Due to the closing of the City of Durham Landfill, all landfill waste, including autoclaved laboratory waste, must adhere to Virginia’s Medical Waste management Regulations.
 

2.  Incineration of biological waste is a viable option for all biological waste; however, coordination with other departments is necessary to utilize this option.  For pick-up of general lab waste, contact Environmental Services' Biomedical Waste Division for pick-up (681-2727).  The Division of Laboratory Animal Resources must be contacted at 684-5212 for animal carcass disposal.

3.  Chemical disinfection is a treatment option for liquid biological waste.

Categories of biological waste, and acceptable treatments:

Sharps — This category includes needles, syringes with attached needles, capillary tubes, slides and cover slips, scalpel blades, and broken glassware that is contaminated with biological material. These items should be placed in a puncture-resistant container (needlebox). There are two acceptable methods for disposal of needleboxes; 1) place in autoclavable bags and autoclaved before disposal or, 2) contact Environmental Services' Biomedical Waste Division for pick-up (681-2727).

Pipettes — Pipettes used to process human body fluids or cultures of infectious agents, should be placed in a “pipette” biohazard box that is lined with a small autoclavable bag. Once filled, these boxes should be placed in autoclavable bags and autoclaved before disposal. Non-infectious pipettes should also be placed in a puncture-proof containers (i.e. cardboard boxes) before disposal; however, it is not necessary to autoclave.

Microbiological Waste — This category includes cultures and stocks of etiologic agents. Culture plates should be placed in autoclavable bags and steam sterilized before disposal. Liquid microbiological wastes are autoclaved or chemically treated (i.e. bleached) before disposal down the drain.

Specimens of human blood/body fluids or human tissue cultures — These items should be discarded in autoclavable bags and autoclaved before disposal.

Tissue Culture Wastes (Animal and Human) — All waste should be discarded in autoclavable bags, and autoclaved before disposal.

Anatomical/Pathological Waste — This category includes organs, limbs, animal carcasses etc., which must be incinerated (Not Autoclaved!) for proper treatment. All large human-derived tissues should be submitted to Environmental Services' Biomedical Waste Division.  Animal carcasses should be disposed of through the Division of Laboratory Animal Resources.

Non-contaminated glassware — These non-infectious materials should be discarded in a heavy-duty cardboard box and taped shut. Do NOT use cardboard boxes with “biohazard” symbols printed on them, which implies biohazardous waste requiring special treatment.

Solid Wastes — This category includes disposable gloves, gauze, paper wrappings, parafilm, etc., that are minimally contaminated. Decontamination is not required before disposal, however these items should be placed in leakproof containers (i.e., a sturdy, plastic bag).

Biosafety Levels

Four biosafety levels (BSLs) are summarized in the table below for proper handling of biohazardous materials. BSLs consist of combinations of laboratory practices and techniques, safety equipment, and laboratory facilities. Each combination is specifically appropriate for the operations performed, the  documented or suspected routes of transmission of the infectious agents, and for the laboratory function tor activity.
 

Summarized from Biosafety in Microbiological and Biomedical Laboratories, 4th Edition, 1999.
http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm

Classification of Agents According to Risk

Biological agents are assigned to biosafety levels (BSL) based on the risk they pose to human health and the environment. Such factors as severity of disease caused by the agent routes of exposure, and virulence are used when determining the most appropriate BSL. The partial list below is provided to assist laboratories in making preliminary decisions on the appropriate biosafety level for particular agents. Ultimately, the Occupational and Environmental Safety Office (OESO) will make the final BSL assignment. If a particular agent is not listed below, or if further assistance is needed in interpreting BSL requirements, contact the OESO-Biological Safety Division at 684-8822.

Biosafety Level 1
BSL-1 is suitable for work involving well-characterized agents not known to cause disease in healthy adult humans. All bacterial, parasitic, fungal, viral, rickettsial, and chlamydial agents which have been assessed for risk but do not belong to a higher risk group can be safely handled at BSL-1. Be aware that many agents not ordinarily associated with disease are opportunistic pathogens and may cause infection in the young, the aged, and immunocompromised individuals. Examples of BSL-1 agents include: Bacillus subtilis, Eschericia coli -K12, Naegleria gruberi, etc.

