OEH&S Biosafety Manual

The purpose of this manual is to define the biological safety policies and procedures for the University of California, San Francisco (UCSF). These policies and procedures were designed to safeguard personnel and the environment from biologically hazardous materials without unduly limiting academic freedom and to comply with federal and state regulatory requirements. All UCSF Principal Investigators (PIs) and laboratory workers must adhere to the campus biosafety policies and procedures in the conduct of their research and the management of their laboratories.

For information about specific biological safety programs for operations not covered in this manual, contact the Biosafety Committee (BSC) office, the Biosafety Officer (BSO), or your Department Safety Advisor (DSA) at the UCSF Office of Environmental Health and Safety (EH&S).

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Table of Contents

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OEH&S Biosafety Manual Chapter 1

Introduction to Biosafety

Biological laboratories are special work environments that could pose infectious disease risks to persons working in these laboratories or entering them. In fact, there is a clear historical record of infections having been acquired in the laboratory. More than 4,000 laboratory-acquired infections have been reported and many others have occurred.

Incidents such as a splash to the face, needle puncture, contamination of open wounds or skin lesions, ingestion or inhalation of an aerosolized infectious agent and other incidents have all produced laboratory-acquired infections. The CDC-NIH booklet Biosafety in Microbiological and Biomedical Laboratories (US Government Printing Office, Washington D. C., May 1993) recommends a system of laboratory practices for protection of laboratory workers. These are designed either for work with or without experimental animals. These practices are grouped under Biosafety Levels (BSL) 1, 2, 3 and 4. (See Chapter 9 and Appendix A1).

The principles of infection need to be kept in mind when appropriate biosafety precautions are selected. Agents vary in their virulence, or capacity to infect and cause disease. Whether disease results from infection is dependent upon the inoculum or the number of infectious agent particles presented to the host, the route of transmission and the innate capabilities of the agent. The susceptibility of the host to infection may vary according to age, prior exposure with development of immunity, deliberate immunization with a vaccine and host immunodeficiency as the result of a genetic trait, infection (e.g. with HIV-1) or therapy, such as corticosteroids, radiation or other suppressive therapy. Infectious agents are grouped under Risk Group (RG) 1 - 4 according to their virulence characteristics (See Chapter 6 and Appendix A2).

Recommendations for biosafety level (BSL) of laboratory operation are often modified according to the number of infectious particles encountered in the project and the usual route of infection. As an example, purified Mycobacterium tuberculosis is classified in RG 3 because of the serious consequences of infection and the large number of viral particles present. BSL2 laboratory precautions, which include the use of personal protective equipment, such as gloves, are recommended for bloodborne pathogens because HIV-1 virus may be present in small amounts in clinical specimens and the common bloodborne pathogens, (e.g. HIV-1, hepatitis B virus, hepatitis C virus, cytomegalovirus) are transmitted through breaks in the usual body barriers. BSL2 precautions are designed to protect against breaks in the skin, such as those from punctures or open skin lesions by providing barriers. BSL3 precautions are designed primarily to protect against aerosols. Although the bloodborne pathogens are not normally transmitted by aerosols of blood or body fluids, BSL3 precautions may be required when working with concentrated cultures or when performing procedures involving aerosolization of blood or body fluids containing small amounts of virus.

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OEH&S Biosafety Manual Chapter 2

BIOLOGICAL SAFETY PROGRAM ADMINISTRATION

The rules and procedures set forth in the Biological Safety (Biosafety) Manual have one single, straightforward purpose: to protect University of California, San Francisco (UCSF) patients, students, and employees against unnecessary and potentially harmful biological exposure. For these rules and procedures to be effective, it is important to have a structured administrative format in place which defines the roles and responsibilities of each person or administrative office.

A. CHANCELLOR

The Chancellor is ultimately responsible for assuring that comprehensive campuswide programs are in place for the safe handling of all hazardous materials at UCSF. The Biosafety Committee (BSC) and the Office of Environmental Health and Safety (EH&S) have been charged with the planning and implementation of the campus Biological Safety Program whose purpose is to ensure the health and safety of all personnel working with biohazardous or infectious agents and recombinant DNA.

B. UCSF BIOSAFETY COMMITTEE (BSC)

The NIH Guidelines for Research Involving Recombinant DNA Section II b.2 requires the establishment of a Biosafety Committee (BSC) at institutions who sponsor or conduct recombinant DNA research. By policy, UCSF has expanded the role of the BSC to include the review and approval of all work involving potential exposure to human pathogens, whether they are an integral part of the research or incidental to it (such as the possible presence of bloodborne pathogens in "normal" human tissues). At UCSF, the BSC is appointed by the Executive Vice Chancellor for Research and consists of the Chairman, Vice Chairman and Members selected from the faculties represented at UCSF. The members are generally appointed for two year terms but frequently serve more than one term, and have been given jurisdiction over ALL biological materials and some toxins. Two community members, with no UCSF affiliation other than membership on the BSC, are required and appointed to represent the interest of the surrounding community with respect to health and the protection of the environment. The Ex Officio members include the Director of EH&S, the Biosafety Officer (BSO), the Director of the Animal Care Facility and the Director of Employee Health Service, or their designated representative. The BSC as a whole represents collective expertise and research experience in recombinant DNA, infectious agents and biological safety in experiments which may pose potential risks to health or the environment.

