MSDSs

7.0 BIOLOGICAL AGENTS

7.1 General
Many laboratory practices and requirements are common to laboratories using chemical and biological agents. The laboratory procedures described in Section 5.0, "General Laboratory Safety," and Section 6.0, "Chemical Hazards," also apply to laboratories using biological agents, and that information will not be repeated in this part. Most of the information in this section is taken from the book Biosafety in Microbiological and Biomedical Laboratories (U.S. Health and Human Services Publication No. CDC93-8395, Public Health Service, Centers for Disease Control and Prevention, and National Institutes of Health, 1999). A copy of the book is available for reference in ORS.

Biological agent hazards in the laboratory are relatively well defined, especially in the case of conventional disease-producing agents. Major exceptions to this general observation are oncogenic agents and "slow virus" infections.

Broadly speaking, two major risk situations can be identified. In the first, known agents are used and are integral to scientific research or teaching; in the second, potentially harmful biological agents are endogenous to humans or laboratory animals or to animal tissues or fluids. Examples of these are zoonotic infections harbored in the research animal population, viral contaminants in human tissues and cell cultures, and lymphocytic or choriomeningitis-infected animal tumor lines.

Other issues that do not relate specifically to personal safety but should be considered include work with agents that infect lower animals and plants, especially if an accident could seriously jeopardize the agricultural sector of the economy. The possibility of cross-contamination by infectious agents in laboratory animals and media preparation areas can also be a significant problem, especially in common resource facilities.

See Table 7.4 for a list of agents that have summary statements available from ORS.

7.2 Responsibilities

7.2.1 General. The responsibilities of the department head, principal investigator, and others working with biological hazards include all those described in Section 2.0 of this document. Additional responsibilities specific to the handling of biological agents include selecting safety practices based on awareness of the particular hazard and training all personnel accordingly.
7.2.2 Laboratory Safety Profile. Investigators shall submit a Laboratory Safety Profile to ORS and/or the Chemical and Biological Safety Committee for research involving biological agents that require containment at Biosafety Level 2 or above. Investigators shall comply with the requirements for each level of containment based on the biological agents used in research projects. ORS can assist in determining the biosafety level of an agent. The classification of the biosafety level is subject to the approval of the CBSC.

An investigator shall also submit an RDNA Registration form within ISIS for research involving human materials and recombinant DNA.

7.3 Containment Methods
The term containment is used to describe safe methods for managing infectious agents in the laboratory environment. The purpose of containment is to reduce exposure of laboratory workers and others to potentially hazardous agents and to prevent their escape into the outside environment. The three elements of containment are laboratory practice and technique, safety equipment, and facility design.

7.3.1 Laboratory Practice. The most important element of containment is strict adherence to standard microbiological practices and techniques. Persons working with infectious agents or infected materials shall be aware of potential hazards and shall be trained and proficient in the practices and techniques required for safely handling such material. When standard laboratory practices are not sufficient to control the hazard associated with a particular agent or laboratory procedure, additional measures may be needed involving safety equipment and facility design.

7.3.2 Safety Equipment (Primary Barriers). Safety equipment includes biological safety cabinets, enclosed containers, and other engineering controls designed to prevent or minimize exposures to hazardous biological materials. The use of vaccines may in some cases provide an increased level of personal protection.

7.3.2.1 Biological Safety Cabinets (BSCs) The biological safety cabinet is the principal device used to provide containment of infectious splashes or aerosols. There are three types of biological safety cabinets: Class I, Class II, and Class III.

Class I is an open-fronted, negative-pressure, vented cabinet with HEPA-filtered exhaust. It may be equipped with a front closure and gloves for use as a glove box. The inward face velocity is a minimum of 75 linear feet per minute. Suitable for work with low- or moderate-risk biological agents, it provides protection for personnel and the environment but not for the product.

Class II cabinets are open-fronted laminar-flow cabinets with a minimum inward face velocity of 75 linear feet per minute. Class II design resembles that of a fume hood but with HEPA-filtered, recirculated mass airflow within the workspace. Exhaust air is also filtered. Class II cabinets provide protection for personnel, product, and the environment. They are designed for work with low- or moderate-risk biological agents.

Class III cabinets provide the highest level of protection. Class III is a totally enclosed glove-box cabinet of gas-tight construction. The cabinet is maintained under negative air pressure of at least 0.5 inches of water gauge. Supply air is drawn into the cabinet through HEPA filters, and the exhaust air is filtered by two HEPA filters in series before it is discharged to the outside. Generally, the ventilation system is separate from the facility's ventilation system. Class III cabinets are suitable for high-risk biological agents.

Biological safety cabinets used to protect workers from hazardous biological agents shall be tested and certified after installation and before use, any time they are moved, and at least annually. The department head shall provide annual certification and maintain certification records for the department. Testing shall meet the criteria in National Sanitation Foundation Standard Number 49. The biological safety cabinet shall be decontaminated prior to certification or performance tests. See "Decontamination of Biological Safety Cabinets" at http://www.research.northwestern.edu/ors/biosafe/parafrm2.htm. Call ORS for information on the standard and a list of companies qualified to certify biological safety cabinets.

For more detailed information about BSCs, refer to the 1995 CDC/NIH publication "Primary Containment for Biohazards: Selection, Installation and Use of Biological Safety Cabinets." It can be found at http://www.cdc.gov/od/ohs/biosfty/bsc/bsc.htm.

