Chapter 26





Work Process B. Work and Risk Assessment

The work scope must be defined and hazards and risks must be assessed before work begins. These work-planning processes are the first two core ISM functions and required by biosafety standards. Biological work and risks at Berkeley Lab are defined using established institutional assessment and authorization processes, a structured approach as required by DOE, and the standard biosafety risk assessment process defined by the CDC and NIH. It is a primary responsibility of workers, work leads, and supervisors to ensure these processes are implemented before work begins.

1.   Berkeley Lab Assessment and Authorization Processes. Berkeley Lab uses the following institutional assessment and authorization processes and documents to define work, identify biological hazards and potential exposures, assess biological risks, and establish biosafety controls:

a.    A Job Hazards Analysis (JHA) is prepared for each worker (see the ES&H Manual Job Hazards Analysis program)

b.   A Subcontractor Job Hazards Analysis (sJHA) and Work Authorization is prepared for each subcontractor, vendor, or affiliate (see the ES&H Manual sJHA Process – Subcontractor Job Hazards Analysis program).

c.   Biosafety Work Authorizations are prepared for work with biological materials in specific operations or projects. In the case of research involving biological materials, the Institutional Biosafety Committee (IBC) reviews and approves the definition of work, risk assessment, and controls as part of the authorization process. See Work Process B.2 below for details.

2.   Biosafety Risk Assessment Process

a.   The institutional assessment and authorization processes and documents noted in Work Process B.1 above incorporate the standard biosafety risk-assessment process defined and required by the CDC, NIH, and DOE in the Biosafety in Microbiological and Biomedical Laboratories (BMBL), the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines), and the Worker Safety and Health Program (WSHP).

b.   The standard biosafety risk-assessment process starts with considering three primary factors: (1) the inherent work hazard posed by the biological material or agent, (2) the susceptible hosts (i.e., receptors) that may be affected by the material or agent, and (3) the exposure pathways between the threat hazard and the susceptible host.

c.   In addition, BMBL outlines the following five-step approach for laboratory supervisors and work leads to assess biological risk and to select controls for laboratory work:

        1. Identify material or agent hazards, and perform an initial risk assessment.
        2. Identify laboratory procedure hazards.
        3. Make a final determination of the appropriate biosafety containment level, and select additional controls indicated by the risk assessment.
        4. Evaluate a worker’s proficiency in safe work practices, and ensure the integrity of safety equipment.
        5. Review the risk assessment with the biosafety professional, subject matter expert, and the IBC.

d.   The remaining sections of Work Process B present in greater detail the key factors underlined above that must be considered when conducting risk assessments and selecting controls. Primary factors include material or agent hazards (perceived or real) and procedure hazards. Secondary factors include staff proficiencies and other personal factors. See Section II of BMBL for more information on biological risk assessment.

3.   Material or Agent Hazards and Requirements

a.   Biological Materials and Agents. The material or agent hazard(s) and associated requirements must be considered at the start of the risk assessment. Terms used to describe biological materials must also be defined and understood before a risk assessment takes place. This is because these terms often have specific meanings, associated requirements, and associated lists:

      1. The term biological materials is used in this program and the risk-assessment process to describe a broad range of organisms, cells, viruses, and other materials of biological origin that pose differing levels of risks to plants, animals, or humans.
      2. The term biological agent or agent is used to describe a specific biological organism or material that is often directly responsible for producing an effect (e.g., disease). Examples of biological agents include a microorganism (e.g., bacterium, fungus, or parasite), virus, prion, or biological toxin. For example, humans are composed of tissues that contain blood; the blood contains fluids and cells; and the blood may contain the viral pathogen hepatitis B. Although the human body, tissues, blood, cells, fluids, and pathogens are all biological materials, only the hepatitis B virus is a biological agent.

b.   Material Viability. In addition, the risk assessment should consider the state or treatment of the biological material that may change or eliminate the hazardous characteristics of the material, and this information should be included in the Biosafety Work Authorization when the information significantly describes the safety aspects of the work. For example, biohazardous characteristics of a biological material may not be present if the material is in a nonviable, fixed, inactive, or decontaminated state. These terms are listed below along with simplified definitions and examples: 

      1. Nonviable means the material or agent is not capable of living or developing under favorable conditions. Examples include sections of plant or animal tissue that are often not capable of propagating, and extracts of biological samples such as DNA or RNA that cannot replicate without cells. These materials may not pose risks as long as there is no potential for the presence of pathogens.
      2. Fixed means the material has been treated so that it has been stabilized and preserved in place. For example, properly fixing cells with paraformaldehyde or glutaraldehyde typically kills the cells and most potential pathogens.
      3. Inactive means the material is not capable of acting or reacting normally. For example, infectious proteins (i.e., prions) may be inactivated by chemical destruction.
      4. Decontaminated means the material has been treated (e.g., sterilized or disinfected) so that biological contaminants or components have been reduced or inactivated to an acceptable level to reduce or eliminate the possibility of transmission of pathogens to undesired hosts. For example, fresh human bones may be decontaminated internally by radiation.

Text Box:      
	Plate A shows microbial growth	Plate B shows no visible colonies

The above pictures show sterility certification of a genomic DNA preparation from a sewage-derived culture. Sample viability is determined by plating, incubating, and comparing the culture before gDNA extraction on plate A (positive control) with the gDNA prepared sample on Plate B. Following incubation: (a) Plate A shows a lawn of microbial growth, and (b) plate B shows no visible colonies and confirms sterility of the prepared sample. Source: Harry Beller, LBNL, JBEI (April 2011). Assessment of viability is often a component of the risk assessment and work process and determines biosafety containment level.

