3.0 Biosafety Levels

There are four basic biosafety levels as determined by CDC and NIH which describe the microbiological techniques, lab practices, safety equipment and lab facilities necessary to protect workers and the environment. A list of common infectious agents arranged by class appears in Section 12, Special Procedures and Information.

Biosafety levels (BSL), agents, factors, and practices are described in the most recent version of the CDC publication Biosafety in Microbiological and Biomedical Laboratories, (BMBL) (See Table 3.0.1).The BMBL is a guide for the use of biological hazards in the laboratory, and it should be consulted regarding use of viruses, fungi, bacteria, and tissues that can harbor hazardous biological agents. This chapter includes information excerpted from the BMBL, and provides a quick means to compare procedures and requirements for biosafety facilities for conducting work at each biosafety level.

Hybrid biosafety levels denote combined physical containment and safe work practice levels. For example, BSL-2/3 as used in the HIV labs denotes Biosafety Level 2 facilities and Biosafety Level 3 work practices.

NIH Guidelines are restricted to the use of recombinant DNA (rDNA) and recombinant RNA in research and clinical trials. NIH lists four infectious agent risk groups. The biological safety levels described in the NIH Guidelines should be used when working with any recombinant virus, bacteria, plant, fungi, plasmid or other DNA/RNA molecules that have been artificially created. See Appendix Gof the NIH Guidelines for more on NIH required biosafety controls. NIH Guidelines also provide guidance for transgenic animal and plant production.

Without knowing other factors and assuming good microbiological laboratory practices, a biosafety level can be determined as follows:

1. Check the BMBL for the agent and the CDC recommendations for it.

2. Check the American Biological Safety Association website’s list of agents and biosafety levels (www.absa.org/resriskgroup.html).

3. Use the risk groups in the NIH Guidelines as default values.

4. Conduct a risk assessment (see Section 3.6).

5. Consult with the Hutchinson Center BSO.

In addition, unknown properties, larger volumes and lack of investigator experience may also influence the choice of biosafety level. In some cases that require BSL-3, it would be possible to use a BSL-2 facility with BSL-3 controls, with IBC approval.

If the use of the agent will generate uncontained aerosols or if work will involve more than 10 liters of the agent, its use and handling procedures requires consultation with the EH&S BSO and IBC approval. 

                                                                             

Table 3.0.1: Biosafety Levels (BSL), Agents, Factors, and Practices (from BMBL, 5th edition)

BSL

Agents

Factors

Practices*

1

Agents that are not associated with disease in healthy human adults

None

Standard lab and management practices, including appropriate medical surveillance programs

2

Associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available

Not known to be inhalationtransmittable

No aerosol production planned

BSL-1 practices plus:

- Limited access

- Biohazard warning signs

- Sharps precautions

- Biosafety manual

Decontamination of all infectious wastes prior to disposal

2/3

Associated with human disease; special precautions required for some agents

BSL-2 facility with BSL-3 controls

BSL-2 practices plus:

- Controlled access

- Decontamination of clothing before laundering

- Equipment decontaminated before removed

3

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

No or limited sharps allowed

All work with infectious agent done in biosafety cabinet

BSL-2 practices plus:

- Controlled access

- Decontamination of clothing before laundering

- Equipment decontaminated before removed

Disinfectant foot bath as needed

4

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

Not allowed at the Hutchinson Center

Not allowed at the Hutchinson Center

*See CDC/NIH Biosafety in Microbiological and Biomedical Laboratories, 5th Edition (BMBL) (or the most current edition) for a complete list of all controls and practices required for each level.

   

Biosafety Level 1 represents a basic level of containment that relies on standard microbiological practices with no special primary or secondary barriers recommended. A sink is required for hand washing. Lab signage is not required.

3.2       Biosafety Level 2 (BSL-2)

Biosafety Level 2 represents hazards primarily related to accidental percutaneous, mucous membrane, non-intact skin exposures, or ingestions of infectious materials. Extreme caution with contaminated needles or sharp instruments is emphasized. Even though organisms routinely manipulated at BSL-2 are not known to be transmissible by the aerosol route, procedures with aerosol or high splash potential that may increase risk of such exposure are conducted in primary containment equipment, or devices such as a biosafety cabinet or safety centrifuge. Use personal protective equipment as appropriate, including splash shields, face protection, disposable gowns, and gloves. Lab signage is required.