7.3.2.2 Other Safety Equipment
Other safety equipment includes enclosed containers. An example of an enclosed container is the safety centrifuge cap, designed to prevent release of aerosols during centrifugation.

Safety equipment also includes personal protective clothing and equipment such as gloves, coats, gowns, shoe covers, boots, respirators, face masks or shields, and safety glasses or goggles. This clothing and equipment is generally used in combination with biological safety cabinets and other devices that contain the agents, animals, or materials in use.

In situations in which it is impractical to work in biological safety cabinets, personal protective devices may form the primary barrier between personnel and the infectious materials. Examples of such situations include certain animal studies, animal necropsy, and activities relating to maintenance, service, or support of the laboratory facility.

7.3.3 Facility Design (Secondary Barriers). Secondary barriers protect the environment within the facility but outside the laboratory-and the community outside the facility-from exposure to infectious materials. The design of the facility provides the secondary barrier. The three facility designs are the basic laboratory, the containment laboratory, and the maximum containment laboratory.

The Basic Laboratory provides general space where work is done with viable agents that are not associated with disease in healthy adults; it includes Biosafety Levels 1 and 2 facilities. This laboratory is also appropriate for work with infectious agents or potentially infectious materials when the hazard levels are low and laboratory personnel can be adequately protected by standard laboratory practice. While work is commonly conducted on the open bench, certain operations are confined to biological safety cabinets. Conventional laboratory designs are adequate.

The Containment Laboratory has special engineering features that enable laboratory workers to handle hazardous materials without endangering themselves, the community, or the environment. The containment laboratory is described as a Biosafety Level 3 facility. The features that distinguish this laboratory from the basic laboratory are the provisions for access control and a specialized ventilation system. In all cases, a controlled access zone separates the laboratory from areas open to the public.

The Maximum Containment Laboratory has special engineering and containment features that allow laboratory workers to safely conduct activities involving infectious agents that are extremely hazardous to humans or capable of causing serious epidemic disease. The maximum containment laboratory is described as a Biosafety Level 4 facility; it is not applicable to activities at the University.

7.4 Biosafety Levels
The following guidelines are recommended by the Centers for Disease Control and Prevention and the National Institutes of Health and have been adopted as required procedure at the University. They are drawn from the book Biosafety in Microbiological and Biomedical Laboratories.

7.4.1 Biosafety Level 1. Biosafety Level 1 (BSL-1) is suitable for work involving agents of no known or minimal potential hazard to laboratory personnel and the environment. The laboratory may be integral to general traffic patterns in the building. Work may be conducted on open bench tops. Special containment equipment is neither required nor generally used. Laboratory personnel shall have specific training in procedures conducted in the laboratory.

7.4.1.1 Standard Microbiological Practices for BSL-1

1. At the discretion of the principal investigator, access to the laboratory shall be limited or restricted while experiments are in progress.
2. A biohazard sign shall be posted at the entrance to the laboratory whenever infectious agents are present. The sign must include the name of the agent(s) in use and the name and phone number of the principal investigator.
3. Work surfaces shall be decontaminated once a day and after any spill of viable material.
4. All contaminated liquid or solid wastes shall be decontaminated before disposal. Contaminated materials that are to be decontaminated at a site outside the laboratory shall be placed in a durable, leakproof, closed container before being removed from the laboratory.
5. The laboratory shall have an established policy for the safe handling of sharps.
6. Mechanical pipetting devices shall be used; mouth pipetting is prohibited.
7. Eating, drinking, smoking, and applying cosmetics are not permitted in the work area. Food may be stored in cabinets and refrigerators designated and used for this purpose only. Food storage cabinets and refrigerators shall be located outside the work area.
8. Laboratory personnel shall wash their hands after they handle viable materials and animals and before leaving the laboratory.
9. All procedures shall be performed carefully to minimize the creation of aerosols.
10. An insect and rodent control program is in effect. At Northwestern, Facilities Management is responsible for the control of pests and should be contacted if insects or rodents not involved in research, or their wastes, are observed in the laboratory.

7.4.1.2 Safety Equipment for BSL-1

1. Special containment equipment is generally not required for manipulation of agents assigned to Biosafety Level 1.
2. It is recommended that laboratory coats, gowns, or uniforms be worn to prevent contamination or soiling of street clothes.
3. Gloves should be worn if skin is broken or afflicted by a rash.

7.4.1.3 Laboratory Facilities for BSL-1

1. The laboratory shall be designed so that it can be easily cleaned.
2. Bench tops shall be impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.
3. Laboratory furniture shall be sturdy. Spaces between benches, cabinets, and equipment shall be accessible for cleaning.
4. Each laboratory shall contain a sink for handwashing.
5. If the laboratory has windows that open, they shall be fitted with fly screens.

7.4.2 Biosafety Level 2. Biosafety Level 2 is similar to Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment. It differs in that (1) laboratory personnel are specifically trained to handle pathogenic agents and are directed by scientists who are experienced in working with these agents, (2) access to the laboratory is limited when work is being conducted, (3) extreme precautions are taken with contaminated sharp items, and (4) certain procedures that may result in the creation of infectious aerosols or splashes are conducted in biological safety cabinets or other physical containment equipment.

The following standard and special practices, safety equipment, and facilities apply to agents assigned to Biosafety Level 2.