Plate A shows microbial growth

Plate B shows no visible colonies

The above pictures show sterility certification of a genomic DNA preparation from a sewage-derived culture. Sample viability is determined by plating, incubating, and comparing the culture before gDNA extraction on plate A (positive control) with the gDNA prepared sample on Plate B. Following incubation: (a) Plate A shows a lawn of microbial growth, and (b) plate B shows no visible colonies and confirms sterility of the prepared sample. Source: Harry Beller, LBNL, JBEI (April 2011). Assessment of viability is often a component of the risk assessment and work process and determines biosafety containment level.

c.    Risk Group Classification

      1. The principal hazardous characteristics of the agents that are present or that may be present in the biological material must be considered while completing the initial risk assessment. This consideration includes an assessment of the agent’s capability to infect and cause disease in a susceptible human or other host, the severity of disease, and the availability of preventive measures and effective treatments. To facilitate this assessment process, the World Health Organization (WHO) and NIH established an agent risk group (RG) classification for laboratories. This RG classification system, which was also adopted by the CDC, describes four general RGs based on the hazardous characteristics of agents, and the transmission route of natural disease in humans.
      2. Berkeley Lab uses the four RG levels and definitions provided in Appendix B of the U.S.-based NIH Guidelines (see Table 2 below). As shown in Table 2, a higher RG level indicates a higher risk for disease in humans. Assignments of RGs to specific agents may be found in various sources, including:
        1. Appendix B, and Work Process B.2: Provide a list of human pathogens and their RG designations as excerpted from Appendix B of the NIH Guidelines (Classification of Human Etiologic Agents on the Basis of Hazard) 
        2. The American Biological Safety Association (ABSA) Risk Group Database


Table 2. Risk Group Classification

Risk Group (RG) Level

Risk Group Definition


Agents that are not associated with disease in healthy adult humans


Agents that are associated with human disease that is rarely serious, and for which preventive or therapeutic interventions are often available


Agents that are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available (high individual risk but low community risk)


Agents that are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available (high individual risk and high community risk)

Source: Adapted from the NIH Guidelines, Appendix B, Table 1.

      1. Berkeley Lab policy requires that each biological material or agent used for research be categorized by RG in the Biosafety Work Authorization, and the RG must be based on the agent’s or material’s potential for causing disease in humans. This categorization should be based on the following principles:
        1. Agents must be assigned the RG designated by NIH, unless a risk assessment in the Biosafety Work Authorization indicates an alternate RG is warranted for the specific agent to be used.
        2. Agents not classified as RG2, 3, or 4 by NIH are not automatically or implicitly classified as RG1. A risk assessment must be conducted for unclassified agents based on their known properties and relationship to agents listed in NIH RGs.
        3. Some information sources for biological agents (e.g., see Work Process B.3.a) only state the recommended biosafety level (BL) to be used for the agent. An agent’s recommended BL is typically the same as its RG (i.e., RG2 agents are handled at BL2). If an agent has not been assigned an RG by NIH, the risk-assessment process must be used to determine its BL. See Work Process C.4.a for information on BLs.
        4. Bloodborne pathogen materials should be designated RG2. This is because BMBL specifies BL2 containment practices for bloodborne pathogen materials and compliance with the OSHA Bloodborne Pathogens Standard (see Work Process B.3.f.i of this program).

d.   Pathogenic Agents and Toxins

      • The risk assessment includes identification and assessment of the pathogenic agents or toxins that are involved with the work, or may be present in the biological material. A pathogen is an infectious microbe (e.g., bacterium, protozoon, fungus, virus, etc.) or other agent (e.g., prion) that causes disease in a healthy host organism such as a human, animal, or plant. A toxin is a poisonous substance produced by a living organism.
      • Depending on potential hosts and impacts (e.g., humans or livestock), pathogens and toxins may be regulated by a variety of agencies. Table 3 below and the remainder of this section identify categories of pathogens and toxins used in biosafety standards and by regulatory agencies to identify agents, toxins, and associated requirements. Appendix B of this manual also provides lists of many pathogens and toxins.

Table 3. Pathogenic Agent and Toxin Categories

Agent or Toxin

Agent or Toxin

General Example or Source

Human Pathogens

Human Etiologic Agents (NIH Guidelines)

Risk Group 2, 3, or 4 agents such as the bacterial, fungal, parasitic, viral, and rickettsial agents listed in Appendix B of the NIH Guidelines

Human Pathogens (BMBL)

Bacterial, fungal, parasitic, rickettsial, viral, and arbovirus agents that are included in BMBL agent-summary statements and require BL2 or greater containment

Biological Etiologic Agents (DOE WSHP)

Human pathogens such as those listed in Appendix B of the NIH Guidelines

Bloodborne Pathogens (Occupational Safety and Health Administration, OSHA)

Pathogens such as the human immunodeficiency virus (HIV), hepatitis B and C viruses (HBV and HCV).

