Skip to main content

Biological Safety Manual

< Back to Biological Safety

1.0 Introduction

The purpose of this document is to increase awareness of biological hazards frequently encountered in research, clinical, and teaching laboratories at Northwestern University.  Additionally, this manual provides guidance on recommended practices when working with and disposing of biological hazards.  Biological hazards include infectious or toxic microorganisms (including viral vectors), biological toxins, and substances from which transmission of infectious agents or toxins could be reasonably anticipated such as tissues from humans and research animals.  Due to the diverse nature of biological hazards, they are often generically referred to as “agents” so that a discussion may encompass all known and unknown hazards.  The intrinsic danger associated with a particular biological hazard may be mitigated or compounded by the presence of recombinant or synthetic nucleic acids (rsNA).

The objective of safety awareness and practice is to assure laboratory personnel that, with proper precaution, equipment, and facilities, most biohazardous materials can be handled without undue risk to themselves, their peers, their families, and the environment.

This document is intended not only for trained microbiologists, but also for individuals handling human clinical materials in other laboratory disciplines, such as biochemistry, genetics, oncology, immunology, and molecular biology.  Individuals with little or no microbiological training might not appreciate the potential hazard involved in handling the types of samples described above.

The safety principles described herein are based on sound safety practices, common sense, current data, good housekeeping, thorough personal hygiene, and a plan for responding to unanticipated events.  Many of the practices and procedures described herein have been adapted from the Biosafety in Microbiological and Biomedical Laboratories, 5th Edition (BMBL) and the National Institutes of Health Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines).  Laboratories that are well organized and procedurally disciplined are not only safe but also scientifically effective.

2.0 Code of Conduct and Culture of Responsibility

All scientists are accountable for the establishment of a culture of responsibility in their labs and at their institutions.  Fundamental to this culture of responsibility are scientific integrity and adherence to ethical codes of conduct.  For the individual scientist, an ethical code of conduct centers on personal integrity.  It embodies, above all, a commitment to intellectual honesty and personal liability for one’s actions, and to a range of practices that characterize the responsible conduct of research.  Such practices include:

  • Intellectual honesty
  • Accuracy
  • Fairness
  • Collegiality
  • Transparency in conflicts of interest
  • Humane care of animals
  • Adherence to mutual responsibilities

When conducting research involving microorganisms and agents with known or unknown pathogenic properties, additional responsibilities include the following:

  • Awareness of and adherence to all safety protocols
  • Awareness of and adherence to all spill and exposure protocols
  • Awareness of and adherence to reporting requirements of the individual researcher related to spills, exposures, or potential releases
  • Awareness of the reporting requirements of Northwestern University and the various research oversight committees
  • Awareness of all emergency response protocols (e.g. fire, tornado, inclement weather)
  • Awareness of all emergency response protocols (e.g. fire, tornado, inclement weather)
  • Completion of all training requirements set forth by Northwestern University and all applicable research oversight committees
  • Completion of and proficiency in all lab-specific training requirements
  • Completion of all Occupational Heath requirements, including documentation of required physicals, medical clearances, and vaccinations, as applicable
  • Immediate reporting to the Principal Investigator (PI) of any situation that compromises an individual’s ability to perform as required in a research laboratory, including physical or psychological issues
  • Immediate reporting to the PI and Northwestern University, where appropriate, of behavior or activities that are inconsistent with safety and security plans
  • Awareness of and adherence to security protocols

From the perspective of the institution, the establishment of support systems for the individual researcher is essential to the development of a culture of responsibility.  At the individual level, one such support system is the Northwestern University Faculty and Staff Assistance Program .  The program is a network of services, including short-term counseling, to help you and your household family members cope with everyday life issues.  Professional counselors can help you with: everyday needs and life events, relationship/marital concerns, workplace concerns, anxiety and stress, family issues, coping with a serious illness, sleeping difficulties, loss of a loved one, emotional concerns, depression, and communication.  The program provides you with counseling sessions with a local, licensed counselor and unlimited, 24/7 telephonic counseling. For program assistance, contact (855) 547-1851.

Full-time matriculated students have opportunities to receive mental health counseling services at no charge through the Counseling and Psychological Services (CAPS) .  CAPS serves as the primary mental health service at Northwestern University and has offices on both the Evanston and Chicago campuses. CAPS provides a set of core services, including clinical services, educational workshops, and consultation with faculty, staff, and parents.  For more information regarding CAPS services, contact (847) 491-2151.

An additional resource available to students in the Feinberg School of Medicine is the Ombudsperson .  This individual has been appointed to serve as an impartial, neutral, and confidential facilitator for students, residents and fellows.  The Ombudsmen will work with students, residents and fellows to describe available options to address the issue and, if desired, help resolve conflicts. Student, resident and fellow interactions with the ombudsperson are handled as discreetly as possible.

Another important mechanism essential to the development of a culture of responsibility is a formal, confidential reporting mechanism in instances of non-compliance with established, Northwestern University or lab-specific, safety and/or security policies.  There are multiple mechanisms for reporting such non-compliance at Northwestern:

  1. Reporting to your PI/supervisor.
  2. Reporting to your Department Chair or Dean
  3. Reporting to Research Integrity at nu-ori@northwestern.edu or call (312) 503-0054
  4. Reporting through Ethics Point, a third-party vendor that will allow you to report your concerns anonymously or call (866) 294-3545.
  5. Reporting to Research Safety at researchsafety@northwestern.edu or call (312) 503-8300 (Chicago) or (847) 491-5581 (Evanston).

3.0 Recombinant and Synthetic Nucleic Acid Molecules

At Northwestern University, research involving recombinant or synthetic nucleic acids must comply with the National Institute of Health’s Guidelines for Research Involving Recombinant and Synthetic Nucleic Acid Molecules (NIH Guidelines).  The NIH Guidelines are applicable to all recombinant and synthetic nucleic acids research within the United States or its territories, which is conducted at or sponsored by an institution that receives any support for recombinant or synthetic nucleic acids research from the NIH.  Furthermore, many federal funding agencies in the United States have similarly adopted the NIH Guidelines and require adherence to them as a condition of funding.  Any individual receiving support for research involving recombinant or synthetic nucleic acids must be associated with or sponsored by an institution that assumes the responsibilities assigned by the NIH Guidelines.  For the most up-to-date version of the NIH Guidelines, please visit NIH.

3.1 Definition

The National Institute of Health (NIH) has defined recombinant and synthetic nucleic acids as (i) molecules that (a) are constructed by joining nucleic acid molecules and (b) that can replicate in a living cell, i.e., recombinant nucleic acids; (ii) nucleic acid molecules that are chemically or by other means synthesized or amplified, including those that are chemically or otherwise modified but can base pair with naturally occurring nucleic acid molecules, i.e., synthetic nucleic acids; or (iii) molecules that result from the replication of those described in (i) or (ii) above.

3.2  NIH Guidelines

The purpose of the NIH Guidelines is to specify the practices for constructing and handling: (i) recombinant nucleic acid molecules, (ii) synthetic nucleic acid molecules, including those that are chemically or otherwise modified but can base pair with naturally occurring nucleic acid molecules, and (iii) cells, organisms, and viruses containing such molecules.

The NIH Guidelines provides oversight of any nucleic acid molecule experiment, which according to the NIH Guidelines requires approval by NIH, must be submitted to NIH or to another Federal agency that has jurisdiction for review and approval.  Once approvals, or other applicable clearances, have been obtained from a Federal agency other than NIH (whether the experiment is referred to that agency by NIH or sent directly there by the submitter), the experiment may proceed without the necessity for NIH review or approval.

For experiments involving the deliberate transfer of recombinant or synthetic nucleic acid molecules, or DNA or RNA derived from recombinant or synthetic nucleic acid molecules, into human research participants (human gene transfer, HGT), no research participant shall be enrolled until (1) the NIH protocol registration process has been completed, (2) Institutional Biosafety Committee (IBC) approval (from the clinical trial site) has been obtained, (3) Institutional Review Board (IRB) approval has been obtained, and (4) all applicable regulatory authorization(s) have been obtained.

3.3 Institutional Biosafety Committee

Section IV-B-2 of the NIH Guidelines mandates the formation of an Institutional Biosafety Committee and the appointment of a Biological Safety Officer (BSO) by the institution.  For more information regarding the NIH-mandated requirements for the IBC and the most up-to-date version of the NIH Guidelines, please visit the NIH website.  The Northwestern University IBC receives its appointment from the President of Northwestern University upon the recommendation of the Vice President for Research as described in the Charter for the Institutional Biosafety Committee issued in January 2009.  The responsibilities of the IBC and its institutional affiliates are described collectively in the Charter for the IBC as well as the IBC Policies and Procedures Manual.

3.3.1 Membership and Procedure of the IBC

The IBC must be comprised of no fewer than five members so selected that they collectively have experience and expertise in recombinant or synthetic nucleic acid molecule technology and the capability to assess the safety of recombinant or synthetic nucleic acid molecule research and to identify any potential risk to public health or the environment.  At least two members shall not be affiliated with the institution (apart from their membership on the IBC) and who represent the interest of the surrounding community with respect to health and protection of the environment. 

According to the NIH Guidelines, the IBC shall include at least one individual with expertise in plant, plant pathogen, or plant pest containment principles when experiments utilizing Appendix P, Physical and Biological Containment for Recombinant or Synthetic Nucleic Acid Molecule Research Involving Plants, require prior approval by the IBC.  Due to the limited amount of research conducted at Northwestern University that is subject to Appendix P, the Northwestern IBC does not retain an individual with such expertise.  Instead, the Northwestern IBC will call upon an individual with the appropriate expertise in the event it is necessary.

The IBC shall include at least one scientist with expertise in animal containment principles when experiments utilizing Appendix Q, Physical and Biological Containment for Recombinant or Synthetic Nucleic Acid Molecule Research Involving Animals, require IBC prior approval. 

The NIH Guidelines identifies several categories of research, such as recombinant or synthetic nucleic acid molecule research at BSL3, BSL4, or Large Scale (greater than 10 liters), that requires a Biological Safety Officer’s presence on the IBC.  Northwestern University maintains a Biosafety Officer and an Associate Biosafety Officer at all times and thus fulfills this mandate.

When the institution participates in or sponsors recombinant or synthetic nucleic acid molecule research involving human research participants, the institution must ensure that:  (1) the IBC has adequate expertise and training (using ad hoc consultants as deemed necessary); (2) all aspects of Appendix M of the NIH Guidelines have been appropriately addressed by the Principal Investigator; (3) no research participant shall be enrolled in a human gene transfer experiment until the NIH protocol registration process has been completed; and (4) final IBC approval is granted only after the NIH protocol registration process has been completed.  Institutional Biosafety Committee approval must be obtained from the clinical trial site.

3.3.2 Functions of the IBC

As outlined by the NIH Guidelines, the IBC is responsible for reviewing recombinant or synthetic nucleic acid molecule research conducted at or sponsored by the institution for compliance with the NIH Guidelines.  At the discretion of the IBC itself, the review includes the following:

  • Assessment of the containment levels for the proposed research
  • Assessment of the facilities, procedures, practices, and training and expertise of personnel involved in the research

For HGT studies, the IBC must fulfill additional regulatory requirements including:

  • Ensuring compliance with Appendix M of the NIH Guidelines
  • Ensuring that no research participant is enrolled in a HGT experiment until the NIH protocol registration process has been completed, IBC approval has been obtained, IRB approval has been obtained, and all applicable regulatory authorizations have been obtained
  • Reviewing the recommendations of the Recombinant DNA Advisory Committee (RAC) for HGT studies, where applicable, and ensuring that the PI has appropriately responded to them
  • Ensuring that final IBC approval is granted only after the NIH protocol registration process has been completed
  • Ensuring compliance with all surveillance, data reporting, and adverse event reporting requirements set forth in the NIH Guidelines.

Following review, it is the responsibility of the IBC to notify the PI of the result of the IBC’s review and subsequent approval, if applicable.  The IBC is also responsible for setting the containment level of research involving recombinant or synthetic nucleic acids in organisms from Risk Groups 2, 3, and 4, as well as such research involving live animals and plants.  The IBC is granted the authority by the NIH to lower the containment level for certain experiments as specified in Section III-D-2-a of the NIH Guidelines.

The IBC is also responsible for periodically reviewing the recombinant and synthetic nucleic acids research conducted at the institution to ensure compliance with the NIH Guidelines.  Finally, the IBC must adopt and ensure implementation of emergency plans covering accidental spills and personnel contamination resulting from recombinant or synthetic nucleic acids research. 

3.3.3 Relationship with Research Safety

At Northwestern University, Research Safety works closely with the IBC to manage many day-to-day operations of the IBC and to ensure IBC policy implementation.  Several members of the Research Safety staff act as administrators to the IBC and maintain accurate records of the IBC meetings.  Some Research Safety staff also sit on the IBC and act as voting members.  The IBC has entrusted Research Safety with IBC policy implementation and ensuring compliance with IBC decisions.  Although the IBC and Research Safety are empowered through different means, they work together to ensure the safety of the Northwestern University research community as well as the broader, Chicago-land community as well.

4.0 General Biosafety Principles

4.1 Risk Assessment

To apply biological safety practices rationally while handling a potential pathogen, one must perform a risk assessment.  A thorough risk assessment considers the following:

  • The agent’s biological and physical nature
  • The sources likely to harbor the agent
  • Host susceptibility
  • The procedures that may disseminate the agent
  • The best method to effectively inactivate the agent

Globally, numerous government agencies have classified microorganisms that are pathogenic to humans into Risk Groups (RG) based on the transmissibility, invasiveness, virulence or capability of causing disease, lethality, and the availability of vaccines or therapeutic interventions.  Risk groupings of infectious agents usually correspond to Biosafety Levels (BSL or BL), which describe the recommended containment practices, safety equipment, and facility design features necessary to safely handle both benign and pathogenic microorganisms.  The list of pathogenic microorganisms includes bacteria, viruses, fungi, parasites, and other infectious entities.  The classification scheme ascends in order of increasing hazard from Risk Group 1 (RG1) agents, which are non-pathogenic for healthy adults to RG4 agents, which display a high morbidity and mortality and for which treatments are not generally readily available.

In the United States, Risk Groups are only assigned to pathogens that pose a risk to humans.  That is, only human and animal pathogens capable of infecting humans are assigned a risk group.  Animal pathogens and plant pathogens are not assigned risk groups in the United States.  The Risk Group listing found in the NIH Guidelines is an accepted standard even when recombinant or synthetic nucleic acids (rNA) technology is not being used.

The American Biological Safety Association, International (ABSA) also provides a comprehensive Risk Group database.  This database also provides references to other global agencies and their Risk Group classification. 

Another reliable source of information regarding human pathogens is available from the Health Canada website.  This site provides detailed Pathogen Safety Data Sheets for a vast number of human pathogens.  These documents describe the hazardous properties of a specific pathogen and recommendations for work involving the agent in a laboratory setting.  These documents are also designed to aid in the risk assessment for working with the described agents. 

Microorganisms that are RG1 require standard laboratory facilities and microbiological practices whereas those in RG4 require elaborate procedures, engineering controls, and facilities for maximum containment.  Many of the agents likely to be handled experimentally at Northwestern University are RG2 or RG3 pathogens, designated as moderate and high hazard, respectively.  These agents typically require more sophisticated engineering controls (such as facilities and equipment) than standard laboratories.  Special handling and decontamination procedures are often required as well.