7.4.2.1 Standard Microbiological Practices for BSL-2

1. At the discretion of the laboratory director, access to the laboratory is limited or restricted while experiments are in progress.
2. Laboratory personnel are to wash their hands after they handle viable materials and animals, after removing gloves, and before leaving the laboratory.
3. Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted in the work areas. Food is stored outside the work area in cabinets or refrigerators designated and used for this purpose only.
4. The laboratory shall have an established policy for the safe handling of sharps.
5. Mouth pipetting is prohibited; mechanical pipetting devices are used.
6. All procedures are performed carefully to minimize the creation of splashes or aerosols.
7. Work surfaces are decontaminated at least once a day and after any spill of viable material.
8. All cultures, stocks, and other regulated wastes are decontaminated by an approved decontamination method, such as autoclaving before disposal. Materials to be decontaminated outside the immediate laboratory are to be placed in a durable, leakproof container that is closed for transport from the laboratory.
9. An insect and rodent control program is in effect. At Northwestern, Facilities Management is responsible for the control of pests and should be contacted if insects or rodents not involved in research, or their wastes, are observed in the laboratory.

7.4.2.2 Special Practices for BSL-2

1. Access to the laboratory is limited or restricted by the principal investigator when work with infectious agents is in progress. In general, persons at increased risk of acquiring infection or for whom infection may be unusually hazardous are not allowed in the laboratory or animal rooms. Persons who are immunocompromised or immunosuppressed may be at unusual risk of acquiring infections.
2. The principal investigator establishes policies and procedures whereby only persons who have been advised of the potential hazard and meet specific requirements (e.g., immunization) enter the laboratory or animal rooms.
3. When an infectious agent requires special provisions (e.g., immunization) for entering a laboratory where it is in use, a hazard warning sign incorporating the universal biohazard symbol is posted on the access door to the laboratory work area. The hazard warning sign identifies the infectious agent, lists the name and telephone number of the principal investigator or other responsible person, and indicates the special requirements for entering the laboratory.
4. Laboratory personnel receive appropriate immunizations for the agents handled or potentially present in the laboratory.
5. When appropriate, baseline serum samples for laboratory and other at-risk personnel are collected and stored. Additional specimens may be collected periodically.
6. A biosafety manual shall be developed for the laboratory. Laboratory personnel are advised of special hazards and are required to read and follow instructions on practices and procedures.
7. Laboratory personnel receive appropriate training on the potential hazards associated with the work involved, the necessary precautions to prevent exposures, and the exposure evaluation procedures. Personnel receive annual retraining and receive additional training when procedures or policies change.
8. A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels. Needles and syringes should be used in the laboratory only when there is no alternative, such as when parenteral injection, phlebotomy, or aspiration of fluids from laboratory animals and diaphragm bottles are conducted. Plasticware should be substituted for glassware whenever possible.
   1. Only needle-locking syringes or disposable syringe-needle units (i.e., the needle is integral to the syringe) are used for injection or aspiration of infectious materials. Used disposable needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal; rather they must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal. Nondisposable sharps must be placed in a hard-walled container for transport to a processing area for decontamination, preferably by autoclaving.
   2. Syringes that resheathe the needle, needleless systems, and other safe devices should be used when appropriate.
   3. Broken glassware must not be handled directly by hand but must be removed by mechanical means such as a brush and dustpan, tongs, or forceps. Containers of contaminated needles, sharp equipment, and broken glass are decontaminated before disposal.
9. Cultures, tissues, and specimens of body fluids are placed in a container that prevents leakage during collection, handling, processing, storage, transport, or shipping.
10. Laboratory equipment and work surfaces should be decontaminated with an appropriate disinfectant on a routine basis as well as after work with infectious material is finished and, especially, after overt spills, splashes, or other contamination by infectious materials. Contaminated equipment must be decontaminated before it is sent for repair or maintenance or packaged for transport.
11. Spills or accidents that result in overt exposures to infectious materials are immediately reported to the laboratory director. Medical evaluation, surveillance, and treatment are provided as appropriate at no cost to employees, and written records are maintained.
12. Animals not involved in the work being performed are not permitted in the lab.

7.4.2.3 Safety Equipment for BSL-2

1. Properly maintained biological safety cabinets, preferably Class II, or other appropriate personal protective equipment or physical containment devices are used whenever:
   1. Procedures with a potential for creating infectious aerosols or splashes are conducted. These may include centrifuging, grinding, blending, vigorous shaking or mixing, sonic disruption, opening containers of infectious materials in which internal pressure may differ from ambient pressure, inoculating animals intranasally, and harvesting infected tissues from animals or eggs.
   2. High concentrations or large volumes of infectious agents are used. Such materials may be centrifuged in the open laboratory if sealed rotor heads or centrifuge safety cups are used and if these rotors or safety cups are opened only in a biological safety cabinet.
2. Face protection (goggles, mask, face shield, or other splatter guards) is used for anticipated splashes or sprays of infectious or other hazardous materials to the face when the microorganisms must be manipulated outside the biological safety cabinet.
3. Protective laboratory coats, gowns, smocks, or uniforms designated for lab use are worn in the laboratory. This protective clothing is removed and left in the laboratory before lab personnel leave for nonlaboratory areas (e.g., cafeteria, library, or offices). All protective clothing is either disposed of in the laboratory or sent to the laundry service (only after being decontaminated). It is never taken home.
4. Lab personnel wear gloves when handling infected animals and when hands may come in contact with infectious materials or contaminated surfaces or equipment. Wearing two pairs of gloves may be appropriate; if a spill or splatter occurs, the hand will be protected after the contaminated glove is removed. Gloves are disposed of when contaminated, removed when work with infectious materials is completed, and not worn outside the laboratory. Disposable gloves are not washed or reused.