The BSC is responsible for ensuring that research conducted at UCSF is in compliance with the NIH Guidelines for Research Involving Recombinant DNA Molecules, drafting campus biosafety policies and procedures, and reviewing individual research proposals for biosafety concerns. The BSC usually meets monthly to review proposals for all UCSF campuses and for faculty members who work off-campus. Veterans Administration-funded projects are specifically excluded from the BSC's charge. The BSC does not oversee biosafety policies for the hospitals, clinics or clinical laboratories on the various campuses. These are the responsibility of the respective Infection Control Committees.

Principal Investigators (PIs) who wish to perform research using biological materials submit an application for Biological Use Authorization to the BSC. The Committee normally reviews applications that involve work at Biosafety Level 2 or above and the BSO reviews Biosafety Level 1 research applications. Biosafety Level 1 applications are considered exempt from BSC review unless the BSO has specific concerns which warrant full Committee involvement. Committee review includes an independent assessment of the containment levels required by the NIH Guidelines for the proposed research, an assessment of the laboratory facilities, procedures, and practices, and of the training and expertise of personnel.

The BSC is authorized by the Chancellor to limit or suspend any research that is not in compliance with UCSF biosafety policies and procedures. The BSC advises and works with the EH&S in administering the various aspects of the campus Biological Safety Program.

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C. OFFICE OF ENVIRONMENTAL HEALTH AND SAFETY (EH&S)

The Office of Environmental Health and Safety (EH&S) administers the Biological Safety Program and other campus safety programs, such as radiation and chemical safety. As designated by the Director of EH&S, the Biosafety Officer is primarily responsible for implementing and overseeing the technical aspects of the campus Biological Safety Program.

1. BIOSAFETY OFFICER (BSO)

The Biosafety Officer's (BSO’s) duties include, but are not necessarily limited to, providing technical advice to the BSC and researchers on laboratory containment and safety procedures, overseeing periodic inspections to ensure that laboratory standards are rigorously maintained, and developing emergency plans for handling spills and personnel contamination. The BSO regularly reports on the Biological Safety Program to the BSC, particularly if any significant problems or violations of the NIH Guidelines or any research-related accidents or illnesses have occurred. The BSO reviews and approves Biosafety Level 1 applications and works closely with the Department Safety Advisors on all aspects of the Biological Safety Program.

2. DEPARTMENT SAFETY ADVISORS (DSAs)

Department Safety Advisors (DSAs) are the primary contact person for all EH&S activities for their assigned departments. The DSA is responsible for conducting laboratory inspections, reviewing safety equipment such as biological safety cabinets ("tissue culture hoods") and autoclaves, and training laboratory personnel in various aspects of biosafety, such as bloodborne pathogens and Q-fever. DSAs also assist researchers in completing their Biological Use Application (BUA) forms and in maintaining an accurate and up-to-date Biological Safety Logbook.

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D. EMPLOYEE HEALTH SERVICE (EHS)

Employee Health Service (EHS) provides occupational health programs to the UCSF Medical Center and campuses. An Employee Health Service representative serves as an ex officio member of the BSC and provides recommendations as to appropriate medical surveillance and preventive programs for personnel using hazardous infectious agents when such programs are deemed necessary. Employee Health Service works with the BSO and BSC to investigate laboratory exposures and laboratory-acquired infections, advises the BSC regarding new developments in regulatory medical surveillance programs and consults with researchers about recommended immunization practices and the availability of vaccines.

E. DEPARTMENTAL CHAIRPERSONS

Departmental Chairpersons are responsible for providing the support to researchers which ensures that appropriate facilities are available to control biological hazards and to enable PIs to comply with pertinent campus policies, for assuring that the PI and all personnel listed on an application have training that is commensurate with the proposed project and that the project design and monitoring methods meet UCSF safety standards. Departmental Chairpersons, or their designated safety representative, are responsible for correcting work errors and conditions that may result in personal injury.

F. PRINCIPAL INVESTIGATORS (PIs)

The Principal Investigator (PI) is directly and primarily responsible for the compliance of all laboratory personnel with all UCSF biosafety policies and procedures and for the safe operation of the laboratory. His or her knowledge and judgment are critical in assessing risks and appropriately applying the campus biosafety guidelines.