3.3       Biosafety Level 3 (BSL-3)

Biosafety Level 3 represents a level of use at which hazards are primarily related to inhalation, and in which the agent in question is known to be or suspected to be spread through inhalation. Work at this level requires that a separate lab safety manual be developed by the lab, including roles and responsibilities, hazards, hazard mitigation and procedures for use and decontamination, emergency response. That manual must be approved by the IBC. It is recommended that no sharps be used in BSL-3 facilities. Procedures with the live agent are to be performed in a Type II A/B biosafety cabinet (BSC). Use personal protective equipment as appropriate, including splash shields, face protection, disposable gowns, and gloves. Lab signage is required. Entry into and out of a BSL-3 facility must be controlled so that only properly trained individuals are allowed unescorted access.

3.4       Biosafety Level 4 (BSL-4)

The Hutchinson Center does not do BSL-4 work at this time.

3.5       Use Proper Facilities for Each Biosafety Level     

Different biosafety levels have different facilities requirements. Before you begin a task, make sure that your lab set-up, personal protective equipment, and posting requirements match your BSL. See Table 3.5.1, Comparison of Biosafety Facilities Required for Different BSLs to determine the requirements for your BSL and review complete list of requirements in the most current edition of the BMBL.

Table 3.5.1: Comparison of Biosafety Facilities Required for Different BSLs

Safety Measures

BSL-1

BSL-2

BSL-3

Presence of animals and plants not related to the experiment

Not Permitted by Center

Prohibited

Prohibited

PI or LSC notifies lab staff of agents used

Required

Required including pathogenicity

Required including pathogenicity

Removal of infectious materials from the lab

Materials are placed in a sealed, leak-proof container

Materials are placed in a sealed, leak-proof container

Materials are decontaminated or placed in a sealed, leak-proof container

Use of procedures to minimize the creation of aerosols

Aerosol production may require increase in biosafety levels

Use BSC when risk of aerosols is present

Use BSC when risk of aerosols is present

Use of protective clothing

Lab coat and gloves recommended (also see chapters on radioactive material and chemical safety)

Lab coat and gloves required; safety glasses or goggles required when splash or spray hazard is present

Lab coat, gloves, safety glasses/ goggles/face protection required; respiratory protection may be required

A puncture-proof container is provided for the safe removal of sharps

Required

Required

Required

Use of insect and rodent control program

Required

Required

Required

Biohazard sign on entry to lab and storage areas

Universal Biohazard Symbol when infectious agent present

Name of PI, lab safety coordinator, and emergency phone numbers must be posted

Name of PI, lab safety coordinator, and emergency phone numbers must be posted

The lab is designed and furniture arranged to facilitate cleaning

Required

Required

Required

Bench tops are impervious to water and are resistant to acids, alkalis, organic solvents, and moderate heat

Required

Required

Required

Hand washing sink is provided inside the lab

Required

Required

Required

Windows inside the lab

Closed and sealed

Closed and sealed

Closed and sealed

Table 3.5.1: Comparison of Biosafety Facilities, Page 2

Safety Measures

BSL-1

BSL-2

BSL-3

Lab fixtures are arranged to collect a minimum of dust

Required

Required

Required

A ducted exhaust air ventilation system is used

Not Required

Not Required

Required

Continuous flow centrifuges or other equipment exhaust air through HEPA filters prior to discharge into lab

Not Required

Not Required

Required

Vacuum lines are protected with liquid disinfectant traps and HEPA filters; routine maintenance or replacement is performed as needed

Not Required

Required

Required

Eyewash station is readily available

Not Required

Required

Required


3.6       Risk Assessment  

Risk factors that need to be evaluated are the agent hazard, the protocol hazard, and the susceptibility to disease of at-risk persons. The agent and protocol hazards to be considered include the following:

Agent Hazards

  1. Pathogenicity
  2. Virulence
  3. Infectious dose
  4. Route of transmission
  5. Agent stability
  6. Host range

Protocol Hazards

  1. Agent concentration
  2. Manipulations that produce droplets and aerosols
  3. Manipulations involving sharps
  4. Manipulations with high potential for spills and splashes
  5. Exposure to zoonotic diseases of experimental animals
  6. Alteration of agent hazard