 

Table 1: Risk Group Classification Definitions and Examples

Definitions and examples for Risk Groups 1-4
  DEFINITION EXAMPLES
Risk Group 1 Agents not associated with disease in healthy adult humans E. coli K-12, Saccharomyces cerevisiae
Risk Group 2 Agents associated with human disease that is rarely serious and for which preventative or therapeutic interventions are often available E. coli O157:H7, SalmonellaCryptosporidium, Hepatitis A, B, C, D, and E viruses
Risk Group 3 Agents associated with serious or lethal human disease for which preventative or therapeutic interventions may be available Yersinia pestis, Brucella abortus, Mycobacterium tuberculosis, Human Immunodeficiency Virus (HIV), Transmissible spongiform encephalopathies (TSE) agents
Risk Group 4 Agents associated with serious or lethal human disease for which preventative or therapeutic interventions are not usually available Ebola virus, Macacine herpesvirus (Monkey B virus)
>

 

Microorganisms classified as RG 2 or higher have been reported to cause infection and disease in otherwise healthy adults.  Many have been associated with laboratory-acquired infections.  Furthermore, lab-acquired infections with RG2 agents are significantly more common that lab-acquired infections with RG3 or RG4 agents.  The progression from invasion to infection to disease following contact with an agent depends upon the route of transmission, inoculum, invasive characteristics of the agent, and resistance of the person exposed.  Not all contacts result in infection and even fewer develop into clinical disease.  Even when disease occurs, severity can vary considerably.  It is prudent to assume virulence and handle such agents at the prescribed biosafety level.

 

4.2 Routes of Infection

Pathogens may be transmitted via one or more routes of infection.  The route(s) depends on the characteristics of the particular pathogen.  The most common routes of infection are inhalation of infectious aerosol, dusts, or small droplets, exposure of mucous membranes to infectious droplets, ingestion from contaminated hands or utensils, or percutaneous inoculation (injection, incision, or animal bite).  Appropriate precautions can be implemented to avoid such experts. 

The Pathogen Safety Data Sheets available from Health Canada are a valuable resource for identifying the most common route(s) of infection for a particular pathogen.  For additional questions or concerns, please contact Research Safety (researchsafety@northwestern.edu) or the Biosafety Officer Rob Foreman (robert.foreman@northwestern.edu).

 

4.3 Exposure Sources
4.3.1 Clinical and Pathological Specimens

Any Specimen from human patients or animals may contain infectious agents.  Specimens most likely to harbor such microorganisms include blood, sputum, urine, semen, vaginal secretions, cerebrospinal fluid, synovial fluid, pleural fluid, pericardial fluid, peritoneal fluid, amniotic fluid, feces, and tissues.  Personnel in laboratories and clinical areas handling human blood, body fluids, and non-human primate material, or even human cell lines that have been screened for pathogens should practice Universal Precautions.  Universal Precautions is an approach to infection control wherein all human blood and certain human body fluids are treated as if known to be infectious and contain blood-borne pathogens such as HIV, Hepatitis B Virus (HBV), and Hepatitis C Virus (HCV).  Such personnel are required by law (OSHA 29 CFR 1910.1030) to undergo blood-borne pathogen training. 

At Northwestern University, this training requirement can be satisfied online in myHR Learn.  Your PI or Safety Designate must indicate in Lumen that you are exposed to bloodborne pathogens or human source materials; this will assign the Bloodborne Pathogens certification to you in myHR Learn. 

For additional questions, please contact researchsafety@northwestern.edu.

Animals may harbor pathogens that are dangerous to humans.  Some pathogens harbored by animals are infectious to humans but do not cause symptoms or a noticeable disease in the natural host animal.  For personnel handling these animals or their tissues or body fluids, it is recommended that an analogous approach to infection control be adopted as when handling human tissue and body fluids.  This approach is often called General Precaution and assumes that these animals, as well as their tissues, blood, and body fluid, are potentially infectious.

4.3.2 Cultures

Accidental spilling of liquid infectious cultures is an obvious hazard due to the generation of aerosols and/or small droplets.  However, even routine manipulations of cultures may release microorganisms via aerosol and/or small droplet formation.  Such manipulations include, but are not limited to:

  • Popping stoppers from culture vessels
  • Opening closed vessels after vigorous shaking/vortexing
  • Spattering from flame-sterilized utensils
  • Expelling the final drop from a pipette
  • Spinning microfuge tubes in a standard microfuge
  • Immediately following vortexing

Manipulate cultures of infectious agent carefully to avoid the uncontrolled release of aerosols or the generation of large droplets or spills.  Centrifugation should involve the use of gasket-sealable tubes, carriers, and rotors, when available.  Seal microplate lids with tape or replace them with adhesive-backed film.  Load, remove, and open tubes, plates, and rotors within a biological safety cabinet or chemical fume hood.  Keep in mind that the fume hood will protect you from your sample but will not protect your sample from potential room contamination.  For more information on the use of biological safety cabinets and chemical fume hoods, please refer to Section 5.3 Engineering Controls.

When preparing aliquots of infectious agent for long-term storage, consider that lyophilization of viable cultures may release high concentrations of dispersed particles if ampules are not properly sealed.  Breakage of ampules in liquid nitrogen dewars may also present hazards because of survival of pathogens in the liquid phase.

Equipment used for manipulations of infectious materials, such as cell sorters and automated harvesting equipment, must be evaluated to determine the need for secondary containment and to consider decontamination issues.  Costly equipment of this type are often operated as multi-user or core facilities; the inherent variability in risk from one project to another makes it imperative that operators and users of these facilities understand risks and methods for risk mitigation.

Use of well-established human or animal cell cultures in laboratories requires special consideration.  Cell or tissue cultures in general present few biohazards, as evidenced by their extensive use and low risk of infection to laboratory personnel.  Indeed, there are very few reports of infection of laboratory personnel associated well-established human or animal cell cultures.  However, when a cell culture is inoculated with or known to contain an etiologic agent, it should be classified and handled at the same biosafety level as the agent itself. 

Biosafety Level 2 containment conditions should be used for cell lines of human origin, even those that are well-established like HeLa and HEK293, and for all human clinical material (such as tissues and fluids obtained from surgery or autopsy).  Non-human primate cell cultures derived from lymphoid or tumor tissue, cell lines exposed to or transformed by a non-human primate oncogenic virus, and all non-human primate tissue should also be handled at BSL2.  Manipulation of large volumes of human or non-human primate tissues or cell lines, or manipulations that have the potential to create aerosols, should all be performed within a biosafety cabinet.

4.3.3 Animals

It is imperative that care and thoughtfulness be exercised when using animals to isolate and propagate microorganisms, study pathology, or produce antibodies.  Laboratory animals may harbor microorganisms that can produce human diseases following exposure through bites, scratches, or excreted material.  In the process of inoculating animals, an investigator can be exposed to infectious material by accidental self-inoculation or inhalation of infectious aerosols.  During surgical procedures, necropsies, and processing of tissues, aerosols can be produced unintentionally. Or the operator can inflict self-injury with contaminated instruments.  Since animal excreta can also be a source of infectious microorganisms, investigators should take precautions to minimize aerosols and dust when changing bedding and cleaning cages.  The Center for Comparative Medicine (CCM) offers training for any personnel working with animals.  For more information on training, visit the CCM website or call CCM at (312) 503-2758.

 

4.4 Laboratory Exposure Potential

While it can be assumed that investigators in research and clinical laboratories have more experience than students in teaching labs, safety consciousness and the establishment of a culture of safety is an ongoing process that cannot be ignored.  Complacency should be avoided in even the most senior scientists.  Risk assessments should be an ongoing process in all laboratories taking into account the individual, training received, research conducted, agents and equipment used, operational controls, and facility limitations.

4.4.1 Teaching Laboratories

Whenever possible, it is recommended that teaching laboratories make use of avirulent strains of infectious microorganisms.  It is critical, however, that even attenuated microorganisms be handled with care.  Students should be cautioned against and train do prevent unnecessary expose, as exposure to an avirulent strain may cause harm to an immunocompromised individual.  Establishment of safety consciousness is integral to the conduct of good science.

4.4.2 Research Laboratories

Experiments in research laboratories using high concentrations or large quantities of pathogens increase the risk of exposure.  The use of animals in research on infectious diseases also presents greater opportunities for exposure.  As recommended for teaching laboratories, attenuated or avirulent strains should be used whenever possible.

4.4.3 Clinical Laboratories

Personnel in laboratories performing diagnostic work-up of clinical specimens from human or animal patients/subjects are often at risk of exposure to infectious agents.  The absence of an infectious disease diagnosis does not preclude the presence of pathogens.  This is especially true of materials from patients who have received immunosuppressive therapy since such treatment may activate latent infections or make hosts more likely to harbor infectious agents.

 

4.5 Health Status

Some circumstances, albeit rare, warrant special considerations or measures to prevent infection of laboratory personnel by certain microorganisms:

It is recommended that all laboratory personnel discuss occupational risks with their personal healthcare provider.  This is especially important when working with biohazardous or potentially biohazardous agents.  Certain medical conditions may increase an individual’s risk of health problems when working with animals and/or pathogenic microorganisms.  These conditions can include, but are not limited to: diabetes, pregnancy, certain autoimmune diseases, immunodeficiency or immunosuppression, animal-related allergies, chronic skin conditions or respiratory disorders, and steroid therapy, even if only temporary.

5.0 Biohazard Containment

Although the most important aspect of biohazard containment is the awareness and care used by personnel in handling infectious materials, certain features of laboratory design, ventilation, and safety equipment can prevent dissemination of pathogens, should their accidental release occur.

 

5.1 Biosafety Levels

Biosafety Levels consist of combinations of laboratory practices and procedures, safety equipment and laboratory facility design features commensurate with laboratory operations performed, and are based on the potential hazards imposed by the agents used and for the specific lab activity.  It is the combination of practice, equipment, and facility that form the basis for physical containment strategies for infectious agents.  There are four biosafety levels with Biosafety Level 1 (BSL1 or BL1) being the least stringent and Biosafety Level 4 (BSL4 or BL4) being the most stringent.  The general recommendations for the four Biosafety Levels are as follows:

  • BSL1 is recommended for agents that are non-pathogenic
  • BSL2 is recommended for potentially pathogenic and pathogenic agents transmitted by direct contact (percutaneous inoculation, ingestion, or mucous membrane exposure)
  • BSL3 is recommended for pathogenic agents with the potential to be transmitted via aerosol
  • BSL4 is recommended when total separation between the pathogenic agent and investigator is critical

Risk Group designations often correlate directly with the physical containment level appropriate for a given research activity.  It is important to note that while Risk Group designations are set by health agencies and inflexible, Biosafety Level designations are set by the local Institutional Biosafety Committee and may be flexible.  A brief description of the requirements (according to the BMBL) for each Biosafety Level is provided as well as a correlation between Risk Group and Biosafety Level is provided below in Table 2 and Table 3, respectively.  It is important to note that this manual focuses on Biosafety Level 2.

 

Table 2: Summary of Biosafety Level Requirements

Biosafety Level Requirements, Levels 1-4
  BIOSAFETY LEVEL
  1 2 3 4
Restricted access1 Yes Yes Yes NA
Controlled access2 No Desirable Yes Yes
Isolation of laboratory No No Desirable Yes
Room sealable for decontamination No No Yes Yes
Inward airflow ventilation No Desirable Yes Yes
Mechanical ventilation via building system No Desirable Desirable No
Mechanical, independent ventilation No No Desirable Yes
Filter air exhaust No No Yes Yes
Double door entry No No Yes Yes
Airlock No No No Yes
Airlock with shower No No No Yes
Effluent treatment No No No Yes
Autoclave on-site Yes Yes Yes Yes
Autoclave in laboratory room No No Yes Yes
Double-ended autoclave No No Desirable Yes
Class I or II BSC3 No Yes Yes Desirable
Class II BSC No No Desirable Yes

1. Restricted access, laboratory doors are closed during work hours and locked after hours

2. Controlled access, only laboratory staff is allowed access at any time

3. BSC, Biological Safety Cabinet


 

Table 3: Relationship of Risk Groups to Biosafety Levels, Practices, and Equipment

Risk groups with biosafety levels, laboratory type, laboratory practices and safety equipment
RISK GROUP BIOSAFETY LEVEL EXAMPLES OF LABORATORY TYPE LABORATORY PRACTICES SAFETY EQUIPMENT
1 Basic – BSL1 Basic teaching SMP1 None; open bench work
2 Basic – BSL2 Primary health services; primary level hospital; diagnostic, teaching, and public health; most biomedical research SMP plus protective clothing; biohazard sign Open bench plus BSC2 for potential aerosols
3 Containment – BSL3 Special diagnostics; highly pathogenic biomedical research BSL2 Practices plus special clothing, respiratory protection, controlled access, directional airflow BSC and/or other primary containment for all activities
4 Maximum Containment – BSL4 Dangerous pathogens units; extremely pathogenic biomedical research BSL3 Practices plus airlock entry/exit, shower exit, special waste disposal Class III BSC or positive pressure suits, double-ended autoclave, filtered air

1. SMP, Standard Microbiological Practices. Standard Microbiological Practices consists of aseptic techniques and other practices that are necessary to prevent contamination of the laboratory with the agents being handled and contamination of the work with agents from the environment. SMP is used to keep the agents being handled inside their primary containers without any other organisms getting in and contaminating the research materials. The main objective of SMP is to ensure that contamination does not affect the research results. (Source: Appendix D. Good Microbiological Practice) For more information on SMP, please refer to Appendix C: Good Microbiological Technique (Page 62).

2. BSC, Biological Safety Cabinet


 

For more information regarding Biosafety Levels, consult the Biosafety in Microbiological and Biomedical Laboratories, 5th Edition (BMBL).

Experiments involving rsNA are also governed by another method of providing containment called biological containment.  For biological containment, highly specific biological barriers are considered in the risk assessment process.  Specifically, biological containment considers natural barriers that limit either (1) the infectivity of a vector or vehicle (plasmid or virus) for specific hosts, or (2) its dissemination and survival in the environment.  For additional information on biological containment, consult the NIH Guidelines.

 

5.2 Practices and Procedures

The following practices correspond to BSL2 containment.  They are important for the prevention of laboratory infection and disease, as well as for the reduction of the potential for contamination of experimental material.  These practices and procedures provide the foundation for the more restrictive containment of RG3 organisms.  If you are considering research with an RG3 organism, or feel as though your research may require BSL3 containment, contact Research Safety at (312) 503-8300 (Chicago) or (847) 491-5581 (Evanston) or researchsafety@northwestern.edu.