1. GENERAL

As part of this responsibility, the PI must assure that:

a. No research using infectious agents or recombinant DNA (rDNA) is initiated unless it has met all the requirements outlined in this manual.

b. Appendices A and B have been consulted to determine the appropriate Risk Group classification of the microorganisms to be used and the prescribed microbiological practices and laboratory techniques required by the Risk Group of the microorganisms to be used in the proposed research have been adopted.

c. He/she will report immediately to the BSO all significant violations of the policies and procedures and all significant research-related accidents (spills, needle-sticks, exposures, injuries, etc.) which result in overt or potential exposure to infectious materials.

d. He/she is prepared to implement methods for dealing with accidental spills and personnel contamination.

e. Appropriate permits required by the United States Department of Agriculture (USDA) and/or the United States Public Health Service (USPHS) for work with certain animal and plant pathogens are obtained.

f. Appropriate importation, exportation and interstate shipping requirements for certain biological materials are followed. See Appendices K.

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2. PRIOR TO INITIATING RESEARCH, THE PRINCIPAL INVESTIGATOR SHALL:

a. Receive appropriate BSC or BSO approval for all research projects.

b. Determine, in consultation with the BSO (476-2097) and Employee Health Service (885-7580), the usefulness of serological screening, the requirements of medical surveillance, and the availability of vaccination for certain Risk Group 2 and 3 agents. Inform the laboratory staff of the reasons and provisions for any precautionary medical practices advised or requested, such as vaccination or serum collection. Where appropriate, the hepatitis B vaccination series must be offered free of charge to the employee. The PI is responsible for keeping records of HBV immunizations and/or declination statements for his or her staff.

c. Communicate in writing to the BSC any protocol changes which substantially modify the research procedures upon which approval was originally based. Other modifications, such as changes in personnel or laboratory sites, should be provided to the BSO either directly or through the DSA.

d. Assure that personnel working with infectious agents or biohazardous materials are appropriately trained so that they are aware of the hazards and proficient in the practices and techniques required for the safe handling of such materials. Instruction in proper laboratory procedures, bloodborne pathogens and Q-fever training, is available through EH&S. For more information, contact the BSO (476-2097).

3. DURING THE CONDUCT OF THE RESEARCH, THE PRINCIPAL INVESTIGATOR SHALL:

a. Supervise the performance of the staff to ensure that the required safety practices and techniques are employed.

b. Investigate and report in writing to the BSC any significant biosafety problems pertaining to the pursuit of the research goals, specifically, new information which was not available at the time of the application. The PI is responsible for correcting any conditions that might release biohazardous materials into the environment.

c. Implement the procedures prescribed for dealing with laboratory accidents.

d. Assure that the biological characteristics of the microorganisms used in experiments haven’t undergone adverse change. Periodic assessment should include the purity and phenotype of the strain. Special restrictive characteristics such as attenuation require regular surveillance. Strain verification should be performed periodically on all replication-defective microorganisms.

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G. LABORATORY PERSONNEL

Safety in activities involving biohazardous agents ultimately depends on the individual conducting these activities. Motivation and good judgment are essentials in the protection of health and the environment. The NIH Guidelines are intended to help the institution, the BSC, EH&S and the PI determine the safeguards that should be implemented. These guidelines will never be complete or final in that all conceivable experiments involving rDNA and other biohazardous agents cannot be foreseen. Therefore, it is the responsibility of each individual employee to adhere to the intent of the Guidelines as well as to their specifics.

Personnel who work with Risk Group 1 agents must have standard training in microbiological practices to ensure proper handling of the agent. Those personnel working with Risk Group 2 or 3 agents must also have specific training in handling pathogenic microorganisms and those individuals working with Risk Group 3 agents must have specific training in handling potentially lethal agents. Annual Bloodborne Pathogens training is required for all UCSF employees with occupational exposure to HIV/HBV/bloodborne pathogens and Q-fever training is required for personnel working with sheep, goats or their tissue or cell lines.

Risk Group 4 agents are not permitted on the UCSF campus and Biosafety Level 4 Facilities are not available.

It may be inadvisable for a person in an immunocompromised condition to work with microorganisms. This includes individuals under systemic corticosteroid therapy, chemotherapy for malignancies, radiation therapy, and those who have certain diseases (e.g., lymphomas, leukemia, and AIDS) which induce severe impairment of immune competence. Medical advice should be sought regarding possible work restrictions.

Additionally, certain microbes such as Toxoplasma gondii, rubella virus, cytomegalovirus, and vesicular stomatitis virus pose a hazard to pregnant women who should carefully evaluate the risk of working with or near these agents. Special hazards and exceptions (e.g., individuals vaccinated against rubella virus) must be determined by the PI who is primarily responsible for establishing the safety of personnel under his/her supervision.

It should also be noted that Ascaris suum and A. lumbricoides produce volatile allergens (ascaricides) that may induce anaphylaxis in sensitized individuals. PIs who propose to work with Ascaris spp. must advise personnel of this biohazard.