Select Agents (CDC)

Pathogens categorized by the CDC as select agents because of their severe threat to humans (e.g., biological weapons)

Plant and Animal Pathogens


Materials, organisms, or agents regulated by USDA-APHIS that may harm domestic or native animals or plants, or natural resources



Bacterial, fungal, algal, and animal toxins

Select Agents and Toxins


Human, animal, and plant pathogens and toxins categorized by the CDC and Animal and Plant Health Inspection Service (APHIS) as select agents and toxins because of their potential severe threat to humans (e.g., biological weapons)



Misfolded proteins and materials potentially containing other misfolded proteins that cause diseases known as transmissible spongiform encephalopathies (TSEs)

                                          i.      Pathogen and Toxin Information and Guidance

1.   Documentation of the hazardous characteristics and controls for well-known pathogens and toxins is usually readily available and should be considered in the risk assessment. This section lists agencies and organizations that provide such information, along with links to the information sources.

2.   Because it may be difficult to find information on lesser-known pathogens or toxins, variants of pathogens, or opportunistic pathogens, their use may require additional risk assessments. For example, special technical information might be needed for avirulent or attenuated agents that have been physiologically modified or genetically altered and therefore several-orders-of-magnitude less likely to produce disease in a healthy host organism. In addition, “opportunistic pathogens” may not be listed as pathogens because they may only infect immunocompromised hosts.

a.    BMBL Agent Summary Statements. Section III of the BMBL provides summary statements for many agents associated with laboratory-acquired infections or increased public health concern. Risk assessments must consider any information from these agent summary statements that apply to specific Berkeley Lab work activities. Categories included in the agent summary statements are listed below:

              • Bacterial agents
              • Fungal agents
              • Parasitic agents
              • Rickettsial agents
              • Viral agents
              • Arboviruses and related zoonotic viruses
              • Alphabetic listing of 597 arboviruses and hemorrhagic fever viruses
              • Toxin agents
              • Prion diseases

b.    Pathogen Safety Data Sheets for Infectious Substances. The Public Health Agency of Canada produces and provides pathogen safety data sheets for infectious substances as a safety resource for Canadian laboratory workers who may be exposed to these agents in research, public health, teaching, and other laboratories.

c.    CDC Health Information. The CDC A-Z Index provides information on topics with relevance to a broad cross section of CDC audiences. The items are representative of popular topics and frequent inquiries, or have critical importance to the CDC’s public health mission. Topics such as diseases and vaccinations are covered.

      1. Human Pathogens. Human pathogens are infectious microbes (e.g., bacteria, protozoa, fungi, viruses, etc.) or other agents (e.g., prions) that cause disease in healthy humans. Pathogens are also often referred to as etiologic agents or infectious agents. “Etiologic” is an adjective that means disease-causing. The terms “infectious agent” and “infectious material” are also used in biosafety standards and in this manual. “Infectious agent” means human pathogen. “Infectious material” means a biological material that potentially contains human pathogens or infectious agents. Listed and linked below are biosafety standards that cover human pathogens.

1.   Human Etiologic Agents (NIH Guidelines). The NIH Guidelines provide a list of human pathogens and their RG2, RG3, and RG4 designations in Appendix B (Classification of Human Etiologic Agents on the Basis of Hazard) of the NIH Guidelines (also see Appendix B of this manual). Work with human pathogens at Berkeley Lab will be conducted in accordance with the agent-specific RG designations in Appendix B of the NIH Guidelines and this Biosafety Program.

2.   BMBL Human Pathogens. BMBL agent summary statements contain BL-specific containment guidance for specific human pathogens (see Work Process B.3.i, Pathogen and Toxin Information and Guidance, of this program). Work with human pathogens at Berkeley Lab will be conducted in accordance with the IBC. The IBC will determine the proper containment level for pathogenic work, and use the recommended BL guidance presented in BMBL agent summary statements when available and applicable to the work activity. See Work Process C.4.a of this manual for additional information on BLs.

3.   DOE WSHP Biological Etiologic Agents. The DOE WSHP regulation (10 CFR 851, Appendix A, Section 7) has specific requirements for “biological etiologic agents.” Berkeley Lab’s program to comply with 10 CFR 851 defines a biological etiologic agent as an agent of biological origin (e.g., bacterium, fungus, parasite, virus, etc.) that causes disease in humans (i.e., is pathogenic to humans). See Appendix B of this manual for the NIH list of human etiologic agents. Work with biological etiologic agents must be documented, reviewed, and authorized as discussed in Work Process D.1.a.iv, Biological Use Authorization, of this program. During and following the review and authorization process, the IBC and EHS Division ensure the implementation of oversight and reporting responsibilities for biological etiologic agents. See Appendix A for specific Berkeley Lab requirements related to biological etiologic agents under 10 CFR 851.

4.   OSHA Bloodborne Pathogens. See Work Process B.3.f, Bloodborne Pathogens and Human Material, and Appendix C for requirements related to human pathogens that are considered bloodborne pathogens (BBPs) under the OSHA Bloodborne Pathogens Standard.

5.   CDC Select Agents. The Health and Human Services (HHS) CDC regulation on select agents and toxins lists agents that are both select agents and human pathogens. See Work Process B.3.d, Pathogenic Agents and Toxins, for more information.

      1. Plant and Animal Pathogens. See Work Process B.3.e, USDA-Regulated Materials, Organisms, and Agents, of this program for information on plant and animal pathogens; and Work Process B.3.d.v, Select Agents and Toxins, for more information on plant and animal pathogens that are also select agents.

      2. Toxins. A biological toxin, biotoxin, or toxin is a poisonous substance produced by a living organism. The poisonous nature of toxins means that they may cause death or severe incapacitation at relatively low exposure levels. Toxins include, for example, bacterial toxins, fungal toxins, algal toxins, and animal toxins. Examples include microcystins produced by freshwater cyanobacteria, or venoms produced by snakes or spiders. The word “toxin” without other descriptors such as “bio” is used in this manual and is a proper technical term to specifically describe toxins of biological origin. Toxic substances that are not of biological origin are properly termed “poisons.”