5.2.1 Personal Hygiene
  • Do not eat, drink, take medicine or dietary supplements, chew gum, use tobacco, apply cosmetics or lotions, or handle contact lenses in the laboratory.
  • Do not store food and drink for human consumption in laboratory.  This includes refrigerators and freezers as well as desks, cabinets, etc.
  • Wash hands frequently after handling infectious materials, after removing latex/nitrile gloves and protective clothing, and always before leaving the laboratory.
  • Keep hands away from the mouth, nose, eyes, face, and hair.
  • Do not store personal items such as coats, boots, bags, and books in the laboratory.
5.2.2 Responsibilities and Pre-requisites for Laboratory Work
  • A laboratory safety manual should be assembled outlining activities and defining standard operating procedures.  In most cases, the lab’s Northwestern Institutional Biosafety Committee (IBC) protocol, together with this Biosafety Manual, will provide the necessary information to work safely.
  • When working with rNA and/or agents at BSL2 or higher, Northwestern IBC approval must be obtained before work can begin.  Please note that, per the NIH Guidelines, the local IBC determines the Biosafety Level for a given research project.  If you are uncertain of the Biosafety Level of your research, please contact Research Safety at (312) 503-8300 (Chicago) or (847) 491-5581 (Evanston) or researchsafety@northwestern.edu.
  • Principal Investigators and/or laboratory supervisors are responsible for training employees and ensuring that all personnel are informed of hazards.
  • When RG2 (or higher) pathogens are used in long-term studies, post a biohazard sign at the laboratory entrance, identifying the Personal Protective Equipment (PPE) necessary for safe entry and emergency contact personnel.
  • Report all accidents and spills to the laboratory supervisor.  All laboratory personnel should be familiar with the emergency spill protocol and the location of cleanup equipment.
  • Good housekeeping practices are essential in laboratories engaged in work with infectious microorganisms.  The establishment of a regular cleaning regimen, including decontamination of all shared equipment and common areas, is highly recommended.
  • Advise custodial staff of hazardous areas and locations that should be off-limits.  Use appropriate signage, including biohazard signs.
5.2.3 Laboratory Procedures for Handling Infectious Microorganisms
  • Plan and organize materials/equipment before starting work.
  • Keep laboratory doors closed; limit access to lab personnel only.
  • Wear a fully fastened laboratory coat when working with infectious agents.  Wear protective gloves whenever handling potentially hazardous materials, including human blood and body fluids.
  • Remove and leave all protective clothing, including gloves and lab coat, within the laboratory before exiting.
  • Never mouth-pipette; use mechanical pipetting devices.
  • When practical, perform all aerosol-producing procedures such as shaking, grinding, sonicating, mixing, and blending in a properly operating biological safety cabinet.  Note that some equipment may compromise the cabinet function by disturbing the air curtain.
  • Centrifuge materials containing infectious agents in shatter-resistant, closable tubes.  Use a centrifuge with a sealed rotor or screw-capped safety cups.  After centrifugation, open the tubes in a biological safety cabinet.
  • Avoid using needles and syringes whenever possible.  When necessary, discard used syringe-needle units in a sharps container without removing or recapping the needles.  NEVER RECAP NEEDLES.
  • Cover countertops with plastic-backed disposable paper where hazardous materials are used to absorb spills and facilitate cleanup.
  • Wipe work surfaces with an appropriate disinfectant after experiments and immediately after spills.  For help selecting the appropriate disinfectant, refer to the Chemical Disinfection section.
  • Decontaminate all contaminated or potentially contaminated materials by appropriate methods before disposal.  Refer to the Disposal of Wastes Contaminated with Infectious Agents section for more information.
5.3 Engineering Controls
5.3.1 Laboratory Design

Generally speaking, more virulent organisms and agents require a greater degree of physical containment.  Physical containment is composed of two main parts: primary containment and secondary containment.  Proper safety equipment provides primary containment.  Laboratory design provides secondary containment.  Research Safety is available for consultation on these matters.  For consultation, please contact Research Safety at (312) 503-8300 (Chicago) or (847) 491-5581 (Evanston) or researchsafety@northwestern.edu.

5.3.2 Laboratory Ventilation

In order to control containment, it is important that laboratory air pressure be lower than that in the adjacent spaces.  This negative air pressure differential ensures that air will enter the laboratory and not egress into the hallway.  Labs with multiple rooms should have the airflow balanced such that the most negative room is where the most hazardous work takes place.  To maintain negative room pressure, laboratory doors must be kept closed.

Exhaust air from biohazardous laboratories should not be recirculated in the building.  It should be ducted to the outside and released from a stack remote from the building air intake.  In certain special situations, including many BSL3 labs, air exhausting from a containment facility should be filtered through High Efficiency Particulate (HEPA) filters, which can capture microorganisms.

5.3.3 Biological Safety Cabinets (BSC)

Biological Safety Cabinets (also called Biosafety Cabinets or BSCs) are the primary means of containment developed for working safely with infectious organisms.  When functioning correctly and used in conjunction with Standard Microbiological Practice (SMP), BSCs are very effective at controlling infectious aerosols.  BSCs are a type of laboratory safety equipment utilizing airflow to maintain the safety of the worker.  BSCs are distinct from other safety equipment of this type, such as chemical fume hoods and clean benches.  BSCs are designed to provide personnel, environmental, and product protection when appropriate practices and procedures are followed.

This section provides a brief overview of the type of BSC most likely to be found in laboratories at Northwestern. 

5.3.3.1 Biological Safety Cabinet Types

There are three kinds of BSCs, designated as Class I, Class II, and Class III, that have been developed to meet varying research and clinical needs.  All BSCs use HEPA filters to ensure no particles enter the working space of the cabinet, get recirculated within the cabinet, or get exhausted to the environment.  Two types of Class II BSCs are commonly used at Northwestern, described below.  Both are appropriate for manipulating and containing RG2 and RG3 pathogens and agents.  There are various sub-types of each class of BSC.  The sub-type depends on the percentage of air recirculated or exhausted.

Class II Type A – Draws air in from the room, 70% of which gets recirculated within the cabinet to create the air curtain and 30% gets filtered and exhausted into the laboratory.

Class II Type B – Draws air in from the room, 30% of which gets recirculated within the cabinet to create the air curtain and 70% gets filtered and exhausted.  Type B cabinets are different from Type A in that 100% of the air gets exhausted through a ducted line to the outdoor environment.

Horizontal laminar flow clean benches are not biological safety cabinets and should never be used for work with hazardous or potentially hazardous materials or agents.  This equipment protects the material in the cabinet but not the worker or the environment. 

Similarly, chemical fume hoods are not biological safety cabinets.  They draw air in from the room, potentially protecting the worker, but do not protect the material in the cabinet and exhaust unfiltered air into the environment.  Chemical fume hoods may be appropriate for some biohazardous agents.

Some BSCs are equipped with an ultraviolet (UV) lamp.  These are installed with the intention of inactivating or destroying microbes.  Ultraviolet lamps have limited ability to inactivate biological agents and their efficiency is dramatically reduced by a variety of factors including age of the bulb, media containing the microbe, distance to the intended target, etc.  Moreover, UV lamps pose a hazard to the worker as exposure to UV light may cause eye and skin damage.  Custodial workers are often unwitting, incidental casualties of an UV lamp left on overnight.  Ultraviolet lamps are not recommended as the sole source of decontamination within a BSC and extreme care should be taken with their use.

5.3.3.2 Biological Safety Cabinet Operation

Loading Materials and Equipment

  • Disinfect interior surfaces of the BSC
  • Load only items needed for the procedure; avoid clutter and excess storage
  • Do not block the front or rear exhaust grilles
  • Disinfect the exterior of all containers prior to commencing
  • Segregate clean and dirty materials on separate sides of the cabinet; material should flow from sterile to non-sterile
  • Materials should be placed at least six inches back from the front grille
  • Never place non-sterile items upstream of sterile items
  • Ensure view screen is at the appropriate height

Start-up

  • Turn on blower and fluorescent light; close drain valve
  • Before loading equipment into the BSC, allow the air to circulate to purge potentially contaminated air.
    • The NIH recommends that the blower run for 15 minutes prior to using the BSC.
    • Research Safety and the CDC recommend waiting at least 4-5 minutes.
  • Check grilles for obstructions and disinfect all interior work surfaces with a disinfectant appropriate for the agent in use
  • Restrict traffic in the vicinity of the BSC

Recommended Work Technique

  • Wash hand thoroughly with soap and hot water before and after procedure
  • Wear sterile gloves and lab coat/gown; use aseptic technique
  • Avoid blocking the front grille; do not work over the grille
  • Adjust chair so armpits are at the same elevation as the lower edge of the view screen
  • Avoid rapid movement during procedures both within and in the vicinity of the BSC
  • Move hands and arms straight into and out of the work area; never sweep hands/arms out of the work area during a procedure

Final Purging and Wipe-down

  • Disinfect the exterior of all containers before removal from the work zone
  • Decontaminate interior work surfaces of the BSC with an appropriate disinfectant effective against the agent being manipulated
  • After completing work and decontamination, run the BSC blower for 2 minutes before unloading materials

Decontamination and Spills

  • All containers and equipment should be surface decontaminated and removed from the cabinet when work is completed.  The final surface decontamination of the cabinet should include a wipe down of the work zone.  Investigators should remove their gloves and gowns and wash their hands as the final step in SMP.
  • Small spills within the BSC
    • Do not turn off the BSC
    • Cover the spill with absorbent paper towels
    • Carefully pour an appropriate disinfectant onto the towel-covered spill; a 10% bleach solution is recommended to reduce the amount of disinfectant needed as a result of dilution by addition to the spill
    • Removing the contaminated toweling
    • Place contaminated material into the biohazard bag
    • Splatter onto items within the cabinet, as well as the cabinet interior, should be immediately wiped with a towel dampened with decontaminating solution
    • Gloves should be changed after the work surface is decontaminated
    • Hands should be washed whenever gloves are changed
  • Large spills within the BSC
    • Do not turn off the BSC
    • Ensure drain valve is closed
    • Decontaminate and remove all items from within the BSC
    • Pour an appropriate disinfectant directly onto the work surface and through the grilles into the drain pan; a bleach solution is recommended to mitigate the effects of dilution when mixed with the spilled material
    • Allow for at least 20-30 minutes to allow for contact time
    • Empty the drain pan into a collection vessel containing more disinfectant
    • Dispose of the effluent as a chemical hazard but be sure to inform Research Safety of it’s source
    • If the spill contains radioactive material, a similar method may be used and Health Physics staff should be alerted

6.0 Disposal of Waste Contaminated With Infectious Agents

The following biohazardous waste disposal guidelines are designed to protect the public, the environment, laboratory and custodial personnel, waste haulers, and landfill/incinerator operators at each stage of the waste handling process.  Generators of biohazardous waste must ensure that the labeling, packaging, and intermediate disposal of waste conforms to these guidelines.  Use the definitions below to facilitate your understanding of appropriate decontamination and disposal guidelines.

  • Decontamination refers to a process of removing disease-causing microorganisms and agents, rendering an object safe for general handling.
  • Disinfection refers to a process that kills or destroys most disease causing microorganisms, except spores.
  • Sterilization refers to a process that destroys all forms of microbial life, including spores, viruses, and fungi.

 

6.1 What is Infectious Waste?

The following are common examples of items usually considered to be infectious waste:

  • Microbiological laboratory wastes such as cultures derived from clinical specimens and pathogenic microorganisms
  • Laboratory equipment which may have come in contact with clinical specimens, pathogenic microorganisms, or cultures derived from them
  • Tissues, large quantities of blood, and/or body fluids from humans
  • Tissues, large quantities of blood, and/or body fluids from one or more animals carrying an infectious agent that can be transmitted to humans
  • Contaminated sharps (needles, broken glass, etc.)

The following items require decontamination prior to disposal even though they are not necessarily considered infectious waste:

  • Regulated rsNA-containing organisms
  • Exotic and/or virulent plant pathogens
  • Exotic and/or virulent animal pathogens

The following are usually not included in the definition of waste but should be placed in containers, such as plastic bags, prior to disposal to contain the waste.  If these items are mixed with infectious wastes, they must be managed as though they were infectious.  For this reason, segregate infectious waste from other waste.

  • Items soiled or spotted, but not saturated, with human blood or bodily fluid (e.g. blood-spotted gloves, gowns, dressings, etc.)
  • Containers, packages, waste glass, laboratory equipment, and other materials that have had no contact with blood, body fluids, clinical cultures, or infectious agents
  • Noninfectious animal waste (e.g. manure, bedding, tissue, blood, and body fluids) or cultures from an animal that is not known to be carrying an infectious agent that can be transmitted to humans

 

6.2 Packaging of Waste

Laboratory materials used in experiments with potentially infectious microorganisms, such as discarded cultures, tissues, media, plastics, sharps, glassware, instruments, and laboratory coats, must either be handed off to a contractor licensed as an infectious waste treatment facility or be decontaminated before disposal or washing for reuse.  Collect contaminated materials in leak-proof containers labeled with the universal biohazard symbol.  Autoclavable biohazard bags are recommended.

For researchers at Northwestern University, autoclaving biohazard waste prior to disposal is neither required nor recommended.  Due to the tendency of biohazard waste to be variable and the potential for failed autoclave runs, all biohazard waste should be disposed of in the large collection bins located in research areas.  A third-party vendor collects all biohazard waste at Northwestern, transports it to a disposal facility, and is responsible for decontamination and disposal.  In the event that biohazard waste must be decontaminated by autoclave prior to disposal, after autoclaving, biohazard symbols on the container should be defaced.  This is to alert custodial and waste disposal personnel that a particular container is safe to handle.  Autoclaved biohazard waste should still be placed in the normal biohazard waste disposal stream to ensure proper disposal regulations are followed.

Uncontaminated sharps and other noninfectious items that may cause injury require special disposal even if they do not need to be decontaminated.  Sharps need to be collected in rigid, puncture-resistant containers to prevent wounding of workers, custodial personnel, and waste handlers.  If a package is likely to be punctured from sharp-edged contents, double bagging or boxing may be necessary.

Most common waste containers are available through the campus stock room or through Research Safety.  Specialized waste containers may need to be ordered if they are not carried by the stock room.  Research Safety provides a number of waste containers at no charge to the laboratory/PI.  The following is a description of supplies how it should be accurately employed for the disposal of waste.  For more information, please refer to Hazardous Waste.

 

Table 4: Hazardous Waste Supplies

Appropriate use and availability of various waste supplies
WASTE SUPPLIES APPROPRIATE USE AVAILABILITY
Biohazard Waste Labels Used to label all Biohazard Bags. Affix label before filling the bag. Available through Research Safety
Biohazard Bags Label with Biohazard Waste Labels and collect biohazard waste for disposal in Research Safety-provided collection bins. Use secondary containment while filling the bag and to transport all waste. Do not put anything in the bag that may puncture it such as pipets or other rigid materials. Available free-of-charge in the campus stock room
Pipet Keeper Use for disposal of serological pipets. Place pipets in Pipet Keeper before placing in a Biohazard Bag. Available free-of-charge in the campus stock room
Biohazard Sharps Container Collect biohazard sharps (e.g., needles, scalpels, razor blades, etc.) for disposal for Research Safety. Label the biohazard sharps container with PI name, lab location, and phone number. Available for purchase in the campus stock room
Vacuum Trap Filter Place a filter between the trap and the vacuum line to prevent liquids from entering the house vacuum system. Available for purchase in the campus stock room
Vacuum Trap Decontamination Schedule Label vacuum traps to document decontamination. Bleach is recommended for decontamination; add enough bleach to achieve a final concentration of 10% bleach. Available through Research Safety
Bleach1 Use for disinfection of liquid biohazard waste. A 10% bleach solution should be prepared daily (e.g., 1 part bleach to 9 parts water). Available for purchase in the campus stock room
Ethanol1 Use for disinfection of work surfaces and lab equipment. A 70% solution of ethanol should be prepared daily (e.g., 7 parts 190+ proof ethanol to 3 parts water). Available for purchase in the campus stock room
Alconox1 Use as directed by the manufacturer. This product is used for cleaning work surfaces and minor biological, chemical, and radiological spills. Available for purchase in the campus stock room
Spill Tray Secondary containment for hazardous liquid waste and reagent storage. Use inside appropriate storage cabinets, fume hood, or hazardous waste accumulation area. Available through Research Safety; provided at waste pickup or by request
Spill Kit The kit includes a clear chemical waste bag, a hazardous waste label, and a box of sorbent pads. Available through Research Safety; provided at waste pickup
Plastic Spray Bottles Use to prepare 1% Alconox detergent solutions for quick and easy clean up of the workspace and minor spills. Available through Research Safety
Broken Glass Box Use for the collection of glass containers and empty reagent bottles. If possible, clean all glass before placing it in the box. If glass is contaminated with biological hazards, chemicals, or radioactive materials, contact Research Safety for help with disposal. Available free-of-charge in the campus stock room
Incinerator Box Use to collect biohazard waste such as serological pipettes, empty culture flasks, etc. Avoid putting any liquids into the box. Dispose of the entire box when it is full; do not remove/replace the plastic liner. Available free-of-charge in the campus stock room
Hazardous Waste Labels Complete and label all hazardous waste. Affix label before filling the container. Available through Research Safety
Waste Jars Label with completed Hazardous Waste Label and use to collect liquid waste (for disposal as chemical waste), Eppendorf tubes, pipet tips, and other solid, chemically-contaminated waste Available free-of-charge in the campus stock room
1. For questions and concerns regarding the selection of an appropriate disinfection solution, please contact Research Safety at researchsafety@northwestern.edu or call (312) 503-8300 (Chicago) or (847) 491-5581 (Evanston).

