bands primarily based on their biological<br />
effects:<br />
• UVA (315 to 400 nm) is long-wave UV or “back light” and is used in dentistry and<br />
tanning. UVA rays can penetrate the middle layer of skin (dermis) and cause darkening<br />
and toughening of the skin. Overexposure to UVA has also been associated with<br />
suppression of the immune system and cataract formation.<br />
• UVB (280 to 315 nm) is medium-wave UV and is used for fade testing and photocuring<br />
of plastics. UVB rays reach the outer layer of skin (epidermis) and cause skin burns,<br />
erythma (reddening of the skin), and darkening of the skin. Prolonged exposures<br />
increase the risk of skin cancer.<br />
• UVC (100 to 280 nm) is short-wave UV and is used as a germicidal (e.g., inside<br />
biosafety cabinets). UVC poses the most risk to skin. Although UVC from the sun is<br />
absorbed by the atmosphere, manmade sources of UVC need to restrict their intensity<br />
and control exposure.<br />
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Electromagnetic spectrum. Source: CCOHS, OSH Answers, Physical Agents, Ultraviolet<br />
Radiation (February 2010).<br />
UV light that penetrates skin. Source: FDA, Radiation-emitting Products, Ultraviolet Radiation<br />
(February 2010).<br />
The eyes are particularly sensitive to UV radiation. Even a short exposure of a few seconds can<br />
result in painful but temporary inflammatory conditions known as photokeratitis and<br />
conjunctivitis. Examples of eye disorders resulting from UV exposure include "flash burn,"<br />
"ground-glass eye ball," "welder's flash," and "snow blindness.” The symptoms are pain,<br />
discomfort similar to the feeling of sand in the eye, and an aversion to bright light.<br />
The eyes are most sensitive to UV radiation from 210 nm to 320 nm (UVC and UVB). Maximum<br />
absorption by the cornea occurs around 280 nm. UVA absorption by the lens may be a factor in<br />
producing a cataract (a clouding of the lens in the eye).<br />
All wavelengths less than 320 nm (UVB and UVC) are actinic, which means they are capable of<br />
causing chemical reactions. Wavelengths below 180 nm are of little practical biological<br />
significance since the atmosphere readily absorbs them.<br />
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F.4.2.2 <strong>Biosafety</strong> Cabinet UV Light<br />
Long-term exposure to UV light may be used for disinfecting surfaces and air; however, UV light<br />
is not recommended or necessary for use inside biosafety cabinets (BSCs). This is because UV<br />
light is limited by many factors (see bulleted list below) as a disinfectant and harmful to human<br />
tissue. Other means of disinfection (e.g., chemical) are recommended for use inside BSCs.<br />
UV light’s ability to disinfect inside BSCs is limited by a number of factors including:<br />
• Penetration: UV light lacks penetrating power. Microorganisms beneath dust particles<br />
or beneath the work surface are not affected by the UV radiation.<br />
• Relative Humidity: Humidity decreases the effectiveness of UV light. Antimicrobial<br />
effects of UV light drops off precipitously above 70% relative humidity.<br />
• Temperature and Air Movement: Optimum temperature for UV light output is 77 to<br />
80°F. Temperatures below this optimum temperature result in reduced output of the<br />
antimicrobial wavelength. Moving air tends to cool the lamp below its optimum operating<br />
temperature and results in reduced output.<br />
• Lamp Cleanliness: Dust and dirt can block the antimicrobial effectiveness of UV lights.<br />
UV lamps need to be cleaned weekly with an alcohol and water mixture.<br />
• Lamp Age: The intensity of UV light emitted from UV lamps decreases with age, and<br />
bulb ratings (hours of use) may vary by manufacturer. UV lamps need to be checked<br />
periodically (approximately every six months) to ensure the intensity and wavelength of<br />
UV light needed for antimicrobial activity is being emitted.<br />
See Appendix E, Section E.5, of this manual for additional information on using UV light inside<br />
BSCs. If UV light is used as an antimicrobial but is not a required biosafety control, then<br />
maintenance and testing of the UV lights is not required for biosafety purposes. For example,<br />
germicides are used as the primary means of BSC disinfection, so maintenance and testing of<br />
the UV light inside the BSC is not required for biosafety purposes.<br />
F.4.3 Ionizing Radiation<br />
Ionizing radiation is radiation of sufficiently high energy to cause ionization in the medium<br />
through which it passes. This radiation may be of a stream of high-energy particles (e.g.<br />
electrons, protons, alpha particles) or short-wavelength electromagnetic radiation (e.g.,<br />
ultraviolet, X-rays, gamma rays). This type of radiation can cause extensive damage to the<br />
molecular structure of a substance either as a result of the direct transfer of energy to its atoms<br />
or molecules, or as a result of the secondary electrons released by ionization. The effect of<br />
ionizing radiation in biological tissue can be very serious, usually as a consequence of the<br />
ejection of an electron from a water molecule and the oxidizing or reducing effects of highly<br />
reactive species. Biological effects on living cells may include DNA damage and mutations.<br />
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Ionizing and nonionizing radiation. Source: Wikipedia, “Nonionizing Radiation” (February 2010).<br />
Different types of ionizing radiation display different degrees of penetration and may be used to<br />
sterilize equipment (e.g., medical instruments) or biological materials (e.g., inside human<br />
cadaver bones). Use of ionizing radiation as an antimicrobial requires established and<br />
specialized methods known to sterilize specific items.<br />
F.4.4 Visible Light<br />
Strong visible light can decrease bacterial viability. Drying laundry on a clothesline is an<br />
example of disinfection by using detergents and strong visible light.<br />
F.4.5 Filtration (HEPA Filters)<br />
Filtration is used as an antimicrobial treatment for air and liquids.<br />
• High-efficiency particulate air (HEPA) filters are used to filter air flowing into aseptic<br />
areas (e.g., the work area inside a BSC) and out of potentially contaminated areas (e.g.,<br />
exhaust from a BSC). See Section 5.6.4.2(a) and Appendix E of this manual for<br />
additional HEPA filter and BSC information.<br />
• Filtration is commonly used when materials are heat labile, but sterilization is not<br />
necessarily achieved unless the filter has very small filter pores. Smaller filter pores will<br />
also slow filtration speed.<br />
F.5 Autoclave Sterilization and Safety<br />
This section provides general information and guidelines on autoclave principles, operation, and<br />
maintenance typically needed to sterilize materials or equipment and ensure operator safety.<br />
Autoclave is a piece of equipment with a chamber that is used to sterilize items by applying wet<br />
heat (i.e., high-pressure steam) at temperatures above the normal boiling point of water and<br />
pressures above normal atmospheric pressure.<br />
Autoclaves are used to sterilize laboratory equipment or materials such as glassware, media,<br />
reagents, or waste. Autoclaves are commonly used because they are a dependable means of<br />
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achieving the necessary level of killing efficacy (or sterility assurance level) for most biological<br />
materials. In addition, autoclaves do not generate other chemical antimicrobial waste or sources<br />
of contamination. See Section F.2.1.1 for general information on sterilization and killing efficacy.<br />
Autoclaves must be operated and monitored properly to achieve sterility and safety. Operator<br />
safety is a concern because autoclaves may pose physical hazards (e.g., heat, steam,<br />
pressure) and biological hazards.<br />
F.5.1 Autoclaves and Sterilization<br />
Autoclaves achieve higher sterilization efficacy in part because they generate wet-heat<br />
temperatures (e.g., 121°C or 250°F) higher than those achieved under standard atmospheric<br />
pressure (i.e., 100°C or 212°F). Exposure of material in an autoclave to 121°C (250°F) for 15 or<br />
more minutes is typically sufficient for sterilization, but the material’s temperature must be<br />
121°C before the time to achieve sterilization is started. Large items, large volumes, and items<br />
that are poorly penetrated by the autoclave’s steam may take much longer than 15 minutes to<br />
sterilize. If penetration of moisture into the item is blocked, sterilization may not be achieved.<br />
Autoclave conditions critical to ensuring reliable sterilization methods are proper temperature<br />
and time and the complete replacement of autoclave chamber air with steam (i.e., no<br />
entrapment of air). Some autoclaves utilize a steam-activated exhaust valve that remains open<br />
during the replacement of air by live steam until the steam triggers the valve to close. Others<br />
utilize a precycle vacuum to remove air prior to steam introduction.<br />
Standard autoclave conditions for the types of materials that need sterilization should be<br />
established. Autoclave treatment conditions to achieve sterility will vary in relation to the volume<br />
of material treated, volume of the autoclave, the contamination level, the moisture content, and<br />
other factors. Treatment conditions for typical materials are listed below:<br />
• Laundry: 121°C (250°F) for a minimum of 30 minutes.<br />
• Trash: 121°C (250°F) for at least 45 minutes per bag. Size of the autoclave and size of<br />
the bags greatly affect sterilization time. Large bags in a small autoclave may require 90<br />
minutes or more.<br />
• Glassware: 121°C (250°F) for a minimum of 25 minutes.<br />
• Liquids: 121°C (250°F) for 25 minutes for each gallon.<br />
• Animals and bedding: Steam autoclaving is not recommended (sterilization time required<br />
would be at least 8 hours). Incineration in an approved facility is the recommended<br />
treatment of these wastes.<br />
F.5.2 Autoclave Operation and Safety<br />
This section provides general autoclave operation information and guidelines that should be<br />
used when applicable to the operation and as needed to ensure operator safety and<br />
sterilization. In addition, specific requirements and operational procedures noted in the<br />
autoclave owner’s manual should be followed since each autoclave may have unique<br />
characteristics. The owner’s manual should be readily available to answer autoclave operational<br />
questions.<br />
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F.5.2.1 Autoclave Instruction<br />
The supervisor and work lead must ensure that the autoclave operator understands the<br />
autoclave hazards, controls needed to protect themselves, and any procedures necessary to<br />
accomplish sterilization for biosafety purposes.<br />
F.5.2.2 Autoclave Operation Modes<br />
Autoclaves typically use different combinations and patterns of high heat, vacuum, and pressure<br />
to sterilize the load. These combinations and patterns are used in autoclave run cycles or runs<br />
and are based on the type of material to be sterilized. General types of runs include liquids for<br />
any type of water-based solutions, dry goods with vacuum, and dry goods without vacuum.<br />
Autoclaves often have an additional drying cycle in which hot air is drawn through the chamber<br />
to dry materials after sterilization. Controls for different autoclaves vary, so the manufacturer’s<br />
instructions regarding loading, load sizes, cycle types, and settings should be carefully followed.<br />
Additional information typical of these different run cycles is listed below:<br />
• Liquids Run. This run is longer than the other two runs, but uses lower temperatures to<br />
minimize evaporation of the liquids being sterilized.<br />
• Dry Goods with Vacuum Run. This run moves steam and heat into the deepest parts of<br />
large bags or bundles of materials and provides the best conditions for killing resistant<br />
organisms. During this type of run, the chamber alternates between cycles of high<br />
pressure, steam, and vacuum. It is important that steam and pressure be able reach the<br />
entire load, so bag closures should be carefully loosened once they are in the autoclave.<br />
• Dry Goods without Vacuum Run. This run pressurizes the chamber with steam for the<br />
duration of the cycle and then returns to normal. This process is used primarily for items<br />
that have been cleaned but need to be sterilized. Materials should be packed so that the<br />
heat and pressure can readily reach the whole load.<br />
F.5.2.3 Autoclave Container Selection<br />
Bags, pans, and other containers are used in the autoclave to provide primary and secondary<br />
containment for the materials and items that need to be autoclaved. Additional considerations<br />
and practices regarding these containers include:<br />
• Polypropylene Autoclave Bags. Autoclave or biohazard bags that may be used to<br />
contain solid materials are tear-resistant but can be punctured or burst in the autoclave.<br />
These bags should therefore be placed in a rigid container during autoclaving. Bags are<br />
available in a variety of sizes, and some are printed with an indicator that changes color<br />
when processed.<br />
• Polypropylene Containers and Pans. Polypropylene is a plastic capable of withstanding<br />
autoclaving, but it is resistant to heat transfer. Materials contained in a polypropylene<br />