Biosafety Level 2

Viral Agents:

Bacterial/Rickettsial Agents:

Fungal Agents:

Parasitic Agents:

Biosafety Level 3

Viral Agents:

Bacterial/Rickettsial Agents:

Fungal Agents:

Biosafety Level 4

Viral Agents:

Laboratory Equipment

Biological Safety Cabinets (BSCs)
BSCs are the most commonly used primary containment devices in microbiological laboratories. There are three classes of BSCs (Class I, II, and III). When combined with appropriate microbiological techniques, each Class provides different levels of protection.

Class I BSC — These cabinets provide both personnel and environmental protection, however, they do not provide product protection such as that needed for sterile tissue culture work. Class I BSCs are suitable for work with low to moderate risk agents.

Class II BSC — These cabinets are the most commonly used BSCs at Duke. Class II BSCs provide environmental, personnel and product protection. The main difference between Class I and II cabinets is the HEPA filtration of the air flow down across the work surface of a Class II cabinet.
 

Class II, Type A BSC A. Blower B. Rear plenum C. Supply HEPA filter D. Exhaust E. Sash F. Work surface

Class III BSC — These gas tight BSCs provide the highest level of environmental, personnel and
product protection. A Class III BSC, (also referred to as a glove box), provides a complete physical
barrier between the product and personnel. These cabinets are used for high risk biological agents
when absolute containment is required.

A high efficiency particulate air (HEPA) filter is the main functional unit of a BSC. The HEPA filter is a device which removes particulates and microorganisms from the air. These filters remove 99.97% of all particulates 0.3 microns in diameter and have a greater efficiency for particles < or > 0.3 microns. HEPA filters are made of boron silicate fiber sheets which are pleated to increase surface area. In order to direct the airflow in the filter, aluminum baffles separate each pleat.
 
 

Certification of Biological Safety Cabinets

BSCs are to be certified by one of the Occupational and Environmental Safety Office’s “approved vendors”. These vendors are National Sanitation Foundation certified, and have demonstrated expertise in maintaining BSCs. For a list of approved certifiers, contact the OESO Biological Safety Division at 684-8822.

All cabinets in which human materials, infectious agents, or other potentially infectious materials are being used must be certified annually. Cabinets in which non-infectious materials are manipulated (i.e. sterile tissue culture) should be certified at least every two years. All newly purchased or recently moved cabinets must be certified before they can be used for any type of work.

Any cabinet being used for work with infectious agents with the potential for aerosol transmission (i.e. vaccinia virus) must be decontaminated with formaldehyde gas prior to maintenance or relocation of the cabinet.
 

Clean Benches
Horizontal laminar-flow clean benches are designed to protect the product from contamination and should never be confused with BSCs! The near-sterile work area makes these devices good for many applications in which the product does not pose a risk to the worker. Clean benches are considered inappropriate for work with potentially infectious agents.

Centrifuges
Centrifuges (including microhematrocrit centrifuges) are commonly used in the laboratory environment.  Centrifuges must be properly used and maintained to ensure safe operation.  The following are suggested practices:

• Refer to the owner's manual for routine maintenance requirements.
• Perform a visual inspection prior to each use (note unusual cracks, irregularities or wear).
• Verify proper loading of specimens to maintain balance.
• After starting, listen for unusual noises or vibrations until programmed speed is reached.
• Perform routine decontamination of interior surfaces using an appropriate disinfectant. Immediate decon is required when visible contamination is noted.
• Prevent the release of aerosols when centrifuging infectious materials that are spread via the aerosol route by using "safety devices", (i.e. sealed buckets, safety trunnion cups, sealed heads).  Safety cups must be opened in a BSC after centrifuging such materials to avoid the release of aerosols into the room.
• Spills should be addressed immediately by following established biological spill procedures.  Special precautions should be taken when broken glass or other sharps may be involved.

Homogenizers and Blenders
These items are commonly used in laboratories, and both are considered producers of aerosols. Safety sealed homogenizers and blenders are commercially available and should be used when working with those agents known or suspected of being transmitted through aerosols. The purpose of these items is to contain any aerosols created during work procedures. These safety devices may be used on the open benchtop; however, they must be opened in a BSC. All non-sealed devices must be used exclusively in a BSC.

Human Blood, Blood Products,
Tissues and Body Fluids

In 1991, the Occupational Safety and Health Administration (OSHA) promulgated a standard to minimize the risk for occupational exposure to bloodborne pathogens (e.g., HIV, Hepatitis B). The regulation, titled Occupational Exposure to Bloodborne Pathogens mandates several provisions for those working with materials that are human-derived such as human blood, blood products, other bodily fluids and any unfixed tissues. The full text of the Duke University Bloodborne Pathogen Exposure Control Plan, including a copy of the Bloodborne Pathogen Standard can be found in the Appendices of this section. The Plan must be readily available to all employees working with those materials mentioned above. This includes all employees working with primary human cell lines, or human cell lines that have not been well-characterized and tested for human pathogens. The following are a few highlights of the Plan.