 

6.3 Methods of Decontamination

Choosing the right method to eliminate or inactivate a biohazard is not always simple.  The choice depends largely on the treatment equipment available, the target agent, and the presence of interfering substances (e.g. media, high organic content, tissues) that may protect the organism from decontamination or mitigate the effects of the decontamination equipment.  A variety of treatment techniques are available, but practicality and effectiveness govern which is more appropriate.

Ideally, biohazardous waste should be decontaminated before the end of each working day unless it is to be collected for treatment off-site.  In the latter case, the waste should be packaged and stored (frozen, if pathological waste) until the scheduled pickup by the third-party contractor.  Biohazardous waste should never be compacted.  Ordinary lab wastes should be disposed of as routinely as possible to reduce the amount requiring special handling.

Regardless of its infectious nature, all organisms containing recombinant or synthetic nucleic acids (rsNA) must be decontaminated before entering the waste stream.  This includes non-pathogenic strains used for cloning, protein expression, diagnostics, etc.

6.3.1 Steam Sterilization

Decontamination by autoclave of biohazardous waste is not common practice at Northwestern University.  There are only a few, specific instances in which biohazardous waste is autoclaved prior to disposal.  All waste laboratory biohazardous waste is collected and disposed of by third-party contractors.  In the event decontamination of biohazardous waste is required prior to collection and disposal, it is best accomplished by steam sterilization.  The autoclave should be properly functioning and routinely monitored for efficacy with a biological indicator, such as spores of Bacillus stearothermophilus.  The tops of the biohazard bags should be opened just slightly to allow steam to enter the bag.  If the material is too dry, it may be necessary to add water to the package or autoclave bin.

When autoclaving biohazardous waste, it is recommended that the cycle be at least one hour.  However, the nature of the batch should determine the cycle duration.  For example, if the batch contains a dense organic substrate such as animal bedding or manure, one hour may not be sufficient to inactivate pathogens within the material.  A considerably longer exposure time may be required to effectively decontaminate such waste.  Since there is a practical limit to the time that can be spent autoclaving waste, alternative treatment options may be more effective and economical.

Use extreme caution when treating waste that is co-contaminated with volatile, toxic, or carcinogenic chemicals, radioisotopes, or explosive substances.  Autoclaving this type of waste may release dangerous gasses into the air (e.g., autoclaving waste previously treated with bleach may release chlorine).  Such waste should be chemically decontaminated, incinerated, or sent to a hazardous waste landfill.

Regardless of its infectious nature, all organisms containing recombinant or synthetic nucleic acids (rsNA) must be decontaminated before entering the waste stream.  This includes non-pathogenic strains used for cloning, protein expression, diagnostics, etc.  For help determining the best method of decontamination and disposal, consult the lab’s Biological Registration in Lumen, contact Research Safety at researchsafety@northwestern.edu, or call (312) 503-8300 (Chicago) or (847) 491-5581 (Evanston).

6.3.2 Sewage Treatment

Most fluid waste can be discarded through the sanitary sewer by pouring it into a sink drain and flushing the drain with water.  This includes human blood and infectious cultures as long as they have been properly decontaminated.  Care should be taken to avoid generation of aerosols.  The routine processing of municipal sewage provides chemical decontamination.  If the fluid is contaminated with infectious agents or biological toxins, however, it must be decontaminated by chemical disinfection or steam sterilization before sewer disposal.

Regardless of its infectious nature, all organisms containing recombinant or synthetic nucleic acids (rsNA) must be decontaminated before entering the waste stream.  This includes non-pathogenic strains used for cloning, protein expression, diagnostics, etc.  For help determining the best method of decontamination and disposal, consult the lab’s Biological Registration in Lumen, contact Research Safety at researchsafety@northwestern.edu, or call (312) 503-8300 (Chicago) or (847) 491-5581 (Evanston).

6.3.3 Chemical Disinfection

Where autoclaving is not appropriate, an accepted alternative is to treat material with a chemical disinfectant.  The disinfectant should be freshly prepared at a concentration known to be effective against the agent in use.  The disinfectant choice should be one that quickly and effectively kills/inactivates the agent at the lowest concentration and with minimal risk to the user.  However, higher concentrations of disinfectant are necessary to clean up large spills. It may not be possible to achieve active concentrations of some chemical disinfectants in the context of a spill. Other considerations, such as economy and shelf life, are also important.  Allow sufficient contact time to ensure complete inactivation.

It is important to be aware that common laboratory disinfectants can pose hazards to users.  For example, ethanol and quaternary ammonium compounds may cause contact dermatitis.  Importantly, once material has been treated with chemical disintectant, it should not be autoclaved. For further information about chemical disinfectants, contact Research Safety at researchsafety@northwestern.edu, or call (312) 503-8300 (Chicago) or (847) 491-5581 (Evanston).

6.3.3.1 Halogenated Chemicals

Halogenated chemicals such as hypochlorite (household bleach) are the least expensive and are also highly effective in decontaminating large spills.  Their drawbacks include short shelf life, easy binding to non-target organic substances, and corrosiveness.  Hypochlorite typically is diluted between 1:10 and 1:100 such that the available halogen is 0.01-5.0%.  A 1:10 dilution of household bleach will achieve this concentration.  Be aware that using chlorine compounds to disinfect some substances may inadvertently release toxic compounds.  For example, using chlorine compounds to disinfect substances co-contaminated with radioiodine may cause gaseous release of the isotope.

6.3.3.2 Alcohols

Alcohols, such as ethanol and isopropanol, are effective against vegetative forms of bacteria, fungi and enveloped viruses.  They will not efficiently destroy spores or non-enveloped viruses.  Alcohols are usually most effective at 70% concentration (a 7:10 dilution of alcohol in water).  Some intrinsic characteristics of alcohols limit their usefulness, such as flammability, poor penetration, presence of protein-rich materials, and rapid evaporation.  The fact that isopropanol and ethanol rapidly evaporate is a reason for 70% being the recommended concentration; the added water allows for increased contact time with the targeted agent.

6.3.3.3 Phenolics

Two phenol derivatives commonly found as constituents in hospital disinfectants are ortho-phenylphenol and ortho-benzyl-para-chlorophenol.  The antimicrobial properties of these compounds and many other phenol derivatives are much improved over those of the parent chemical.  Porous materials absorb phenolics and the residual disinfectant can become an irritant.  In high concentrations, phenol acts as a gross protoplasmic poison: penetrating and disrupting the cell wall and precipitating cellular proteins.  Phenolics are effective against nearly all microbes including bacteria, mycobacteria (e.g., M. tuberculosis), fungi, and viruses.  Many phenolic germicides are Environmental Protection Agency (EPA)-registered as disinfectants for use on environmental surfaces (e.g. laboratory surfaces).

6.3.3.4 Quaternary Ammonium Compounds

Quaternary ammonium compounds, or quaternaries, are a diverse class of disinfectants with varied chemical structure.  The microbial properties are unique to each compound.  Quaternaries are good cleaning agents but their microbicidal properties can be diminished by high water hardness and absorption or inactivation of the active ingredient by some cleaning materials.  Quaternaries have been shown to be effective against fungi, gram-positive bacteria, and enveloped viruses.  Generally, quaternaries are not effective in disinfection of spores, many gram-positive bacteria, mycobacteria, or enveloped viruses.

6.3.3.5 Formaldehyde

Formaldehyde is used as a disinfectant in both its liquid and gaseous states.  Liquid formaldehyde is used most commonly whereas gaseous formaldehyde is used only in specific circumstances such as high containment labs or when the entire atmosphere within a large space must be decontaminated.  Formaldehyde is most commonly sold and used in a water-based solution called formalin.  This aqueous solution is effective at decontaminating bacteria, including mycobacteria, fungi, viruses, and spores.  Depending on the target agent and other factors such as media and additional organic matter, effective concentrations can range from 2% to 8% formalin in water.  It is important to also note that the contact time required varies widely.  Formaldehyde poses a number of health risks to laboratory staff that are not seen with other disinfectants.  Consult with Research Safety if you are considering using formaldehyde as a disinfectant at researchsafety@northwestern.edu, or call (312) 503-8300 (Chicago) or (847) 491-5581 (Evanston).

6.3.3.6 Peroxides

Hydrogen peroxide is the most common peroxide-forming chemical used in healthcare and biomedical laboratory settings for decontamination.  Hydrogen peroxide is active against bacteria, spores, viruses, and fungi by producing destructive hydroxyl free radicals that attack membrane lipids, nucleic acids, and other essential cellular components.  Cellular catalase, produced by aerobic and facultative anaerobic microbes to protect them from metabolically produced hydrogen peroxide, is overwhelmed by concentrations used for disinfection.  Effective concentrations for decontamination range from 6% to 25%, depending on the target agent.  Stability of the peroxide during storage is a consideration; disinfection solutions containing peroxide should be prepared fresh.  Concentrations of peroxide as low as 3% have been shown to cause dermal irritation when direct exposure occurs.

6.3.3.7 Iodophors

An iodophor is a combination of iodine and a solubilizing agent.  The resulting complex provides a sustained release reservoir of iodine and releases small amounts of free iodine in aqueous solution.  Iodophors have been used as both an antiseptic and a disinfectant.  The best-known and most commonly used iodophor is povidone-iodine.  The Food and Drug Administration (FDA) has not cleared any liquid chemical sterilant or high-level disinfectant with iodophors as the main active ingredient.  Iodophors are thought to function by allowing iodine to penetrate the cell wall and disrupt protein and nucleic acid structure and synthesis.  Iodophors are effective in decontaminating bacteria, mycobacteria, and viruses but may require prolonged contact times to kill certain fungi and bacterial spores.

 

Table 5: Chemical Disinfectants and their Efficacy

Efficacy of disinfectants with recommended dilutions
DISINFECTANT CLASS EFFECTIVE AGAINST RECOMMENDED DILUTION
  Fungi Bacteria (Gram-positive and Gram-negative) Mycobacteria Spores Enveloped Viruses Non-enveloped Viruses  
Phenolics Good Good Good Ineffective Mild Varies 1%-5%
Hypochlorites Mild Good Fair Fair Mild Mild 0.005-0.5% free chlorine
1-10% household bleach
Alcohols Ineffective Good Good Ineffective Mild Mild 70-80%
Formaldehyde Good Good Good Good1 Good Mild 2-8%
Glutaraldehyde Good Good Good Good2 Mild Mild 2%
Iodophors Good Good Good Mild Mild Mild 0.5%
1. above 40°C
2. above 20°C

 

Table 6: Chemical Disinfectants and their Properties

Efficacy of disinfectants, with toxicity, stability and corrosive/flammability information
DISINFECTANT CLASS INACTIVATED BY TOXICITY STABILITY1 CORROSIVE FLAMMABLE
  Protein Hard Water Detergent Skin Eyes Lungs      
Phenolics Mild Mild MildC+ Yes Yes No Yes Yes No
Hypochlorites Good Mild MildC+ Yes Yes Yes No Yes No
Alcohols Mild Mild Not inactivated No Yes No Yes No Yes
Formaldehyde Not inactivated Mild Not inactivated Yes Yes Yes Yes No Possibly2
Glutaraldehyde Not inactivated Mild Not inactivated Yes Yes Yes Yes No No
Iodophors Good Mild MildA- Yes Yes No Yes Yes No

1. Stability for more than one week, stored away from light and air exposure

2. Formaldehydes can be flammable depending on the physical form and other conditions

C+. Can be inactivated by a cationic detergent

A-. Can be inactivated by an anionic detergent

7.0 Emergency Plans and Reporting

Regardless of how carefully one works, laboratory accidents occur and necessitate emergency response.  Emergency plans should be tailored for a given biohazardous situation and may vary from one lab to another.  The laboratory supervisor should prepare instructions specifying immediate steps to be taken.  These instructions should be displayed prominently in the laboratory and periodically reviewed with laboratory personnel.  No single plan will apply to all situations but the following general principles should form the basis of laboratory-specific plans.

 

7.1 Spill Protocols
  1. In the event of an extensive or explosive spill of virulent pathogen, everyone should leave the affected area immediately.  Contaminated clothing should be removed.  Exposed skin should be washed thoroughly.
  2. Close the laboratory door and post a “No Entry” sign indicating the presence of a hazard.  Notify the laboratory supervisor, PI, and Research Safety at (312) 503-8300 (Chicago) or (847) 491-5581 (Evanston).
  3. Determine the necessity and extent of medical treatment for individuals exposed to infectious microorganisms.  In the event of life-threatening injuries, contact emergency personnel; call 911.  For non-life-threatening injuries, personnel accidentally exposed via ingestion, skin puncture, or obvious in halation of an infectious agents should be given appropriate first aid and, if necessary, transported to Northwestern Memorial hospital emergency room.
  4. Contact Risk Management at 1-5582 from a campus phone.  Risk Management will make an appointment for individuals with injuries through Corporate Health (Chicago Campus) or Omega (Evanston Campus). 
  5. Do not reenter the room until large droplets have settled and aerosols have been cleared by the building ventilation system (a minimum of 30 minutes), and the extent of the hazard and its dissemination has been determined.
  6. Each person who enters the laboratory for cleanup should wear proper PPE.
  7. Use an appropriately concentrated disinfectant to clean up and decontaminate the area.  A supply of stock disinfectants should always be available.  For help determining the appropriate chemical disinfectant, refer to Tables 5 and 6 (page 30) or contact Research Safety at researchsafety@northwestern.edu, or call (312) 503-8300 (Chicago) or (847) 491-5581 (Evanston).
  8. Dispose of all contaminated, non-reusable materials in biohazard bags and discard as biohazard waste.
  9. Decontaminate all reusable materials used in cleanup procedures.

In an emergency situation, attention to immediate personal danger overrides containment considerations.  Currently, there is no known biohazard on either of the Northwestern University campuses that would prohibit properly garbed and masked fire or security personnel from entering any biological laboratory in an emergency.

 

7.2 Exposure Protocols

Determine the necessity and extent of medical treatment for persons exposed to infectious microorganisms.  Personnel accidentally exposed via ingestion, skin puncture, or obvious inhalation of an infectious agent should be given appropriate first aid and, if necessary, taken to the nearest emergency room.  For exposures to the eyes or mucous membranes, the exposed area should be flushed with running water for 15-20 minutes.  Seek medical attention as soon as possible following exposure: Corporate Health for Chicago campus and Omega for Evanston campus.  For both campuses, contact Risk Management at 1-5582.

 

7.3 Reporting

The importance of reporting accidental spills or exposure events is not to identify fault or failure within the laboratory or on the part of the laboratory personnel.  The goal of reporting such incidents is to identify opportunities to refine standard operating procedures for the laboratory and improve laboratory safety.  Importance is always placed on personal health as well as the health and safety of coworkers, the research community, and the general public.

