This document has been provided to assist researchers in preparing recombinant DNA BAF’s for review by the Institutional Biosafety Committee (IBC). Also, to assist researchers with meeting NIH Guideline requirements “in practice”, specific portions of the NIH Guidelines are summarized in an effort to assist. Included Topics:
Applicability of the NIH Guidelines
NIH’s Definition of Recombinant DNA Molecules
Recombinant DNA molecules are defined as either molecules that are constructed outside living cells by joining natural or synthetic DNA segments to DNA molecules that can replicate in a living cell, or molecules that result from the replication of those previously described.
All Researchers who are using rDNA molecules as part of their research must file a BAF with the WSU IBC regardless of funding source. The IBC reviews all BAF’s. They provide recommendations and approvals as appropriate. The WSU Biosafety Officer supports the review process by assisting researchers with BAF questions, Biosafety manuals and training as appropriate.Principle Investigator’s Responsibilities
Under the NIH Guidelines, both the institution and the researchers are charged with specific responsibilities for compliance that involve elements that go beyond general laboratory safety requirements.NIH Guidelines for Research Involving the Use of Recombinant DNA Molecules (Section lV-B-7)
"On behalf of the institution, the Principle Investigator is responsible for full
compliance with the NIH guidelines in the conduct of recombinant DNA research"
As part of this general responsibility, the Principle investigator shall:
- Not initiate or modify recombinant DNA research which requires Institutional Biosafety Committee (IBC) approval prior to initiation until that research or the proposed modification thereof has been approved by the Institutional Biosafety Committee and has met all other requirements of the NIHGuidelines;
- Report any significant problems, violations of the NIH Guidelines, or any significant research-related accidents and illnesses to the IBC, NIH/OBA and other appropriate authorities as applicable within 30 days;
- Report any new information bearing on the NIH Guidelines to the IBC and to NIH/OBA;
- Be adequately trained in good microbiological techniques (including standard microbiological practices);
- Adhere to IBC-approved emergency plans for handling accidental spills and personnel contamination and;
- Comply with applicable shipping requirements for recombinant DNA molecules. To register rDNA molecule use with the IBC, the PI shall:
- Make an initial determination of the required levels of physical and biological containment in accordance with NIH Guidelines;
- Select appropriate microbiological practices and laboratory techniques to be used for the research;
- Submit the initial research protocol and any subsequent changes (e.g. changes in the source of DNA or host-vector system) to the IBC for review and approval or disapproval and;
- Remain in communication with the IBC throughout the conduct of the project.
- Make available to all laboratory staff the protocols that describe the potential biohazards and the precautions to be taken;
- Instruct and train laboratory staff in the practices and techniques required to ensure safety (including standard microbiological practices), and the procedures for dealing with accidents and;
- Inform the laboratory staff of the reasons and provision for any precautionary medical practices advised or requested, (e.g., vaccinations or serum collection).
- Supervise the safety performance of the laboratory staff to ensure that the required safety practices and techniques are employed;
- Investigate and report any significant problems pertaining to the operation and implementation of containment practices and procedures in writing to the IBC, NIH/OBA, and other appropriate authorities as applicable;
- Correct work errors and conditions that may result in the release of rDNA materials and;
- Ensure the integrity of the physical containment (e.g., biological safety cabinets) and the biological containment (e.g., purity and genotypic and phenotypic characteristics).
Biosafety Level 1 is suitable for work involving agents not reasonably expected to be a hazard, or those agents of minimal potential hazard to laboratory personnel and the environment. The laboratory is not separated from the general traffic patterns in the building. Work is generally conducted on open bench tops. Special containment equipment is not required or generally used. Laboratory personnel have specific training in the procedures conducted in the laboratory and are supervised by a scientist with general training in microbiology or a related science.*Note: This level of containment is the minimum standard for all recombinant DNA work at WSU.
Standard Microbiological Practices (BL1)
- Access to the laboratory is limited or restricted at the discretion of the PI when experiments are in progress.
- Work surfaces are decontaminated once a day and after any spill of viable material. Biosafety Note: Cross-contamination can result if work surfaces are not regularly decontaminated. Effective decontamination of work surfaces is achieved through the proper use of disinfectants that are effective for destruction of the microorganism of concern.