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OEH&S Biosafety Manual Chapter 3

CRITERIA FOR DETERMINING WHO NEEDS BIOSAFETY COMMITTEE APPROVAL

• Recombinant DNA
• Infectious Agents
• Toxins
• Human blood, body fluids, or unfixed tissue
• Tissues, organ or cell cultures of human origin
• Human Gene Therapy
• Old World primates or sheep; Old World primates can transmit herpes B virus and sheep can transmit Coxiella burnetii, the causative agent of Q-fever.

If you are working with potentially infectious agents and human subjects or experimental animals, BSC review is necessary in addition to review by the Committee on Human Research (CHR) and/or the Committee on Animal Research (CAR).

Note: The CHR and the CAR do not consider biosafety issues during their respective reviews.

PIs whose research comes under the governance of any of the following campus rules or governmental regulations are required to complete a BUA, and where applicable, maintain a medical surveillance program for laboratory employees:

• NIH Guidelines for Research Involving Recombinant DNA Molecules
• Cal-OSHA Bloodborne Pathogens Standard
• Cal-OSHA Tuberculosis Standard
• Cal-OSHA Special Orders for Q-Fever
• Medical Waste Management Act (generate medical (red bag) or sharps waste)
• International, Federal and State Transport Regulations

BUA application forms entitled Application for Biological Use Authorization are included in Appendix E. For additional forms or information, call the BSC office at 476-2198 or your Department Safety Advisor (DSA). For more detailed information about the BUA review process, see Chapter 4. Please note that the foregoing requirements will include virtually all laboratory research performed at UCSF. If you are uncertain whether your research falls into any of the above categories, contact the BSO at 476-2097.

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OEH&S Biosafety Manual Chapter 4

THE BIOLOGICAL USE AUTHORIZATION (BUA) APPLICATION PROCESS

A. NEW APPLICATIONS

Principal Investigators (PIs) who meet the criteria outlined in Chapter 3 for submitting a Biological Use Authorization (BUA) should submit a completed application form (including a separate Principal Investigator Form) to the Biosafety Committee Coordinator for administrative screening and subsequent forwarding to the Biosafety Committee (BSC) or the Biosafety Officer (BSO) for review. All applicants must complete all pages of the form and any additional forms or narratives requested.

1. INFORMATION TO INCLUDE IN YOUR BUA:

a. A clear statement of the goal of the experiments.

b. A description of the actual experiments; sufficient detail should be provided so that the BSC can understand what procedural steps are involved in your experiments.

c. Your estimate of the biohazards associated with your experiments or project and the required Biosafety Level; if you have any questions regarding this estimation, contact the BSO at 476-2097.

d. A description of your biosafety facilities.

e. A description of your plan for biosafety; the BSC must be able to judge the adequacy of your biosafety precautions.

f. For research involving recombinant DNA (rDNA), identification of the specific hosts, vectors, genes and sources of DNA; when using new or uncommon hosts, vectors, genes or DNA molecules, describe them briefly.

g. For research using experimental animals, completion of the form called Animal Involvement in the Animal Care Facility after discussing arrangements with your consulting Aninal Care Facility (ACF) veterinarian.

h. For research using sheep or Old World primates (genus Macaca) or their tissue or cell lines, indication that all personnel have had the appropriate training provided by the Office of Environmental Health and Safety (EH&S).

i. A description of your health surveillance plan. If you are drawing human blood or collecting human body fluids or tissue and processing, transporting or storing these samples, you must have a medical surveillance program in accordance with the Cal-OSHA Bloodborne

Pathogens (BBP) Standard. As part of the medical surveillance plan, specify that hepatitis B immunization and post-exposure follow-up and treatment will be provided to laboratory personnel free of charge and that records of immunization or declination will be retained. Indicate that laboratory personnel will attend initial BBP training and annual retraining offered by EH&S.

j. A description of the biosafety training that you provide (i.e., informing personnel of the hazards associated with the research, training in the safe conduct of specific experimental procedures, initial training and periodic retraining in laboratory and BUA-specific hazards.)

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2. BUAs EXEMPT FROM FULL COMMITTEE REVIEW

Studies in which the research involves:

1. only the use of Risk Group 1 or otherwise unclassified biological materials and procedures or
   
2. Biosafety Level 2 containment requirements solely because of the use of a human cell line will be reviewed and approved by the BSO rather than the full BSC. An Approval Letter signed by the BSO will be forwarded to the PI. Such BUAs must be renewed after three years if the study will be continuing.

   Note: If a study meets the criteria for exemption from BSC review, a BUA must be completed for review and approval by the BSO.

3. BUAs SENT TO FULL COMMITTEE

If the applicant is required to submit an application for BSC review, the BUA must include sufficient information and details on the experimental procedures to assist the BSC in determining the appropriateness of the proposed facilities and safety procedures. As part of the review process, the assigned Department Safety Advisor (DSA) will conduct a laboratory site visit to review the information on the application and to determine the appropriate biosafety level, training, medical surveillance, personal protective equipment and other relevant safety requirements for the study. All of the information provided by the PI in the BUA, such as the biosafety training status of personnel, will be confirmed by the DSA during the site visit to the laboratory, which occurs prior to BSC review.