pan will therefore take longer to autoclave than the same materials in a stainless steel<br />
pan. The time required to sterilize material in a polypropylene container may be reduced<br />
by removing the container’s lid, turning the container on its side, or selecting a container<br />
with the lowest sides and widest diameter that will fit in the autoclave.<br />
• Stainless Steel Containers and Pans. Stainless steel is a good conductor of heat and is<br />
less likely to increase sterilizing time, but it is more expensive than polypropylene.<br />
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F.5.2.4 Autoclave Preparation and Loading<br />
• Wear long pants, closed-toe shoes, body protection such as a lab coat, gloves, and<br />
safety glasses or goggles.<br />
• Before loading the autoclave, check inside the autoclave for any items left behind by the<br />
previous user that could pose a hazard (e.g., sharps), and then clean the drain strainer.<br />
• Load the autoclave properly according to manufacturer’s recommendations. Typical<br />
loading practices are listed below.<br />
• Do not autoclave items containing materials such as corrosives, solvents, volatiles, or<br />
radioactive materials that may contaminate the autoclave, create an inhalation hazard,<br />
or explode.<br />
• Use autoclave bags and autoclavable polypropylene or stainless steel pans. Other<br />
plastics may melt.<br />
• Load liquids as follows:<br />
o Fill liquid containers only half full.<br />
o Loosen caps or use vented closures so that heated and expanding liquids and<br />
vapors do not cause explosion of bottles or tubes.<br />
o Use only borosilicate glass (e.g., Pyrex TM or Kimax TM ) that can withstand the high<br />
autoclave temperature.<br />
o Use a pan with a solid bottom and walls to contain the liquid and catch spills.<br />
• Load autoclave bags as follows:<br />
o Put bags into pans to catch spills.<br />
o Gather bags loosely at the top and secure the top with a large rubber band or<br />
autoclave tape. This will create an opening through which steam can penetrate. Bags<br />
are impermeable to steam and therefore should not be twisted and taped shut.<br />
• Load dry goods such as glassware as follows:<br />
o Check plastic materials to ensure they are compatible with the autoclave.<br />
o Put individual glassware pieces within a heat-resistant plastic tray on a shelf or rack<br />
and not on the autoclave bottom or floor.<br />
o Add 1/4 to 1/2 inch of water to the tray so the bottles will heat evenly.<br />
• Leave space between items in the load to allow steam circulation.<br />
F.5.2.5 Autoclave Cycle and Time Selection<br />
Ensure the door to the autoclave is fully closed and latched, and the correct cycle and time has<br />
been selected before starting the cycle. Cycle selection should be based on the type of items<br />
and packs to be autoclaved:<br />
• Use liquid cycle with slow exhaust when autoclaving liquids to prevent contents from<br />
boiling over.<br />
• Use fast exhaust cycle for glassware.<br />
• Use fast exhaust and dry cycle for wrapped items.<br />
Time selection should be based on the items’ sizes, volumes, insulating capacity, and other<br />
characteristics as follows:<br />
• Take into account the size of the items to be autoclaved. Larger items with more volume<br />
take longer to autoclave. For example, a 2-liter flask containing 1 liter of liquid takes<br />
longer to sterilize than four 500 ml flasks that each contain 250 ml of liquid.<br />
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• Materials with a high insulating capacity such as animal bedding or high-sided<br />
polypropylene containers increase the time needed for the load to reach sterilizing<br />
temperatures.<br />
• Autoclave bags containing biological waste should be autoclaved for 50 minutes to<br />
ensure decontamination.<br />
F.5.2.6 Removing Autoclave Loads<br />
Practices that should be used to prevent the operator from being injured or burned while<br />
removing the load from the autoclave include:<br />
• Wear long pants, closed-toe shoes, body protection such as a lab coat, safety glasses or<br />
goggles, and heat-resistant gloves to open the autoclave door and remove nonliquid<br />
items from the autoclave.<br />
• When handling large volumes of liquid, wear waterproof boots (e.g., rubber), a rubber or<br />
plastic apron that extends past the top of the boots, and sleeve protectors in addition to<br />
the clothing and personal protective equipment listed above.<br />
• Check that the run cycle is finished and the chamber pressure is zero.<br />
• Open the door in the following manner to prevent burns caused by steam rushing out the<br />
door: Stand behind the door, slowly open the door a crack, and keep head and hands<br />
away from the opening.<br />
• Allow liquids to cool for 10 to 20 minutes before removing the load from the autoclave.<br />
Liquids removed too soon may boil up and out of the container and burn the operator.<br />
Then let the liquids cool for an extended period (e.g., one hour) before touching the load<br />
with ungloved hands. Be sure others in the area know a heat hazard is present.<br />
• Allow loads containing only dry glassware to cool for 5 minutes before removing the load<br />
from the autoclave. Then let the glassware cool for about 15 minutes before touching<br />
with ungloved hands.<br />
F.5.2.7 Autoclave Material Staging<br />
The following guidelines apply to staging materials for autoclaving and cleaning:<br />
• Materials or equipment that will be reused and are contaminated with biohazardous<br />
material or waste should be autoclaved before being washed and stored.<br />
• Laboratories and other areas where materials or equipment are staged for autoclaving or<br />
cleaning should have separate areas or containers for items designated as<br />
“Biohazardous—To Be Autoclaved” and “Not Biohazardous—To Be Cleaned.”<br />
• Biohazardous materials or equipment being staged for autoclaving should be sterilized<br />
or safely confined and identified at the close of each workday. Such items should not be<br />
placed in autoclaves overnight in anticipation of autoclaving the next day.<br />
F.5.2.8 Burn Emergencies<br />
If you are burned, seek medical treatment as soon as possible. Burns to the face, third-degree<br />
burns, or burns over large areas of the body should be treated as emergencies. The LBNL<br />
emergency phone number should be called. Minor burns should be treated by using first aid<br />
procedures. These procedures include immersing the burn immediately in cool water, removing<br />
clothing from the burn area, and keeping the injured area cool for at least 5 minutes and<br />
preferably longer. Any burns to the face or eye or any burns that blister should be seen by a<br />
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physician. Regardless of the degree of severity, report the burn to your supervisor and Health<br />
Services as an occupational injury.<br />
F.5.3 Autoclave Maintenance and Monitoring<br />
Assurance is needed that the autoclave is operating properly and sterilizing the load. Assurance<br />
includes routine autoclave maintenance, monitoring autoclave conditions, and maintaining<br />
documentation.<br />
Maintenance described in the autoclave owner’s manual should be performed to ensure the<br />
autoclave is operating properly. This maintenance typically includes periodic maintenance<br />
performed by a qualified technician and more frequent maintenance procedures performed by<br />
the operator.<br />
Monitoring the sterilization process and efficacy typically includes the use of different monitoring<br />
methods including:<br />
• Mechanical Monitoring. Mechanical monitoring, a secondary method for ensuring<br />
sterilization, involves observing and recording physical aspects of the cycle such as<br />
temperature, pressure, or time. Thermometers, pressure gauges, clocks, and logs are<br />
commonly used to observe and record the run’s physical parameters. Some autoclaves<br />
have recording devices to assist in recording run cycle conditions.<br />
• Chemical Monitoring. Chemical monitoring uses chemical indicators that change color or<br />
physical form when an autoclave bag or pack is exposed to certain autoclave<br />
temperatures. Examples include autoclave tape and special markings on autoclave bags<br />
that are used as external indicators on the outside of the load. These indicators are<br />
typically considered process indicators since they only show that the item has been<br />
processed through the autoclave at a certain temperature, but they do not show that:<br />
o Sterilization has been achieved or that a complete sterilization cycle has occurred.<br />
o Temperature was achieved in the innermost parts of the load unless they are<br />
carefully placed in the load. An easy way to check interior temperature is to wrap an<br />
item such as a plastic test tube or pipette tip with autoclave tape, attach string to the<br />
item, and put the item deep into the load. Then, tape the other end of the string to the<br />
outside of the bag so that the indicator can be pulled out of the bag. Recover the<br />
indicator after the run and confirm that it has also changed color. Warning: do not<br />
open a bag of material that may present a hazard to the operator (e.g., Risk Group 2<br />
material) to bury an indicator inside.<br />
• Biological Monitoring. Biological monitoring (or spore testing) uses live, resistant<br />
bacterial spores on strips or in self-contained vials as biological indicators that<br />
sterilization has been achieved as demonstrated by the death of the bacterial spores.<br />
Use of appropriate biological indicators at locations throughout the autoclave is<br />
considered the best and most direct indicator of sterilization. The biological indicator<br />
most widely used for wet heat sterilization is Bacillus stearothermophilus spores.<br />
Biological indicators must be used to test the efficiency of the autoclave when the<br />
autoclave is used as the final treatment of the item prior to disposal as medical<br />
waste/biohazardous waste, or when the item will be reused and is contaminated with<br />
RG2 biological materials. In these cases, tests should be performed periodically, and<br />
test records should be maintained for three years.<br />
The autoclave and process should be evaluated and corrected if monitoring indicates that the<br />
autoclave run conditions were not correct, temperatures were not sufficient as shown by<br />
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chemical indicators, or spores on biological indicators were not killed. Discontinue use of the<br />
autoclave if it is not working properly and post a “do not use” sign. Mechanical failures need to<br />
be attended by a qualified autoclave technician. When the problem is corrected, the load should<br />
be re-autoclaved to ensure sterility.<br />
F.6 References<br />
• Abedon, Stephen T., Ohio State University, supplemental lecture Web site on Control of<br />
Microbial Growth dated November 21, 1998, and Web page on Sterilization and<br />
Disinfection dated March 28, 2003<br />
• Alfa Medical article: Sterilization – Instrumental in Patient Safety, Chris H. Miller,<br />
accessed 2010<br />
• America Biological Safety Association (ABSA) Position Paper on the Use of Ultraviolet<br />
Lights in Biological Safety Cabinets, December 2000<br />
• <strong>Biosafety</strong> in Microbiological and Biomedical Laboratories (BMBL), fifth edition, CDC and<br />
NIH, Appendix B: Decontamination and Disinfection<br />
• Canadian Centre for Occupational Health and Safety (CCOHS), OSH Answers, Physical<br />
Agents, Ultraviolet Radiation Web site, February 2010<br />
• CDC Morbidity and Mortality Week Report (MMWR), Recommendations and Reports,<br />
Guidelines for Infection Control in Dental Care Health Care Settings, Appendix A –<br />
Regulatory Framework for Disinfectants and Sterilants, December 19, 2003, 52(RR17),<br />
62-64<br />
• Cornell University, Office of EH&S, Autoclave Safety, accessed 2010<br />
• Food and Drug Administration (FDA), radiation-emitting products, ultraviolet radiation<br />
Web site, February 2010<br />
• <strong>Lawrence</strong> <strong>Berkeley</strong> <strong>National</strong> <strong>Laboratory</strong>, <strong>Biosafety</strong> <strong>Manual</strong>, 1998<br />
• Leadley, Sam, Pam Sojda, and N.Y. Pavilion, Using Bleach as a Germicide for <strong>Manual</strong><br />
Washing Feeding Equipment, Cooperative Extension System Web page, November<br />
2008.<br />
• OSHA Standards Interpretation and Compliance Letters, EPA-registered disinfectants<br />
for HIV/HBV, February 28, 1997<br />
• OSHA Instruction CPL 02-02-069, Enforcement Procedures for the Occupational<br />
Exposure to Bloodborne Pathogens, January 27, 2001. Section D (Methods of<br />
Compliance), Section 23 regarding appropriate disinfectant<br />
• University of California at <strong>Berkeley</strong>, EH&S Fact Sheet No. 33 – Using Autoclaves Safely,<br />
February 19, 2003<br />
• University of Maryland, Biological Safety – Autoclave Safety and Procedures, accessed<br />
2010<br />
• University of South Carolina, Autoclave Safety Policy, March 3, 2008<br />
• Wikipedia articles: iodophor, ionizing radiation, irradiation, nonionizing radiation,<br />
surfactant, ultraviolet, ultraviolet germicidal irradiation, accessed 2010<br />
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Appendix G<br />
Biological Spills and Cleanup<br />
G.1 Introduction and Scope<br />
Hazards need to be assessed and a safe response must be implemented for each spill<br />
situation. This appendix provides general guidelines for decontamination and cleanup of various<br />