The secure and responsible conduct of life sciences research depends, in part, on observation and reporting by peers, supervisors, and subordinates.  Individuals working with potentially infectious material and/or rsNA constructs with either direct or indirect, acute or latent disease potential (e.g., insertional mutagenesis due to exposure to a viral vector) must understand and acknowledge their responsibility to report activities that are inconsistent with a culture of responsibility or are otherwise troubling.  Likewise, institutional and laboratory leadership must acknowledge their responsibility to respond to reports of concerning behavior and undertake actions to prevent retaliation stemming from such reports.

There are numerous methods for reporting concerning behavior, as described above in the Code of Conduct and Culture of Responsibility section (page II).  Where appropriate, an individual should report to his/her PI, supervisor, Department Chair, or Dean.  In instances where confidentiality is of importance, reports may be made to Research Integrity (nu-ori@northwestern.edu or call (312) 503-0054), Ethics Point (a third-party vendor that will allow you to report your concerns anonymously or call (866) 294-3545), or Research Safety (researchsafety@northwestern.edu or call (312) 503-8300 (Chicago) or (847) 491-5581 (Evanston)).

Persons with health conditions, whether chronic or acute, that have the potential to place themselves or others at risk in the laboratory should self-report to their personal physician and/or Corporate Health (Chicago) or Omega (Evanston).  A decision about continued involvement with research involving infectious agents must be an informed decision that includes appropriate medical expertise.

8.0 Transporting Hazardous Biological Materials

For the purposes of transportation, dangerous goods are those substances or articles that have the potential to cause harm to individuals, property, or the environment in the event of an accident or incident.  Since infectious substances pose a risk to health (in the form of disease) if an individual is exposed to them during transport, they are considered dangerous goods when transported.  This is important to note, as the hazard level of a particular substance may not be the same in transport as it is when being manipulated in a laboratory.  In order to avoid such exposures, national and international regulations govern how shipments of infectious substances are to be prepared and transported. 

National and international regulations dictate that any individual involved in the transport of hazardous materials must be trained, tested, certified, and retain a record of their training.  This include any individuals who are responsible for the preparation and packaging of a shipment, marking and labeling packages, preparing shipping documents, loading and unloading transport vehicles, and supervising any of the afore mentioned activities.

There are various national and international regulatory bodies that have provided guidance and requirements for the shipment of dangerous goods.  The United States Department of Transportation (DOT) regulates the transport of dangerous goods via roadways and railways.  Dangerous goods that are shipped internationally via air are subject to the International Air Transport Association (IATA) Dangerous Goods Regulations (DGR).  Compliance with IATA DGR meets or exceeds the requirements set forth by the US DOT. 

Northwestern University provides access to training through Research Safety.  The Safe Shipping of Biological Materials and Dry Ice course provides shipping training in accordance with IATA DGR.  Alternative training may be obtained through other agencies; however, it is the recommendation of Northwestern University that all personnel involved in shipping of dangerous goods be trained in compliance with IATA DGR.  IATA DGR training must be renewed every two years.

For more information regarding the Safe Shipping of Biological Materials and Dry Ice course, or any other training offered by Research Safety, please visit Training.

9.0 Viral Vectors

Viral vectors have become standard tools for molecular biologists.  Their usefulness is attributed to the abilities these vectors retain from their parent virus: the abilities to enter a host cell and deliver its payload of genetic material.  The fusion or entry of a viral vector into a host cell and subsequent delivery of genetic material foreign to the virus is referred to as transduction.  Despite the fact that viral vectors can be purchased from commercial vendors, key features of the original nature of the virus remain intact.  For this reason, it is necessary that researchers using these biological agents are aware of their origins and the consequences of their use or misuse.

Various mechanisms are used to enhance safety in manipulating viral vectors.  Many viral vectors have been rendered replication incompetent, meaning the virus not capable of driving its own replication within the host cell.  It is critical to note that regardless of the mechanism(s) used to enhance the safety of a viral vector, the agent is specifically designed to transduce a target host cell, which may indeed be a human cell.  These agents are equally capable of transducing an incidental host in the event of exposure.  Caution is required when manipulating these agents.

The following contains pertinent information for commonly used viral vectors at Northwestern University.

 

9.1 Adenovirus
9.1.1 Virology

There are at least 57 different human adenovirus types (HAdV-1 through 57) in seven different species (Human adenovirus A through G).  Adenoviruses are moderate in size (90-100 nm) and do not have a lipid envelope.  Adenoviruses have a linear, double-stranded DNA genome between 26 and 48 Kb harboring approximately 22-40 genes.  The structure of the genome is relatively simple with an early- and late-phase transcription.   These viruses are capable of infecting both replicating and non-replicating cells and typically replicate in the nucleus of vertebrate cells.  Following replication of the genome, the virus is assembled into its protein capsid and is released from the cell as a result of virally induced cell lysis.

9.1.2 Epidemiology

Different adenoviruses are associated with diverse symptoms and disease, depending on the type of virus.  Conditions caused by adenoviruses include respiratory disease, conjunctivitis, gastroenteritis, or adipogenesis (obesity).  Infection with some types of adenovirus may have no overt symptoms at all.  Adenoviruses are stable outside of the host cell for prolonged periods of time.  Human-to-human transmission occurs primarily via respiratory droplet, but may be spread via fecal routes.

9.1.3 Treatment

There are no treatments or vaccines for adenoviral infection; treatments for infection are directed toward alleviating the symptoms.  Antibiotics may be given with some infections, such as conjunctivitis, to eliminate a possible bacterial infection or prevent a secondary bacterial infection.

9.1.4 Use as a Vector

These viruses are useful as a viral vector due to their ability to transduce both replicating and non-replicating cells, accommodate large transgenes, code for proteins without integrating into the host cell genome and the capsid proteins can be modified to specifically target certain cell types.  Because adenoviruses are commonplace, many humans already possess neutralizing antibodies thereby rendering them ineffective.  Instead, non-human adenoviruses are often used as viral vectors as humans are less likely to have immunity.

Typical routes of exposure in the lab include accidental ingestion and droplet exposure of the mucous membrane (eyes, nose, etc.).  Wild type and naturally occurring adenovirus are capable of rescuing replication-incompetent viral vectors by co-infection.

9.1.5 Disinfection

Effective mechanisms of decontamination include 10% bleach (1:10 dilution, prepared fresh), 2% glutaraldehyde, and standard autoclave run.  It is important to note that alcohol compounds are not effective due to the virus’ protein coat.1

 

9.2 Adeno-Associated Virus (AAV)
9.2.1 Virology

Adeno-associated virus (AAV) is a group of small viruses that infect humans and some primates.  There are at least 11 serotypes, all of which can infect cells from various tissue types.  AAV is capable of infecting replicating and non-replicating cells.  The virus is named as such because it is not capable of replicating by itself and requires the presence of adenovirus, or some other virus types, in order to replicate.  As such, it is often found in host cells co-infected with adenovirus.  The virus is small (about 20 nm), non-enveloped, and has a single-stranded, 4.7Kb DNA genome that is either positive- or negative-sense. 

9.2.2 Epidemiology

AAV has not been definitively shown to cause disease with infection of humans resulting in a mild immune response.  AAV may have a role in male infertility.  The virus can integrate into the host genome.

9.2.3 Treatment

Because wild-type AAV is not known to cause disease, there are no available treatments.  Due to the ability of the virus to infect any host cell and integrate into the genome (see below), it is important to consider the trans gene(s) put into the virus when it is used as a vector.

9.2.4 Use as a Vector

AAV has become an attractive vector for human gene therapy (HGT) research due to the apparent lack of pathogenicity of the virus.  The virus can transduce both dividing and non-dividing cells and stably integrate into the host cell genome at a specific site.  There are limitations to the use of AAV as a vector as well: the amount of genetic material that can be packaged in the virus is limited and, as such, often requires replacement of the virus’ entire genome.

Typical routes of exposure in the lab include accidental ingestion and droplet exposure of the mucous membrane (eyes, nose, etc.).  Wild type and naturally occurring adenovirus are capable of providing the machinery necessary to generate additional AAV virions if a cell becomes co-infection.

9.2.5 Disinfection

Effective mechanisms of decontamination include 10% bleach (1:10 dilution, prepared fresh), 2% glutaraldehyde, and standard autoclave run.  It is important to note that alcohol compounds are not effective due to the virus’ protein coat.1

 

9.3 Epstein-Barr Virus (EBV)
9.3.1 Virology

Epstein-Barr Virus (EBV), also called human herpesvirus 4 (HHV-4) is one of eight viruses in the herpes family and is one of the most common viruses in humans.  The virus particle is 122 to 180 nm in diameter, the genome is double-stranded DNA, and possesses a lipid envelope.  Like other herpesviruses, EBV has a complicated replication cycle including a latent and lytic cycle depending on if the virus was generated during primary or persistent infection cycle, respectively, as well as various environmental factors.

9.3.2 Epidemiology

EBV is the causative agent of infectious mononucleosis (glandular fever, colloquially “mono”).  Transmission of EBV occurs by the oral transfer of saliva and genital secretions. Symptoms in infants, children, and pre-adolescents are mild and flu-like.  In adolescents, the disease presents as fever (lasting one to two weeks), sore throat, swollen glands, and fatigue.  Presentation in adults is less characteristic but many of the symptoms are the same as in adolescents.  Over 90% of adults will have acquired immunity by the age of 40.

EBV is also associated with certain forms of cancer, conditions associated with human immunodeficiency virus (HIV), and higher risk of certain autoimmune diseases.

9.3.3 Treatment

There is no specific treatment of EBV infection; treatment focuses on alleviating the symptoms associated with the specific disease presentation.  A vaccine for EBV is not yet available but is currently in clinical trials.

9.3.4 Use as a Vector

EBV is not widely used as a vector to express exogenous genes in a host cell.  This is likely because the virus has a complex replication cycle including both lytic- and latent-phase gene expression.  More commonly, EBV is used to immortalize human B cell lymphocytes through means of simple transduction of a culture of cells with a largely wild-type virus.

Common routes of exposure in the lab may include ingestion, accidental parenteral injection, droplet exposure of the mucous membranes and inhalation of concentrated aerosolized virus particles.

9.3.5 Disinfection

As a result of its lipid envelope, EBV is susceptible to a wide array of disinfectants.  70% ethanol is sufficient for disinfection but a 1:10 dilution of household bleach is recommended when working with EBV in conjunction with human cell lines.  Also effective in decontamination of EBV is 2% glutaraldehyde/formaldehyde.  A freshly-prepared solution of bleach (prepared the same day it is to be used) is recommended for decontamination of EBV.1

 

9.4 Lentivirus
9.4.1 Virology

Lentivirus describes a genus of viruses in the retroviridae family.  They are characterized by long incubation periods and the ability to produce a persistent infection.  Lentiviruses are non-oncogenic retroviruses, though they may be associated with some types of cancer, capable of producing multi-organ diseases.  Lentiviruses are unique among other retroviruses in that they are capable of infecting dividing and non-dividing cells, making them a powerful and potentially dangerous gene delivery vector.

There are five serogroups of lentiviruses, categorized by the vertebrate host with which they are associated: primates, sheep and goats, horses, cats, and cattle).  Viruses within the genus have a concentric and rod-shaped nucleocapsid (core) surrounded by a spherical lipid envelope, and roughly 80-100 nm in diameter.  The lentivirus genome is a dimer of single-stranded, positive-sense RNA molecules contained within the nucleocapsid.  As with all retroviruses, lentiviruses have an unorthodox replication cycle in that the RNA genome is first reverse-transcribed into DNA before being transcribed into messenger RNA and subsequently translated into protein.

9.4.2 Epidemiology

Transmission of lentiviruses from person to person (or animal to animal) occurs through direct contact with infected body fluids (blood, semen, cerebrospinal fluid, etc.), direct inoculation with used hypodermic needles following intravenous drug use, and infection following organ transplant or blood transfusion.

As a genus, lentiviruses can cause a wide variety of disease that is specific to the natural host organism.  Human, simian, and feline immunodeficiency viruses (HIV, SIV, and FIV, respectively), as well as equine infectious anaemia virus (EIAV) and visna virus are all examples of lentiviruses.  The diseases caused by these viruses can range from severely compromised immune system in humans and felines (HIV and FIV) to fever, anemia, and death in horses (EIAV) to encephalitis and chronic pneumonia in sheep (visna virus).  Interestingly, SIV does not often cause significant disease in the host simians (commonly sooty mangabeys and chimpanzees) but may cause serious illness in humans, should they become infected.

9.4.3 Treatment

As with many viruses, there are little to no treatment options available for the lentiviruses described above.  Most treatment options involve mitigating the symptoms, treatment and prevention of possible secondary/opportunistic infections, augmenting the host immune system, and limiting the ability of the virus to complete its life cycle.  Vaccines for these viruses are not currently available. 

9.4.4 Use as a Vector

Lentiviruses used in research may be derived from any one of the viruses described above, although they are stripped of the genetic elements known to cause disease in the host organism.  The risk posed by these viral vectors lies in the possibility for insertional mutagenesis in the host genome and the risks associated with the trans gene inserted into the virus.  When used as a vector, lentiviruses are often pseudotyped, meaning the host range or cell specificity has been altered to suit the needs of the research.  Lentivirus vectors are typically replication incompetent but the possibility of the virus regaining its ability to replicate, either spontaneously or recombining with another retrovirus, should not be discounted.

Possible routes of exposure in the laboratory include direct contact with skin and mucous membranes of the eye, nose, and mouth, accidental parenteral injection, and ingestion.  The risk associated with aerosols is unknown; generation of aerosols and small droplets may increase the risk of exposure to mucous membranes.

9.4.5 Disinfection

Lentiviruses are susceptible to a broad array of disinfectants due to their lipid envelope.  Appropriate disinfectants include 70% ethanol, 2% glutaraldehyde/formaldehyde, and a freshly-prepared (prepared the same day it is to be used) solution of bleach (1:10 dilution of household bleach).  A solution of bleach is recommended for decontamination of lentivirus.1

 

9.5 Retrovirus (Non-Lentivirus)
9.5.1 Virology

Retroviruses, as a family, possess a lipid envelope sourced from the host cell membrane as well as virus-encoded proteins.  Viral particles contain two copies of a single-stranded, positive sense RNA genome.  Depending on the virus, the genome may be from 7 to 10 kb in length.  Unlike lentiviruses, retroviruses are only capable of infecting dividing cells.  Most non-lentivirus retroviruses used as vectors have a simple replication cycle, relative to lentiviruses.  One unique feature of the replication cycle of retroviruses is that the RNA genome goes through a double-stranded DNA intermediate, called proviral DNA.  The proviral DNA must then be integrated into the host cell genome to complete the replication cycle of the virus.

9.5.2 Epidemiology

Retroviruses are a diverse family of viruses that are able to infect a variety of species and exhibit a range of pathogenic effects in their hosts.  As a result, it can be difficult to conduct a risk assessment on non-lentivirus retroviruses.  For the majority of retroviruses used in biomedical research, humans are not the traditional host and, as such, are not likely to become infected.  However, many retroviruses have been pseudotyped which may have altered the host range to now include humans.  When conducting a risk assessment on a retrovirus used in biomedical research, it is critical to consider the virus as a whole: parent virus, pseudotyping, and trans-gene(s). 

Transmission of retroviruses can occur through various means including exposure to mucous membranes, accidental self-injection, and aerosol transmission.  The mode of transmission is largely dependent on the type of parental virus.

9.5.3 Treatment

Various anti-retroviral agents are available for the treatment of retrovirus infection.  The majority of these have been developed for the purposes of treating HIV infection.  Treatment is rarely recommended in the event of exposure to non-lentivirus retrovirus in the laboratory setting.  Special attention should be paid to any trans-gene(s) the virus is carrying.