The BSC meets monthly to review BUA applications.The Committee’s approval options include:

• Approval - approved as submitted;

• Contingent Approval - approved when contingencies levied by the BSC are met. Often, contingencies are met by the submission of additional information;

• Return for additional information - not approved; when an application is very incomplete or seriously flawed, the BSC may return it to the PI for additional work;

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B. RENEWALS

C. MODIFICATION OF AN APPROVED BUA

D. TERMINATION OF A BUA

If a PI intends to terminate experiments approved under a BUA, EH&S must be notified in writing. This notification will ensure that the inspection, training and surveillance programs instituted for that BUA are appropriately concluded. EH&S provides assistance in the disposal or transfer of any biological materials which are no longer needed and the biohazard warning signs will be removed from the facility. The BUA approval number would then be retired in the EH&S database.

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OEH&S Biosafety Manual Chapter 5

BIOHAZARDS AND POTENTIALLY INFECTIOUS MATERIAL

A. CATEGORIES OF BIOHAZARDS OR POTENTIALLY INFECTIOUS MATERIALS

B. RECOMBINANT DNA (rDNA)

Biohazards are infectious agents or biologically derived infectious materials that present a risk or potential risk to the health of humans or animals, either directly through infection or indirectly through damage to the environment. Infectious agents have the ability to replicate and give rise to potentially large populations in nature when small numbers are released from a controlled situation.

The following is a listing of the potentially hazardous biological materials and agents. Principal Investigators (PIs) should follow the instructions in the Biosafety Application Package carefully to ensure that all appropriate sections of the application are completed. If a PI intends to use agents which are not listed in this section, he or she should contact the Biosafety Committee (BSC) or the Biosafety Officer (BSO) for advice regarding proper completion of the application.

A. CATEGORIES OF BIOHAZARDS OR POTENTIALLY INFECTIOUS MATERIALS

1. Human, animal and plant pathogens

1. Viruses, including oncogenic and defective viruses
2. Rickettsiae
3. Chlamydiae
4. Bacteria, including those with drug resistance plasmids
5. Fungi
6. Parasites
7. Undefined or other infectious agents, such as prions

2. All human blood, blood products, tissues and certain body fluids

3. Cultured cells (all human or certain animal) and potentially infectious agents these cells may contain

4. Allergens

5. Toxins (bacterial, fungal, plant, etc.)

6. Certain recombinant nucleic acid products

7. Clinical and diagnostic specimens

8. Infected animals and animal tissues

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B. RECOMBINANT DNA (rDNA)

1. GENERATION OR USE OF RECOMBINANT DNA

2. HUMAN GENE THERAPY

3. USE OF ANIMALS

The use of animals in research requires compliance with the "Animal Welfare Act", Office of Laboratory Animal Welfare (OLAW) of Public Health Service, and all applicable state or local regulations covering the care and use of animals. All protocols involving the use of animals must be reviewed and approved by the Committee on Animal Research (CAR), before their implementation. All PIs planning to use biological agents in animals must complete the form called Animal Involvement in the Laboratory Animal Resource Center (LARC) for Biosafety Committee review and approval prior to final approval of the protocol.

The PI must notify the LARC in writing prior to initiation of experimentation and post a copy of the Animal Involvement Form at a location specified by the LARC staff.

Because of the possibility of exposure to potentially infectious agents (i.e., rabies virus, herpes B virus, Coxiella burnetii), the CAR has developed policies to protect all individuals who have animal contact. These policies should be consulted and followed when working with animals that may harbor these agents. For further information, the CAR office at 476-2197.

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4. TRANSGENIC ANIMALS

5. TISSUE CULTURE/CELL LINES

a. Risk Group 1/Biosafety Level 1

Cell lines which are non-primate or are of normal non-human primate origin, which do not harbor a primate virus, which are not contaminated with bacteria, mycoplasma or fungi and which are well established, may be considered Risk Group 1 cell lines and handled at Biosafety Level 1.

b. Risk Group 2/Biosafety Level 2

When cell cultures are known to contain an etiologic agent or an oncogenic virus, the cell line can be classified at the same level as that recommended for the agent or virus. The Centers for Disease Control and Prevention (CDC) has recommended that all cell lines of human origin be handled at Biosafety Level 2. PIs who can demonstrate by testing or other certification that their human cell lines are free of bloodborne pathogens as defined by the Bloodborne Pathogens Standard may request permission from the BSC to handle those lines at Biosafety Level 1. Such requests are handled on a case-by-case basis.

Primate cell lines derived from lymphoid or tumor tissue, all cell lines exposed to or transformed by a primate oncogenic virus, all clinical material (e.g., samples of human tissues and fluids obtained after surgical resection or autopsy for use in organ culture or establishment of primary cell cultures), all primate tissue, all cell lines new to the laboratory (until proven to be free of all adventitious agents) and all virus and mycoplasma-containing primate cell lines are classified as Risk Group 2 and must be handled at Biosafety Level 2.