types of biological materials, including:<br />
• Precleanup considerations<br />
• Biological spill outside a biosafety cabinet (BSC)<br />
• Biohazardous spill inside a BSC<br />
• Centrifuge malfunction or spill<br />
• Radioactive and biohazardous spill outside a BSC<br />
• Chemical and biohazardous spill outside a BSC<br />
• Small dead animal, nest, or droppings cleanup<br />
Note the following <strong>Biosafety</strong> <strong>Manual</strong> sections and guidelines for additional information related to<br />
biological spills and cleanup:<br />
• Incident, Accident, and Emergency Response (Section 5.10)<br />
Especially note the LBNL Emergency Response Guide flip chart posted in your area or<br />
online for overall response guidelines for a variety of common emergencies including<br />
biological spills and personal injury. This guide also provides both emergency and<br />
nonemergency telephone numbers.<br />
• Decontamination, Waste, and Decommissioning (Section 5.7)<br />
Especially note the Medical and Biohazardous Waste Generator’s Guide (PUB-3095) for<br />
disposal of medical/biohazardous waste. Also note the Guidelines for Generators to<br />
Meet HWHF Acceptance Requirements for Hazardous, Radioactive, and Mixed Wastes<br />
at <strong>Berkeley</strong> Lab (PUB-3092).<br />
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G.2 Precleanup Considerations<br />
Generally, you may clean a biological spill when the conditions listed below are present. If these<br />
conditions do not exist, request assistance from your supervisor or call the LBNL emergency<br />
and nonemergency telephone numbers listed in the Emergency Response Guide as<br />
appropriate.<br />
Precleanup conditions:<br />
• You understand the biological and other hazards and cleanup procedures.<br />
• Your Job Hazards Analysis (JHA) and training sufficiently cover the work to be<br />
completed.<br />
• There is no potential for personal exposure, injury, or environmental damage.<br />
• The appropriate spill cleanup materials and equipment are available.<br />
• Two people can cleanup the spill thoroughly within an hour.<br />
G.3 Biological Spill outside a <strong>Biosafety</strong> Cabinet<br />
1. If you spilled a Risk Group 1 (RG1) material, or a small dilute amount of an RG2<br />
material, remove any contaminated clothing, wash contaminated body areas with soap<br />
and water, and proceed to Step 6.<br />
2. If you spilled a significant amount (e.g., 100 ml or more) of a RG2 or higher material,<br />
hold your breath, leave the room immediately, and close the door.<br />
3. Warn others not to enter the contaminated area. Get help as needed and call the LBNL<br />
emergency or nonemergency phone numbers in the Emergency Response Guide. If you<br />
leave the area, post a sign warning others to not enter the area.<br />
4. Remove and put contaminated clothing into a container for biohazardous waste disposal<br />
or autoclaving, and thoroughly wash hands and face.<br />
5. Wait 30 minutes before re-entering the area to allow dissipation of airborne biological<br />
materials (aerosols) created by the spill. Put on personal protective equipment (PPE)<br />
before re-entering the room.<br />
6. Put on the following PPE: lab coat or gown, safety glasses, and double gloves. If the risk<br />
of the material or contamination is high, wear additional appropriate PPE such as a<br />
respirator, jumpsuit with tight-fitting wrists, or shoe covers.<br />
7. Cover the spill with paper towels or other absorbent material to prevent liquid migration<br />
and aerosol production.<br />
8. Gently pour or squirt a freshly prepared solution of 10% household bleach or other<br />
appropriate disinfectant around the edges and then into the center of the spill area until<br />
the towels are soaked with the disinfectant.<br />
9. Let the disinfectant stay in contact with the spilled material for at least 10 minutes, and<br />
up to 20 minutes for larger volumes or RG2 materials.<br />
10. Use paper towels to wipe up the spill, working from the edges into the center of the spill.<br />
If sharps or sharp fragments such as glass might be in the spill, do not touch the spill<br />
materials with gloved hands. In this case, use a dustpan and squeegee or disposable<br />
cardboard to scoop up the spill materials and sharps.<br />
11. Clean the spill areas with paper towels soaked with disinfectant, and then with paper<br />
towels wetted with water.<br />
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12. Dispose of or autoclave contaminated items. Dispose of contaminated items using<br />
biohazardous waste containers, biohazard bags, sharps containers, and other means<br />
specified in the Medical and Biohazardous Waste Generator’s Guide (PUB-3095).<br />
Reusable and autoclavable items may be decontaminated using an autoclave bag and<br />
pan in an on-site autoclave.<br />
13. Remove and dispose of PPE, or place coats in lab coat laundry bin. Wash hands with<br />
soap and water.<br />
14. Report spill, exposure, and injury incidents to your work lead or supervisor and in<br />
accordance with Section 5.10 of this manual.<br />
Biohazardous and sharps spill cleanup. Source: Health and Human Services (HHS) Centers for<br />
Disease Control and Prevention (CDC) Office of Health and Safety, (, <strong>Biosafety</strong> in the<br />
<strong>Laboratory</strong>) presentation (accessed from the Web in May 2010)<br />
G.4 Biohazardous Spill inside a <strong>Biosafety</strong> Cabinet<br />
This procedure assumes the spill of biohazardous material of significant quantity or risk inside a<br />
biosafety cabinet (BSC).<br />
1. Ensure the BSC is operating and continues to operate during this procedure so as to<br />
prevent airborne contaminants from escaping the cabinet.<br />
2. Put on the following PPE: lab coat or gown, safety glasses, and chemical-resistant<br />
double gloves. Wear additional PPE (e.g., respirator or goggles) as needed based on<br />
the risk of the material, contamination, or splashing.<br />
3. Spray or wipe walls, work surfaces, and equipment with a disinfectant that is effective<br />
against the agents that may be present. A 1% solution of an iodophor decontaminant<br />
(Wescodyne or equivalent) is effective against most viruses, fungi, vegetative bacteria,<br />
and most nonencysted amoeba. A decontaminant detergent has the advantage of<br />
detergent activity, which is important because extraneous organic substances frequently<br />
interfere with the reaction between microorganisms and the active agent of the<br />
decontaminant.<br />
4. Flood the BSC’s top work surface tray with disinfectant. In a Class II BSC, also flood with<br />
disinfectant the drain pans and catch basins below the work surface. Allow the<br />
disinfectant to stand for 10 to15 minutes.<br />
5. Remove excess disinfectant from the tray by wiping with a sponge or cloth soaked in<br />
disinfectant. In a Class II BSC, drain the BSC’s top work surface into the BSC catch<br />
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basin, lift out the work surface and removable exhaust grilles, and wipe the top and<br />
bottom (underside) surfaces with a sponge or cloth soaked in disinfectant. Replace the<br />
work surface and grilles. Drain the disinfectant from the BSC base into an appropriate<br />
container. Place the container with disinfectant, gloves, cloth, or sponge in an autoclave<br />
pan, and then autoclave according to standard procedures.<br />
6. Report spill, exposure, and injury incidents to your work lead or supervisor and in<br />
accordance with Section 5.10 of this manual.<br />
G.5 Centrifuge Malfunction or Spill<br />
This procedure assumes that the following types of centrifuge events have occurred, especially<br />
if RG2 materials are involved: the spill of biological material in the centrifuge, significant<br />
mechanical failure (e.g., rotor failure), or centrifuge tube or container breakage. Evidence of<br />
such conditions might include noises during centrifuge operation or visual signs of failure or<br />
leakage when the centrifuge is opened. Note that breakage of tubes and leakage of fluid into the<br />
centrifuge wells or cups during centrifugation may release relatively few agents into the air.<br />
However, if a tube breaks and leaks in the centrifuge chamber, then aerosols and droplets may<br />
be created and dispersed.<br />
In the event of a centrifuge malfunction or spill, follow the following steps:<br />
1. Turn centrifuge off immediately. Keep the centrifuge lid closed and latched.<br />
2. Notify others.<br />
3. Evacuate the laboratory if hazardous aerosols may have been generated. Close the<br />
door, post a biohazard spill sign at the lab door, and stay out of the laboratory for 30<br />
minutes.<br />
4. For spill cleanup, the operator should wear PPE (i.e., gloves, lab coat, eye protection),<br />
remove debris, and clean and disinfect centrifuge interior, rotors, safety cups, or buckets<br />
in accordance with the manufacturer's instructions.<br />
5. Place any contaminated PPE and all cleanup materials in a biohazardous waste<br />
container. Wash hands and any exposed skin surfaces with soap and water.<br />
6. Report spill, exposure, and injury incidents to your work lead or supervisor in accordance<br />
with Section 5.10 of this manual.<br />
G.6 Radioactive and Biohazardous Spill<br />
This procedure assumes the spill of material outside a biosafety cabinet that has both<br />
radioactive and biohazardous concerns. In this case, the biological component of the spill<br />
should be inactivated prior to disposal of the spilled materials as radioactive waste. Call the<br />
Radiation Protection Group at extension 7277 or 510-486-7277 for instruction and assistance.<br />
Spill of RG1 material or small amount (e.g., less than 100 ml) of dilute RG2 material:<br />
1. Warn others not to enter the contaminated area. Post a sign on the door as needed.<br />
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2. Remove any contaminated PPE (e.g., lab coat, gloves) if there is a risk of exposure to<br />
biohazardous agents, and isolate PPE in a plastic bag or appropriate container.<br />
3. Contact the Radiation Protection Group (RPG) 24/7 at extension 7277 or 510-486-7277<br />
to report the incident. If RPG is responding to the spill location, wait until RPG arrives<br />
before proceeding with the steps below.<br />
4. Monitor yourself for radioactive contamination. If contaminated, wait for RPG assistance.<br />
5. Thoroughly wash your hands and face if there is a risk of exposure to biohazardous<br />
agents.<br />
6. Put on the following PPE: lab coat or gown, safety glasses, and double gloves. If the risk<br />
of the material or contamination is high, wear additional appropriate PPE such as<br />
respirator, jumpsuit with tight-fitting wrists, or shoe covers.<br />
7. Cover the spill with paper towels or other absorbent material to prevent liquid migration<br />
and aerosol production.<br />
8. Gently pour or squirt a freshly prepared solution of 10% household bleach or other<br />
appropriate disinfectant around the edges and then into the center of the spill area until<br />
the towels are soaked with the disinfectant.<br />
9. Let the disinfectant stay in contact with the spilled material for at least 10 minutes, and<br />
up to 20 minutes for larger volumes or RG2 materials.<br />
10. Use paper towels to wipe up the spill, working from the edges into the center of the spill.<br />
If sharps or sharp fragments such as glass might be in the spill, do not touch the spill<br />
materials with gloved hands. In this case, use a dustpan and squeegee or disposable<br />
cardboard to scoop up the spill materials and sharps.<br />
11. Clean the spill areas with paper towels soaked with disinfectant, and then with paper<br />
towels wetted with water.<br />
12. Place all contaminated materials into a plastic bag, and place the bag in the appropriate<br />
radiation waste container. Monitor for radiation contamination all potentially<br />
contaminated items that are not placed in the radiation waste container. Decontaminate<br />
and resurvey these items as necessary.<br />
13. Report spill, exposure, and injury incidents to your work lead or supervisor and in<br />
accordance with Section 5.10 of this section.<br />
Spill of Risk Group 2 material greater than 100 ml:<br />
1. If you spilled a significant amount (e.g., 100 ml or more) of an RG2 material, hold your<br />
breath, leave the room immediately, and close the door.<br />
2. Warn others not to enter the contaminated area.<br />
3. If possible, remain stationary and request assistance from others to contact the<br />
Radiation Protection Group (RPG). Contact RPG 24/7 at extension 7277 or 510-486-<br />
7277 for assistance.<br />
4. Remove any contaminated PPE (e.g., lab coat, gloves) if there is a risk of exposure to<br />
biohazardous agents, and isolate PPE in a plastic bag or appropriate container.<br />
5. Thoroughly wash your hands and face if there is a risk of exposure to biohazardous<br />
agents.<br />
6. Proceed with the remaining steps after arrival of RPG.<br />
7. Wait 30 minutes before reentering the area to allow dissipation of airborne biological<br />
materials (aerosols) created by the spill. Put on PPE before reentering the room.<br />
8. Follow Steps 6 through 13 noted in the previous section titled “RG1 materials or small<br />
amounts (e.g., less than 100 ml) of dilute RG2 materials.”<br />
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G.7 Chemical and Biohazardous Spill<br />
This procedure assumes the spill of material outside a biosafety cabinet, the material has both<br />