7.4.2.4 Laboratory Facilities (Secondary Barriers) for BSL-2

1. Provide lockable doors for facilities that house restricted agents (see Table 7.1).
2. Each laboratory contains a sink for handwashing.
3. The laboratory is designed so that it can be easily cleaned. Rugs are not appropriate in laboratories.
4. Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.
5. Laboratory furniture is sturdy, and spaces between benches, cabinets, and equipment are accessible for cleaning.
6. If the laboratory has windows that open, they are fitted with fly screens.
7. An eyewash facility is readily available.
8. Biological safety cabinets shall be installed in such a manner that fluctuations of the room supply and exhaust air do not cause them to operate outside their parameters for containment. Biological safety cabinets shall be located away from doors, from windows that can be opened, from heavily traveled laboratory areas, and from other potentially disruptive equipment so as to maintain the biological safety cabinets' air flow parameters for containment.
9. Illumination shall be adequate for all activities, avoiding reflections and glare that could impede vision.

7.4.3 Biosafety Level 3. All work to be conducted with agents assigned to Biosafety Level 3 must be approved in advance by the Chemical and Biological Safety Committee. If you desire to work with BSL-3 agents, contact ORS in advance.

BSL-3 work must be conducted in accordance with the facility safeguards, standard microbiological practices, special practices, and safety equipment described in Biosafety in Microbiological and Biomedical Laboratories. See Tables 7.7 and 7.8 for lists of biohazardous agents that must be handled at BSL-3.

Biosafety Level 3 is applicable to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents that may cause serious or potentially lethal disease as a result of exposure by inhalation. Laboratory personnel have specific training in handling pathogenic and potentially lethal agents and are supervised by scientists experienced in working with these agents.

All procedures involving the manipulation of infectious materials are conducted within biological safety cabinets or other physical containment devices, or by personnel wearing appropriate personal protective clothing and equipment. The laboratory has special engineering and design features such as access zones, sealed penetrations, and directional airflow.

Many laboratories may not have all the facility safeguards recommended for Biosafety Level 3. In these circumstances, acceptable safety may be achieved for routine or repetitive operations (e.g., diagnostic procedures involving the propagation of an agent for identification, typing, and susceptibility testing) in Biosafety Level 2 facilities. However, the recommended standard microbiological practices, special practices, and safety equipment for Biosafety Level 3 must be rigorously followed.

7.4.4 Biosafety Level 4. University use of biological agents requiring Biosafety Level 4 containment practices is not anticipated. Should an investigator wish to conduct research with these agents, a specially developed Laboratory Safety Profile shall be submitted to the Chemical and Biological Safety Committee for review and approval prior to initiation of research. Approval is also required by the vice president for research prior to initiation of research.

7.5 Biological Spills
A biological spill shall be followed by prompt action to contain and clean up the spill. When a spill occurs, warn everyone in the area and call for assistance as needed. The degree of risk involved in the spill depends on the volume of material spilled, the potential concentration of organisms in the material spilled, the hazard of the organisms involved, the route of infection of the organisms, and the diseases caused by the organisms.

Spills of biological agents can contaminate areas and lead to infection of laboratory workers. Prevention of exposure is the primary goal in spill containment and cleanup, exactly as in chemical spills. In evaluating the risks of spill response, generation of aerosols or droplets is a major consideration.

If an accident generates droplets or aerosols in the laboratory room atmosphere, especially if the agent involved requires containment at Biosafety Level 2 or higher, the room shall be evacuated immediately.

Doors shall be closed and clothing decontaminated. Call ORS to supervise the cleanup. In general, a 30-minute wait is sufficient for the droplets to settle and aerosols to be reduced by air changes. Longer waiting periods may be imposed depending on the situation. Laboratory personnel and/or ORS must exercise judgment as to the need for outside emergency help in evacuation.

If a spill of a biological agent requiring containment at Biosafety Level 2 or higher occurs in a public area, evacuation of the area shall be immediate. The principal investigator shall be responsible for designating the extent of evacuation until ORS or emergency personnel arrive. Prevention of exposure to hazardous aerosols is of primary importance.

Anyone cleaning a spill shall wear personal protective equipment (for example, laboratory coat, shoe covers, gloves, and possible respiratory protection) to prevent exposure to organisms. An air-purifying negative-pressure respirator with P-100 filter cartridges is generally adequate protection against inhalation of most biological agents. However, there may be exceptions. Contact ORS for advice in choosing the correct respiratory protection and for information regarding the requirements that must be met to wear a respirator.

An appropriate chemical disinfectant should be chosen that is effective against the organisms involved in the spill (see Tables 7.3A and 7.3B).

7.5.1 Sterilization, Disinfection, and Decontamination. The Environmental Protection Agency recognizes the following categories of chemical germicides (a germicide is an agent that kills pathogenic organisms). The information in this section is drawn from Protection of Laboratory Workers from Instrument Biohazards and Infectious Disease Transmitted by Blood, Body Fluids, and Tissue, Approved Guideline, NCCLS Document M29-A, Vol. 17, No.20 (National Committee for Clinical Laboratory Standards, December, 1997).

