Printed and spiral bound copies of the manual are available for pickup upon request, please call (650) 723-0448 for more information.
This revision of the Biosafety Manual was prepared under the auspices of the Administrative Panel on Biosafety (APB) by the Office of Environmental Health and Safety (EH&S) after careful review of pertinent federal and state government regulatory documents, along with reference guidelines from the Centers for Disease Control and the National Institutes of Health. This manual will:
Address the most commonly asked questions from faculty, staff and students on general Biosafety, pathogens, and recombinant DNA (rDNA) or synthetic nucleic acid (sNA) issues;
Provide information about training, safe work practices, safety equipment and personal protective equipment; and,
Provide guidance for investigators who need to submit an application for review by the Administrative Panel on Biosafety.
Printed and spiral bound copies of the manual are available for pickup upon request, please call (650) 723-0448 for more information.
This revision of the Biosafety Manual was prepared under the auspices of the Administrative Panel on Biosafety (APB) by the Office of Environmental Health and Safety (EH&S) after careful review of pertinent federal and state government regulatory documents, along with reference guidelines from the Centers for Disease Control and the National Institutes of Health. This manual will:
Address the most commonly asked questions from faculty, staff and students on general Biosafety, pathogens, and recombinant DNA (rDNA) or synthetic nucleic acid (sNA) issues;
Provide information about training, safe work practices, safety equipment and personal protective equipment; and,
Provide guidance for investigators who need to submit an application for review by the Administrative Panel on Biosafety.
Message From the Vice Provost and Dean of Research of Stanford University
To: The Stanford Academic Community
This Biosafety Manual represents the institutional practices and procedures for the safe use and handling of biological materials, recombinant DNA and synthetic nucleic acids at Stanford University. The Administrative Panel on Biosafety and the Biosafety Manager have revised this document based on the latest government regulatory requirements, guidelines and current professional standards. It is designed to inform the laboratory worker of good work practices and safe procedures which are found in most biosafety manuals; however, this manual also emphasizes the regulatory requirements that must be followed and the need for all related research to be conducted in a responsible manner.
The Environmental Health and Safety Office, through the Biosafety Manager, is responsible for monitoring individual principal investigators and laboratory facilities for adherence to the practices and procedures described in this manual. However, it is the responsibility of each principal investigator to ensure that all lab workers are familiar with the contents of this manual and that these workers and employees are trained to recognize potential related hazards prior to initiation of the research work. Your cooperation with the Administrative Panel on Biosafety and the Environmental Health and Safety Office is essential to comply with the regulatory requirements that our University must follow in order to continue the success of our research endeavors.
If you have any questions regarding this document, please call the Research Compliance Administrator at 723-4697 or the Biosafety Manager at 725-1473.
Sincerely,
Ann Arvin
Vice Provost and Dean of Research
1.2Major Updates
Biosafety at a Glance
A snapshot of minimum requirements to work with rDNA/Infectious Agents, and associated lab requirements
Approvals (Chapter 4)
Panel
Oversight
Website
APB
rDNA/ Infectious Agents BSL2, 3 (in vitro, in vivo)
Please contact Biosafety with questions or to request a consultation:
Email: biosafety-owner@lists.stanford.edu
Telephone: (650) 725-1473
1.3Introduction
Introduction
This revision of the Biosafety Manual was prepared under the auspices of the Administrative Panel on Biosafety (APB) by the Office of Environmental Health & Safety (EH&S) after careful review of pertinent federal and state government regulatory documents, along with reference guidelines from the Centers for Disease Control and the National Institutes of Health.
This manual will:
Discuss components of creating and working in a research laboratory with a robust safety culture
Address the most commonly asked questions from faculty, staff and students on general Biosafety, pathogens, and recombinant DNA (rDNA) or synthetic nucleic acid (sNA) issues;
Provide information about training, safe work practices, safety equipment and personal protective equipment; and
Provide guidance for investigators who need to submit an application for review by the Administrative Panel on Biosafety.
Due to the ever-changing regulatory environment that we all live and work in, updates to this manual will be made as needed; these changes will also be made on the EH&S Biosafety web site (https://ehs.stanford.edu/ topic/biosafety-biosecurity).