chemical and biological hazards, the chemical in the material is considered a hazardous waste,<br />
and the chemical has not already rendered the biological material nonviable or inactive.<br />
1. Prior to starting your research, determine which chemical disinfectant(s) and absorbent<br />
materials are compatible with the chemical(s) that may become biologically<br />
contaminated and whether the contaminated chemical(s) can be autoclaved. Autoclaves<br />
heat materials at high temperatures and pressures, and the autoclave operator may be<br />
exposed to chemical vapors when the autoclave is opened.<br />
2. If you spilled a significant amount (e.g., 100 ml or more) of a RG2 material, hold your<br />
breath, leave the room immediately, and close the door.<br />
3. Warn others not to enter the contaminated area. Get help as needed. If you leave the<br />
area, post a sign warning others to not enter the area.<br />
4. Remove and put contaminated clothing in container lined with a plastic bag for eventual<br />
decontamination, autoclaving, or disposal. Thoroughly wash hands and face. If clothing<br />
is chemically contaminated, autoclaving may not be advisable.<br />
5. If you evacuated the laboratory as stated in Step 2, call the LBNL emergency or<br />
nonemergency phone numbers in the Emergency Response Guide and wait 30 minutes<br />
before reentering the area to allow dissipation of airborne biological materials (aerosols)<br />
created by the spill. Put on PPE before reentering the room.<br />
6. Consult the LBNL Chemical Hygiene and Safety Plan for chemical spill response<br />
procedures. If the chemical(s) in the spill present a greater hazard than the biological<br />
agent(s), proceed with chemical decontamination first.<br />
7. Put on at least the following PPE: lab coat or gown, safety glasses, and chemicalresistant<br />
double gloves. If the risk of the material or contamination is high, wear<br />
additional appropriate PPE such as respirator, jumpsuit with tight-fitting wrists, or shoe<br />
covers.<br />
8. Cover the spill with an absorbent material or towel that will not react chemically with the<br />
spilled chemical. Towels will prevent liquid migration and aerosol production.<br />
9. Use a disinfectant that is compatible with the chemical(s) in the spill. Gently pour or<br />
squirt the disinfectant around the edges and then into the center of the spill area until the<br />
absorbent material or towel is soaked with the disinfectant.<br />
10. Let the disinfectant stay in contact with the spilled material for at least 10 minutes, and<br />
up to 20 minutes for larger volumes or RG2 materials.<br />
11. Use chemically compatible towels, dustpan, squeeges, or cardboard to scoop and wipe<br />
up the spill, working from the edges into the center of the spill. If there may be sharps or<br />
sharp fragments such as glass in the spill, do not touch the spill materials with gloved<br />
hands.<br />
12. Clean the spill areas with towels soaked with disinfectant, and then with towels wetted<br />
with water.<br />
13. If the chemical(s) are compatible with autoclaving, contaminated materials (paper<br />
towels, absorbent, glass, liquid, gloves, dustpan, squeegee, etc.) may also be placed<br />
into autoclave bags and an autoclave pan. Cover the pan with aluminum foil and<br />
autoclave according to standard directions. After autoclaving, the now-sterile materials<br />
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may require being disposed of as hazardous chemical waste via the LBNL Waste<br />
Management Group.<br />
14. If the chemical(s) are not autoclavable (or if you do not know whether they are<br />
autoclavable), then transfer the disinfected materials into a screw cap container, and<br />
place the container in the Satellite Accumulation Area.<br />
15. Report spill, exposure, and injury incidents to your work lead or supervisor and in<br />
accordance with Section 5.10 of this section.<br />
G.8 Cleanup of Small Dead Animals, Nests, or Droppings<br />
The following general procedure should be used as a guideline for cleanup of small dead<br />
animals, nests, or droppings. This procedure may need to be adapted depending on the nature<br />
of the materials and situation. Contact the Facilities Division via the Facilities Work Request<br />
Center if assistance is needed due to a pest infestation or to the nature or size of the concern.<br />
1. Wear PPE such as reusable or disposable rubber gloves and safety glasses when<br />
handling decontaminant solutions, dead animals, or cleaning up contaminated<br />
materials. Use double disposable gloves if possible and appropriate. Determine if<br />
disposable or cleanable protective clothing is also needed.<br />
2. Clean up dead animals, nests, droppings or contaminated food by first spraying or<br />
soaking the item with an appropriate disinfectant such as 10% household bleach,<br />
Lysol ® , or other appropriate janitorial disinfectant (see Appendix F, Sections F.2.3 of<br />
this manual). Allow the disinfectant sufficient time to decontaminate the item (e.g., 10<br />
minutes).<br />
3. If possible and appropriate, pick up the decontaminated item with an impervious<br />
barrier such as a plastic bag placed over the item. Place the decontaminated item into<br />
a plastic bag, tie the bag shut, place the bag into a second bag, and tie the second<br />
bag shut.<br />
4. Clean up localized gross surface contamination as needed by spraying or soaking<br />
with disinfectant and using disposable paper towels. Place waste materials in a plastic<br />
bag, remove outer contaminated disposable gloves, and double bag the waste<br />
materials.<br />
5. Dispose of the bags of waste in the general trash. Use an outside dumpster as<br />
needed to prevent odor problems.<br />
6. Clean contaminated surfaces or floors as needed. Use a solution of water, detergent,<br />
and disinfectant to mop floors or wipe surfaces. Steam clean or shampoo carpets and<br />
upholstered furniture. Do not vacuum or dry sweep surfaces before wet cleaning. Pour<br />
mop or cleaning wastewater into a drain that is connected to the building sanitary<br />
sewer system.<br />
7. Remove PPE, and then clean it or dispose of it.<br />
8. Remove any potentially contaminated clothing and launder separately with detergent<br />
and hot water.<br />
9. Wash hands with soap and water.<br />
The State of California encourages the reporting of dead birds and squirrels to assist state<br />
agencies in tracking disease. This reporting is optional at LBNL and involves keeping the<br />
animal for 24 hours without decontamination or freezing. Note the California West Nile Virus<br />
Web site for additional information and online reporting.<br />
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Appendix H<br />
Transportation and Shipping<br />
H.1 Introduction and Scope<br />
This appendix provides requirements, guidelines, and direction on transporting and shipping<br />
biological materials as needed to safely move the material from one location to another. This<br />
includes:<br />
• Employee transport of biological materials between laboratories, between buildings, in<br />
motor vehicles, and on LBNL buses<br />
• Use of LBNL Receiving, Transportation, and Shipping<br />
• Shipping through LBNL Shipping by a contracted shipping company (e.g., common<br />
carrier such as FedEx or UPS)<br />
• Packaging, transportation, and shipping in accordance with:<br />
o U.S. Department of Transportation (DOT) Hazardous Material Regulations<br />
(HMR) for movement of biological materials in public right-of-ways within the U.S.<br />
o International Air Transport Association (IATA) Dangerous Goods Regulations<br />
(DGR) for shipment of biological materials (e.g., infectious substances) by air.<br />
Employees who wish to transport or ship a biological material should use this appendix (starting<br />
in Section H.2) to assess if the material is a regulated biological material and select a mode and<br />
process for moving the material. Modes and processes detailed in this appendix cover safe<br />
movement of all biological materials and potential shipping and transportation regulatory issues,<br />
although most LBNL biological materials that need to be moved are not regulated. Regulatory<br />
requirements for packaging, transporting, and shipping are applicable only if the material is:<br />
• Moved in vehicles, airplanes, railcars, or vessels via public right-of-ways such as<br />
roadways, airways, railways, and sea lanes that are accessible to the public, and<br />
• A regulated biological material (i.e., categorized by DOT HMR or IATA DGR as an<br />
infectious substance or genetically modified organism).<br />
This appendix does not cover the following topics:<br />
• Transportation and shipping of nonbiological hazardous materials. These topics are<br />
covered in the following LBNL documents:<br />
o The PUB-3000, Sections 5.8.11 and 5.8.13: Provides overview of services provided<br />
by the LBNL Environment, Health, and Safety (EH&S) Division to transport<br />
radioactive and hazardous materials, and by LBNL Transportation Services to ship<br />
them.<br />
o The Chemical Hygiene and Safety Plan (CHSP), “Chemical Procurement,<br />
Transportation, and Inventory”: Provides instructions for moving hazardous material<br />
research samples and small quantities by hand or in a passenger vehicle.<br />
• Other regulatory requirements related to the import, export, and transfer of biological<br />
materials. See Appendix I of this manual for information on these topics.<br />
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H.2 How to Determine Transportation Mode and Requirements<br />
LBNL employees should use the following steps to determine the transportation mode and<br />
requirements needed to transport or ship a biological material:<br />
1. Determine the desired mode of transportation or shipping.<br />
2. Use Table H-1 to determine if the desired transportation mode can be used. If needed,<br />
use Section H.4 to determine if the material is subject to IATA or DOT shipping<br />
regulations. Section H.4 can also be used for definitions of terms.<br />
3. Use Section H.3 to determine the requirements or process for packaging, labeling,<br />
transporting, or shipping the material.<br />
Table H-1<br />
Transportation Modes and Biological Materials Not Allowed<br />
General<br />
Transport Mode<br />
Personal<br />
Transportation<br />
Licensed<br />
Transporter<br />
Specific<br />
Transport Mode<br />
Hand carry between<br />
laboratories<br />
Hand carry between<br />
buildings<br />
Personal motor<br />
vehicle*<br />
LBNL bus or other<br />
public transportation<br />
LBNL Transportation<br />
Department<br />
Common carrier<br />
Biological Materials that are<br />
Not Allowed<br />
No restrictions on types of biological<br />
materials.<br />
No restrictions on types of biological<br />
materials.<br />
Regulated** biological materials are not<br />
allowed except for regulated materials<br />
contained in human or animal samples<br />
(including, but not limited to, secreta, excreta,<br />
blood and its components, tissue and tissue<br />
fluids, cells, and body parts) being<br />
transported for research, diagnosis,<br />
investigational activities, or disease treatment<br />
or prevention; or that are biological products.<br />
Samples containing Category A infectious<br />
substances are not allowed.<br />
Regulated** biological materials or other<br />
biological materials that may present a<br />
detrimental risk to the health of humans or<br />
other organisms either directly through<br />
infection or indirectly through damage to the<br />
environment are not allowed.<br />
No restrictions on types of biological<br />
materials.<br />
No restrictions on types of biological<br />
materials unless restricted by the carrier.<br />
Footnotes:<br />
* Personal transport in a motor vehicle means transportation in a private or government<br />
passenger vehicle such as a car, van, or pickup truck.<br />
** Materials that are and are not subject to DOT and IATA regulations are described in Section<br />
H.4 of this appendix.<br />
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Here is an example of how to apply Steps 1, 2, and 3 above:<br />
An LBNL research employee wants to transport his established human cells in a personal<br />
vehicle between two LBNL sites in direct support of his research project. According to Table<br />
H-1, this is allowable because it is a human sample being transported solely for the purpose<br />
of research, regardless of whether or not the human cells are a regulated biological material.<br />
According to the second bullet in Section H.4.1, these cells would not be considered<br />
regulated biological materials unless they contained infectious agents or were collected from<br />
individuals suspected of having an infectious disease; however, this determination does not<br />
matter, because this is a human sample being transported in direct support of a research<br />
project. The researcher must package and label the human cells according to Section H.3.1.3<br />
(Personal Transport in Motor Vehicle). The researcher may then give the packaged cells to<br />
another person who is affiliated with the research for transport in a personal vehicle if this<br />
individual knows the cells are in the vehicle, is informed of the applicable requirements in this<br />
appendix, and is doing the transport solely for the purpose of supporting the research.<br />
H.3 Requirements and Processes for<br />
Receiving, Transporting, and Shipping<br />
This section presents requirements and processes related to receiving, transporting, and<br />