Sterilizer or Sterilant: An agent intended to destroy all microorganisms and their spores on inanimate surfaces.

Disinfectant: An agent intended to destroy or irreversibly inactivate specific viruses, bacteria, or pathogenic fungi, but not necessarily their spores, on inanimate surfaces. Most disinfectants are not effective sterilizers.

Hospital Disinfectant: An agent shown to be effective against specific organisms such as Staphylococcus aureus, Salmonella choleraesuis, and Pseudomonas aeruginosa. It may also be effective against other organisms and some viruses. The labels of all commercially available hospital disinfectants contain a claim (which must be documented) of effectiveness for specific agents.

Antiseptic: A chemical germicide formulated for use on skin or tissue. Antiseptics should not be used as disinfectants.

Decontamination: A procedure that eliminates or reduces microbial contamination to a safe level with respect to the transmission of infection. Sterilization and disinfection procedures are often used for decontamination.

The OSHA Bloodborne Pathogens Standard requires that all equipment and environmental and working surfaces shall be cleaned and decontaminated after contact with blood or other potentially infectious materials. The standard also requires decontamination of contaminated work surfaces after completion of procedures, immediately or as soon as feasible after any overt contamination of surfaces or any spill of potentially infectious material, and at the end of the work shift if the work surface has become contaminated. All reusable equipment shall be decontaminated immediately or as soon as feasible upon visible contamination.

It should be emphasized that, for any infectious material, adequate precleaning of surfaces is important for any disinfection or sterilization procedure. Ten minutes of exposure to a disinfectant may not be adequate to disinfect objects that have narrow channels or other areas that can harbor microorganisms. Alcohols, for example, are effective for killing hepatitis B virus (HBV) but are not recommended for this purpose because of their rapid evaporation and the consequent difficulty of maintaining proper contact times.

Chlorine compounds are probably the most widely used disinfectants in the laboratory. You can easily prepare an inexpensive, broad-spectrum disinfectant by diluting common household bleach.

Bleach is a 5.25% sodium hypochlorite solution-this is equal to approximately 50,000 ppm of free available chlorine. This level of chlorine can be harmful to skin and eyes. Lower concentrations are effective in disinfection and are less hazardous for the worker. The concentration to be used is based on your assessment of the severity of the contamination or spill of infectious materials.

• For small spills of infectious agents or for contamination on hard, smooth surfaces, a 1:100 dilution of commercial bleach is adequate. This is equivalent to 500 ppm of free chlorine.
• In the case of large or concentrated spills of infectious agents, a higher level of chlorine is needed to be effective in destroying the microorganisms. Use a 1:10 dilution (5,000 ppm of free chlorine) and flood the contaminated area with the solution. Alternatively, you can mix the disinfectant with the spilled material. This higher concentration is more suitable for porous surfaces that may harbor organisms in tiny cracks or pits.

Make the solution fresh each day. Be aware that chlorine compounds may corrode metals, especially aluminum. While a 10% household bleach solution is a commonly used decontaminant concentration, it is probably stronger than necessary for ordinary uses. It can be extremely irritating to personnel. Therefore, the use of higher concentrations of bleach in chemical fume hoods, and the autoclaving of materials that have been treated with bleach, should be reserved for significant contamination.

Note that bleach will react with water to form hypochlorous acid (HOCl), which will decompose to chlorine (Cl₂) and hydrogen chloride (HCl). Special care should be taken when autoclaving hypochlorite solutions because the procedure can generate chlorine gas, which will corrode steel. To avoid evolution of chlorine, the hypochlorite solution should be neutralized with sodium thiosulfate prior to autoclaving.

Formaldehyde is an OSHA-regulated chemical that is a suspect carcinogen, so its use as a disinfectant is not recommended.

Iodophors that are registered with the EPA may be effective hard-surface decontaminants when used per manufacturer's instructions, but iodophors formulated as antiseptics are not suitable for use as disinfectants.

Peracetic (peroxyacetic) acid and hydrogen peroxide mixtures minimize the negative effects of corrosiveness sometimes seen with chlorine compounds and high concentrations of peracetic acid alone. A limited number of trade-name products containing <0.1% peracetic acid and <1.0% hydrogen peroxide and registered with the EPA as sterilants/disinfectants are available. The benefit of these products is their rapid action and broad-spectrum of germicidal activity, in addition to the reduced corrosiveness.

In Section 5.0, the use of any chemicals with the prefix "per" is discouraged for cleaning glassware due to the reactivity of oxidizing materials. Peracetic acid is generally a strong irritant. The low percentage in these products reduces this danger. Nonetheless, these products are intended only for highly concentrated spills of biological materials.

Quaternary ammonium compounds are low-level disinfectants and are not recommended for spills of human blood, blood products, or other potentially infectious materials.

7.5.2 Decontamination of Spills. The following procedure is recommended for decontaminating spills of agents used at BSL-2.