1.   Typical laboratory work with very small quantities of most toxins can be performed with minimal risk to the worker. Toxins do not replicate, are not infectious, and are difficult to transmit mechanically or manually from person to person. Other characteristics that further limit the spread of toxins include the fact that many commonly employed toxins are relatively unstable in the environment (especially in the case of protein toxins) and have very low volatility.

2.   Toxins must be handled using the general and “particularly hazardous substance” sections of the Berkeley Lab Chemical Hygiene and Safety Plan (CHSP). In addition, safety and security controls (presented below) based on a risk assessment must be used for each specific laboratory operation. The main laboratory risks are accidental exposure by direct contamination of the mouth, eyes, or other mucous membranes; inadvertent aerosol generation; and needlestick or other accidents that may compromise the normal barrier of the skin.

3.   Requirements and guidelines for storage and work with toxins in the laboratory are covered and summarized below. See Work Process B.3.d.v.3, Select Agents and Toxins, for additional information on toxins listed in the National Select Agent Registry.

a.    BMBL Guidelines for Work with Toxins. According to Appendix I of BMBL, toxins of biological origin must be reviewed and should be incorporated into work with toxins based on a risk assessment approved by the IBC. Key criteria in the guidelines and Berkeley Lab policies:

              1. A risk assessment should be conducted to develop safe operating procedures and a specific chemical plan. It is Berkeley Lab policy that this toxin assessment and plan should be documented in the Biosafety Work Authorization and should cover applicable topics and guidelines presented in Appendix I of BMBL. General topics should include: description of work; safety and security risks, hazards, or concerns; and safety and security controls.
              2. Each worker must be trained in the theory and practice of toxins, with emphasis on practical hazards associated with laboratory operations. This training includes how to handle transfers of toxins or liquids containing toxin, where to place waste solutions and contaminated materials or equipment, and how to decontaminate work areas after routine operations as well as after accidental spills.
              3. An inventory-control system should be in place to account for toxin use and disposition. At Berkeley Lab, original primary containers of toxins must have Berkeley Lab chemical barcodes and be entered into the Berkeley Lab Chemical Management System.
              4. Access to work areas should be controlled.
              5. Routine operations with dilute toxin solutions should be conducted under BL2 containment with the aid of personal protective equipment, laboratory hoods, biosafety cabinets, or comparable engineering controls.
              6. Work with dry toxins should be minimized or eliminated.

b.    BMBL Toxin Agent Summary Statements. Section VIII-G of BMBL contains information and guidance on specific toxins. When specific toxins listed in this section of the BMBL are used, this guidance must be reviewed and should be incorporated into the work in accordance with the IBC-approved risk assessment.

                                   v.      Select Agents and Toxins

1. agents and toxins are specific pathogenic agents and toxins regulated by the HHS-CDC and the USDA-APHIS due to their potential threat (e.g., as biological weapons) to human, animal, and plant health. Specific genetic elements, recombinant nucleic acids, and recombinant organisms that may pose a similar threat are also regulated. Appendix B, Section B.2, of this manual provides the list of select agents and toxins and additional toxin information.

2.   Possession, use, storage, or transfer of select agents and toxins must be conducted in compliance with the HHS-CDC and USDA-APHIS regulations related to human, plant, and animal select agents and toxins. Specific controls for select agents must be detailed in a Biosafety, Security, and Incident Response Plan for Select Agents. In addition, select agent registrations, transfers, and destructions will be reported to the DOE Berkeley Site Office when and as described in Appendix A. Contact the EHS Biosafety Officer for guidance and assistance. Controls for select agents have also been integrated into the overall Biosafety Program.

3.   See the National Select Agent Registry (NSAR) Program Web site for additional information on select agents provided by HHS-CDC and USDA-APHIS. The NSAR Program oversees possession of select agents and toxins for the HHS-CDC Division of Select Agents and Toxins and the USDA-APHIS Agricultural Select Agent Program.

                                   vi.      Prions

Text Box:  
Normal and diseased (misfolded) prions. Source: ScienceBlogs, Basic Concepts: Prions, by Shelley Batts (February 11, 2007).
Normal and diseased (misfolded) prions. Source: ScienceBlogs, Basic Concepts: Prions, by Shelley Batts (February 11, 2007).

1.   A prion is an infectious agent composed of protein. All such agents discovered to date propagate by transmitting a misfolded protein; the protein does not itself self-replicate and the process is dependent on the presence of the polypeptide in the host organism. The misfolded form of the prion protein has been implicated in prion diseases known as transmissible spongiform encephalopathies (TSEs). TSEs are neurodegenerative diseases that affect humans and a variety of domestic and wild animal species. Examples are Creutzfeldt-Jakob disease (CJD) in humans and bovine spongiform encephalopathy (BSE), also known as mad cow disease, in cattle. All known prion diseases affect the structure of the brain or other neural tissue, are currently untreatable, and are always fatal.

2.   Prion diseases are transmissible by inoculation or ingestion of infected tissue or homogenates. Prion infections usually occur in brain or other central nervous system tissues, and to a lesser extent in lymphoid tissues including spleen, lymph nodes, gut, bone marrow, and blood.