- All contaminated liquid or solid wastes are decontaminated before disposal. Biosafety Note: Liquid wastes may be pretreated with chemical disinfectants before disposal via a lab sink that is connected to the sanitary sewer. Alternatively, liquid wastes (without chemical disinfectants) may be autoclaved prior to disposal via a lab sink that is connected to the sanitary sewer. Solid wastes contaminated with infectious agents or recombinant DNA must be managed as solid biohazardous waste for treatment and disposal purposes. Surface disinfection of such wastes is not an appropriate method of treatment.
- Mechanical pipetting devises are used; mouth pipetting is prohibited.
- Eating, drinking, smoking, and applying cosmetics are not permitted in the work area. Food may only be stored in cabinets or refrigerators designated for this purpose.
- Persons wash their hands: (i) after they handle materials involving organisms containing recombinant DNA molecules and animals, and (ii) before exiting the laboratory.
- All procedures are performed carefully to minimize the creation of aerosols.
- In the interest of good personal hygiene, facilities (e.g., hand washing sink, shower, changing room) and protective clothing (e.g., uniforms, laboratory coats) shall be provided that are appropriate for the risk of exposure to viable organisms containing recombinant DNA molecules.
- Contaminated materials that are to be decontaminated at a site away from the laboratory are placed in a durable leak-proof container which is closed before being removed from the laboratory.
- An insect and rodent control program is in effect. Biosafety Note: An insect and rodent control program is only as effective as the lab personnel are. That is when problems are noted the lab personnel need to take action by reporting the problem to the appropriate party. See your supervisor to obtain the appropriate contact party.
Special containment equipment is generally not required for manipulations of agents assigned to BL1.
Laboratory Facilities (BL1)
- The laboratory is designed so that it can be easily cleaned. Biosafety Note: The ability to effectively clean a lab space is essential for work with viable biological materials. Open storage of supplies, personal items and nonessential equipment increases the amount of surface area that is susceptible to contamination in the event of a spill or lab practices that create uncontained aerosols. These items should be placed in closed storage whenever possible.
- Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat. Biosafety Note: If bench paper is used on bench tops where infectious agents or viable materials containing recombinant DNA molecules are manipulated, this bench paper must be discarded at the conclusion of the procedures and the bench top disinfected at least once a day as noted previously under the “Standard Microbiological Practices” section.
- Laboratory furniture is sturdy. Spaces between benches, cabinets, and equipment are accessible for cleaning.
- Each laboratory contains a sink for hand washing.
- If the laboratory has windows that open, they are fitted with fly screens.
Experiments in these categories are exempt from the NIH Guidelines. However, at WSU the IBC committee has made the determination that this research work requires the submittal of a completed Biological Application Form (BAF) to the IBC. This allows the IBC to verify exemption status and to generate documentation for the PI that may be required by funding agencies relative to NIH compliance.Exempt experiments include:
- Those not in organisms or viruses.
- Those consisting entirely of DNA segments from a single non-chromosomal or viral DNA source.
- Those consisting entirely of DNA from a prokaryotic host, including its indigenous plasmids or viruses, when propagated in that host (or a closely related strain of the same species).
- Those consisting of DNA from eukaryotic hosts, including chloroplasts, mitochondria or plasmids (excluding viruses), when propagated only in that host (or a closely related host).
- Natural exchanges (see NIH Guidelines Appendices A1-AVI)
- Those that do not pose a significant risk to health or the environment as
summarized below: (See Appendix C of NIH Guidelines for full text).
- Recombinant DNA in tissue culture. Recombinant DNA molecules containing < 1/2 of any eukaryotic viral genome that are propagated and maintained in cells are exempt, with the following exceptions – Risk Group 3 & 4 or restricted organisms.
- E. Coli K-12 host-vector systems (BL1 practices recommended, IBC
can specify higher containment if it deems necessary). These
experiments are exempt provided that:
- The E. coli host does not contain conjugation proficient plasmids or generalized transducing phages (unless a non-conjugative vector is used).