Universal Precautions
Universal precautions is defined as handling all human blood, body fluids, and tissues as if they are infectious. This calls for the use of appropriate protective measures to reduce or eliminate the risk of occupational exposure.

Hepatitis B Vaccination
All employees working with human blood, blood products, fresh tissues or bodily fluids shall be offered the Hepatitis B vaccine at no cost to them. If an employee should decline the vaccine, they must sign a waiver which is kept on file in the Employee Occupational Health and Wellness (EOHW). For more  information about the vaccine, contact the EOHW at 684-3136.

EOHW Blood and Body Fluid Exposure Hotline
All occupational exposures to potentially infectious materials are to be reported immediately by calling 115 if on campus and 684-8115 if off campus. An EOHW representative will discuss with the employee the appropriate follow up of the exposure. It is important that exposures are reported as soon after the incident as possible because some post exposure treatments are considered time sensitive.

Safety Training
All new employees who are to work with materials covered by OSHA’s Bloodborne Pathogen Standard are to receive initial safety training and annually thereafter. General Laboratory Safety Training is available as an online training module. On-site genereal laboratory safety training can be requested. Laboratory-specific training is the responsibility of the Primary Investigator.

Recombinant DNA

Recombinant DNA (rDNA) can be defined as molecules that are constructed outside living cells by joining natural or synthetic DNA segments to DNA molecules that can replicate in a living cell. Since the inception of rDNA technology, scientists have been concerned over the possibility that artificially constructed rDNA could be biologically hazardous if not handled appropriately or released into the environment. These concerns prompted the development of the NIH Guidelines on rDNA research in May of 1976. The most recent revision was published in April of 2002 and is available for review at http://oba.od.nih.gov/oba/rac/guidelines_02/NIH_Guidelines_Apr_02.htm.

Experiments involving the generation of rDNA may require approval by the Duke University Institutional Biosafety Committee (IBC) prior to submission to outside agencies and the initiation of experimentation. Principal Investigators should obtain proper rDNA forms for submittal to the Institutional Biosafety Committee. On rare occasions, other groups are to be involved in the review of  DNA research proposals. They include the OBA (NIH Office of Biotechnology Activities) and the RAC (Recombinant DNA Advisory Committee). The OBA has the responsibility for reviewing and coordinating all activities of NIH relating to the Guidelines. The RAC is a public advisory committee that advises the Secretary, the Assistant Secretary for Health and the Director of the NIH, rDNA research.

Generally, experiments requiring the use of recombinant biological agents should be handled under the same BSL requirements as the wild type agent. For example, handling of adenoviral vectors should be performed under BSL 2 conditions.

Part III of the Guidelines divides experiments into five categories based on pathogenicity of the source DNA, the vector used and the recipient host. The following is a summary of the five categories.

Experiments exempt from Guidelines and registration with IBC is not required  

• rDNA molecules that are not in organisms or viruses 
• rDNA molecules consisting entirely of DNA segments from a single non-chromosomal or viral DNA source 
• rDNA molecules consisting entirely of DNA from a prokaryotic host including its indigenous plasmids or viruses when propagated only in that host or when transferred to another host by well established physiological means 
• rDNA molecules consisting entirely of DNA from an eukaryotic host including its chloroplasts, mitochondria, or plasmids when propagated only in that host 
• rDNA molecules consisting entirely of DNA segments form different species that exchange DNA by known physiological processes 
• rDNA molecules that do not present a significant risk to health or the environment
   

Those experiments requiring IBC notice only simultaneous with intiation  

• experiments involving the formation of rDNA molecules containing no more than two-thirds of the genome of any eukaryotic virus, BSL 1 and no helper virus 
• experiments involving rDNA modified whole plants, BSL 1
   