shipping biological materials by an LBNL employee, LBNL Transportation or Shipping Groups,<br />
or a common carrier. See Section H.2 to determine if the desired mode of transportation or<br />
shipping can be used to transport the biological material.<br />
H.3.1 Employee Transportation of Materials<br />
This section covers minimum requirements for transporting biological materials by an LBNL<br />
employee without the use of the LBNL Transportation Group or a common carrier. General<br />
objectives that should be accomplished whenever employees transport biological materials<br />
include:<br />
• Biological materials will not be spilled in the event of accident (e.g., due to a person<br />
tripping or a vehicle accident).<br />
• The identity of biological materials, their hazards or lack of hazards, and owners may be<br />
explained by people transporting the materials and determined by other people who may<br />
find the materials.<br />
• Exterior surfaces of containers will not be contaminated with biological materials.<br />
• Regulated biological materials being transported in public right-of-ways (e.g., in vehicles<br />
on roads or in airplanes) will be packaged and transported in accordance with DOT and<br />
IATA regulations.<br />
H.3.1.1 Hand-Carry Transport between Laboratories<br />
Hand-carry transport between laboratories generally means an LBNL employee is hand-carrying<br />
the biological material in a container and walking between laboratories in the same building or<br />
buildings that are closely connected and designed for pedestrian traffic. Requirements and<br />
precautions for such transport include:<br />
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• Primary or secondary containers that prevent leakage are required. When Risk Group<br />
(RG) 2 or bloodborne pathogen (BBP) materials are transported, a biohazard label must<br />
be displayed on the exterior of the outermost container. When possible and appropriate<br />
for the work and risk:<br />
o Primary containers of biological material should be break-resistant (e.g., plastic),<br />
leakproof, have secure caps or lids, and be disinfected on the outside.<br />
o Primary containers of biological material should be placed in a secondary container<br />
that prevents leakage. Racks or packing should be used inside the secondary<br />
container as needed to keep the primary containers upright and prevent breakage.<br />
• The primary or secondary containers should be labeled with the identity of the contents,<br />
ownership information, and any appropriate biohazard information. Such labeling may<br />
not be needed if the primary container(s) and secondary container will remain in<br />
continuous possession of the person(s) transporting and processing the materials.<br />
• Remove gloves and wash hands after preparing biological materials for transport. Lab<br />
coat, clean gloves, and eye protection should be worn during transport if there is a risk of<br />
unexpected exposure, contamination, or spillage.<br />
• Medical/biohazardous waste must be transported in accordance with the container and<br />
labeling requirements in Medical and Biohazardous Waste Generator’s Guide (PUB-<br />
3095).<br />
H.3.1.2 Hand-Carry Transport between Buildings<br />
Hand-carry transport between buildings generally means the packaged biological material is<br />
carried by an LBNL employee who is walking between nonadjacent LBNL or University of<br />
California, <strong>Berkeley</strong> (UCB) buildings. Requirements and precautions for such transport include:<br />
• Biological materials transported by this means are not subject to DOT and IATA<br />
regulations, but the biological materials should be transported according to the<br />
packaging and labeling criteria described in Section H.3.1.3 (Personal Transportation in<br />
Motor Vehicle) of this appendix.<br />
• Employees transporting materials by this means should take precautions to ensure they<br />
can walk safely between buildings. Precautions may include having one hand free to<br />
open doors and hold stair rails, use of a hand truck, and wearing slip-resistant shoes.<br />
• Medical/biohazardous waste cannot be transported off LBNL sites (e.g., between<br />
discontinuous LBNL locations or different institutions). Medical/biohazardous waste must<br />
be transported in accordance with the container and labeling requirements in Medical<br />
and Biohazardous Waste Generator’s Guide (PUB-3095).<br />
H.3.1.3 Personal Transportation in a Motor Vehicle<br />
Personal transportation in a motor vehicle means transportation by an LBNL employee in a<br />
private or government passenger vehicle such as a car, van, or pickup truck. Requirements for<br />
such transport of biological materials are described in this section. These requirements meet the<br />
DOT HMR requirements for transporting materials of trade:<br />
Materials allowed. Materials that may be transported in a motor vehicle include unregulated<br />
biological materials noted in Section H.4.1, the regulated materials noted as an exception in<br />
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Table H-1, and dry ice. Other regulated biological materials or medical/biohazardous waste are<br />
not allowed. Transportation of any regulated biological material must be in direct support of a<br />
principal business (e.g., research project), and the principal business must not be motor vehicle<br />
transportation (e.g., a company paid to transport items).<br />
Packaging and labeling. An inner container and outer package are required.<br />
• Manufacturer’s packaging. When applicable, each regulated biological material must be<br />
contained and packaged in the manufacturer’s original container and packaging, or a<br />
container and packaging of equal or greater strength and integrity.<br />
• Inner containers:<br />
o Use break-resistant (e.g., plastic) containers, if possible.<br />
o Liquids must be in a leakproof container. Lids on inner containers must have a<br />
positive means of closure. For example, a screw-type cap should be used instead of<br />
parafilm, aluminum foil, or a stopper.<br />
o Container(s) must be disinfected as needed for safety and should be placed in a<br />
Ziploc ® bag or an equivalent secondary spill container.<br />
o Information must be placed on or with the container(s) as needed to clearly<br />
communicate the container’s contents, hazards, and ownership. Each individual<br />
container must be labeled with enough information to identify its contents. In<br />
addition, the container(s) or secondary bag(s) must also be labeled with the identity<br />
of the material, the name and phone number of the sender, the name and phone<br />
number of the recipient (if different than the sender), and hazard information. Hazard<br />
information includes a biohazard label if the material is biohazardous (e.g., RG2),<br />
any words needed to explain the hazard, or words indicating the material is not<br />
hazardous.<br />
o Containers for sharps (i.e., sharps container) must be constructed of a rigid material<br />
resistant to punctures and securely closed to prevent leaks or punctures.<br />
Leakproof plastic containers<br />
with screw caps. Source: VWR.<br />
Containers inside break-resistant<br />
and leakproof carrier. Source: VWR (May<br />
2010).<br />
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Ice chest with secure lid as an outer<br />
package. Source: unidentified.<br />
Biohazard label for inner and outer<br />
containers. Source: 29 CFR 1910.1030(g)(1)<br />
• Outer packaging:<br />
o The outer packaging must be a strong and tight packaging made of a rigid material. It<br />
must also be securely closed. Examples include a cardboard, plastic, or metal box or<br />
pail with a secure lid. A plastic carrier that is leakproof, easy to clean, and has a<br />
secure lid is typically the best package for biological materials (e.g., ice chest or<br />
enclosed laboratory tube carrier).<br />
o Packing material or racks must be used between the inner container(s) and outer<br />
packaging as needed to keep the container(s) upright, cushion the container(s), and<br />
prevent the container(s) from shifting or damage.<br />
o Sufficient absorbent material must be inside the outer packaging to absorb the entire<br />
contents of all inner liquid container(s).<br />
o The exterior of the outer packaging must be labeled with the same information<br />
required for the inner container. The common name(s) or shipping name(s) of the<br />
materials must be used.<br />
o Outer packaging must be secured against shifting inside the vehicle during transport.<br />
Generally, the safest place to secure biological materials is in a vehicle trunk. If<br />
hazardous materials are also transported, these materials must be placed in the<br />
trunk or truck bed.<br />
Material quantity of regulated biological material:<br />
• Each inner container must not be more than 0.5 kg (1.1 lbs) or 0.5 L (17 ounces), and an<br />
aggregate contained within the entire outer package must not be more than 4 kg (8.8<br />
lbs) or 4 L (1 gallon), or<br />
• A single inner container containing not more than 16 kg (35.2 lbs) or 16 L (4.2 gallons)<br />
that is inside a single outer package.<br />
Ice and dry Ice. Ice and dry ice may be used inside the package to keep the biological materials<br />
cold. Ice must be packaged so that any melting water will be contained inside the outer<br />
packaging. Dry ice is frozen carbon dioxide that will sublimate into gas, so dry ice must be<br />
placed in packaging that is not gas-tight (e.g., ice chest). Dry ice is only regulated as a<br />
hazardous material in air transport, but is not regulated in ground (e.g., motor vehicle) transport<br />
in the U.S.<br />
Hazard communication. The operator of a motor vehicle that contains a regulated biological<br />
material must be informed of the presence of the material, and must be informed of the<br />
requirements in this section.<br />
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H.3.1.4 Personal Transportation on an LBNL Bus<br />
Personal transportation on an LBNL bus means the packaged biological material is carried by<br />
an LBNL employee on an LBNL shuttle bus. The following materials must not be transported on<br />
an LBNL bus: regulated biological materials, medical/biohazardous waste, or other biological<br />
materials that may present a detrimental risk to the health of humans or other organisms, either<br />
directly through infection or indirectly through damage to the environment. Any other biological<br />
materials transported by this means are not subject to transportation regulations, but the<br />
biological materials should be transported according to the packaging and labeling criteria<br />
described in Section H.3.1.3 (Personal Transportation in a Motor Vehicle) above.<br />
H.3.2 LBNL Receiving, Transportation, and Shipping<br />
Receiving, transportation, and shipping of biological materials are conducted institutionally from<br />
Building 69 by Resource Services in the Facilities Division. These services are conducted in<br />
accordance with PUB-3000, Section 5.8 (Traffic and Transportation), DOT HMR, IATA DGR,<br />
and by personnel with appropriate regulatory qualifications. For questions about shipping or<br />
receiving biological materials, contact LBNL Shipping at 510-486-5084 or LBNL Receiving at<br />
510-486-4935.<br />
H.3.2.1 LBNL Receiving<br />
Biological materials that are shipped by a contracted shipping company (i.e., common carrier) to<br />
LBNL must be received by LBNL Receiving and are typically delivered to the requestor via<br />
LBNL Transportation in the packaging and with the documentation that was received from the<br />
common carrier.<br />
H.3.2.2 LBNL Transportation<br />
This section covers the pickup and delivery of biological materials or items that contain<br />
biological materials (e.g., freezers) within LBNL by LBNL Transportation or a carrier authorized<br />
by Transportation. Transportation of materials must be requested through the Facilities Work<br />
Request Center, and a completed Transportation Authorization Form (TAF) must be<br />
attached to each item to be transported. Additional directions include:<br />
• When placing a work request for transportation, the requestor will be asked if the item to<br />
be transported contains hazardous materials. The requestor should declare that the item<br />
does not contain hazardous materials if the item to be transported does not contain<br />
regulated biological material as described in Section H.4 or other hazardous materials.<br />
• If the item does not contain a regulated biological or other hazardous material, the<br />
requestor should package and label the biological materials as described in Section<br />
H.3.1.3 (Personal Transportation in Motor Vehicle) of this appendix.<br />
• If the item contains a regulated biological or other hazardous material, the requestor<br />
should consider personal transportation of the item in a motor vehicle (see Section<br />
H.3.1.3 of this appendix) if allowed (see Table H-1), or contact LBNL Shipping for advice<br />
and directions.<br />
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• See Section H.3.2.3 below if the item will also be shipped by a common carrier after<br />
transportation within LBNL.<br />
H.3.2.3 LBNL and Common Carrier Shipping<br />
Shipment of biological materials by a common carrier out of LBNL must be conducted by LBNL<br />
Shipping. Information and assistance must be provided by the sender. Use the following<br />
guidelines for shipping:<br />
• Note directions for transportation and pickup of materials in Section H.3.2.2.<br />
• An LBNL Shipping Document must also accompany all material leaving LBNL.<br />