3.   Section VIII-H of BMBL provides an agent summary statement that includes guidelines for prion diseases. When applicable, this guidance must be used to incorporate controls based on a risk assessment into the Biosafety Work Authorization.

e.   USDA-Regulated Materials, Organisms, and Agents

      1. The USDA-APHIS defends America’s animal and plant resources from agricultural pests and diseases by regulating materials, organisms, or agents that may harm domestic or native animals or plants, or natural resources. These materials, organisms, or agents may cause harm directly (e.g., predator or pathogen) or indirectly (e.g., vector). General examples include specific animals, plants, genetically engineered organisms, animal pathogens, plant pathogens, soil that may contain such pathogens, and agents that pose a severe threat.
      2. The transfer, storage, use, and disposal of APHIS-regulated materials at Berkeley Lab must be conducted in accordance with APHIS regulations. Generally, APHIS requires a permit or other document to import, export, or store regulated materials from or to locations outside the continental United States or between U.S. states. APHIS permits are issued to individuals and are not transferrable to others. The APHIS permit and sometimes an accompanying “compliance agreement” dictate specific controls and limitations when working with regulated materials. Individuals responsible for the transfer, storage, use, or disposal of such materials will obtain permits when required, ensure that the materials and permits are covered in the Berkeley Lab Biosafety Work Authorization, and ensure that specific requirements in the permit and compliance agreement are implemented.
      3. Materials, organisms, and agents that threaten animal and plant health are regulated by branches of the USDA-APHIS and examples are listed below in Table 4. Additional agency requirements and Web links for more information are detailed in Appendix I, Section I.2.2, of this manual. See Work Process B.3.d.v of this program for more information on select agents and toxins.

Table 4. Materials Regulated by USDA-APHIS


Examples of USDA-APHIS-Regulated Materials, Organisms, and Agents

Plant Protection and Quarantine (PPQ)

Plant pests such as soil, plant pathogens, plants, plant products, weeds, insects, mollusks, and nematodes

Veterinary Services (VS)

Material, organisms, vectors, animal pathogens, animal products, cell cultures and their products, live animals, semen, embryos, and veterinary biologics (e.g., vaccines, antibodies, and diagnostic kits) that may harm animal health

Biotechnology Regulatory Services (BRS)

Certain genetically engineered organisms that may pose a plant pest risk, including organisms that are plants, insects, or microbes

Agricultural Select Agent Program

Animal and plant pathogens that are select agents

f.    Bloodborne Pathogens and Human Materials

      1. The federal OSHA Bloodborne Pathogens Standard has comprehensive requirements for workers who are or may be exposed to BBPs or designated materials assumed to contain BBPs. Berkeley Lab uses the term “BBP materials” to describe the pathogens and materials covered by the OSHA standard. These BBP materials are summarized in Table 5 and discussed in the next paragraph. BMBL guidelines for working with human and mammalian cells and tissues are also discussed below.

Text Box:    
BBP materials, including human blood, human tissue, and human cells. Source: Berkeley Lab.
BBP materials, including human blood, human tissue, and human cells. Source: Berkeley Lab.

Table 5. Materials Covered by the OSHA Bloodborne Pathogens Standard*


  • Bloodborne pathogens such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV)
  • Human blood. Includes blood, blood components, and products made from human blood
  • Other potentially infectious materials (OPIM):
    • Unfixed human tissue or organ (other than intact skin) from a living or dead human
    • Primary human tissue cultures. Explants of living human tissue placed in a medium for tissue culture
    • Primary human cell strains.** Propagated in vitro from primary explants of human tissue or body fluids that have a finite lifetime (i.e., nontransformed) in tissue culture for 20 to 70 passages
    • Established human cell lines.** Immortalized cells that have been transformed by spontaneous mutation or natural or laboratory infection with an immortalizating agent, and then propagated or passed many times (e.g., in vitro or in animals such as mice)
    • Human body fluids. Fluids assumed to be potentially infectious, including semen, vaginal secretions, cerebrospinal fluid, synovial fluid, pleural fluid, pericardial fluid, peritoneal fluid, amniotic fluid, saliva in dental procedures, any body fluid visibly contaminated with blood, and all body fluids in situations where it is difficult or impossible to differentiate between body fluids. Some human secretions that do not contain visible blood are not considered OPIM (e.g., urine, feces, vomit, tears, sweat, sputum, nasal secretions, and saliva).
    • HIV or HBV infected materials. HIV-containing cell or tissue cultures, organ cultures, and HIV- or HBV-containing culture medium or other solutions; and blood, organs, or other tissues from humans or experimental animals infected with HIV or HBV


Text taken from OSHA Bloodborne Pathogens Standard 29 CFR 1910.1030 and the OSHA Standard Interpretation on Applicability of 1910.1030 to Establish Human Cell Lines.



Most primary human cell strains and established human cell lines at Berkeley Lab (e.g., American Type Culture Collection cell lines) are OPIM as required by the OSHA Standard Interpretation on such cells. If the researcher does not want to consider the cells OPIM, the cells must be “characterized.” Characterization must include documented screening of the cell lines or strains for viruses specified as BBPs in the OSHA standard, including human immunodeficiency viruses, hepatitis viruses, and herpes viruses (e.g., Epstein-Barr virus) if the cells are capable of propagating such viruses. Documentation that the cell line in culture is free of BBPs must be reviewed and approved by the Biosafety Officer and the Institutional Biosafety Committee.