9.5.4 Use as a Vector

Non-lentivirus retroviruses are frequently used as a mechanism for gene delivery in tissue culture.  Common retroviruses that are modified for this purpose include Moloney Murine Leukemia Virus (MoMuLV) and Murine Stem Cell Virus (MSCV).  These viruses are typically modified to limit the ability of the virus to replicate within a host cell without the presence of a helper virus or helper plasmid.  These viruses are often further modified to express glycoproteins derived from other enveloped viruses.  Common glycoproteins include Vesicular Stomatitis Virus (VSV-g) as well as other viruses capable of adhering to and entering human cells including measles, Ebola, and Marburg.

9.5.5 Disinfection

Retroviruses, similar to lentiviruses, are susceptible to a broad array of disinfectants due to their lipid envelope.  Appropriate disinfectants include 70% ethanol, 2% glutaraldehyde/formaldehyde, and a freshly-prepared (prepared the same day it is to be used) solution of bleach (1:10 dilution of household bleach).1

 

9.6 Poxvirus/Vaccinia
9.6.1 Virology

Virus particles in the poxviridae family are exceptionally large (about 200 nm by 300nm) and brick- or ovoid-shaped.  Their genome is composed of a single, linear, double-stranded DNA segment.  All viruses within this family have a lipid bilayer envelope, although the intracellular mature virion form of the virus has a different envelope and is also infectious.  The virus binds to the host cell via cell surface receptor thought to be glycosaminoglycans (GAGs).  Following binding, the virus enters the cell and sheds its outer lipid membrane.  Next the virus particle fuses with the cellular membrane which releases the core of the virus into the cytoplasm.  Replication and assembly of pox viruses occurs entirely in the cytoplasm, a trait which is unusual for viruses with a double-stranded DNA genome.  The virus is able to do this because it encodes for all of its own replication machinery and only relies on host cell machinery for the initial stages of entry and replication.  The manner in which virus particles are released from the cell depends of the specific virus but may include lysis, budding, and/or occlusion. 

9.6.2 Epidemiology

The poxviridae is a family of viruses comprised of 69 species that are capable of infecting vertebrate and arthropod hosts.  There are four genera of poxviruses that are capable of infecting humans: orthopoxvirus, parapoxvirus, yatapoxvirus, and molluscipoxvirus.  Specifically, within the orthopoxvirus genera are variola virus (the causative agent of smallpox), vaccinia virus (the active constituent of the vaccine used to eradicate smallpox), cowpox (which provided the inspiration for the first smallpox vaccine), and monkeypox virus (a common zoonotic infection in Central and West Africa that presents as a disease nearly indistinguishable from smallpox). 

Viruses within the poxviridae family typically causes a disease of the outermost surface layer of cells (the skin).  The virus is transmitted via contact with mucosal membranes, exposure to broken skin, or inoculation.  The disease presents as a vesicular or pustular lesion.  For vaccinia virus, there is typically an area of induration or erythema surrounding a scab or ulcer at the inoculation site.  The disease may progress and spread to other areas of the skin and body.  Major complications of the disease include encephalitis, progressive vaccinia (in immunocompromised individuals), and eczema vaccinatum.  The virus may also be transferred to the fetus of a pregnant woman.  Minor complications include generalized vaccinia with multiple lesions, benign rash, and secondary infections.  Complications can be more serious for individuals with eczema and those who are immunocompromised.  Individuals who are pregnant, may become pregnant, are immunocompromised, or have underlying skin conditions are strongly encouraged not to work with vaccinia virus or any other virus in the poxviridae family.

9.6.3 Treatment

Treatment of the disease caused by vaccinia virus entails administration of anti-vaccinia immunoglobulin antibodies.  Antiviral medication may also be of value in treating complications.  Other medications may be administered to mitigate secondary complications.  

Vaccination against these viruses is a consideration.  Vaccinia virus is itself a vaccine against smallpox.  There are multiple vaccine strains available.  Consideration for receiving the vaccine should be taken if the individual is pregnant, may become pregnant, is immune compromised, or has an underlying skin condition.  The vaccine itself is not without complications for healthy individuals, as described above.

9.6.4 Use as a Vector

Vaccinia virus and other poxviruses can make useful viral vectors in part because they can accept as much as 25 kb of foreign DNA, making it useful for expressing large eukaryotic and prokaryotic genes.  Foreign genes are integrated stably into the viral genome resulting in efficient replication and expression of biologically active molecules.  Furthermore, post-translational modifications, such as methylation and glycosylation, occur normally in transfected cells.  Additionally, the virus elicits a strong host immune response in vivo making it useful for vaccine development.

Vaccinia is used to generate live recombinant vaccines for the treatment of other illnesses.  Modified versions of vaccinia virus have been developed for use as recombinant vaccines.  For example, the modified Ankara strain (MVA) of vaccinia virus was developed by repeated passage in a line of chick embryo fibroblasts in order to generate an attenuated virus from the original, pathogenic wild type virus.  Other vaccinia virus vectors achieve attenuation through more direct genetic manipulation.  For example, NYVAC is another attenuated form of the vaccinia virus that has been used in the construction of live vaccines that has a deletion of 18 vaccinia virus genes that render it less pathogenic. 

9.6.5 Disinfection

Poxviruses are susceptible to a broad array of disinfectants due to their lipid envelope.  Appropriate disinfectants include 70% ethanol, 2% glutaraldehyde/formaldehyde, and a freshly-prepared (prepared the same day it is to be used) solution of bleach (1:10 dilution of household bleach).  A solution of bleach is recommended for decontamination of poxviruses.1

 

9.7 Rabies Virus
9.7.1 Virology

Rabies virus is the causative agent of rabies in humans and animals.  It is a neurotropic virus whose transmission can occur through the saliva of infected animals and humans, although the latter is less common.  Rabies virus has a broad host range: the virus has been known to naturally infect many mammalian species, has been found to infect birds in a laboratory setting, and can infect cell cultures derived from mammals, birds, reptiles, and insects.2-4

The rabies virus has a lipid envelope and a cylindrical morphology resembling a bullet.  The viral genome is a single negative-sense strand of RNA which is tightly bound by the viral nucleoprotein.  The genome is typically just under 12 kilobases in length and encodes five genes whose order is highly conserved: nucleoprotein, phosphoprotein, matrix protein, glycoprotein, and the viral RNA polymerase.  Virus transcription and replication takes place in the cytoplasm of the host cell which can be identified by histology and be used as definite proof of a rabies infection. 2

9.7.2 Epidemiology

Following entry into the host through a wound, the virus travels through the peripheral nervous system to the central nervous system (CNS).  Retrograde axonal transport of the virus to the CNS may be mitigated by viral P protein.  The virus is also capable of decreasing the host immune response using the same P protein.  Once the virus has infected the CNS it can further spread to other organs including salivary glands which maintain a particularly high concentration of the virus.  Thus, the virus can be easily transmitted through the saliva of the host organism following a bite. 2-4

Infection with a rabies virus causes inflammation in the brain in mammals.  Symptoms can include fever, tingling at the site of exposure, violent movements, uncontrolled excitement, fear of water, an inability to move parts of the body, confusion, and loss of consciousness.3,5  Once symptoms appear, death is nearly a certainty, between 2 and 10 days later.6  The virus can be fatal in as little as two days or as many as five years depending on the species of the host animal.  Long-term incubation periods may even be asymptomatic.  Most infected mammals die within weeks.7

9.7.3 Treatment

The recommended treatment for disease is human rabies immunoglobulin (HRIG) administered as soon as possible following a known or suspected exposure.  An additional four doses of HRIG should be administered over a 2-week period.4,8 Thoroughly washing the wound with soap and water immediately following exposure is effective in reducing the number of virus particles at the site of exposure.9  Treatment of the wound site with povidone-iodine or rubbing alcohol can reduce the number of virus particles further.10 It is recommended that laboratory workers be vaccinated against the virus before working with it or its derivatives.11

9.7.4 Use as a Vector

Several neurotropic viruses are employed as tools to understand neuronal networks.12 Rabies virus is frequently used due to the specific nature of its transport through neuronal cells, the limited spread to non-neuronal cells, and low cytopathogenicity in infected neurons.  There are several drawbacks to using rabies virus as a viral vector.  As an RNA virus, it has an inherently higher mutation rate than DNA viruses and has a strictly cytoplasmic replication cycle which makes cell type-specific expression difficult or impossible.13   Another obvious drawback to rabies virus as a tool for studying neuronal pathways is that it is infectious to humans.

One way rabies virus has been made safer for use as a vector is by deletion of the glycoprotein (G) gene.  Deletion of rabies virus G prevents the virus from spreading beyond the initially infected cell. 13,14  Currently, one of the major uses of rabies virus as a viral vector is for vaccine generation.15

9.7.5 Disinfection

Due to the presence of a lipid envelope, a broad array of disinfection methods are available for rabies virus.  Appropriate disinfectants include 70% ethanol, 2% glutaraldehyde/formaldehyde, and a freshly-prepared (prepared the same day it is to be used) solution of bleach (1:10 dilution of household bleach). A solution of bleach is recommended for decontamination of rabies virus.1

 

9.8 Baculovirus
9.8.1 Virology

Baculoviruses preferentially infect and are capable of causing disease in insects, arthropods, and other invertebrates.  Over 600 host species have been identified including moths, mosquitoes, and shrimp.  The most commonly studied baculoviruses are in the genus Nucleopolyhedrovirus which contains a 134-kilobase, circular, double-stranded DNA genome with 154 open reading frames.  The virion is enveloped in a membrane acquired from the host cell peppered with viral proteins.16,17

9.8.2 Epidemiology

Baculoviruses, particular of the Nucleopolyhedrovirus genus, are often studied for their application as species-specific insecticide.  Insects are infected by consuming the virus as it sits on a plant leaf.  The virus replicates within the host cells, kills the host organism, and ultimately facilitates the complete breakdown of the host organism thereby depositing more virus on the surface of the plant.16,17

While baculoviruses are capable of causing disease in insects, arthropods, and other invertebrates, they are incapable of replicating in mammalian cells and plants.  It is this feature that makes baculoviruses one of the safest viral vectors available for biomedical research.  Although baculoviruses are not capable of causing an infection in mammalian cells, the virus is still capable of entering a mammalian cell and delivering its genetic material.16,17  Thus, a baculovirus carrying a human oncogene should be handled with the same caution as a lentivirus vector.

9.8.3 Treatment

Baculoviruses are not capable of causing disease in mammals.  When baculoviruses are being used as a viral vector, consideration should be given to the genetic material being carried.

9.8.4 Use as a Vector

Baculoviruses will readily replicate in cultured anthropod cell lines but not in mammalian cells.  The virus is, however, capable of delivering genetic material to mammalian cells.  Thus, baculoviruses are considered one of the safer viral vectors despite being fully replication competent.18  These agents are generally safe to work with at BSL1 containment.

9.8.5 Disinfection

Although baculoviruses are not pathogenic, cultures must still be inactivated by heat or chemical means because they contain recombinant nucleic acid.  Due to the presence of a lipid envelope, a broad array of disinfection methods are available for rabies virus.  Appropriate disinfectants include 70% ethanol, 2% glutaraldehyde/formaldehyde, and a freshly-prepared (prepared the same day it is to be used) solution of bleach (1:10 dilution of household bleach).1

10.0 Biological Toxins

Biological toxins are natural, poisonous substances produced as by-products of microorganisms (exotoxins, endotoxins, and mycotoxins such as T-2 and aflatoxins), plants (plant toxins such as ricin and abrin), and animals (zootoxins such as marine toxins and snake venom).  Unlike pathogenic microorganisms, including those that produce toxins, the toxins themselves are not contagious and do not replicate.  In this regard, toxins behave more like chemicals than infectious agents.  However, unlike many chemical agents, biological toxins are not volatile and are odorless and tasteless.  The stability of toxins varies greatly, depending on the toxin structure (low molecular weight toxins are very stable).

Most biological toxins, with the exception of T-2 Mycotoxin, are not dermally active; intact skin is an excellent barrier against most toxins.  In contrast, mucous membranes of the eyes, nose, and mouth, and broken skin all serve as points of entry into the body.  Aerosol transmission, ingestion, and percutaneous transmission are also a concern for most biological toxins.

Bacterial toxins can be exotoxins (including enterotoxins or endotoxins.  Exotoxins are cellular products excreted from certain Gram-positive and –negative bacteria, highly toxic, and are relatively unstable.  These types of toxins are lethal at microgram quantities and are destroyed rapidly when heated to greater than 60°C.  Bacterial endotoxins are lipopolysaccharide complexes derived from the cell membrane of Gram-negative bacteria which are released upon bacterial death.  Endotoxins are moderately toxic and relatively stable.  Endotoxins are lethal at tens- to hundreds-micrograms quantities and can withstand heating at 60°C for hours without losing activity.

The modes of action of biological toxins vary but include damage to cell membranes or cell matrices (e.g. Staphylococcus aureus alpha toxin), inhibition of protein synthesis (e.g. Shiga toxin), or via activation of secondary messenger pathways (e.g. Clostridium botulinum and C. difficile toxins).

 

Table 7: Comparison of Biological Toxins versus Chemical Agents

Comparison of Biological Toxins versus Chemical Agents
TOXINS ARE USUALLY… CHEMICALS ARE USUALLY…
Natural in origin Man-made
Non-volatile Many are volatile
Not dermally active Dermally active
More toxic than chemicals Less toxic than many toxins
Odorless and tasteless Have odor and taste
Diverse toxic effects Narrow range of effects
Generate an immune response No immune response

 

 

10.2 Laboratory Requirements and Safety Operations

Most work with biological toxins can be safely managed in a BSL2 setting.  In some cases, such as large-scale production, manipulation of large quantities of a toxin’s powder form, management at BSL3 may be required, depending on the toxin in question and the quantities used.  The most hazardous form of any toxin is the dry, powder form.  Manipulations of dry forms of toxins should be performed in a biological safety cabinet or in a fume hood.  In some cases, a glove box may be recommended for such operations. 

Once reconstituted into an aqueous form, BSL2 management is usually sufficient for work with most biological toxins.  Your lab should maintain negative, directional air-flow and, like all BSL2 labs, have a hand-washing sink available within the lab.  Access to the lab should be controlled with the toxin is in use.  Biohazard warning signs displaying the biosafety level and emergency contact information should be posted at the lab entrance.  Additionally, it is recommended that a sign indicating that a toxin is being used be posted on the door.  This should also include a description of the minimal PPE required for entrance.  Personal protective equipment should include a lab coat, gloves, and mucous membrane protection.  If vacuum lines are used, it is advisable to protect the vacuum system with an in-line disposable HEPA filter (available through Research Safety).  All personnel in the lab should be trained regarding the specific hazards associated with the toxin in use.  All supplemental training documents should be uploaded to Lumen.

Some toxins, when stored in large enough quantities, are regulated under the Federal Biological Select Agents and Toxins Regulations.  All research with these toxins must be registered with the IBC before work can begin.  These toxins are listed below.  For more information, please visit the Select Agents website.

 

Table 8: Select Agent Toxins, Maximum Permissible Non-regulated Amounts, and Mean Lethal Dose

Toxins by maximum permissible amount and LD50
TOXIN MAX. PERMISSIBLE AMOUNT (MG) LD50(ΜG/KG)1
Abrin 1,000 0.7
Botulinum toxin A 1 0.0012
Botulinum toxin B 1 0.0012
Botulinum toxin C1 1 0.0011
Botulinum toxin C2 1 0.0012
Botulinum toxin D 1 0.0004
Botulinum toxin E 1 0.0011
Botulinum toxin F 1 0.0025
Conotoxin3 100 12-30
Diacetoxyscirpenol (DAS) 10,000 1,000-10,000
Ricin 1,000 2.7
Saxitoxin 200 8
S. aureus enterotoxin B 100 25
S. aureus enterotoxin F 100 2-10
S. aureus enterotoxin A, C, D and E 100 20-50
T-2 toxin 10,000 5,000-10,000
Tetrodotoxin 500 8

1. Note that LD50 values are from various sources. For specifics on rout of application, animal used, and variations on the listed toxins, consult the cited sources.