3.6.1     Reviewing an At-risk Person’s Susceptibility to Disease

When reviewing susceptibility to disease of at-risk persons, factors that should be considered include:

1. Wide variation in infectious dose in humans;

2. Availability of vaccines, treatments;

3. Occupational medical evaluation and surveillance; and

4. Experience and skill level of at-risk personnel.


3.6.2 NIH Classification of Infectious Agents for rDNA Work

1. NIH has a slightly different classification scheme than the CDC’s for biosafety hazards, but the NIH focus is on rDNA. Agents are classified by risk group and broken down into bacterial, viral, fungal and parasitic agents. For details and examples of risk group agents, see the NIH Guidelines Appendix B, available at: http://www4.od.nih.gov/oba/rac/guidelines/guidelines.html

1. Risk Group 1 (RG1):RG1 agents are not associated with disease in healthy adult humans. Examples of RG1 agents include asporogenic Bacillus subtilis or Bacillus licheniformis, Escherichia coli-K-12, and adeno-associated virus types 1 through 4, all bacterial, parasitic, fungal, viral, rickettsial, and chlamydial agents not included in a higher class Recombinant DNA with less than 2/3 genome of Risk Group II or III Agents (eukaryotic virus).

2. Risk Group 2 (RG2): An agent associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available.

3. Risk Group 3 (RG3): An agent that is associated with serious or lethal human disease for which preventive or therapeutic interventions may be available (high individual risk but low community risk). RG3 agents include recombinant DNA, human or animal pathogens using RG3 agents, human or animal pathogens cloned in RG4 nonpathogenic eukaryotic or prokaryotic host-vectors DNA/RNA, and helper viruses.

4. Risk Group 4 (RG4): An agent that is 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). Several RG4 agents are also covered under Section 10, Select Agents and Toxins.

5. Other: Agents not listed in Risk Groups 2, 3, and 4 are not automatically or implicitly classified in RG1. A risk assessment must be conducted based on the known and potential properties of the agents and their relationship to agents that are listed.


   

         

3.7 Exemptions from NIH Guidelines

The following etiological agents are exempt from the requirements listed in this chapter (i.e., the NIH Guidelines); however, an EMUA must still be filed with EH&S. Exempt status should be indicated on the EMUA at the time of submission. Please refer to the Appendix C of the NIH Guidelines for more detailed information.

1. Recombinant DNA molecules containing less than one-half of any eukaryotic viral genome (all viruses from a single family being considered identical) that are propagated and maintained in cells in tissue culture.

2. Experiments which use E. coli K-12 host-vector systems, with the exception of those experiments listed below are exempt, provided:

a. The E. coli host shall not contain conjugation proficient plasmids or generalized transducing phages.

b. Lambda or lambdoid or Ff bacteriophages or nonconjugative plasmids are used as vectors.

c. "...experiments involving the insertion into E. coli K-12 of DNA from prokaryotes that exchange genetic information with E. coli may be performed with any E. coli K-12 vector (e.g., conjugative plasmid). When a nonconjugative vector is used, the E. coli K-12 host may contain conjugation-proficient plasmids, either autonomous or integrated, or generalized transducing phages."

d. Experiments using certain genera of Saccharomyces, Bacillus, Clostridium, Lactobacillus, Listeria, Pediococcus, Staphlyococcus, and Streptococcus, may be exempt. (Check the NIH Guidelines, Appendix C, for details.)

   

Research involving animals requires IACUC approval, and if producing transgenic animals, IBC registration. Experiments with recombinant DNA or the use of an infectious agent in animals also require IBC approval.

The IBC specifies containment and work practices for research involving infectious agents and recombinant DNA in animals. A special designation may be used for research involving whole animals in which the animal’s genome has been altered by stable introduction of recombinant DNA into the germ-line (transgenic animals) and experiments involving viable recombinant DNA-modified microorganisms tested in whole animals.

    

The "A" designation is used for research involving whole animals infected with viable pathogens. For the purpose of infected animal research, three levels of containment are used at the Center. Containment levels are: ABSL-1, ABSL-2, and ABSL-3. Research involving the use of whole animals containing recombinant DNA and viable pathogens is identified using the "A" designation.

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