Those experiments requiring IBC approval  

• human or animal pathogens as host-vector systems 
• introduction of rDNA into Class 2 or 3 agents 
• DNA from human or animal pathogens is cloned into nonpathogenic prokaryotic or lower eukaryotic host-vector systems 
• DNA from Class 2 or 3 agents is transferred into nonpathogenic prokaryotes or lower eukaryotes 
• infectious animal or plant DNA or RNA viruses or defective animal or plant 
• DNA or RNA viruses in the presence of helper virus in tissue culture systems 
• infectious or defective Class 2 or 3 animal viruses in the presence of helper virus 
• experiments involving infectious or defective animal or plant viruses in the presence of helper virus not covered above 
• experiments involving whole animals in which the animal’s genome has been altered by stable introduction of rDNA, or DNA or RNA derived therefrom 
• experiments involving genetically engineered plants by rDNA methods, BSL 2 or 3
   

Those experiments requiring IBC and NIH approval  

• cloning of toxin molecules with LD 50 <100ng/kg body weight 
• accelerated review of human gene transfer 
• minor modifications to human gene transfer
   

Those experiments requiring IBC, NIH and RAC approval  

• the deliberate transfer of a drug resistance trait to microorganisms that are not known to acquire the trait naturally 
• the deliberate transfer of rDNA, or DNA or RNA derived from rDNA, into humans. (must simultaneously be submitted to the Food and Drug Administration for review)
   

Packaging and Shipping Biological Materials
Although several agencies have published regulations or guidelines for the proper packaging and shipment of biological materials, the International Air Transport Association's (IATA) Dangerous Goods Regulations (DGR) governs all international shipments. Futhermore, all air transport of regulated biological materials (including domestic flights) must strictly adhere to the DGR.  For this reason, the OESO provided training is primarily focused on compliance with these regulations.

Training
All personnel involved in the process of shipping biological materials must receive proper training initially and at least every two years thereafter. Training is provided through the OESO website's "online-training" link.  The program is titled Shipping Biological Materials.  The Training Supplement Guide includes a summary of the most relevant training content for properly classifying, packing and labeling a shipment.  Note:  The information provided in the checklists below may not include all relevant shipping criteria, and are not intended to be used without first completing the official training.
 
 

Checklist for Shipping Patient Specimens (for which there is minimal likelihood that pathogens are present)

Specimen Packaging

__        Specimen in leak-proof primary container
__        Absorbent material is sufficient to absorb entire contents of primary container(s)
__        Primary containers are wrapped individually
__        Leak- proof secondary container

Labeling Outer Container

__        Statement: “Exempt human specimen” or “Exempt animal specimen”
__        Miscellaneous Class 9 label⁽²⁾ if shipment contains dry ice, "UN 1845" and amount used in kg

Completing the Airbill

__        Name and address of shipper and recipient
__        Check “Saturday Delivery” box if applicable
__        In Section 6 (Special Handling) of the airbill, indicate that the shipment is NOT a dangerous good
__        Check the “Dry Ice” box if applicable and indicate “UN 1845” and the quantity of dry ice in kg
__        Shipper’s signature (optional)

Checklist for Shipping Category B Infectious Substances

Specimen Packaging

__        Specimen in leak-proof primary container
__        Absorbent material is sufficient to absorb entire contents of primary container(s)
__        Primary containers are wrapped individually
__        Leak- proof secondary container
__        Itemized list of contents placed between secondary and outer container

Labeling Outer Container

__        UN 3373 label⁽¹⁾
__        Statement: “Biological Substance, Category B” adjacent to UN 3373 label
__        Miscellaneous Class 9 label⁽²⁾ if shipment contains dry ice, "UN 1845" and amount used in kg

Completing the Airbill

__        Name and address of shipper and recipient
__        Check “Saturday Delivery” box if applicable
__        In Section 6 (Special Handling) of the airbill, indicate that the shipment is a dangerous good, which does NOT require a Shipper’s Declaration
__        Check the “Dry Ice” box if applicable and indicate “UN 1845” and the quantity of dry ice in kg
__        Shipper’s signature (optional)

 (1) -     (2) -
 
 

Checklist for Shipping Category A Infectious Substances

Specimen Packaging

__        Specimen in watertight primary container
__        Absorbent material is sufficient to absorb entire contents of primary container(s)
__        Primary containers are wrapped individually
__        Watertight secondary container
__        Itemized list of contents placed between secondary and outer container 

Labeling Outer Container

__        Infectious Substance, Class 6 label
__        “Infectious Substance Affecting Humans”, identification of agent in parentheses, “UN 2814” and net quantity of infectious substance
__        Miscellaneous Class 9 label if shipment contains dry ice, “UN 1845” and amount used in kg
__        Name and telephone number of a responsible person
__        If shipment includes >50mL or 50g of a Category A infectious substance, then add a “Danger, do not load in passenger aircraft” label to the outer container