2. Only short, paralytic alpha conotoxins with specific sequences are considered Select Agents.


 
10.3 Security

It is important that stocks of biological toxins be maintained in locked freezers and refrigerators.  Since biological toxins are not self-replicating, it is prudent to maintain an inventory of toxins present in the lab at any given time.  This inventory should display the current quantity of a particular toxin in site, the date and amount removed from storage, the person removing the aliquot from storage, the purpose of use, and the quantity remaining.  Note that an inventory is required for all Select Agent Toxins and should be maintained through NSIS.

 

10.4 Decontamination Methods

The majority of biological toxins can be inactivated or decontaminated with household bleach (a 1:2 dilution of household bleach or a 2.5 sodium hypochlorite solution) or autoclaving.  The tables below describe the recommended inactivation regimens for biological toxins in common use.

 

Table 9: Complete inactivation of toxins with a 30-minute exposure time to varying concentrations of sodium hypochlorite (NaOCL) with and without sodium hydroxide (NaOH)

Inactivation of toxins by various concentrations of sodium hypochlorite (NaOCL) with and without sodium hydroxide (NaOH)
TOXIN 2.5% NAOCL + 0.25 N NAOH 2.5% NAOCL 1.0% NAOCL 0.1% NAOCL
T-2 Mycotoxin Yes No No No
Brevetoxin Yes Yes No No
Microcystin Yes Yes Yes No
Tetrodotoxin Yes Yes Yes No
Saxitoxin Yes Yes Yes Yes
Palytoxin Yes Yes Yes Yes
Ricin Yes Yes Yes Yes
Botulinum Yes Yes Yes Yes

 

Table 10: Complete inactivation of toxins by autoclaving or 10-minute exposure to varying temperatures of dry heat

Complete inactivation of toxins by autoclaving or 10-minute exposure to varying temperatures of dry heat
TOXIN AUTOCLAVING DRY HEAT (IN FAHRENHEIT)
    200 500 1,000 1,500
T-2 Mycotoxin No No No No Yes
Brevetoxin No No No No Yes
Microcystin No No Yes Yes Yes
Tetrodotoxin No No Yes Yes Yes
Saxitoxin No No Yes Yes Yes
Palytoxin No No Yes Yes Yes
Ricin Yes Yes Yes Yes Yes
Botulinum Yes Yes Yes Yes Yes

For skin exposure involving minute quantities of toxin, soap and water are effective in removing the toxin burden (only T-2 mycotoxin are dermally active).  For significant exposures to biological toxins, contact Corporate Health immediately.

11.0 Select Agents and Toxins

The “Antiterrorism and Effective Death Penalty Act of 1996” established new provisions to regulate transfer of certain biological agents and toxins (i.e., select agents). To comply with this rule, Northwestern University has registered with the Centers for Disease Control and Prevention (CDC). If principal investigators wish to transfer material in or out of the university, they must provide information in advance so the registration can be updated.

Laboratories registered with the CDC are subject to inspection by that agency. Contact Research Safety for further information.

List of Select Agents and Toxins

Select Agent Exclusions

12.0 Dual Use Research

Broadly defined, “dual use” refers to the malevolent misapplication of technology or information initially developed for benevolent purposes.  In the realm of life sciences, “dual use” refers to the potential misuse of microorganisms, toxins, recombinant/synthetic DNA technology, or research results to threaten public health or national security.  “Dual Use Research of Concern,” referred to as DURC, is research that has the potential to be directly misapplied.

Northwestern University has developed and implemented a formal policy regarding DURC and is reproduced below.  For further information and questions regarding this policy, please contact the Institutional Contact for Dual Use Research (ICDUR), Robert Foreman at robert.foreman@northwestern.edu.

 

12.1 Policy Statement

Northwestern University has adopted the United States Government Policy for Institutional Oversight of Life Sciences Dual Use Research of Concern which was released on September 24, 2014 and takes effect on September 24, 2015.  This policy, which will be referred to as the September 2014 DURC Policy, states that:

Life sciences research that meets the scope specified in this policy is subject to institutional oversight, as described in this Policy.  The purpose of this oversight is to preserve the benefits of such research while minimizing the risk that the knowledge, information, products, or technologies generated by Dual Use Research of Concern (DURC) could be used in a manner that results in harm to public health and safety, agricultural crops and other plants, animals, the environment, material, or national security; and

Oversight includes the identification of life sciences research that raises dual use concerns as well as the implementation of measures to mitigate the risk that DURC is used in a manner that results in harm.  Measures that mitigate the risks of DURC should be applied in a manner that minimizes, to the extent possible, adverse impact on legitimate research, is commensurate with the risk, includes flexible approaches that leverage existing processes, and endeavors to preserve and foster the benefits of research. 

 

12.2 Reason for Policy/Purpose

Research in the life sciences is critical for the advancement of medicine, public health and safety, agricultural improvements, environmental improvements, and even national security.  The benefits of life sciences research and the techniques that allow for such advancements inherently create risks, as well.  The primary risk of life sciences research is that the findings and/or techniques, although developed for benevolent purposes, may also be employed with malevolent intent.  Research and techniques of this type are referred to as “dual use research” and can encompass a broad range of research initiatives.  Not all research programs defined as dual-use are of concern to public health, agricultural sustainability, and national security.

Research that can pose a threat to public health, agricultural sustainability, and national security through its misapplication is referred to as dual use research of concern.  The United States Government Policy for Institutional Oversight of Life Sciences Dual Use Research of Concern, published in September 2014 defines DURC as follows:

Life sciences research that, based on current understanding, can be reasonably anticipated to provide knowledge, information, products, or technologies that could be directly misapplied to pose a significant threat with broad potential consequences to public health and safety, agricultural crops and other plants, animals, the environment, materiel, or national security.

The designation of a life science research project as DURC is not intended to become a negative moniker nor should it imply that the research should not be conducted at all.  Moreover, this designation does not imply that, once completed, the results of a DURC project should not be communicated to the scientific community or the general public.  On the contrary, the designation of research as DURC and the policies implemented by the United States Government (USG) and the institution are intended to be a method of supporting such research while providing additional oversight to ensure the safety of the researchers as well as the general public.

In order to mitigate the risks of conducting and disseminating the results of DURC in the life sciences, the USG has put into effect two policies to provide a comprehensive oversight system that includes both the USG and the institution at which the research is being conducted.  The scope of the policy is limited to a well-defined subset of life sciences research that involves one or more of15 different biological agents and toxins and one or more of 7 different categories of experiments with the afore mentioned biological agents and toxins.

The policies implemented by the USG and adopted by Northwestern University are designed to provide additional oversight and support to research being conducted with any of the biological agents and toxins described in this policy.  However, possession of and research on one or more of the biological agents and toxins is insufficient for the determination of DURC.  In order to be categorized as DURC, the research with one or more of the biological agents and toxins must fall within one or more of the seven research categories described in this policy.

In order to meet the requirements of the policies implemented by the USG regarding DURC, Northwestern University will establish a Dual Use Research of Concern Review Committee (DRC) that will work in concert with Principal Investigators (PIs) and the Northwestern University Institutional Review Board (IBC) in order to identify research projects that could be considered DURC, compile a risk mitigation strategy tailored to the specific research project, and ensure compliance by the research staff. 

 

12.3 Who Approved this Policy

This policy was initially approved by the President of Northwestern University, the Provost, the Associate Vice President for Research, the Institutional Biosafety Committee (IBC), and the Executive Director of Research Safety.  This policy is reviewed and revised as the United States Government revises its own DURC Policy and/or provides additional guidance concerning its implementation.  The policy is then re-approved by the IBC.

 

12.4 Who Needs to Know this Policy

All faculty, staff, students, and individuals employed by Northwestern University who manage or conduct life sciences research projects or programs at any Northwestern University campus or affiliated building.  All visiting or temporary faculty, staff, students, and individuals who manage or conduct life sciences research projects or programs at any Northwestern University campus or affiliated building.

 

12.5 Contacts

If you have any questions on the Conduct and Oversight of Dual Use Research of Concern Policy, you may call the Associate Biosafety Officer of Research Safety at 312-503-8300, or send an e-mail to robert.foreman@northwestern.edu.

 
12.6 Policy/Procedures
12.6.1 Applicability of the Northwestern University DURC Policy

The September 2014 DURC Policy applies to any institution within the United States that both (1) receives USG funds to conduct or sponsor life sciences research; and (2) conducts or sponsors research that involves one or more of the 15 biological agents or toxins listed in Section 6.2.1 of the September 2014 DURC Policy, reproduced herein.  Additionally, the September 2014 DURC Policy applies to institutions outside of the United States that receive USG funds to conduct or sponsor research that involves one or more of the 15 biological agents or toxins listed in Section 6.2.1 of the September 2014 DURC Policy, reproduced herein.

Northwestern University and its faculty, staff, and students receive funding from a broad range of local, state, and federal funding agencies for a broad array of research projects and programs.  It is the Policy of the USG that research conducted at an institution which receives any amount of funding from the USG be subject to the DURC Policy.  Therefore, Northwestern University has adopted the following with regards to the applicability of this Policy for research conducted by the faculty, staff, and students.

All life sciences research projects and programs conducted at any Northwestern University campus, building, or affiliated locations will be subject to the September 2014 DURC Policy and Northwestern University DURC Policy.  All faculty, staff, students, and any individual employed by Northwestern University who conduct life sciences research will be subject to the DURC Policies of the USG and Northwestern University.  All visiting or temporary faculty, staff, students, and any other individuals who are conducting life sciences research projects or programs on any Northwestern University campus, building, or affiliated location will be subject to the September 2014 DURC Policy and Northwestern University DURC Policy.

12.6.2 Scope of the Northwestern University DURC Policy

Consistent with the September 2014 DURC Policy, under the Northwestern University Policy, research that uses one or more of the biological agents or toxins listed in this Policy and produces, aims to produce, or can reasonably be anticipated to produce one or more of the effects listed in this Policy will be evaluated for DURC potential.

Biological agents and toxins requiring oversight by this Policy:

Avian influenza virus (highly pathogenic)

Bacillus anthracis

Botulinum neurotoxin

Burkholderia mallei

Burkholderia pseudomallei

Ebola virus

Foot-and-mouth disease virus

Francisella tularensis

Marburg virus

Reconstructed 1918 influenza virus

Rinderpest virus

Toxin-producing strains of Clostridium botulinum

Variola major virus

Variola minor virus

Yersinia pestis

12.6.3 Categories of experiments requiring oversight by this Policy

There are seven categories of experiments that require oversight under this policy.  Experiments that fall into one or more of these categories are not necessarily forbidden.  Instead, experiments within these categories require additional oversight.  These categories are:

  1. Enhances the harmful consequences of the biological agent or toxin
  2. Disrupts immunity or the effectiveness of an immunization against the biological agent or toxin without clinical and/or agricultural justification
  3. Confers to the biological agent or toxin resistance to clinically and/or agriculturally useful prophylactic or therapeutic interventions against that agent or toxin or facilitates their ability to evade detection methodologies
  4. Increases the stability, transmissibility, or the ability to disseminate the biological agent or toxin
  5. Alters the host range or tropism for the biological agent or toxin
  6. Enhances the susceptibility of a host population to the agent or toxin
  7. Generates or reconstitutes an eradicated or extinct biological agent or toxin listed above
12.6.4 Procedure for Review

This section describes the organizational framework for review of research with dual use potential and the oversight of DURC and articulates the roles and responsibilities of Northwestern University, the Northwestern University DRC, and Northwestern University’s PIs under the September 2014 DURC Policy and Northwestern University DURC Policy.  Components of the review and oversight system for DURC include:

  1. Identification, by the PI, of life sciences research that involves one or more of the 15 biological agents or toxins listed above.
  2. The institutional review process by the DRC for assessing whether research that uses one or more of the biological agents or toxins listed above also produces, aims to produce, or is reasonably anticipated to produce one or more of the effects listed above.
  3. For research anticipated to produce at least one of the seven effects, determination of whether the research meets the definition of DURC, stated above.  A risk assessment should underpin the determination of DURC.
  4. Identification of the anticipated benefits of the research identified as DURC.  The anticipated benefits should be considered in conjunction with the previously identified risks in order to develop a draft risk mitigation plan to guide the conduct and communication of the DURC.  The risk mitigation plan must be approved by the USG funding agency.  Plans will be evaluated by the Northwestern University DRC annually and modified as necessary for the duration of the research.  The Northwestern University DRC is responsible for ensuring that the DURC is conducted in accordance with the risk mitigation plan.  Research that has already been determined to be DURC under the March 2012 DURC Policy, and for which a risk mitigation plan has already been developed, does not need a new risk mitigation plan but the extant risk mitigation plan will be subject to ongoing review and modification, as necessary, by the Northwestern University DRC.
  5. Notification of the results of this review process to the relevant USG funding agency and, in instances when the research is determined to be DURC, provision of the draft risk mitigation plan by the Northwestern University DRC to the USG funding agency.  For non-USG funded research, notification will be made to the National Institutes of Health (NIH) which will receive the notification for administrative purposes and will in turn refer the notification to an appropriate agency based upon the nature of the research.
  6. The September 2014 DURC Policy requires that each institution subject to the Policy certify that it will comply with the Policy.  As such, passage of this Northwestern University DURC Policy will constitute certification of compliance with the September 2014 DURC Policy.
  7. Oversight by USG funding agencies and the USG as articulated in the March 2012 DURC Policy with additional responsibilities with respect to the September 2014 DURC Policy as described herein.
12.6.5 Responsibilities of Principal Investigators (PIs)

The PI must notify the Northwestern University DRC as soon as (1) the PI’s research involves one or more of the biological agents or toxins listed above; (2) the PI’s research with one or more of the biological agents or toxins listed above also produces, aims to produce, or can be reasonably anticipated to produce one or more of the seven effects listed above, or (3) the PI’s research that is within the scope of this policy may otherwise meet the definition of DURC.  The notification must include the PI’s assessment of whether any research involving these biological agents or toxins produces, aims to produce, or is reasonably anticipated to produce one or more of the seven effects listed above.

The PI must work with the Northwestern University DRC to assess the dual use risks and benefits of the DURC and to develop risk mitigation measures.

The PI must conduct DURC in accordance with the provisions in the risk mitigation plan.

The PI must be knowledgeable about and comply with all Northwestern University and USG policies and requirements for oversight of DURC.

The PI must ensure that laboratory personnel conducting life sciences research with one of the biological agents listed above have received education and training on DURC.  Laboratory personnel include those under the supervision of laboratory leadership such as graduate students, postdoctoral fellows, research technicians, laboratory staff, and visiting scientists.

The PI must communicate DURC in a responsible manner.  Communication of research and research findings is an essential activity for all researchers, and occurs throughout the research process, not only at the point of publication.  Researchers planning to communicate DURC should do so in compliance with the approved risk mitigation plan.

12.6.6 Responsibilities of Northwestern University as a USG-Funded Research Institution

Northwestern University must establish and implement internal policies and practices that provide for the identification and effective oversight of DURC.