Directions for completing this form can be found on the LBNL Shipping Web site. This<br />
form requires the sender to describe the item and material to be shipped, and asks if the<br />
item and material is a regulated hazardous material (i.e., contains Dangerous Goods).<br />
• The sender may use the lists of unregulated and regulated materials in Section H.4 to<br />
answer the Dangerous Goods question on the form in regards to biological materials.<br />
Section H.4 can also be used to determine what information should be included in the<br />
form’s description section. The sender is responsible for providing a description of the<br />
item and biological material and its potential biological or hazardous materials risks so<br />
that LBNL Shipping can correctly categorize and ship the material.<br />
• Trained personnel in LBNL Shipping determine if the material is subject to DOT and<br />
IATA shipping regulations. They also ensure the material is correctly packaged, labeled,<br />
and documented for shipment. If the material is a regulated biological material, LBNL<br />
Shipping will work with the sender to ensure the shipping requirements are implemented<br />
at the sender’s LBNL location.<br />
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Packaging and labeling as an infectious substance. Transporting Infectious Substances<br />
Safely, US DOT Document PHH50-0079-0706 (October 1, 2006).<br />
H.4 Unregulated and Regulated Materials<br />
This section provides information on which biological materials are or are not subject to DOT<br />
HMR and IATA DGR infectious substance and genetically modified organism shipping<br />
regulations. LBNL employees should use this information to assist in selecting or requesting<br />
appropriate modes of transport for their biological materials.<br />
H.4.1 Unregulated Biological Materials<br />
The following materials are not subject to DOT and IATA infectious substance shipping<br />
regulations:<br />
• Substances that do not contain infectious substances or that are unlikely to cause<br />
disease in humans or animals.<br />
• Noninfectious biological materials from humans, animals, or plants. Examples include<br />
noninfectious cells, tissue culture, blood, or plasma from individuals not suspected of<br />
having an infectious disease, DNA, RNA, or other genetic elements.<br />
• Substances containing microorganisms that are nonpathogenic to humans or animals.<br />
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• Substances that have been neutralized or inactivated so that they no longer pose a<br />
health risk.<br />
• Environmental samples that are not considered to pose a significant risk of infection<br />
(e.g., food and water samples).<br />
• Dried blood spots.<br />
• Fecal occult blood screening tests.<br />
• An infectious substance (other than a Category A infectious substance) contained in a<br />
patient sample being transported for research, diagnosis, investigational activities, or<br />
disease treatment and prevention; or a biological product when such materials are being<br />
transported by a private carrier in a motor vehicle used exclusively to transport such<br />
materials.<br />
• Blood or blood components that have been collected for the purpose of transfusion or<br />
the preparation of blood products to be used for transfusion or transplantation.<br />
• Tissues or organs intended for use in transplantation.<br />
• A material with a low probability of containing an infectious disease, or where the<br />
concentration of the infectious substance is at a level that naturally occurs in the<br />
environment and cannot cause disease when exposure to it occurs. Examples of these<br />
materials include foodstuffs and environmental samples (e.g., samples of water, dust, or<br />
mold).<br />
• A biological product, including an experimental or investigational product or component<br />
of a product, subject to federal approval, permit, review, or licensing requirements such<br />
as those required by the Food and Drug Administration (FDA) or U.S. Department of<br />
Agriculture (USDA).<br />
H.4.2 Regulated Biological Materials<br />
The materials presented below are subject to DOT and IATA shipping regulations for infectious<br />
substances and genetically modified organisms:<br />
Infectious substances are materials regulated for shipping. These materials are known to be,<br />
or are reasonably suspected to contain, an animal or human pathogen. A pathogen is a virus,<br />
microorganism (including bacteria, plasmids, or other genetic elements), proteinaceous<br />
infectious particle (prion), or a recombinant microorganism (hybrid or mutant) that is known or<br />
reasonably expected to cause disease in humans or animals. Microorganisms that are unlikely<br />
to cause human or animal diseases are not subject to biological shipping regulations.<br />
• Category A infectious substances are materials capable of causing permanent<br />
disability, or a life threatening or fatal disease in humans or animals when exposure to<br />
them occurs. Category A infectious substances are shipped as infectious substances<br />
affecting humans (UN2814) or infectious substances affecting animals (UN2900).<br />
Examples of Category A infectious substances are listed in a table in the infectious<br />
substances section of the IATA Dangerous Goods Regulations.<br />
• Category B infectious substances are materials that do not meet Category A criteria.<br />
Category B infectious substances are shipped as UN3373.<br />
Patient specimens or diagnostic specimens are any human or animal materials including but<br />
not limited to excreta, secreta, blood, blood components, tissue, and tissue fluids being shipped<br />
for the purpose of diagnosis. Patient specimens that have a minimal likelihood of containing<br />
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pathogens are regulated materials, but they are also exempt from many shipping requirements.<br />
Professional judgment is used to determine if a specimen contains pathogens and should be<br />
based on the patient’s medical history, symptoms, local conditions, and individual<br />
circumstances. The outer package must be marked “Exempt human specimen” or “Exempt<br />
animal specimen.” If there is more than a “minimal likelihood” that a patient specimen contains<br />
pathogens, it must be shipped as a Category A or Category B infectious substance.<br />
Biological products are materials that are derived from living organisms and manufactured for<br />
use in the prevention, diagnosis, treatment, or cure of disease in humans or animals and are<br />
certified by the USDA, FDA, or other national authority. Examples of biological products include<br />
certain viruses, therapeutic serums, toxins, antitoxins, vaccines, blood, and blood products.<br />
Biological products transported for final packaging, distribution, or use by medical professionals<br />
are not subject to biological shipping regulations. Biological products that do not meet these<br />
criteria must be shipped as UN2814, UN2900, or UN3373 when appropriate.<br />
Genetically Modified Organisms (GMO) or microorganisms (GMMO) are organisms whose<br />
genetic material has been purposely altered through genetic engineering in a way that does not<br />
occur naturally. GMOs and GMMOs that are not infectious but that can alter animals, plants, or<br />
microorganisms in a way that is not normally the result of natural reproduction are considered a<br />
miscellaneous hazard (Class 9) and are shipped as UN3245. GMOs and GMMOs that are<br />
infectious must be shipped as UN2814, UN2900, or UN3373.<br />
H.5 References and Resources<br />
• International Air Transport Association (IATA) Dangerous Goods Regulations (DGR),<br />
Section 3.6.2, “Division 6.2 : Infectious Substances,” and Section 3.9, “Class 9:<br />
Miscellaneous Dangerous Goods, Genetically Modified Microorganisms and Genetically<br />
Modified Organisms”<br />
• PUB-3095, Medical and Biohazardous Waste Generator Guidelines, LBNL, latest<br />
revision<br />
• Transporting Infectious Substances Safely, guide to changes effective October 1, 2006,<br />
US DOT Document PHH50-0079-0706<br />
• UNH Shipment of Biological Materials <strong>Manual</strong>, University of New Hampshire, updated<br />
March 30, 2007<br />
• UNH Guide to Shipping with Dry Ice, April 9, 2007<br />
• U.S. Department of Transportation (DOT) Hazardous Material Regulations (HMR), 49<br />
CFR 171.8 (Definitions), 173.134 (Infectious Substances), and 173.6 (Materials of<br />
Trade)<br />
• U.S. Postal Service (USPS) Domestic Mail <strong>Manual</strong> Section 10.17 (Infectious<br />
Substances)<br />
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Appendix I<br />
Import, Export, and Transfer Restrictions<br />
I.1 Introduction and Scope Borders<br />
Materials being transferred (i.e., imported, exported, or transferred) from one location or person<br />
to another may be subject to regulatory restrictions or permit requirements. United States (U.S.),<br />
state, and foreign government agencies restrict and permit the movement of certain biological<br />
materials across borders to prevent threats to public health, agriculture, environment, and<br />
national security.<br />
This appendix provides an outline of U.S.-based regulatory restrictions, permits, and lists related<br />
to the transfer (i.e., import, export, or transfer) of biological and related materials. This outline<br />
provides LBNL personnel with a starting point for determining whether such materials are<br />
potentially regulated by U.S. agencies, and whether there are restrictions or permits applicable<br />
to transfer of the material or equipment. Contact the LBNL <strong>Biosafety</strong> Office for additional advice.<br />
This appendix does not provide comprehensive information about restricted materials, or<br />
transfer or shipping requirements. Additional LBNL policy information may be found in the<br />
following documents:<br />
• Web links and references to external agencies provided in this appendix<br />
• Appendix H of this manual for transportation and shipping requirements<br />
• The <strong>Berkeley</strong> Lab Export Control <strong>Manual</strong> for general LBNL export control requirements<br />
The supervisor, work lead, person transferring the biological material, person requesting<br />
transfer of the biological material, and permit holder all have LBNL or legal responsibilities for<br />
complying with transfer requirements, obtaining any required permits, and following the<br />
conditions of the permit. Regulatory requirements, permits, and permit conditions related to the<br />
transfer of biological materials should also be included in the <strong>Biosafety</strong> Work Authorization. The<br />
LBNL <strong>Biosafety</strong> Office and Institutional <strong>Biosafety</strong> Committee (IBC) will review the researcher’s<br />
assessment and documentation of transfer requirements during the work authorization review<br />
process.<br />
I.2 Importing or Transfer into the U.S. and California<br />
There may be restrictions or permits required for the transfer of biological material between<br />
collaborators, or for importing material into the U.S. from foreign countries or in some cases into<br />
California or the San Francisco Bay Area.<br />
Shipments and persons entering the U.S. are processed by the U.S. Customs and Border<br />
Protection (CBP), which is a branch of the Department of Homeland Security. The CBP checks<br />
materials transported by travelers and shipments for proper import permits, packaging, and<br />
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labeling. This check may include opening and inspecting the package. Noted concerns may be<br />
reported to other U.S. agencies. In addition, the California Department of Food and<br />
Agriculture (CDFA) and the U.S. Department of Agriculture-Animal and Plant Health Inspection<br />
Service (USDA-APHIS) do not allow the import of certain materials that may be infested with<br />
invasive species identified as pests by the state. CDFA also has border protection stations that<br />
inspect vehicles for commodities that may be infested with pests. The person importing the<br />
material (the “importer”) should therefore:<br />
• Obtain an import permit from the appropriate government organization prior to shipment,<br />
if required.<br />
• Package and label the material according to permit and shipping requirements.<br />
• Consider including a courtesy letter (e.g., a letter that describes the contents in detail<br />
and any hazards, concerns, permit requirements, or lack thereof) with the shipment.<br />
Prior to shipment of the material, the person importing the material (the “importer”) should<br />
contact the appropriate government organization to determine its transfer requirements. The<br />
importer is legally responsible for ensuring that personnel package, label, and ship regulated<br />
material from the foreign country according to the regulating agency’s requirements and<br />
shipping regulations. Shipping labels are often also issued to the importer with the permit. The<br />
importer must send the labels and one or more copies of the permit to the shipper. The permit<br />
and labels inform CBP and other agencies of the package contents.<br />
USDA-APHIS label for shipping soil samples under a soil permit. Source: LBNL Environment,<br />
Health, and Safety (EH&S).<br />
I.2.1<br />
CDC and APHIS Select Agent and Toxin Restrictions<br />
Select agents and toxins are specific pathogenic agents and toxins that pose a severe threat to<br />
human, animal, and plant health because of their potential for use as biological weapons. They<br />