      1. BBPs are infectious agents capable of causing human disease and are transmitted through human blood and tissues. Examples include HBV and HIV. According to the OSHA Bloodborne Pathogens Standard, materials that are regulated based on their potential to contain BBPs include human blood, human blood components, products made from human blood, and OPIM listed in Table 5. Berkeley Lab uses the term “BBP materials” to describe all of these materials covered by the OSHA standard. Dried blood and some human secretions (e.g., urine, feces, vomit, tears, sweat, sputum, nasal secretions, and saliva) that do not contain visible blood are not considered OPIM even though they may contain other types of infectious agents or present health concerns.

Text Box:  BBP work activity, blood collection. Source: HHS CDC Office of Health and Safety, Biosafety in the Laboratory presentation (Web accessed May 2010)
BBP work activity, blood collection. Source: HHS CDC Office of Health and Safety, Biosafety in the Laboratory presentation (Web accessed May 2010)

Text Box:  
BBP work activity, culturing human cells. Source: Berkeley Lab EHS.
BBP work activity, culturing human cells. Source: Berkeley Lab EHS.

      1. Appendix H of BMBL states that a risk assessment should be conducted for human and primate cells based on the origin and source of cells or tissues, and such cells should be handled using BL2 practices and containment (see Work Process C.4.a for further discussion of BLs). While many requirements in the BMBL and OSHA Bloodborne Pathogens Standard are similar to one another, the OSHA standard additionally requires initial and annual BBP training, availability of hepatitis B vaccination at no cost to employees, and a written Exposure Control Plan (ECP). Researchers satisfy documentation requirements for a risk assessment, BL2 containment, and an ECP once they have an approved Biological Use Authorization (BUA). BL2 containment must be used unless the BUA risk assessment indicates that alternative controls are sufficient. BUAs are further discussed in Work Process D.1.a.iv, Biological Use Authorization.

      2. Berkeley Lab work that involves BBP materials will be performed in compliance with the OSHA Bloodborne Pathogens Standard and BMBL. Berkeley Lab’s program for compliance with these standards is integrated into the larger Berkeley Lab Biosafety Program that is described in this manual.

g.   Recombinant Materials, Organisms, and Agents

                                        i.      Recombinant Terms, Processes, and Risks

1.   Genetic material plays a fundamental role in determining the structure and nature of cell substances. It exists in the nucleus, mitochondria, and cytoplasm of a cell or organism, and is capable of self-propagation and genetic variation. The genetic material of a cell can be a gene, a part of a gene, a group of genes, a deoxyribonucleic acid (DNA) molecule, a fragment of DNA, a group of DNA molecules, or the entire genome of an organism. A nucleic acid is a macromolecule composed of chains of monomeric nucleotides. In biochemistry, nucleic acids carry genetic information or form structures within cells. The most common nucleic acids are DNA and ribonucleic acid (RNA). Nucleic acids are universal in living things, as they are found in all cells and viruses. The term “genetic recombination” is used to describe the process by which the strand of genetic material (usually DNA, but can also be RNA) is broken and then joined to a different DNA molecule to create recombinant genetic material. The NIH Guidelines defines recombinant and synthetic nucleic acid molecules as molecules constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell or molecules that result from the replication of such molecules.

2.   Vectors are commonly used in genetic engineering to create recombinant materials, organisms, agents, or cells. In molecular biology, a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. Such a vector usually does not cause disease itself, but may change the properties and risks associated with the host cell. The four major types of vectors are plasmids, bacteriophages and other viruses, cosmids, and artificial chromosomes. Two common vectors are plasmids and viral vectors.

a.    Plasmid vectors are commonly used to multiply or express particular genes. Many plasmids are commercially available for such uses. Plasmids are DNA segments that are separate from chromosomal DNA and are capable of replicating independently of the chromosomal DNA. In many cases, a plasmid is circular and double-stranded. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms. Plasmids are considered transferable genetic elements, capable of autonomous replication within a suitable host. Plasmid host-to-host transfer requires direct, mechanical transfer by "conjugation" or changes in host gene expression, allowing the intentional uptake of the genetic element by "transformation." Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. For example, plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances. If these plasmids are inserted into a different host bacterium, the new host may acquire antibiotic resistance or produce toxic protein.

b.    Viral vectors are a viral tool commonly used to deliver genetic material into cells. This process can be performed inside a living organism (in vivo) or in cell culture (in vitro). Viruses have evolved specialized molecular mechanisms to efficiently transport their genomes inside the cells they infect. Delivery of genes by a virus is termed transduction, and the infected cells are described as transduced. Although viral vectors are occasionally created from pathogenic viruses, they are modified in such a way as to minimize the risk of handling them. This usually involves the deletion of a part of the viral genome critical for viral replication. Such a virus can efficiently infect cells, but once the infection has taken place, it requires a helper virus to produce new virions. Examples of recombinant viral vectors include:

              1. Retroviral vectors from retroviruses such as the Moloney murine leukemia virus
              2. Lentiviral vectors from lentiviruses (a subclass of retroviruses) such as HIV
              3. Adenoviral vectors from adenoviruses
              4. The adeno-associated virus (AAV)

c.   Transgenic organisms may also be used or created in genetic engineering. A transgenic organism is an organism whose genome has been altered by the transfer of a gene or genes from another species or breed. Examples of transgenic organisms include vertebrates such as mice, plants, and microbes.

d.   Recombinant Risks. Work with or the creation of recombinant organisms or specific recombinant genomic materials and nucleic acids may create new risks to humans, animals, plants, or the environment. These potential recombinant risks must be identified and evaluated during the risk-assessment process. Examples of genetic modifications that may increase risk Untitled event include modifications that increase an agent’s pathogenicity or susceptibility to effective treatments (e.g., antibiotics), or increase an organism’s ability to compete in the natural environment.