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6. TUBERCULOSIS

Since 1985, the incidence of tuberculosis in the United States has been increasing steadily, reversing a 30 year downward trend. Recently, drug-resistant strains of Mycobacterium tuberculosis have become a serious concern. Outbreaks of tuberculosis, including drug-resistant strains, have occurred in health-care environments.

In October 1994, the CDC published its "Guidelines for Preventing the Transmission of Tuberculosis in Health-Care Facilities, 1994". The guidelines contain specific information on ventilation requirements, respiratory protection, medical surveillance and training for those personnel who are considered at-risk for exposure to tuberculosis. For more information, contact the BSO or your Department Safety Advisor (DSA).

PIs intending to work with Mycobacterium tuberculosis in the laboratory must obtain written approval from the BSC via the BUA application process before beginning work. Propagation and manipulation of Mycobacterium tuberculosis cultures must be performed at Biosafety Level 3.

7. USE OF VACCINIA VIRUS

PIs wishing to use vaccinia virus must obtain written approval from the BSC via the BUA application process. Biosafety Level 2 practices and procedures must be followed. All employees who directly handle cultures or animals contaminated or infected with vaccinia virus, recombinant or defective vaccinia viruses or other orthopox viruses that infect humans, should be offered the small pox vaccine. Additional information about the use of vaccinia virus at the University of California, San Francisco (UCSF) may be found in Appendix L6.

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OEH&S Biosafety Manual Chapter 6

BIOLOGICAL AGENTS AND BIOHAZARD CLASSIFICATION

Biological agents are classified into four Risk Groups on the basis of the following characteristics:

A. RISK GROUP 1 (RG1)

Agents that are not associated with disease in healthy human adults.

B. RISK GROUP 2 (RG2)

Agents that are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available. This Risk Group includes agents which may produce disease of varying degrees of severity from accidental inoculation or injection or other means of cutaneous penetration but which are contained by ordinary laboratory technique.

C. RISK GROUP 3 (RG3)

Agents that are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available (high individual risk but low community risk). This Risk Group includes agents involving special hazards or agents derived from outside the United States which require a federal permit for importation unless they are specified for higher classification. Many Risk Group (RG3) pathogens require special conditions for containment.

D. RISK GROUP 4 (RG4)

Agents that are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available (high individual and high community risk). These agents require the most stringent containment because they are extremely hazardous to laboratory personnel or may cause serious epidemic disease. This risk group includes RG3 agents from outside the United States when they are employed in entomological experiments or when other entomological experiments are conducted in the same laboratory area.

Note:

The use of agents in Risk Group 4 is not permitted at the University of California, San Francisco (UCSF).

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E. RESTRICTED FOREIGN ANIMAL PATHOGENS

While not a Risk Group per se, these are animal pathogens that are excluded from the United States by law or whose entry is restricted by United States Department of Agriculture administrative policy.

NOTES:

Federally licensed vaccines containing live bacteria or viruses are not subject to these Risk Group classifications. These classifications are applicable, however, to cultures of the strains used for vaccine production, or further passages of the vaccine strains.

Importation of biological samples from outside the United States is subject to strict Federal and State licensing, and transportation and packaging requirements. All requests must be processed through the Office of Environmental Health and Safety (EH&S) for importation and exportation permits.

A list of biological agents classified according to risk may be found in Appendix A2. CDC agent summary statements on retroviruses (including HIV), hepatitis viruses (including HBV) and Mycobacterium tuberculosis may be found in the CDC-NIH guide Biosafety in Microbiological and Biomedical Laboratories, 3^rd Edition. Copies of this and other guides are available on-line; see Appendix M.

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OEH&S Biosafety Manual Chapter 7

A Containment

B Laboratory Practice and Technique

C Safety Equipment (Primary Containment)

D Facility Design (Secondary Containment)

A. CONTAINMENT

The term "containment" is used in describing safe methods for managing infectious agents in the laboratory environment where they are being handled or maintained. The purpose of containment is to reduce or eliminate exposure of laboratory workers, other people and the outside environment to potentially hazardous agents. The three elements of containment include laboratory practice and technique, safety equipment, and facility design.

1. PRIMARY CONTAINMENT

The protection of personnel and the immediate laboratory environment from exposure to infectious agents is provided by good microbiological technique and the use of appropriate safety equipment, such as biological safety cabinets. The use of vaccines may provide an increased level of personal protection for certain pathogens.

2. SECONDARY CONTAINMENT

The protection of the environment external to the laboratory from exposure to infectious materials is provided by a combination of facility design and operational practices. The risk evaluation of the work to be done with a specific agent will determine the appropriate combination of these elements.

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B. LABORATORY PRACTICE AND TECHNIQUE

The most important element of containment is strict adherence to standard microbiological practices and techniques.