When research is identified by a PI as using one of the biological agents or toxins listed above, Northwestern University must initiate an institutional review and oversight process that includes the steps described below, as applicable.  Research that has already been determined to be DURC under the USG March 2012 DURC Policy, and for which a risk mitigation plan has already been developed, is not required to undergo the review process from the beginning but will be subject to ongoing review and notification, as described below.

  1. Verification, by the Northwestern University DRC, that the research identified by the PI uses one or more of the biological agents or toxins listed above.
  2. Review, by the Northwestern University DRC, of the PI’s assessment of whether the research produces, aims to produce, or is reasonably anticipated to produce one or more of the seven effects listed above and final determination of their applicability.  If the DRC determines that the research in question does not involve one or more of the seven categories of experiments, the research is not subject to additional review or oversight. But shall continue to be assessed by the PI.
  3. If the research has been assessed to meet the scope of the September 2014 DURC Policy and the Northwestern University DURC Policy, determination by the DRC of whether the research meets the definition of DURC must take place.  A risk assessment will underpin both the determination of DURC and the subsequent development of a draft risk mitigation plan.  The PI will be included in these activities, as appropriate.  If the DRC determines that the research in question does not meet the definition of DURC, the research is not subject to additional DURC oversight but Northwestern University shall notify the appropriate USG funding agency of the DRC’s findings.  If the DRC determines that the research in question meets the definition of DUC, all additional review and DURC oversight steps shall be followed.  Research that has been determined to be DURC should not be conducted until an approved risk mitigation plan is in place.
  4. Within 30 calendar days of the DRC review of the research for DURC potential, Northwestern University will notify the USG funding agency of any research that involves one or more of the 15 listed biological agents and one or more of the seven listed experimental effects, including whether it meets or does not meet the definition of DURC.  For non-USG-funded research, notification will be made to the NIH, which will in turn refer the notification to an appropriate USG funding agency, based upon the nature of the research.  The initial notification will include the following: (1) the grant or contract number related to the research (if the research is funded by the USG), (2) the name(s) of the PI(s), (3) the name(s) of the biological agent(s) listed above, and (4) a description of why the research is deemed to produce one or more of the seven experimental effects listed above.  For research that is determined by the DRC to meet the definition of DURC, the notification will also include the following: the name of the investigator (if different from the PI) responsible for the performance of the DURC and a description of the DRC’s basis for its determination.
  5. Identification by the DRC of the anticipated benefits of the research identified as DURC.  The anticipated benefits should be considered in conjunction with the previously identified risks in order to develop a draft risk mitigation plan to guide the conduct and communication of the DURC.  The Northwestern University DRC will work with both the PI and the USG funding agency, of for non-Federally funded DURC, the NIH-designated USG agency to develop a risk mitigation plan.  Research that has already been determined to be DURC under the March 2012 DURC Policy, and for which a risk mitigation plan has already been developed, does not need a new risk mitigation plan but the extant risk mitigation plan will be subject to ongoing review and modification, as necessary, by the DRC.
  6. Within 90 calendar days of the DRC’s determination that the research is DURC, the DRC will provide the draft mitigation plan to the USG funding agency for final review and approval.  In the case of non-USG funded research, draft mitigation plans should be provided to the USG agency designated by the NIH.  USG agencies will provide an initial response within 30 calendar days and should finalize the plan within 60 calendar days of receipt of the draft plan.
  7. After a risk mitigation plan is developed and is approved by the USG funding agency, the DURC must be conducted in accordance with that plan immediately following approval.
  8. The DRC will review, at least annually, all active risk mitigation plans.  If the research in question still constitutes DURC, the DRC should modify the plan as needed.
  9. The DRC will provide notification to the USG funding agency within 30 calendar days of (1) any change in the status of a DURC project at Northwestern University (including whether the research is determined by the DRC to no longer meet the definition of DURC), and (2) details of any changes to risk mitigation plans (which need to be approved by the funding agency).  Such notification should be made to the USG funding agency or, in the case of non-USG funded research, to the USG agency designated by the NIH.

According to the September 2014 DURC Policy, it is the responsibility of Northwestern University to ensure that internal policies are established which provide a mechanism for the PI to immediately refer a project to the DRC as soon as:

  1. The PI’s research involves one or more of the biological agents or toxins listed above,
  2. The PI’s research with one or more of the biological agents or toxins listed above also produces, aims to produce, or can be reasonably anticipated to produce one or more of the seven effects listed above, or
  3. The PI’s research that falls within the scope of this policy may meet the definition of DURC.

It is the responsibility of Northwestern University, as mandated by the September 2014 DURC Policy, to designate an Institutional Contact for Dual Use Research (ICDUR).  This individual will serve as an institutional point of contact for questions regarding compliance with and implementation of the requirements for the oversight of research that falls within the scope of the September 2014 DURC Policy and/or meets the definition of DURC.  If questions arise regarding compliance, implementation of this Policy, or the March 2012 DURC Policy, or when guidance is needed about identifying DURC or developing risk mitigation plans, the ICDUR serves as the liaison (as necessary) between Northwestern University and the relevant program officers at the USG funding agencies, or for non-USG funded research, between the Northwestern University and the NIH (or the USG agency to which the NIH has referred Northwestern University).

Northwestern University is also charged, by the September 2014 DURC Policy, to establish an Institutional Review Entity to execute the managerial requirements of this policy, as described above.  The Institutional Review Entity at Northwestern University shall be referred to as the DURC Review Committee (DRC) and will be composed of a subset of the members of the Institutional Biosafety Committee (IBC).  In accordance with the September 2014 DURC Policy, the DRC shall be composed of at least five members and:

  1. Be sufficiently empowered by Northwestern University to ensure it can execute the managerial requirements of this policy,
  2. Include persons with sufficient breadth of expertise to assess the dual use potential of the range of relevant life sciences research conducted by a given research laboratory, program, core facility, or department,
  3. Include persons with knowledge of relevant USG policies and understanding of risk assessment and risk management considerations, including biosafety and biosecurity.  The DRC may also include, or have available as consultants, at least one person knowledgeable in Northwestern University’s commitments, policies, and standard operating procedures,
  4. On a case by case basis, recuse any member of the DRC who is involved in the research project in question or has direct financial interest, except to provide specific information requested by the DRC, and
  5. Engage in ongoing dialogue with the PI of the research in question when conducting a risk assessment and developing a risk mitigation plan.

Northwestern University and the DRC shall, in accordance with the September 2014 DURC Policy, maintain records of institutional DURC reviews and completed risk mitigation plans for the term of the research grant or contract plus three years after its completion, but no less than eight years, unless a shorter period is required by law or regulation.

Northwestern University and the DRC shall provide education and training on DURC for individuals conducting life sciences research with one or more of the biological agents listed in this policy and maintain records of such education and training for the term of the research grant or contract plus three years after its completion.  The Northwestern University ICDUR shall be responsible for developing, coordinating, and administering the DURC education and training program.

The DRC shall, as necessary, assist the PIs who are conducting life sciences research when questions arise about whether their research may require further review or oversight.

The DRC shall develop, implement, and establish its own policies and internal mechanisms for PIs to appeal decisions regarding research that is determined by the DRC to meet the definition of DURC.

The DRC will make information about the process for review of research subject to the September 2014 DURC Policy and the Northwestern University DURC Policy available upon request, as consistent with applicable law, and in accordance with other Northwestern University Policy.

In accordance with the September 2014 DURC Policy, Northwestern University must certify that it is in compliance with all aspects of the September 2014 DURC Policy when applying for USG funds for life sciences research.  The DRC shall be the committee responsible for ensuring compliance and providing such certification.

In the event DURC is being conducted as a part of a collaboration between Northwestern University and another Institution, the primary institution (the institution which holds the grant for the USG funding) shall be responsible for identifying DURC and developing and implementing the risk mitigation plan.  In the event that Northwestern University is not the primary institution, the Northwestern University DRC shall review the primary institution’s draft risk mitigation plan.  The Northwestern University DRC must agree to adopt the primary institutions risk mitigation plan before the DURC may proceed.  Northwestern University will not relinquish compliance oversight of DURC conducted at any Northwestern University campus or affiliated location.  The Northwestern University DRC will be responsible for ensuring compliance with the risk mitigation plan developed by the primary institution.

12.6.7 Responsibilities of the Northwestern University Institutional Biosafety Committee

While it is the responsibility of the PI to identify research with dual use potential, the Northwestern University IBC shall inform the DRC of any research which may meet the definition of DURC as discovered through the IBC research review process.  The DRC shall be responsible for communicating with the PI in order to determine if the research is DURC.  The IBC will also inform the DRC of any suspected non-compliance by a PI or any laboratory personnel suspected of not maintaining compliance with the approved risk mitigation plan.

The DRC shall be responsible for developing the draft risk mitigation plan.  Once the risk mitigation plan is approved by the appropriate USG funding agency, the IBC shall uphold the requirements of the risk mitigation plan and work with the DRC to ensure compliance by the PIs.  The IBC shall take under advisement all recommendations put forth by the DRC regarding the continuation or cessation of DURC and research with dual use potential.

12.6.8 Compliance with the Northwestern University DURC Policy

Non-compliance with the September 2014 DURC Policy may result in the suspension, limitation, or termination of USG funding, or loss of future USG funding opportunities for the non-compliant USG-funded research project and of USG funds for other life sciences research at the institution, consistent with existing regulations and policies governing USG-funded research, and may be subject the institution to other potential penalties under applicable laws and regulations.  While each USG funding agency is responsible, in accordance with its relevant statutory and regulatory authorities, for determining how best to ensure compliance with the oversight requirements set forth in the September 2014 DURC Policy for research it funds, the USG has developed and promulgates consistent processes for this purpose.

Due to the severity of the potential consequences for the entirety of the Northwestern University community, it is of the utmost importance that Northwestern University itself holds its faculty, staff, students, and employees, permanent, visiting or temporary, to the same stringent requirements with its DURC Policy as the USG does for the institutions in its own Policy.  Accordingly, should the DRC be made aware of non-compliance with this Policy of any faculty, staff, student, or employee, the DRC will make a strong and immediate recommendation to the IBC to suspend the approval of all protocols associated with the non-compliant research project or program.  The DRC will recommend that the suspension of all protocols remain in effect until the DRC resolves the issue with the non-compliant laboratory, research project, and/or program.  The DRC will also determine actions to be taken in the future to ensure ongoing compliance with the September 2014 DURC Policy and the Northwestern University DURC Policy.

 

12.7 Forms, Instructions and Additional References

Tools for the Identification, Assessment, Management, and Responsible Communication of Dual Use Research of Concern: A Companion Guide

www.phe.gov/s3/dualuse/Documents/us-policy-durc-032812.pdf

 

Implementation of the U.S. Government Policy for Institutional Oversight of Life Sciences DURC: Case Studies

www.phe.gov/s3/dualuse/Documents/us-policy-durc-032812.pdf

 

Training on the US Government Policy for Institutional Oversight of Life Sciences Dual Use Research of Concern

www.phe.gov/s3/dualuse/Documents/durc-us-policy-trng.pdf

 

United States Government Policy for Institutional Oversight of Life Sciences Dual Use Research of Concern (Released September 24, 2014)

www.phe.gov/s3/dualuse/Documents/durc-policy.pdf

 

United States Government Science, Safety, Security (S3) website:

http://www.phe.gov/s3/dualuse/Pages/default.aspx

 

United States Government Policy for Oversight of Life Sciences Dual Use Research of Concern (Released March 29, 2012)

www.phe.gov/s3/dualuse/Documents/us-policy-durc-032812.pdf

13.0 Biological Safety Resources

Appendices

Works Cited
  1. Biological Safety: Principles and Practices, 4th Edition. Biological Safety: Principles and Practices, 4th Edition, 1-623 (2006).
  2. Dietzgen, R. G., Kondo, H., Goodin, M. M., Kurath, G. & Vasilakis, N. The family Rhabdoviridae: mono- and bipartite negative-sense RNA viruses with diverse genome organization and common evolutionary origins. Virus Res 227, 158-170, doi:10.1016/j.virusres.2016.10.010 (2017).
  3. Jackson, A. C. Human Rabies: a 2016 Update. Curr Infect Dis Rep 18, 38, doi:10.1007/s11908-016-0540-y (2016).
  4. Zhu, S. & Guo, C. Rabies Control and Treatment: From Prophylaxis to Strategies with Curative Potential. Viruses 8, doi:10.3390/v8110279 (2016).
  5. WHO. Rabies Fact Sheet, (2016).
  6. Mortality, G. B. D. & Causes of Death, C. Global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet 385, 117-171, doi:10.1016/S0140-6736(14)61682-2 (2015).
  7. Kumar, V., Abbas, A. K., Fausto, N., Robbins, S. L. & Cotran, R. S. Robbins and Cotran pathologic basis of disease. 7th edn,  (Elsevier Saunders, 2005).
  8. Rupprecht, C. E. et al. Use of a reduced (4-dose) vaccine schedule for postexposure prophylaxis to prevent human rabies: recommendations of the advisory committee on immunization practices. MMWR. Recommendations and reports : Morbidity and mortality weekly report. Recommendations and reports 59, 1-9 (2010).
  9. Rabies, Australian bat lyssavirus and other lyssaviruses, (2013).
  10. National Association of State Public Health, V. et al. Compendium of Animal Rabies Prevention and Control, 2016. J Am Vet Med Assoc 248, 505-517, doi:10.2460/javma.248.5.505 (2016).
  11. Rupprecht, C. E. & Gibbons, R. V. Clinical practice. Prophylaxis against rabies. N Engl J Med 351, 2626-2635, doi:10.1056/NEJMcp042140 (2004).
  12. Callaway, E. M. Transneuronal circuit tracing with neurotropic viruses. Curr Opin Neurobiol 18, 617-623, doi:10.1016/j.conb.2009.03.007 (2008).
  13. Gomme, E. A., Wanjalla, C. N., Wirblich, C. & Schnell, M. J. Rabies virus as a research tool and viral vaccine vector. Adv Virus Res 79, 139-164, doi:10.1016/B978-0-12-387040-7.00009-3 (2011).
  14. Wickersham, I. R., Finke, S., Conzelmann, K. K. & Callaway, E. M. Retrograde neuronal tracing with a deletion-mutant rabies virus. Nat Methods 4, 47-49, doi:10.1038/nmeth999 (2007).
  15. Wirblich, C. et al. One-Health: a Safe, Efficient, Dual-Use Vaccine for Humans and Animals against Middle East Respiratory Syndrome Coronavirus and Rabies Virus. J Virol 91, doi:10.1128/JVI.02040-16 (2017).
  16. Kelly, B. J., King, L. A. & Possee, R. D. Introduction to Baculovirus Molecular Biology. Methods Mol Biol 1350, 25-50, doi:10.1007/978-1-4939-3043-2_2 (2016).
  17. Possee, R. D. & King, L. A. Baculovirus Transfer Vectors. Methods Mol Biol 1350, 51-71, doi:10.1007/978-1-4939-3043-2_3 (2016).
  18. Kost, T. A. & Kemp, C. W. Fundamentals of Baculovirus Expression and Applications. Adv Exp Med Biol 896, 187-197, doi:10.1007/978-3-319-27216-0_12 (2016).
  19. Gill, D. M. Bacterial toxins: a table of lethal amounts. Microbiol Rev 46, 86-94 (1982).
  20. Stirpe, F., Barbieri, L., Battelli, M. G., Soria, M. & Lappi, D. A. Ribosome-inactivating proteins from plants: present status and future prospects. Biotechnology (N Y) 10, 405-412 (1992).
  21. Sweet, D. V.    (ed Centers for Disease Control and Prevention United States Department of Health and Human Services) (National Institute for Occupational Safety and Health, Cincinnati, OH, 1997).