are therefore regulated by the Department of Health and Human Services, Centers for Disease<br />
Control and Prevention (HHS-CDC) and the , USDA-APHIS. See Section 3.3.2.5 of this manual<br />
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for additional information, and Appendix B, Sections B.2 and B.3, for a list of select agents and<br />
toxins. Consult the most recent online list at http://www.selectagents.gov/.<br />
Only facilities registered with and individuals approved by CDC or APHIS are allowed to<br />
possess, have access to, or transfer the specific agents and strains or toxins for which they are<br />
approved. These activities must be conducted in accordance with the LBNL <strong>Biosafety</strong>, Security,<br />
and Incident Response Plan for Select Agents. Transfers of select agents or toxins must be<br />
conducted with approval and involvement of the LBNL EH&S <strong>Biosafety</strong> Office.<br />
I.2.2<br />
APHIS Agricultural Permits<br />
The USDA-APHIS defends America’s animal and plant resources from agricultural pests and<br />
diseases by regulating materials, organisms, or agents that may harm domestic or native<br />
animals or plants, or natural resources. These materials, organisms, or agents may cause harm<br />
directly (e.g., predator or pathogen) or indirectly (e.g., vector). Generally, APHIS requires a<br />
permit or another document issued to an individual to import, export, or store regulated<br />
materials from or to locations outside the continental U.S. or between U.S. states.<br />
Section 3.3.3 of this manual provides an overview of APHIS agency branches along with<br />
categories and examples of regulated materials, organisms, and agents. The following sections<br />
provide additional agency details, requirements, and Web links for more information.<br />
I.2.2.1 APHIS Plant Health Permits<br />
The Plant Protection and Quarantine (PPQ) branch of APHIS safeguards agriculture and<br />
natural resources from the risks associated with the entry, establishment, or spread of animal<br />
and plant pests and noxious weeds to ensure an abundant, high-quality, and varied food supply.<br />
PPQ provides the following resources:<br />
• PPQ Permits Web page: Provides permit applications for soil, plant pests, plants, plant<br />
products, weeds, etc.<br />
• The PPQ Soil Circular: Defines what is and is not soil, and provides<br />
information about soil treatments and permits. Soil is a mixture of inorganic<br />
and organic materials, when the organic materials are unidentifiable plant<br />
and/or animal parts. This mixture can support biological activity and therefore<br />
carry and introduce harmful pests or diseases from one location to another.<br />
• The PPQ Plant Pest Program: Provides a list of select insects, mollusks,<br />
nematodes, plant diseases, or noxious weeds that are considered pests.<br />
• The PPQ Cooperative Agricultural Pest Survey Program: Provides lists of<br />
<strong>National</strong> Pests of Concern and State Pests of Concern.<br />
• A list of fungal plant pathogens for each U.S. state is currently being developed by PPQ<br />
to help expedite the permit process for obtaining research isolates. The list will be based<br />
on the Widely Prevalent Fungi of the United States Web site.<br />
Appendix B, Section B.4, of this manual also provides lists of bacterial, fungal, and viral plant<br />
pathogens that may be regulated by USDA.<br />
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I.2.2.2<br />
APHIS Animal Health Permits<br />
The Veterinary Services (VS) branch of APHIS protects and improves the health, quality, and<br />
marketability of our nation's animals, animal products, and veterinary biologics by preventing,<br />
controlling, and/or eliminating animal diseases, and monitoring and promoting animal health and<br />
productivity. VS provides the following information on permits, types of materials, and diseases:<br />
• VS animal health permits for importing controlled material, organisms, vectors, animal<br />
products, cell cultures and their products, live animals, semen, and embryos.<br />
• Center for Import Export (NCIE) in APHIS VS regulates the import, export, and<br />
interstate movement of all animals and animal products (e.g., tissues, blood, and<br />
semen), including those that are genetically engineered.<br />
• Center for Veterinary Biologics (CVB) in APHIS VS regulates and requires veterinary<br />
biologics permits for veterinary biologics. Examples of veterinary biologics include<br />
vaccines, antibodies, diagnostic kits, and certain immunomodulators, including those<br />
developed using genetically engineered organisms.<br />
• Animal health disease information.<br />
• Animal diseases by animal species.<br />
I.2.2.3 APHIS Genetically Engineered Organisms Permits<br />
APHIS uses the term biotechnology to mean the use of recombinant DNA technology, or<br />
genetic engineering (GE) to modify living organisms. APHIS regulates certain GE organisms<br />
that may pose a risk to plant or animal health. In addition, APHIS participates in programs that<br />
use biotechnology to identify and control plant and animal pests. Below is a list of the regulatory<br />
agency branches and requirements for genetically engineered organisms and facilities.<br />
• Biotechnology Regulatory Services (BRS) in APHIS uses permits, notifications, and<br />
petitions to regulate the importation, interstate movement, or environmental release of<br />
certain GE organisms including plants, insects, or microbes that may be plant pests.<br />
When transgenic Drosophila developed for research need to be moved, BRS requires a<br />
Drosophila Courtesy Permit Application or an APHIS 2000 Form to confirm they are not<br />
plant pests and therefore do not need to be regulated.<br />
• See NCIE and CVB in Section I.2.2.2 above.<br />
I.2.3<br />
CDC Agents or Vectors of Human Disease Permits<br />
CDC requires a U.S. Public Health Service permit to import an etiologic agent, or material<br />
containing an etiologic agent, host, or vector of human disease. A permit is also required for<br />
interstate transfer if the original CDC import permit was issued on the condition that any<br />
subsequent transfer of the material would require a permit. According to the CDC Etiologic<br />
Agent Import Permit Program, the materials listed below require a permit.<br />
• Etiologic agents. Etiologic agents that are microorganisms, infectious agents, and<br />
toxins that cause disease in humans (e.g., bacteria, bacterial toxins, viruses, fungi,<br />
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rickettsiae, protozoans, and parasites) require a CDC permit. Etiologic agents also<br />
include naturally occurring, bioengineered, or synthesized components of an etiologic<br />
agent when the component causes human disease. Examples of etiologic agents are<br />
listed in Appendix B, Sections B.2 and B.3.<br />
• Biological materials. Biological materials that are known or suspected of containing an<br />
etiologic agent also require a CDC permit. Examples include unsterilized specimens of<br />
human and animal matter (e.g., tissue, blood, body discharges, fluids, excretions or<br />
similar material) known or suspected of containing an etiologic agent.<br />
• Hosts and Vectors<br />
o Animals. Any animal known or suspected of being infected with an organism<br />
capable of causing disease that is transmissible to humans may require a CDC<br />
permit. See the CDC animal importation Web site for more information.<br />
o Bats. All live bats require an import permit from the CDC and the U.S. Fish and<br />
Wildlife Services.<br />
o Arthropods. Any living insect or other arthropod that is known or suspected of<br />
containing an etiologic agent requires a CDC permit.<br />
o Snails. Snail species capable of transmitting a human pathogen require a CDC<br />
permit.<br />
I.2.4<br />
Food and Drug Administration Import Program<br />
With the exception of most meat and poultry, all food, drugs, biologics, cosmetics, medical<br />
devices, and electronic products that emit radiation are subject to examination by the U.S. Food<br />
and Drug Administration (FDA) when they are being imported or offered for import into the U.S.<br />
Most meat and poultry products are regulated by USDA. FDA requires various notifications or<br />
approvals prior to importing. See the FDA Import Program Web site for more information.<br />
I.2.5<br />
Fish and Wildlife Service Permits<br />
The import, export, or re-export of a wildlife or plant specimen may be regulated by a<br />
conservation law or treaty (e.g., Endangered Species Act) that is implemented by the U.S. Fish<br />
& Wildlife Service (FWS). These laws are part of domestic and international conservation efforts<br />
to protect wildlife and plants subject to international trade. Wildlife is any living or dead wild<br />
animal, its parts, and products made from the animal. Wildlife not only includes mammals, birds,<br />
reptiles, amphibians, and fish, but also invertebrates such as insects, crustaceans, arthropods,<br />
mollusks, and coelenterates. The FWS Permits Web site should be used to determine whether<br />
a wildlife or plant specimen requires a permit and how to obtain a permit. Table I-1 provides<br />
examples of wildlife or plant specimens that may require a permit to export or import.<br />
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Table I-1<br />
Wildlife or Plant Specimens That May Require an FWS Permit<br />
Export<br />
• African elephant ivory<br />
• Animals<br />
• Artificially propagated plants<br />
• Asian elephant ivory<br />
• Biological samples<br />
• Captive-born export<br />
• Circuses/traveling animal exhibitions<br />
• Goldenseal<br />
• Ginseng<br />
• Marine mammals<br />
• Museum specimens<br />
• Personal pet<br />
• Plants<br />
• Raptors<br />
• Trophies by taxidermist<br />
• Wildlife<br />
Import<br />
• African elephant<br />
• African elephant ivory<br />
• African leopard<br />
• Argali<br />
• Asian elephant ivory<br />
• Biological samples<br />
• Birds<br />
• Bontebok<br />
• Circuses/traveling animal exhibitions<br />
• Marine mammals<br />
• Museum specimens<br />
• Personal pet<br />
• Plants<br />
• Polar bears<br />
• Scientific and zoological breeding or display<br />
• Sport hunted trophy<br />
• White rhinoceros<br />
• Wildlife<br />
Source: adapted from the UNH Shipment of Biological Materials <strong>Manual</strong>, University of New<br />
Hampshire, March 30, 2007.<br />
I.3 Exporting or Transfer from the U.S.<br />
Controls for exporting from LBNL are outlined in the <strong>Berkeley</strong> Lab Export Control <strong>Manual</strong>. These<br />
export controls are designed to protect items and information that are important to the U.S. The<br />
controls are based on government rules and regulations that govern the transfer of the following<br />
items to non-U.S. entities or individuals, regardless of where or how the transfer takes place:<br />
• Goods (systems, components, equipment, or materials)<br />
• Technologies (technical data, information, or assistance)<br />
• Software/codes (commercial or custom)<br />
The <strong>Berkeley</strong> Lab Export Control <strong>Manual</strong> should be consulted for general export control<br />
requirements. This section of the <strong>Biosafety</strong> <strong>Manual</strong> only outlines U.S.-based regulatory<br />
restrictions and lists related to the export of biological materials.<br />
Depending on the nature of the biological material, there may be restrictions or U.S. export<br />
permits required for the transfer of material to foreign countries. The country to which the<br />
material is being transferred may also require an import permit. If the material requires an export<br />
permit, the permit must be obtained from the appropriate government agency prior to transfer or<br />
shipment.<br />
When leaving the U.S., travelers may be questioned or packages may be opened and inspected<br />
by any inspection service provided by other countries. The person exporting the material should<br />
therefore:<br />
• Obtain an export permit from the appropriate government organization prior to shipment,<br />
if required.<br />
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• Package and label the material according to permit and shipping requirements.<br />
• Consider including a courtesy letter (e.g., a letter that describes the contents in detail<br />
and any hazards, concerns, permit requirements or lack thereof) with the shipment.<br />
Several agencies and export control lists outlined in the next sections are involved in controlling<br />
exports of biological agents that may be used as biological weapons. Since LBNL is not a<br />
Department of Energy (DOE) Defense Programs laboratory, the export controls of most<br />
relevance at LBNL are those administered by the Department of Commerce, Bureau of Industry<br />
and Security, under the Commerce Control List (see Section I.3.1).<br />
I.3.1<br />
Commerce Control List<br />
The Department of Commerce controls the export of all goods, technologies, and software not<br />
regulated by another government agency. Because LBNL is not a DOE Defense Programs<br />
laboratory, the most relevant export controls are those administered by the Department of<br />
Commerce Bureau of Industry and Security (BIS), which maintains the Export<br />
Administration Regulations (EAR) Database. An important component of EAR is the<br />
Commerce Control List (CCL), a section of the regulations that lists specific goods,<br />
technologies, and software, the countries to which those items may or may not be exported, and<br />