Persons working with infectious agents or infected materials must be aware of potential hazards, and must be trained and proficient in the practices and techniques required for handling such material safely. The Principal Investigator (PI) is responsible for ensuring that laboratory personnel are properly trained; the PI may delegate the provision of training to the Laboratory Supervisor, but the responsibility remains with the PI.

Each laboratory should develop an operational manual which identifies specific hazards that will or may be encountered, and which specifies practices and procedures designed to minimize or eliminate risks. Personnel should be advised of special hazards and should be required to read and to follow the required practices and procedures. A scientist trained and knowledgeable in appropriate laboratory techniques, safety procedures and hazards associated with the handling of infectious agents must direct laboratory activities.

When standard laboratory practices are not sufficient to control the hazard associated with a particular agent or laboratory procedure, additional measures may be needed. The PI is responsible for selecting additional safety practices, which must be in keeping with the hazard associated with the agent or procedure.

Laboratory personnel safety practices and techniques must be supplemented by appropriate facility design and engineering features, safety equipment and management practices.

C. SAFETY EQUIPMENT (PRIMARY CONTAINMENT)

Safety equipment includes biological safety cabinets, enclosed containers and other engineering controls designed to remove or minimize exposures to hazardous biological materials. The biological safety cabinet (sometimes called a tissue culture hood) is the principal device used to provide containment of infectious splashes or aerosols generated by many procedures.

1. BIOLOGICAL SAFETY CABINETS (BIOSAFETY CABINETS)

Biological safety cabinets are designed to contain aerosols generated during work with biological material through the use of laminar air flow and high efficiency particulate air (HEPA) filtration. Three types of biological safety cabinets (Class I, II and III) are used in laboratories. Open-fronted Class I and Class II biological safety cabinets are partial containment devices which provide a primary barrier offering significant levels of protection to laboratory personnel and to the environment when used in combination with good laboratory technique.

The Class I biological safety cabinet is suitable for work involving low to moderate risk agents, where there is a need for containment, but not for product protection. It provides protection to personnel and the environment from contaminants within the cabinet but does not protect the work within the cabinet from "dirty" room air.

The Class II biological safety cabinet protects the material being manipulated inside the cabinet (e.g., cell cultures, microbiological stocks) from external contamination. It meets requirements to protect personnel, the environment and the product. There are three basic types of Class II biological safety cabinets: Type A, Type B and 100% Exhaust. The major differences between the three types may be found in the percent of air that is exhausted or recirculated, and the manner in which exhaust air is removed from the work area.

The gas-tight Class III biological safety cabinet, or glove box, provides the highest attainable level of protection to personnel, the environment and the product. It is the only unit which provides a total physical barrier between the product and personnel. It is for use with high risk biological agents and is used when absolute containment of highly infectious or hazardous material is required.

It is important to note that horizontal laminar flow benches must not be utilized for work with biohazardous or chemically hazardous agents. These units provide product protection by ensuring that the product is exposed only to HEPA-filtered air. They do not provide protection to personnel or the ambient environment.

Biological safety cabinets used as primary barriers must be certified annually by a qualified vendor. Contact the Office of Environmental Health and Safety (EH&S) for information about vendors or other biological safety cabinet-related information.

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2. PERSONAL PROTECTIVE EQUIPMENT (PPE)

This equipment may include items for personal protection such as protective clothing (e.g., gowns, gloves), respirators, face shields, safety glasses or goggles. Personal protective equipment (PPE) is often used in combination with other safety equipment when working with biohazardous materials. In some situations, protective clothing may form the primary barrier between personnel and the infectious materials.

D. FACILITY DESIGN (SECONDARY CONTAINMENT)

The design of a facility is important in providing a barrier to protect people working inside and outside the laboratory, and to protect people or animals in the community from infectious agents which may be accidentally released from the laboratory. Facility design must be commensurate with the laboratory's function and the recommended biosafety level for the agent being manipulated.

The recommended secondary barrier(s) will depend on the risk of transmission of specific agents. For example, the exposure risks for most laboratory work in Biosafety Level 1 and 2 facilities will be direct contact with the agents, or inadvertent contact exposures through contaminated work environments. Secondary barriers in these laboratories may include separation of the laboratory work area from public access, availability of decontamination equipment (e.g., autoclave) and handwashing facilities.

As the risk for aerosol transmission increases, higher levels of primary containment and multiple secondary barriers may become necessary to prevent infectious agents from escaping into the environment. Such design features could include specialized ventilation systems to assure directional airflow, air treatment systems to decontaminate or remove agents from exhaust air, controlled access zones, an airlock at the laboratory entrance, or separate buildings or modules for physical isolation of the laboratory building itself.

EH&S has a plan review program to assist PIs in proper laboratory design, equipment need and selection.