                                        ii.      Recombinant Requirements        


1.   NIH Requirements. Requirements and specific practices for constructing and handling recombinant DNA molecules, and organisms and viruses containing recombinant DNA molecules, are specified in the NIH Guidelines. The NIH Guidelines have broad requirements for biosafety risk assessment and control similar to BMBL requirements. In addition, the NIH Guidelines have special institutional and line-management requirements such as an Institutional Biosafety Committee (IBC) review process and responsibilities specifically defined for principal investigators (PIs). Institutions such as Berkeley Lab that receive NIH funding are required to comply with the NIH Guidelines. As a condition for NIH funding, Berkeley Lab and lead investigators (e.g., PIs) must ensure that all nonexempt recombinant DNA research experiments conducted at or sponsored by the Berkeley Lab, regardless of the source of funding, comply with the NIH Guidelines. The work review and authorization processes and documents detailed in Work Process D.1.a.i, General Review and Authorization Process,  are key tools that assist researchers to comply with the NIH Guidelines.

2.   APHIS Requirements. APHIS permits may also be required for the importation, interstate movement, or environmental release of certain genetically engineered organisms that may be plant pests (see Appendix I of this manual).

3.   Recombinant research requires a risk assessment, establishment of containment levels and controls, and a Biosafety Work Authorization (for more information, see Work Processes A through E of this manual).

h.   Animals

1.   Working with animals in research, caring for animals in animal care facilities, or coming in contact with animals or vectors in the field may cause zoonotic or other diseases. A zoonosis or zoonose is an infectious disease that can be transmitted (in some instances, by a vector) from nonhuman animals, both wild and domestic, to humans, or from humans to nonhuman animals (the latter is sometimes called reverse zoonosis). Human diseases caused by a noninfectious, etiological agent derived from animals or their vectors are not considered a zoonosis (e.g., allergic reactions to animal products such as dander or urine). Work involving animals may expose workers to etiologic agents in a variety of ways such as wound infections, inhalation of aerosols (e.g., dust from animal bedding), and animal bites or scratches. See Table 6 for examples of zoonotic diseases and other diseases related to animals.

Text Box:         
Dirt or compost containing	Non-human primates
feces and tetanus.	and B virus infection.
Mice, bedding, and allergies. Source: Berkeley Lab EHS. Aquarium fish and skin infections or gastroenteritis. Source: Wikipedia unrestricted distribution. Dirt or compost containing feces and tetanus.  Ticks and Lyme disease. Source: Calif. Dept. of Public Health Web site, 2012.

2.   Worker safety, agricultural, and recombinant risks related to working with animals must be evaluated during the risk assessment, and proper containment measures must be employed. See the following sections and standards for additional information:

            • Work Processes B.3.e and B.3.g discuss agricultural and recombinant risks, respectively.
            • Work Process C.4.a provides an overview of laboratory and animal biosafety level containment categories and criteria.
            • Section VIII of BMBL provides agent summary statements for zoonotic agents. It also recommends containment levels for laboratory use of a zoonotic agent and containment levels for handling animals infected with an agent.

Table 6. Examples of Zoonotic and Other Diseases Related to Animals


Reservoir Vectors

Causative Agent

Exposure Routes



Skin Contact


Vertebrate animals

Animal allergens

Dander, urine, or saliva in dust or bedding



Bacillus anthracis

Contaminated dust with spores

Contaminated with spores

Contaminated materials with spores

Hantavirus pulmonary syndrome

Rodents/deer mice

Sin nombre virus

Contaminated dust from dried urine, saliva, droppings

Herpes B virus infection

Nonhuman primates, particularly endemic in rhesus and cynomolgus members of the Macaque genus

Herpesvirus simiae or
B virus

Aerosolized macaque saliva

Mucosal splashes (e.g., monkey fluids contact the worker’s eyes or mouth)

Monkey bites, monkey scratches, or cage scratches; direct contamination of a preexisting wound with macaque saliva; needle-stick injuries following needle use in macaques

Lyme disease


Borrelia burgdorferi

Ixodid tick bite



Yersinia pestis

Flea (Xenopsylla cheopis, Pulex irritans) bite

Q fever

Sheep, goats, cattle

Coxiella buretii

Barnyard dust contaminated by birth material and excreta

Milk ingestion, regurgitation, and perspiration


Rabid animals

Rabies virus

Bites and saliva from an infected animal

Rocky Mountain spotted fever


Rickettsia rickettsii

Tick bites or skin contact with contaminated materials



Clostridium tetani

Wounds contaminated with dirt or objects containing animal or human feces or saliva

Various diseases such as skin infections or gastro-enteritis

Fish aquarium water

Mycobacterium marinum,
M. fortuitum, Aeromonas hydrophila, other bacteria, and Cryptosporidium spp. protozoa

Skin contact with aquarium water, especially if skin has cuts or abrasions

4.   Laboratory Procedure Hazards

a.   The BMBL five-step approach to assessing biological risk and selecting controls for laboratory work was initially presented in Work Process B.2.c of this program. Step 2 (identifying laboratory procedure hazards) of this approach is discussed in this section.

b.   Historical data on laboratory acquired infections (LAIs) are an indicator of laboratory procedure hazards that have resulted in disease. As discussed in the BMBL, historical data show that past LAIs have occurred from:

      • Parenteral inoculation by a contaminated sharp or syringe needle
      • Spills or splashes of contaminated materials directly onto the skin and mucous membranes
      • Ingestion through mouth pipetting
      • Animal bites and scratches
      • Inhalation of infectious aerosol

c.   See Section II of BMBL for more information regarding LAIs. Prevention of LAIs depends on the conscientious and proficient use of standard microbiological practices and special practices (see Work Process C.1) and the correct use of laboratory equipment. Table 7 below lists examples of hazards that may be found in laboratories using biological materials.