- Lambda or lambdoid bacteriophages or Ff non-conjugative
plasmids are used as vectors (unless the DNA inserted in E. coli
K-12 is from a prokaryote that naturally exchanges genetic
information with E. coli, in which case any E. coli K-12 vector
may be used).
Note: This exemption is for the system; if foreign DNA is introduced into the system, then these experiments do not meet the exemption criteria.
Exceptions include (but may not be limited to):
- Experiments involving Risk Group 3, 4 or restricted organisms
- Large-scale experiments (i.e., >10 liters)
- Cloning of toxin molecule genes coding for biosynthesis of molecules toxic for vertebrates.
Exempt experiments also include:
- Saccharomyces host-vector systems (BL1 practices recommended, IBC can specify higher containment if it deems appropriate). Exceptions: See #2.
- Bacillus subtilis or Bacillus licheniformis host-vector systems (Bl1 practices recommended, IBC can specify higher containmen if it deems appropriate).
- Extrachromosomal elements of Gram positive microorganisms (refer to list in NIH Guidelines). Exceptions: See #2.
- The purchase or transfer of transgenic rodents (applicable to experiments requiring only BL1 containment).
The first 3 categories of studies are those which require a review by the NIH in addition to the local IBC before the work can be initiated. These categories are not applicable to most recombinant DNA molecule research at WSU so they are only briefly explained below. If you feel that your work may fit into one of these categories, please contact the IBC chair or the WSU Biosafety Officer for assistance.
Experiments Requiring IBC Approval, RAC Review and NIH Director Approval Before Initiation
This category is limited to studies that involve the deliberate transfer of drug resistance to microorganisms (not known to acquire the trait naturally) that can compromise the use of the drug to control the microorganism and its disease in humans, veterinary medicine or agriculture.
Note: Antibiotic resistance of low risk hosts for genetic screening purposes is not included in this category.
Experiments Requiring NIH/OBA and IBC Approval Before Inititiation
This category is limited to cloning of genes that encode for toxin molecules with LD-50 < 100 nanograms/kg body weight (e.g., botulinum, tetanus, diphtheria toxins).
Note: Specific approvals have been granted by NIH for cloning in Escherichia coli K-12 for toxins with LD-50 between 100 nanograms/kg and 100 micrograms/kg body weight. Refer to the NIH Guidelines for further information.
Experiments Requiring IBC and IRB Approval and RAC Review Before Participant Enrollment
This category is limited to deliberate transfer of recombinant DNA, or DNA or RNA derived from recombinant DNA, into one or more human subjects. Before enrollment, the following must be submitted to NIH/OBA for each clinical trial site:
- IBC Approval (from clinical trial site location)
- IRB Approval
- IRB-approved informed consent document
- CV of PI’s
- NIH Grant Numbers (if applicable)
The next 2 categories include the recombinant DNA molecule use that is likely to be most applicable to WSU researchers. The information regarding the various applications in these 2 categories is presented in the table format for quick reference.
Experiments Requiring IBC Approval Before Initiation
|NIH Subcategory||Research Application Description||Notes & Specific Considerations|
|D-1||Experiments using Risk Group 2. Risk Group 3, Risk Group 4 or restricted agents as host-vector systems.||This refers to the introduction of recombinant DNA into the pathogenic agent. Containment levels will generally be based on the risk of the unmodified agent.|
|D-2||Experiments in which DNAfrom Risk Group 2, Risk Group 3, Risk Group 4 or restricted agents is cloned into non-pathogenicprokaryotic or lower eukaryotic host-vector systems.||When cloning is from Risk Group 2 or 3 pathogens, BL2 containment is generally prescribed, but specific lowering to BL1 may be approved by IBC.|
|D-3||Experiments involving the use of infectious DNA or RNA viruses or defective DNA or RNA viruses in the presence of a helper virus in tissue culture systems.||
Experiments involving Risk Group 2 viruses (infectious or defective) in the presence of helper virus are generally conducted at BL2)
Experiments involving Risk Group 3 viruses (infectious or defective) in the presence of helper virus are generally conducted at BL3.