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OEH&S Biosafety Manual Chapter 8

BIOSAFETY LEVELS

A. Biosafety Level 1 (BSL1)

B. Biosafety Level 2 (BSL2)

C. Biosafety Level 3 (BSL3)

D. Biosafety Level 4 (BSL4)

E. Vertebrate Animal Biosafety Levels

There are four biosafety levels (BSLs) which represent combinations of laboratory practices and techniques, safety equipment, and laboratory facilities. Each combination is specifically appropriate for the operations performed, the documented or suspected routes of transmission of the infectious agents, and for the laboratory function or activity. The recommended biosafety level for an organism represents the conditions under which the agent can be ordinarily handled safely.

As a general rule, a biosafety level should be used that matches the highest Risk Group (RG) classification of the organisms involved. For example, work with vaccinia virus, a Risk Group 2 (RG2) agent, should be conducted at Biosafety Level 2 (BSL2) or higher; simultaneous work with E. coli (RG1), Epstein-Barr virus (RG2) and Mycobacterium tuberculosis (RG3) should be conducted at Biosafety Level 3 (BSL3).

A. BIOSAFETY LEVEL 1 (BSL1)

Biosafety Level 1 (BSL1) is appropriate for work done with defined and characterized strains of viable microorganisms not known to cause disease in healthy adult humans. It represents a basic level of containment that relies on standard microbiological practices with no special primary or secondary barriers required, other than a sink for handwashing.

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B. BIOSAFETY LEVEL 2 (BSL2)

Biosafety Level 2 (BSL2) is applicable to work done with a broad spectrum of indigenous moderate-risk agents present in the community and associated with human disease of varying severity. Agents may be used safely on the open bench, provided the potential for producing splashes or aerosols is low. Primary hazards to personnel working with these agents relate to accidental percutaneous or mucous membrane exposures or ingestion of infectious materials. Procedures with high aerosol or splash potential must be conducted in primary containment equipment such as biological safety cabinets. Primary barriers such as splash shields, face protection, gowns and gloves should be used as appropriate. Secondary barriers such as handwash, eyewash and waste decontamination facilities must be available.

C. BIOSAFETY LEVEL 3 (BSL3)

Biosafety Level 3 (BSL3) is applicable to work done with indigenous or exotic agents with a potential for respiratory transmission and which may cause serious and potentially lethal infection. Primary hazards to personnel working with these agents (i.e., Mycobacterium tuberculosis, St. Louis encephalitis virus and Coxiella burnetii) include autoinoculation, ingestion and exposure to infectious aerosols. Greater emphasis is placed on primary and secondary barriers to protect personnel in adjoining areas, the community and the environment from exposure to infectious aerosols. For example, all laboratory manipulations should be performed in a biological safety cabinet or other approved enclosed equipment. Secondary barriers include controlled access to the laboratory and a specialized ventilation system that minimizes the release of infectious aerosols from the laboratory.

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D. BIOSAFETY LEVEL 4 (BSL4)

BSL4 is applicable for work with dangerous and exotic agents which pose a high individual risk of life-threatening disease, which may be transmitted via the aerosol route and for which there is no available vaccine or therapy. Agents with close or identical antigenic relationship to Biosafety Level 4 agents should also be handled at this level. Primary hazards to workers include respiratory exposure to infectious aerosols, mucous membrane exposure to infectious droplets and autoinoculation. All manipulations of potentially infected materials and isolates pose a high risk of exposure and infection to personnel, the community and the environment.

Isolation of aerosolized infectious materials is accomplished primarily by working in a Class III biological safety cabinet or a full-body, air-supplied positive pressure personnel suit. The facility is generally a separate building or a completely isolated zone within a complex with specialized ventilation and waste management systems to prevent release of viable agents to the environment.

There are no Biosafety Level 4 (BSL4) facilities at the University of California, San Francisco (UCSF).

E. VERTEBRATE ANIMAL BIOSAFETY LEVELS

There are four animal biosafety levels, designated Animal Biosafety Level 1 through 4, for work with infectious agents in mammals. The levels are combinations of practices, safety equipment and facilities for experiments on animals infected with agents which produce or may produce human infection. In general, the biosafety level recommended for working with an infectious agent in vivo and in vitro is comparable.

NOTE:

Summaries of Biosafety Levels and Animal Biosafety Levels may be found in Appendices A, B and C.

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OEH&S Biosafety Manual Chapter 9

PROTECTIVE BEHAVIOR

A. Prudent Practices and Good Technique

B. Personal Laboratory Housekeeping and Hygiene

C. Universal Precautions

The foundations of protective behavior lie in an individual’s laboratory experience, personal work habits, technical knowledge and attitude toward laboratory safety. Unlike administrative controls, which are behaviors dictated by regulation or laboratory policy, protective behavior is an inate part of each individual worker’s personal approach to the laboratory environment. As such, protective behaviors form the first and most important line of defense against injury or exposure in the biomedical workplace.

A. PRUDENT PRACTICES AND GOOD TECHNIQUE

Prudent practices and good technique are of primary importance in laboratory safety. Both are based on sound technical