1. Wear gloves and a laboratory coat or gown. Heavyweight, puncture-resistant utility gloves, such as those used for housecleaning and dishwashing, are recommended.
2. Do not handle sharps with the hands. Clean up broken glass or other sharp objects with sheets of cardboard or other rigid, disposable material. If a broom and dustpan are used, they must be decontaminated later.
3. Avoid generating aerosols by sweeping.
4. Absorb the spill. Most disinfectants are less effective in the presence of high concentrations of protein, so absorb the bulk of the liquid before applying disinfectants. Use disposable absorbent material such as paper towels. After absorption of the liquid, dispose of all contaminated materials as waste.
5. Clean the spill site of all visible spilled material using an aqueous detergent solution (e.g., any household detergent). Absorb the bulk of the liquid to prevent dilution of the disinfectant.
6. Disinfect the spill site using an appropriate disinfectant, such as a household bleach solution. Flood the spill site or wipe it down with disposable towels soaked in the disinfectant.
7. Absorb the disinfectant or allow it to dry.
8. Rinse the spill site with water.
9. Dispose of all contaminated materials properly. Place them in a biohazard bag or other leakproof, labeled biohazard container for sterilization.

7.5.3 Biological Spill in the Open Laboratory. For a spill in the open laboratory outside a biological safety cabinet, the spill response depends on the size of the spill and hazard of the material. A minimally hazardous material spilled without generating appreciable aerosols can be cleaned with a paper towel soaked in a chemical disinfectant.

A spill of a larger volume of hazardous material with aerosol generation requires evacuating the room, waiting for aerosol reduction, donning personal protective gear (including appropriate respiratory protection), selecting a disinfectant effective against the organisms involved, and cleaning as described above. Following cleanup, response personnel shall wash or shower with a disinfectant soap.

7.5.4 Biological Spill Within a Biological Safety Cabinet. A spill that is confined within a biological safety cabinet generally presents little or no hazard to personnel in the area. However, chemical disinfection procedures are to be initiated at once while the cabinet continues to operate. The disinfectant shall be one that is active against the organisms of potential hazard. Flammable liquids, such as ethanol or isopropanol, shall not be used, even if effective, because of the fire hazard of generating dangerous vapor concentrations within the cabinet that could be ignited by an electrical spark or other source.

Spray or wipe the walls, work surfaces, and equipment with the chosen disinfectant. Allow the disinfectant to remain on the surface for the appropriate contact time (refer to Table 7.3A and 7.3B for recommended contact times).

Minimize the generation of aerosols and use sufficient disinfectant to ensure that drain pans and catch basins below the work surface contain disinfectant. The front exhaust shall also be wiped and the disinfectant drained into a container.

7.5.5 Biological Spill in a Centrifuge or Other Equipment. A biological spill in a centrifuge has the potential for producing large volumes of aerosols. On becoming aware that a spill may have occurred within a centrifuge or other piece of equipment, turn off the equipment, warn others in the area, notify the principal investigator, allow aerosols to settle, and decontaminate following the principles described above.

7.5.6 Biological Spill on a Person. If a biological material is spilled on a person, emergency response is based on the hazard of the biological agent spilled, the amount of material spilled, and whether significant aerosols were generated. If aerosol formation is believed to have been associated with the spill, a contaminated person shall leave the contaminated area immediately. If possible, (s)he should go to another laboratory area so that hallways and other public areas do not become contaminated.

Contaminated clothing is removed and placed in red or orange biohazard bags for disinfecting. Contaminated skin shall be flushed with water and thoroughly washed with a disinfectant soap. Showering may be appropriate, depending on the extent of the spill.

7.6 Human Blood, Blood Products, and Other Potentially Infectious Materials

Northwestern University is required to comply with the OSHA Occupational Exposure to Bloodborne Pathogens Standard found in Title 29, Code of Federal Regulations, Part 1910.1030. The requirements of the standard are covered in the Bloodborne Pathogens Program. This document is available from ORS or on the Web at http://www.research.northwestern.edu/ors/biosafe/index.htm.

You should refer to the Bloodborne Pathogens Program if your work requires occupational exposure to any of the following human materials:

• Blood (human blood, human blood components, and products made from human blood)
• These human body fluids:
  semen
  vaginal secretions
  cerebrospinal fluid
  synovial fluid
  pleural fluid
  pericardial fluid
  peritoneal fluid
  amniotic fluid
  saliva (in dental procedures)
  any body fluid that is visibly contaminated with blood
  all body fluids in situations where it is difficult or impossible to differentiate between body fluids
• Any unfixed tissue or organ (other than intact skin) from a human, living or dead.
• HIV-containing cell, tissue, or organ cultures; HIV- or HBV-containing culture medium or other solutions; and blood, organs, or other tissues from experimental animals infected with HIV or HBV.

Occupational exposure means reasonably anticipated skin, eye, mucous membrane, or parenteral contact with blood or other potentially infectious materials that may result from the performance of an employee's duties.

7.7 Recombinant DNA Research
Recombinant DNA research shall comply with the National Institutes of Health's "Guidelines for Research Involving Recombinant DNA Molecules," as published in the Federal Register, July 5, 1994, Volume 59, No. 127, pages 34,496 through 34,547, and any subsequent amendments thereto. The Recombinant DNA Safety Committee is responsible for implementing the guidelines and overseeing recombinant DNA research.