Recombinant DNA or RNA molecules containing < 2/3 of the genome of any eukaryotic virus are considered defective. These viruses, in the absence of a helper virus, are categorized under III-E.
|D-4||Experiments involving whole animals||
This category applies to whole animals in which the genome has been altered by stable introduction into the germline (transgenic animals). This category also applies to experiments involving viable recombinant DNA-modified microorganisms tested on whole animals
Viral vectors used on animals that do not lead to transmissible infection, either directly or indirectly a result of recombination in animals, may generally be propagated at BL1. Recombinant DNA, or DNA derived from recombinant DNA, from any source except for>2/3 of eukaryotic viral genome may generally be transferred to animals and propagated at BL1.
The purchase of transgenic rodents is exempt under Section III-F and WSU IBC policy.
|D-5||Experiments involving whole plants||
This category applies to experiments that produce genetically engineered plants by way of recombinant DNA methods. This category also applies to experiments using plants together with microorganisms or insects containing recombinant DNA.
This category is restricted to experiments involving exotic infectious agents with recognized potential for serious, detrimental impact on ecosystems and experiments involving sequence encoding potent vertebrate toxins introduced into plants or associated organisms. All other plant experiments are likely to fall under Section III-E.
|D-6||Experiments involving > 10 liters of culture||Appendix K outlines large-scale containment practices. The IBC will decide upon appropriate containment.|
Experiments Requiring IBC Notice Simultaneous with Initiation NOTE: At WSU the IBC has made the determination that all BAF forms are to be submitted prior to initiation of work.
This category includes several different types of applications, which can typically be carried out using biosafety level 1 containment facilities, equipment and practices.
|NIH Subcategory||Research Application Description||Notes & Specific Considerations|
|E||Experiments in which all components are derived from non-pathogenic prokaryotes and nonpathogenic eukaryotes.||BL1 containment|
|E-1||The formation of recombinant DNA molecules containing no more than 2/3 of the genome of any eukaryotic virus.||Propagation and maintenance in tissue cell culture can be done at BL1, provided that cells lack helper virus of concern.|
|E-2||Experiments involving whole plants||
This category covers all whole plant experiments except those that involve exotic infectious agents with recognized potential for serious detrimental impact on ecosystems (i.e., those covered under Categoory III-D).
Includes: Plant-related experiments involving recombinant DNA-modified organisms that have not recognized potential for rapid and widespread dissemination or detrimental impact on ecosystems (e.g., Agrobacterium, rhizobium).
BL1 may be used where modified plants are not noxious weeds and cannot interbreed with noxious weeds of geographic interest.
|E-3||Experiments involving transgenic rodents.||This category applies to experiments involving generation of rodents in which the genome has been altered by the stable introduction of recombinant DNA. The purchase of commerciallyavailable transgenic rodents is exempt.|
Biosafety Approval Form (BAF) Process
Who should complete this form? All WSU-Affiliated PI’s including USDA/ARS PI’s who currently have or plan to possess, store, transport or work with recombinant DNA/RNA, or work with human tissues/fluids or other potentially biohazardous material. Additionally, BAF’s are required for teaching, diagnostic and extension activities that work with potentially biohazardous materials. See below for a complete description of potentially biohazardous materials:Potentially Biohazardous Material
All infectious organisms, (bacteria, chlamydiae, fungi, parasites, prions, rickettsias and viruses) which can cause disease in humans, animals or plants, or cause significant environmental or agricultural impact. In addition, work with materials that may harbor infectious organisms, such as human or primate tissues, fluids, cells or cell cultures are also included.
Potentially biohazardous materials include all of the categories below. Projects involving material(s) included in any of these categories must be submitted for IBC approval prior to starting work.