any special restrictions or exceptions that may apply.<br />
A permit may be required from the Commerce Department when exporting biological agents<br />
such as human, animal, and plant pathogens or toxins; genetic elements and genetically<br />
modified organisms; and products that might be used for culturing large amounts of agents. See<br />
Table I-2 for an example list of biological agents on the CCL. Consult the most recent online list<br />
in CCL Supplement No. 1 to Part 774 Category 1. Consult the BIS export controls Web site and<br />
<strong>Berkeley</strong> Lab Export Control <strong>Manual</strong> for additional information.<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.<br />
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Table I-2<br />
Commerce Control List of Biological Agents<br />
Human Pathogens and Toxins<br />
Bacteria<br />
• Bacillus anthracis<br />
• Brucella abortus<br />
• Brucella melitensis<br />
• Brucella suis<br />
• Burkholderia mallei (Pseudomonas mallei)<br />
• Burkholderia pseudomallei<br />
(Pseudomonas pseudomallei)<br />
• Chlamydia psittaci<br />
• Clostridium botulinum<br />
• Clostridium perfringens, epsilon toxin<br />
producing types<br />
• Enterohaemorrhagic Escherichia coli; serotype<br />
O157 and other verotoxin producing serotypes<br />
• Francisella tularensis<br />
• Salmonella typhi<br />
• Shigella dysenteriae<br />
• Vibrio cholerae<br />
• Yersinia pestis<br />
Toxins<br />
• Abrin<br />
• Aflatoxins<br />
• Botulinum toxins<br />
• Cholera toxin<br />
• Clostridium peifringens toxins<br />
• Conotoxin<br />
• Diacetoxyscirpenol toxin<br />
• HT-2 toxin<br />
• Microcystin (Cyanginosin)<br />
• Modeccin toxin<br />
• Ricin<br />
• Saxitoxin<br />
• Shiga toxin<br />
• Staphylococcus aureus toxins<br />
• T-2 toxin<br />
• Tetrodotoxin<br />
• Verotoxin and other Shiga-like ribosome<br />
inactivating proteins<br />
• Volkensin toxin<br />
• Viscum Album Lectin 1 (Viscumin)<br />
Fungi<br />
• Coccidioides immitis<br />
• Coccidioides posadasii<br />
Viruses<br />
• Chikungunya virus<br />
• Congo-Crimean haemorrhagic fever virus<br />
• Dengue fever virus<br />
• Eastern equine encephalitis virus<br />
• Ebola virus<br />
• Hantaan virus<br />
• Hendra virus (Equine morbillivirus)<br />
• Japanese encephalitis virus<br />
• Junin virus<br />
• Kyasanur Forest virus<br />
• Lassa fever virus<br />
• Louping ill virus<br />
• Lymphocytic choriomeningitis virus<br />
• Machupo virus<br />
• Marburg virus<br />
• Monkey pox virus<br />
• Murray Valley encephalitis virus<br />
• Nipah virus<br />
• Omsk haemorrhagic fever virus<br />
• Oropouche virus<br />
• Powassan virus<br />
• Pulmonary and renal syndrome-haemorrhagic<br />
fever viruses (Seoul, Dobrava, Puumala, Sin<br />
Nombre)<br />
• Rabies virus cultures<br />
• Rift Valley fever virus<br />
• Rocio virus<br />
• South American haemorrhagic fever virus<br />
(Sabia, Flexal, Guanarito)<br />
• St. Louis encephalitis virus<br />
• Tick-borne encephalitis virus (Russian Spring-<br />
Summer encephalitis virus)<br />
• Variola virus<br />
• Venezuelan equine encephalitis virus<br />
• Western equine encephalitis virus<br />
• White pox<br />
• Yellow fever virus<br />
Rickettsiae<br />
• Barlonella quintana (Rochalimea quintana,<br />
Rickettsia quintana)<br />
• Coxiella burnetii<br />
• Rickettsia prowasecki<br />
• Rickettsia rickettsii<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.<br />
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Table I-2<br />
Commerce Control List of Biological Agents<br />
(Continued)<br />
Animal Pathogens and Toxins<br />
Bacteria<br />
• Mycoplasma mycoides as: Mycoplasma<br />
mycoides subspecies mycoides SC (small<br />
colony) (a.k.a. contagious bovine<br />
pleuropneumonia); and Mycoplasma<br />
capricolum subspecies capripneumoniae<br />
(“strain F38”)<br />
Viruses<br />
• African horse sickness virus<br />
• African swine fever virus<br />
• Avian influenza (AI) viruses identified as highly<br />
pathogenic (HP) strains - see the EAR CCL<br />
• Bluetongue virus<br />
Viruses (continued)<br />
• Foot and mouth disease virus<br />
• Goat pox virus<br />
• Lumpy skin disease virus<br />
• Lyssa virus<br />
• Newcastle disease virus<br />
• Peste des petits ruminants virus<br />
• Porcine enterovirus type 9 (swine vesicular<br />
disease virus)<br />
• Porcine herpes virus (Aujeszky's disease)<br />
• Rinderpest virus<br />
• Sheep pox virus<br />
• Swine fever virus (Hog cholera virus)<br />
• Teschen disease virus<br />
• Vesicular stomatitis virus<br />
Plant Pathogens<br />
Bacteria<br />
• Xanthomonas aibliineans<br />
• Xanthomonas campestris pv. citri including<br />
strains referred to as Xanthomonas campestris<br />
pv.citri types A,B,C,D,E or otherwise classified<br />
as Xanthomonas citri, Xanthomonas<br />
campestris pv. aurantifolia or Xanthomonas<br />
campestris pv. Citrumelo<br />
• Xanthomonas oryzae pv. oryzae<br />
(Pseudomonas campestris pv. oryzae)<br />
• Clavibacter michiganensis subspecies<br />
sepedonicus (Corynebacterium michiganensis<br />
subspecies sepedonicum or Corynebacterium<br />
sepedonicum)<br />
• Ralstonia solanacearum Races 2 and 3<br />
(Pseudomonas solanacearum Races 2 and 3,<br />
or Burkholderia solanacearum Races 2 and 3)<br />
Viruses<br />
• Potato Andean latent tymovirus<br />
• Potato spindle tuber viroid<br />
Fungi<br />
• Colletotrichum coffeanum var.virulans<br />
(Colletotrichum kahawae)<br />
• Cochliobo!us miyabeanus (Helminthosporium<br />
oryzae)<br />
• Magnaporthe grisea (pyricularia grisea/<br />
pyricularia oryzae)<br />
• Microcyclus u!ei (Dothidella u!ei)<br />
• Puccinia graminis (Puccinia graminis f.sp.<br />
tritici)<br />
• Puccinia striiformis (Puccinia g!umarum)<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.<br />
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Table I-2<br />
Commerce Control List of Biological Agents*<br />
(Continued)<br />
Genetic Elements and Genetically Modified Organisms<br />
• Genetic elements that contain nucleic acid<br />
sequences associated with the pathogenicity of<br />
controlled microorganisms<br />
• Genetic elements that contain nucleic acid<br />
sequences coding for any controlled "toxins" or<br />
"sub-units of toxins"<br />
• Genetically modified organisms that contain<br />
nucleic acid sequences associated with the<br />
pathogenicity of controlled microorganisms<br />
• Genetically modified organisms that contain<br />
nucleic acid sequences coding for any<br />
controlled “toxins” or “sub-units of toxins”<br />
Technical Note: Genetic elements include,<br />
inter alia, chromosomes, genomes, plasmids,<br />
transposons, and vectors, whether genetically<br />
modified or unmodified.<br />
Source: adapted from CCL Supplement No. 1 to Part 774 Category 1, pages 59 to 66 (April 20,<br />
2010); and UNH Shipment of Biological Materials <strong>Manual</strong> (March 30, 2007).<br />
I.3.2<br />
U.S. Munitions List<br />
It is unlikely that agents and substances on this munitions list would be used or exported from<br />
LBNL, but this section is provided so that personnel can understand what is covered by this list.<br />
The U.S. Department of State controls the export of "defense articles and defense services"<br />
under the International Traffic in Arms Regulations (ITAR). Items in this category to be<br />
export controlled are placed on the U.S. Munitions List (USML), a section of ITAR (Part 121)<br />
maintained by the U.S. State Department in conjunction with the U.S. Department of Defense.<br />
The USML contains many categories of articles, including Category XIV (Toxicological Agents,<br />
Including Chemical Agents, Biological Agents, and Associated Equipment). Section (b) of this<br />
USML category states that biological materials include “Biological agents and biologically<br />
derived substances specifically developed, configured, adapted, or modified for the purpose of<br />
increasing their capability to produce casualties in humans or livestock, degrade equipment, or<br />
damage crops.” Such agents and substances are not typically used at LBNL, but the export of<br />
any item on the USML requires an export license issued by the U.S. State Department. Exports<br />
of all other products not covered by the USML are subject to the export jurisdiction of the U.S.<br />
Department of Commerce, BIS, as discussed in Section I.3.1.<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.<br />
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I.3.3<br />
Biological Weapons Convention Lists<br />
The Convention on the Prohibition of the Development, Production, and Stockpiling of<br />
Bacteriological (Biological) and Toxin Weapons and on their Destruction, commonly known as<br />
the Biological Weapons Convention (BWC), has been in force since 1975. The BWC is the<br />
first multilateral disarmament treaty banning an entire category of weapons. It effectively<br />
prohibits the development, production, acquisition, transfer, retention, stockpiling, and use of<br />
biological and toxin weapons. The BWC is also a key element in the international community’s<br />
efforts to address the proliferation of weapons of mass destruction. The U.S. and other<br />
countries participating in the Australia Group (AG) are States Parties to the BWC. The AG is<br />
an informal forum of countries that, through the harmonization of export controls, seeks to<br />
ensure that exports do not contribute to the development of chemical or biological weapons.<br />
The AG maintains the following Common Control Lists of equipment and agents that require<br />
export control:<br />
• Chemical weapons precursors<br />
• Dual-use chemical manufacturing facilities and equipment and related technology and<br />
software<br />
• Dual-use biological equipment and related technology and software<br />
• Biological agents<br />
• Plant pathogens<br />
• Animal pathogens<br />
U.S. export permits or licenses are not directly regulated by the AG nor covered by the BWC<br />
lists, since the BWC lists are related to international treaty and are not derived from U.S.<br />
regulations. It appears to the author of this LBNL <strong>Biosafety</strong> <strong>Manual</strong> section that the Department<br />
of Commerce BIS and U.S. Department of State are the U.S. agencies that have primary<br />
responsibility for enforcing U.S. exports related to the BWC. Sections I.3.1 and I.3.2 above<br />
should therefore be used to determine U.S. regulatory requirements related to the BWC lists.<br />
The “Core List” of agents on the AG Common Control List appears to be the same or very<br />
similar to the agents on the BIS CCL presented above in Table I-2. Therefore, the Core List of<br />
agents on the BWC list is not relisted in this <strong>Biosafety</strong> <strong>Manual</strong>. However, the AG Common<br />
Control Lists also include a few additional agents that are not on the Core List. These additional<br />
agents are listed in Table I-3. It is not clear to the author of this <strong>Biosafety</strong> <strong>Manual</strong> section how or<br />
if these additional agents are regulated for U.S. export control.<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.<br />
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Table I-3<br />
BWC Agents Not On the Commerce Control List<br />
Plant Pathogens –<br />
Items for Inclusion in Awareness-Raising Guidelines<br />
Bacteria<br />
• Xylella fastidiosa<br />
Viruses<br />
• Banana bunchy top virus<br />
Fungi<br />
• Deuterophoma tracheiphila<br />
(syn. Phoma tracheiphila)<br />
• Monilia rorei (syn. Moniliophthora rorei)<br />
Bacteria<br />
• Clostridium tetani 2<br />
• Legionella pneumophila<br />
• Yersinia pseudotuberculosis<br />
Human Pathogens – Warning List 1<br />
Source: The AG Common Control List of biological agents (October 2009) and plant pathogens<br />
(April 2005).<br />
Table Footnotes:<br />
1<br />
Biological agents are controlled when they are an isolated live culture of a pathogen agent, a<br />
preparation of a toxin that has been isolated or extracted from any source, or material including living<br />
material that has been deliberately inoculated or contaminated with the agent. Isolated live cultures of a<br />
pathogen agent include live cultures in dormant form or in dried preparations, whether the agent is<br />
natural, enhanced, or modified. An agent is covered by this list except when it is in the form of a<br />
vaccine. A vaccine is a medicinal product in a pharmaceutical formulation licensed by, or having<br />
marketing or clinical trial authorization from, the regulatory authorities of either the country of<br />
manufacture or of use, which is intended to stimulate a protective immunological response in humans<br />
or animals in order to prevent disease in those to whom or to which it is administered.<br />
2<br />
AG recognizes that this organism is ubiquitous. However, since it has been acquired in the past as part<br />
of biological warfare programs, it is worthy of special caution.<br />
I.4 References<br />
• <strong>Berkeley</strong> Lab Export Control <strong>Manual</strong><br />
• Commerce Control List, Supplement No. 1 to Part 774 Category 1<br />
• DOE Guidelines on Export Control and Nonproliferation, July 1999<br />
• UNH Shipment of Biological Materials <strong>Manual</strong>, University of New Hampshire, updated<br />
March 30, 2007<br />
• Web sites and Wikipedia articles of referenced government agencies and topics,<br />
accessed April 2010<br />
Printed copies are not official versions of this manual. Before using the printed copy, verify that it is the most current version.<br />
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