Principal investigators intending to use recombinant DNA molecules shall follow the directions in the University's Recombinant DNA Safety Program, available from ORS or on the Web at http://www.research.northwestern.edu/ors/biosafe/index.htm. All recombinant DNA research falls into one of six classes, described in the NIH guidelines as follows:

• Section III-A Experiments that require Institutional Biosafety Committee approval, Recombinant DNA Advisory Committee (RAC) review, and NIH Director approval before initiation
• Section III-B Experiments that require NIH/ORDA and Institutional Biosafety Committee approval before initiation
• Section III-C Experiments that require Institutional Biosafety Committee and Institutional Review Board approval and NIH/ORDA registration before initiation
• Section III-D Experiments that require Institutional Biosafety Committee approval before initiation
• Section III-E Experiments that require Institutional Biosafety Committee notice simultaneous with initiation
• Section III-F Exempt experiments

If an experiment falls into section III-A, III-B, or III-C and one of the other sections as well, the rules pertaining to section III-A, III-B, or III-C shall be followed. If an experiment falls into section III-F alone, or into section III-F and into section III-D or III-E as well, the experiment is considered exempt from the NIH guidelines.

In general, the containment practices to be used for recombinant DNA research shall follow those described for Biosafety Levels 1, 2, and 3 in the CDC-NIH Biosafety in Microbiological and Biomedical Laboratories. However, the NIH Recombinant DNA guidelines take precedence.

7.8 Animal Studies
Animal studies involving the use of hazardous biological agents represent special problems in containment. Policies and operational practices governing the use of animal containment facilities are under the direction of the Animal Care and Use Committee and the Center for Experimental Animal Resources. In general, practices for Animal Biosafety Levels 1, 2, and 3 presented in the CDC-NIH manual, Biosafety in Microbiological and Biomedical Laboratories, are followed.

Experiments involving the use of infectious biological agents in animals are generally conducted in containment facilities. Research in laboratory facilities shall be reviewed and approved, prior to the initiation of work, by the CBSC in conjunction with the Animal Care and Use Committee and the Center for Experimental Animal Resources.

7.9 Handling of Sheep Cells
The University has adopted special policies for the handling of sheep cells due to the potential risk associated with Q fever.

All potentially infectious sheep cells shall be handled at BSL-2, similar to requirements for human cell cultures (as written in the Laboratory Safety Profile).

Lab workers shall assume that all sheep cells are infectious unless there is acceptable serologic confirmation that they are not. Where vendors offer verification of testing of cells before shipment, the following criterion must be met: a biological safety professional or other qualified scientist with background and experience to review potential contamination and risk shall comment, in writing, on the test methods and molecular technology applied to a cell line sample to identify or screen for infectivity.

BSL-3 practices shall be applied in handling cells from sheep birth products (e.g., amniotic fluid, placenta).

7.10 Importation or Transport Permits
The US Department of Agriculture (USDA), Animal and Plant Health Inspection Service (APHIS), Veterinary Services (VS), regulates

• the importation of all animal-origin materials (animal products and byproducts) that could present a disease risk to US livestock
• the import and transport of infectious organisms and vectors of disease agents
• biological materials that contain or have been in contact with certain organisms and animal materials (including cell cultures).

Generally, an USDA veterinary permit is needed for materials derived from animals or exposed to animal-source materials. Materials which require a permit include animal tissues, blood, cells or cell lines of livestock or poultry origin, RNA/DNA extracts, hormones, enzymes, monoclonal antibodies for IN VIVO use in non-human species, certain polyclonal antibodies, antisera, bulk shipments of test kit reagents, and microorganisms including bacteria, viruses, protozoa, and fungi. Exceptions to this requirement are human and nonhuman primate tissues, serum, and blood.

Permit applications are available through the Web (http://www.aphis.usda.gov/animal_health/permits/) or the local USDA port office in Des Plaines, IL. The phone number is (847) 298-5220. Alternatively, you may call the national import-export products staff at (301) 734-7885 for further information.

There are certain materials (listed below) allowed to be imported without an USDA permit; however, USDA inspectors will nonetheless review these materials at the port of entry. ORS maintains a copy of the USDA "Guidelines for Importation" on file for a discussion of the necessary shipping documents that must accompany the materials. These particular guidelines (http://www.aphis.usda.gov/NCIE/fac_imp.html) cover:

• Human and nonhuman primate material
  Includes: tissues, blood, extracts, proteins, DNA, human vaccines in final dosage form
• Feline and canine material
  Includes: blood, tissues, serum, feces, extracts, fluids, canine semen for research purposes
  Does Not Include: cell cultures, tissue cultures, cell culture products, canine semen for reproductive purposes
• Live laboratory mammals and their material-for research purposes
  Includes: transgenic/knock-out mice and rats, hamsters, gerbils, guinea pigs, rabbits, and their blood, tissue, DNA, extracts, antibodies, feces, sera, and antisera
  Does Not Include: primates, dogs, cats, livestock*, poultry, hedgehogs, tenrecs, monoclonal antibodies, hybridomas, cell lines, and material for commercial purposes
• Amphibians, fish, reptiles, shellfish and aquatic species
  Includes: blood, tissues, serum, feces, extracts, fluids, venom, urine from these species
  Does Not Include: antivenom
• Chemically synthesized materials
  Includes: biochemicals, materials not containing or derived from animal products
• Microbially produced materials
  Includes: enzymes, plasmids, proteins, antibiotics, hormones, extracts, phages, DNA
• Recombinant microbes and their products
  Includes: microbes (bacteria, viruses, yeasts/fungi), proteins, hormones, extracts, plasmids, DNA, RNA
  Does Not Include: materials produced by cell culture techniques
• Nonpathogenic microorganisms (and their extracts)
  Includes: environmental or water organisms, such as algae
• Cell cultures/lines, recombinant cell cultures/lines, and their products (for IN VITRO use)
  Includes: monoclonal antibodies, cell culture supernatants, ascitic fluid, cell extracts, hybridomas, cell cultures/lines which are not derived from livestock* or avian species
  Does Not Include: cell lines of livestock* or avian species origin and their products, microbial cultures and their products
• Test kits
  Includes: pre-packaged kits and kit components (reagents, calibraters, controls) packaged for final use
  Does Not Include: kits used to diagnose infectious diseases of animals (excluding humans)

*Livestock includes any bovine, ovine, caprine, porcine, and equine animal.