Dangerous Goods Declaration Form

__        Name, address and phone number of shipper and recipient
__        Mark out non-applicable “Aircraft Box”
__        Mark out non-applicable “Radioactive” box
__        24-hour emergency response telephone number
__        Name and title of signatory, place, and date
__        Shipper’s signature

“Nature and Quantity of Dangerous Goods” section of the Declaration Form

__        Complete this section using the information provided on Pages 5, 9 & 10 of the Supplemental Training Guide

Importation of agents or vectors of human disease
Importation of infectious materials and vectors that may contain them is regulated by federal law. When an infectious agent is being imported into the United States, it must be accompanied by an importation permit which is issued by the United States Public Health Service (USPHS). Permits are issued only to the importer who must be located in the United States. Importation permit applications are available through the OESO-Biological Safety Division. (684-8822), or the following: http://www.cdc.gov/od/ohs/biosfty/imprtper/htm

Shipping labels containing the universal biohazard symbol, the address of the importer, the permit number and expiration date are issued to the importer with the permit. The importer must send the labels and one or more copies of the permit to the shipper. The importation permit, with the proper packaging and labeling, will expedite clearance of the package of infectious materials through the USPHS Division of Quarantine and release by US Customs.

Importation of etiologic agents of animals, and plant pests
The United States Department of Agriculture's Animal and Plant Health Inspection Service (APHIS) regulates the importation and domestic transfer of agents, which may pose a risk to animals or plants.  A permit must be obtained prior to the receipt of any material that could pose a potential risk to animals or plants.  The permitting procedures and forms are found at the following: http://www.aphis.usda.gov/forms/index.html

Select Agents
The Department of Health and Human Services’ 42 CFR Part 73, titled Possession, Use, and Transfer of Select Agents and Toxins, became law on February 7, 2002. All researchers who possess or plan to possess select agents, must be registered with the Centers for Disease Control and Prevention.  For a list of restricted agents and other Select Agent Program requirements, see the following: http://www.cdc.gov/od/sap/

The Director of OESO's Biological Safety Division will serve as Duke's Responsible Official (RO) for select agents.  All CDC registrations must be facilitated through the RO.  To contact the RO, call 684-8822.

Proper Shipment of Non-Regulated Liquids
Provisions must be made to ensure that all non-regulated liquids (i.e. buffers, water, etc.) are properly packaged to prevent leakage during transport.  The packaging must be of good quality, strong enough to withstand shocks normally encountered during transport.  A triple-packaging system similar to that prescribed for patient specimens (above) must be utilized.  The following must be met.

• Liquid is placed in a leak-proof primary container
• Absorbent material must be placed around the primary container (sufficient amount to absorb entire contents of primary container
• Primary container(s) and absorbent material(s) are placed into leak-proof secondary container.
• Inner packagings (primary and secondary container) are placed into a sturdy outer container (i.e. cardboard box).  Cushioning material is added between secondary container and outer shipper if deemed necessary.

 References
Biological Safety: Principles and Practices; Fleming D, Hunt D, 3rd ed., ASM, 2000
Biosafety in Microbiological and Biomedical Laboratories; 4th ed., CDC/NIH, 1999
Primary Containment for Biohazards: Selection, Installation and Use of Biological Safety Cabinets;
2nd ed., CDC/NIH, 2000
North Carolina Administrative Code 10G.1201-.1207, General Statute 130A-309.26, 1990
Regulated Medical Waste Management Regulations; Virginia Department of Environmental Quality:
VR 672-40-01:1, 1994
Occupational Exposure to Bloodborne Pathogens, Final Rule; 29CFR 1910.1030, OSHA, 1991
Occupational Health and Safety in the Care and Use of Research Animals; NRC, 1997
2005 Dangerous Good Regulations; International Air Transport Association, 46th Ed.
Possession, Use, and Transfer of Select Agents and Toxins; USDHHS, 42 CFR Part 73, 2002
North Carolina Administrative Code G.S. 130A-149, Biological Agent Registry, 2002

Appendicies
Bloodborne Pathogens Exposure Control Plan
OSHA Occupational Exposure to Bloodborne Pathogens Standard
Duke University Blood and Body Fluid Precautions Policy
HIV and HBV Research Laboratories Policy
Post Exposure Evaluation and Follow-Up Procedures
Training Program Contents
Instructions For Completing the Bloodborne Pathogens Exposure Determination Form
Duke University's Select Agent Policy
Duke Viral Vector Policy