Table 7. Equipment Hazard Examples

Equipment Type



Aerosol generating

The diameter of aerosols generated from certain types of equipment will vary from 0.1 to 100 microns.

  • Bacterial cells and spores are 0.3 to 10 microns in diameter.
  • Viruses are 0.02 to 0.3 micron in diameter.
  • Biological particles generated from liquid or powder form particles that are 0.5 micron diameter.
  • Blender: 2-micron-diameter particles
  • Sonicator: 4.8-micron-diameter particles
  • Dropping bacterial flask: 3.5-micron-diameter particles
  • Dropping lyophilized culture: 10-micron-diameter particles
  • Pipette blow out: 4.9-micron-diameter particles
  • Vortex culture: 4.8-micron-diameter particles
  • Centrifuge: 4-micron-diameter particles

Cryogenic temperatures

Cryogenic temperatures of -80°C are used to remove moisture from materials and contain low-temperature refrigerants. If protective equipment is not used, exposure to low temperature may cause cryogenic burns and frostbite.

  • Freezers
  • Lyophilizers (freeze dryers)
  • Use of dry ice in shipping and receiving

High temperatures

The use of heat to decontaminate or sterilize materials is widely used in the biological research laboratory. Physical injury from burns may occur from sudden accidental releases of heat sources or from the handling of hot items.

  • Dry heat temperatures used for sterilization range from 80°C to 200°C.
  • Wet heat is utilized by autoclaves to sterilize materials and can range between 80°C and 500°C.
  • Saturated steam operates at 121°C.

High pressure

Compressed gas cylinders and pressurized equipment are commonly used in the laboratory. Injury may occur from ruptured high-pressure lines.

Autoclaves operate at high pressures of 1,000 kilo Pascal (145 psig).

Oxygen deficiencies

Low-temperature freezers may include a backup system involving the use of a cryogenic liquid. Backup systems may consist of 50–200 liters of liquid nitrogen or liquid carbon dioxide under pressure. Liquid helium is also used in nuclear magnetic resonance (NMR) laboratories.

Oxygen deficiency environment may result from:

  • The displacement of oxygen by expanding gases (i.e., 700 parts of air to 1 part liquid nitrogen)
  • The linear displacement of oxygen from carbon dioxide (gas) generated from the use of dry ice
  • Compressed gas cylinders or tanks

Rotational energies

Sudden release of such rotational energies can cause serious physical injury from unbalanced equipment or flying shrapnel.

Tabletop and floor-mounted low, high, and ultracentrifuges rotate at speeds ranging from less than 5,000 to more than 100,000 rpm with rotor masses up to several kilograms.


Any device having corners, edges, or projections capable of cutting or piercing the skin. Berkeley Lab's definition of sharps includes regulated sharps (medical waste), unregulated biohazardous sharps, and unregulated uncontaminated sharps that pose a safety hazard to custodians and other personnel.

  • Needles with or without syringes
  • Needles with vacutainers
  • Needles with attached tubing
  • Blades (razors, scalpels, X-Acto knives)
  • Broken glass
  • Glassware with sharp edges or points
  • Pasteur pipettes and glass slides

Ultraviolet (UV) C radiation

UVC radiation is used for inactivating microorganisms. Its usefulness, however, is limited by a variety of factors (e.g., low penetrating power). The eyes and skin can be damaged by exposure to direct or strongly reflected UV radiation.

UV lights must be evaluated to determine whether the benefits outweigh the potential hazards. UV radiation is sometimes used in conjunction with:

  • Unoccupied tissue culture rooms
  • Biological safety cabinets
  • UV light boxes

5.   Worker Competence and Health

a.   General. The BMBL five-step approach to assessing biological risk and selecting controls for laboratory work was initially presented in Work Process B.2.c of this program. Step 4 of this approach (i.e., the evaluation of a worker’s proficiencies or competence) and the evaluation of a worker’s health are discussed in this section. Step 4 is an ongoing process in which the supervisor or work lead evaluates a worker’s training, instructions, qualifications, behavior, and health. Worker training and health requirements are also a component of the Biosafety Work Authorization.

b.   Workers are the first line of defense for protecting themselves, others in the laboratory, and the public from exposure to biohazardous agents. Laboratory staff must therefore be properly trained, instructed, and qualified before conducting work. Supervisors and work leads should train and evaluate staff to the point where knowledge of the agent and procedure hazards, aseptic techniques, safety practices, use of safety equipment, caution, and attentiveness become second nature. Knowledge and experience prior to job assignment may also be necessary qualifications. See Work Process D.2, Training, Instruction, and Qualification, for more information on training, instruction, and qualification.

c.   In addition, a worker’s health status may affect his or her susceptibility to an infection or ability to receive immunizations or prophylactic intervention. Workers who know they have an illness or medical condition that affects their immune system or their ability to receive vaccines or medications should seek an evaluation by Health Services in Building 26. See Work Process D.3, Occupational Health and Immunization, for additional information regarding worker health and immunization.



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