7.11 Transfer of Select Agents
For current information on the University's Select Agents Program, please see http://www.research.northwestern.edu/ors/biosafe/selectagents.htm.

7.12 Infectious Waste Management

Infectious waste materials shall be treated properly to eliminate the potential hazard that these wastes pose to human health and the environment. Treatment commonly involves steam sterilization or incineration.

7.12.1 Separation and Packaging of Infectious Waste. Infectious wastes shall be separated from general, noninfectious waste materials and from wastes containing radioactive, carcinogenic, or toxic materials. Some wastes may contain multiple hazards. These shall be handled such that priority is given to the greatest hazard present. A procedure for handling mixed biological/radioactive waste has been prepared by ORS and is available on request. It is also available in the ORS publication Radiation Safety Handbook(pdf).

Disposable infectious materials shall be placed in red or orange plastic bags. The bags shall be seamless, tear-resistant, and autoclavable. Single bags shall have a minimum thickness of 3.0 mils and double bags, 1.5 to 2.0 mils. Bags shall be closed by folding or tying when full, at the end of the day, or before transporting.

To minimize formation of aerosols, infectious wastes shall not be compacted prior to decontamination.

7.12.2 Storage and Transport of Infectious Waste. Infectious wastes that are removed from a laboratory or stored temporarily shall be closed and double-bagged or placed inside a covered, unbreakable outer container.

7.12.3 Infectious Waste Treatment. Infectious wastes are generally rendered noninfectious by autoclaving, although incineration is occasionally used. After sterilization, previously infectious wastes are disposed of as noninfectious. They may be placed in noninfectious trash collection containers and sent to a sanitary landfill. Treated wastes in red or orange bags shall be overpacked into opaque plastic bags of another color (not yellow) for noninfectious disposal. The custodial staff has been instructed not to touch or remove red, orange, or yellow bags. Sterilized liquid wastes may be discarded to the sewer.

As described under procedures for Biosafety Levels 2 and 3 and in the Bloodborne Pathogens Program, syringes and needles shall be handled with extreme caution to avoid autoinoculation and the generation of aerosols. Needles shall not be bent, sheared, replaced in the sheath or guard, or removed from the syringe following use. The needle and syringe shall be promptly placed in a puncture-resistant container. Please contact ORS for further information regarding needle disposal.

All human blood, blood products, nonfixed human tissues, and other potentially infectious materials are considered infectious and shall be disinfected, steam sterilized, or incinerated. Sterilized blood-related waste materials are discarded as nonhazardous.

Infectious wastes, including cultures and stocks of etiologic agents, shall be made noninfectious by steam sterilization. Sterilized wastes are disposed of as nonhazardous.

Pathological wastes are generally incinerated. Steam sterilization is acceptable, although after sterilization, pathological wastes shall be either incinerated or ground and flushed to a sewer.

Animal carcasses, bedding, and wastes are generally incinerated as directed by the Center for Experimental Animal Resources.

7.12.3.1 Steam Sterilization

Most infectious wastes are sterilized, based on the type of waste, load volume, packaging material, and load configuration. It is recommended that the efficacy of the autoclave be monitored using Bacillus stearothermophilis. The frequency of monitoring depends on the hazard of the organism being used and the frequency of waste sterilization.

Infectious wastes that also contain volatile chemicals should be autoclaved only if a chemical (hydrophobic) filter is on line. ORS shall be contacted before steam sterilizing wastes containing carcinogens or radionuclides.

7.12.3.2 Incineration

Incineration is preferred for pathological and animal wastes. At the University, incineration is handled by the Center for Experimental Animal Resources. ORS may be contacted for additional information.

7.12.3.3 Chemical Disinfection

Chemical treatment is usually a disinfection rather than sterilization. Thus it is usually intended as a temporary measure to control infectious wastes until sterilization can treat the hazard. Disinfection may be used as final treatment on a case-by-case basis following a petition by the principal investigator and approval by the Chemical and Biological Safety Committee.

Section 7.5.1 and Tables 7.3A and 7.3B summarize information on practical disinfectants.

Previous | Home

Internal ORS Links: What's New | Emergency Response | Administration | Biological Safety | Chemical Safety | Hazard Communication | Laboratory Safety | New to NU? | Radiation Safety

External ORS Links: Northwestern Home | Vice President for Research | Big 10 EH&S Links | Risk Management | Employee Safety Handbook

ORS - Evanston • 2145 Sheridan Road • Tech NG71 • Evanston, IL 60208
ORS - Chicago • 303 E. Chicago Avenue • Ward B106 W223 • Chicago, IL 60611
Phone 847/491-5581 or 312/503-8300 • FAX 847/467-2797 or 312/503-0547
e-mail researchsafety@northwestern.edu
Last Revision 05/16/2007
© 2007 Northwestern University. World Wide Web Disclaimer and University Policy Statements.