- Recombinant DNA (rDNA)
- Genetically modified organisms. Including, but not limited to:
- Animals, plants, invertebrates, and/or other organisms created by WSU employees or in/on WSU property,
- Genetically modified whole plants (even those commercially available and not requiring APHIS permits)
- Transgenic field trials, any genetically modified organism to be introduced into the environment (by WSU personnel and/or on WSU property)
- Field testing of plants engineered to produce pharmaceutical and industrial compounds,
- Any organisms requiring federal permits (APHIS, CDC,FDA,EPA,…),
- Pathogens/infectious agents (human, animal, plant and other),
- Select/Biological Agents and Toxins (CDC and USDA),
- Biological Toxins
- Human blood and blood products, human bodily fluids, and/or human tissue,
- Work with animals or vectors known or suspected to be reservoirs of BL2 or BL3 infectious agents when such work increases potential exposure risks to personnel or other animals,
- Oncogenic viruses used in conjunction with animals.
The IBC also serves as an advisory committee for University projects that involve possible biohazards that do not appear to fall into one of these areas. When it is unclear as to whether a material constitutes a potential biohazard, the IBC should be consulted. Questions should be directed to the Office of Research Assurances, IBC Coordinator, or WSU Biosafety Officer.
BAF Submittal Process
You must complete a Biosafety Approval Form (BAF) and submit it to the Office of Research Assurances, fax 509-335-6410. To have the BAF reviewed in the month that it is submitted it must be received ten days prior to that month’s scheduled IBC meeting. Website for submittal schedules: www.bio-safety.wsu.edu/meeting.asp.Click here to access the BAF form.
The "BAF Preparation Pointers" section of this document will provide you with guidance on completing the form. Additionally to help ensure a quick review process a pre-IBC meeting is held one week in advance of the IBC meeting. At this time the form is reviewed for completeness. If more information is needed the BSO will attempt to meet with the PI prior to the IBC meeting to ensure that the BAF is complete and ready for consideration by the IBC committee.
Once approval is granted by the IBC, an approval notice will be sent to the PI from the IBC Coordinator. Generally these notices are sent within ten working days of the IBC meeting.
IBC approval for all BSL-1 and BSL-2 work will be valid for three years. All approvals for BSL-3 work will be valid for one year. However, the PI is responsible for notifying the IBC of any changes in the recombinant DNA materials or techniques, facilities used or research staff involved.
Please note that if your recombinant DNA work changes to the degree where the work falls into a different review category, or presents new considerations for containment requirements, you must notify the IBC Chair before initiating these changes to determine if further review actions are required.
It is your responsibility and in your best interest to take measures to assure that your work is properly documented and currently approved to assure compliance with regulations that can impact funding monies.BAF Form Preparation Pointers
|Section 1: “Basic contact information”|
The NIH Guidelines refer to the researcher who is responsible for documentation, training and reporting relative to the recombinant DNA molecule use as the “principle Investigator”. For BAF submittal to the WSU IBC, the Principle Investigator is someone who has a faculty appointment with WSU and meets funding agency requirements as a lead researcher. If you do not have such an appointment, you must partner with someone who does and work with that individual to have the work appropriately documented through that individual on your behalf.
Both the signature of the Principle Investigator and the Department Chair are required on all submitted BAF forms. If lab space is shared with another principle investigator this or these peoples signatures are required also.
|Section 2: "Description of research and facilities"|
Research Project Title
This title should represent the protocol to be used for generation and use of the recombinant DNA molecules. It should not be specific for a particular grant proposal if the protocol is intended to cover multiple grant proposals. IBC approval letters will list the approved general protocol. If you submit grant proposals that would be covered by the approved general protocol but are not listed on the original general protocol, you can have the general protocol updated by submitting an amendment to the IBC.
Yes or No section
This section must be completely filled out. This is the basis for filling out all the rest of the form as is described on the BAF form itself.
Practical Disinfectants for Recombinant DNA
|Liquid Disinfectants||Practical Requirements||Inactivates||Important Characteristics|
|Use/Dilution||Contact time minutes||Vegetative Bacteria||Lipo-viruses||Nonlipid Viruses||Bacterial Spores||Effective Shelf life > 1 week||Effective for Surface Decontamination||Liquids For Discard|
|Category||Lipo virus||Broad Spectrum|
|Quat. Ammon. Cpds.||0.1-2.0%||10||NE||+||+||+||+|
|NE||- Not Effective|
|b||- Variable results dependent on virus|
|Reference: NIH Guidelines for working with Recombinant DNA Lab Safety Monograph (Appendix D updated)|