Please feel free to comment on this manual. If you have questions regarding this manual, please contact the Biosafety & Biosecurity Program at (650) 725-1473 or email biosafety-owner@lists.stanford.edu.
Sincerely,
Ann Arvin,
M.D. Vice Provost and Dean of Research
Mark Holodniy,
M.D. Chair, Administrative Panel on Biosafety
Lawrence Gibbs Associate
Vice-Provost, Environmental Health & Safety
Russell Furr
Director of Research Safety and Deputy Director, Environmental Health & Safety
As this manual addresses biosafety, it should be stated that a Safety Culture is not a secured biological growth media. Safety culture is a part of organizational culture and is often described by the phrase “the way we do things around here”. According to the American Chemical Society, safety culture at an academic institution is a “reflection of the actions, attitudes, and behaviors” demonstrated by the faculty, staff and students concerning safety”.
Several high-profile accidents in the research world have led to the realization that ensuring excellence in research requires a strong, positive safety culture throughout the University. This means that safety is viewed as an operational priority, because of the benefits thoughtful, safe procedures and attitudes bring to research.
2.2Safe Research at Stanford
As an academic institution, safety culture is part of the educational foundation that will accompany our students into their future careers, preparing them to be skilled scientists in academia or industry. Safety’s intrinsic value is seen in better reproducibility and productivity of research, as well as preventing tragic lab accidents that cost lives and knowledge. This chapter is part of a larger conversation about shaping and defining a shared cultural approach, which integrates safety and health seamlessly with the work of our laboratories and classrooms.
Important Information
Continuous Learning
Research is not a static endeavor; managing safety requires ongoing reassessment, feedback and reinforcement. Encourage reporting by members when identifying and reviewing lessons learned after an incident and using these as teaching opportunities. Involve all lab incidents and near misses.
To some degree, as researchers, we all have experienced rules, regulations, compliance approvals, and inspections. It is generally understood that these are part of an established research environment. However, because of this experience, it is easy to incorrectly equate safety rules with safety and come to believe that adhering to a list of rules equates to being good at safety.
In science, researchers think according to the principles: mathematical, physical and chemical laws; biological paradigms. Frameworks and logic, rather than memorization are used, to gain understanding and further knowledge. Safety should be no different. This starts with recognizing that safety is a fundamental part of the scientific process, adding value by exerting greater control, reducing uncertainty, and increasing the safety and quality of your results or product.
While reading information on Safety Culture I came across an article published in Occupational Health & Safety entitled Stop Trying to Create a Safety Culture. Yes, the title did catch my eye so I read on. The article began with:
Safety culture has become the new catch phrase, program focus, and desire of global executives, verbalized in the often expressed, “We need a safety culture!” Safety culture is not new. Stop trying to create it.
OK, I said to myself; I see where they are going with this. Researchers just need to do what they are supposed to be doing, what they are told to do, and we will all be safe. I read on.
Safety practices, risk perceptions, and mitigation techniques have been and always will be a part of human conversation, probably more so among those who are more successful in navigating life’s risks and able to pass this knowledge to their offspring and descendants. Safety is a part of every culture. Everyone to some degree has, or is influenced by, multiple safety cultures.
So they are saying that Darwin was right – be safe or go home. I read on.
Cultures are not a program; they are the interconnectedness that explains why efforts work, don’t work, succeed, and fail.
This was it, the take away that actually made sense. All researchers know that safety trainings, classes, guidance’s, regulations, compliance approvals, and inspections currently exist and are part of the established research environment. Do these not comprise a Safety Culture?
We – researchers, laboratories, Universities- have safety attributes but they have been traditionally broken up into pieces, some more obvious than others, some not totally acknowledged and some just ignored. We must have a Safety Culture gestalt, a whole that is perceived as more than the sum of its parts, that is second nature to all participants, one that will influence the individual decisions carried out when no one is watching- the most important part of cultural reality, safety or otherwise.
For this transition to succeed we all need to be aware of the issues, be open to suggestions, communicate, and work together to change beliefs and behaviors. Not easy, not impossible, certainly doable and vastly rewarding.
– E. Segal PhD, Stanford EH&S Biosafety and Biosecurity Manager</em<
2.3Research Safety Expectations
The University expects that all members of our research community integrate safety into their research activities and go beyond minimum compliance. The following elements (Fig 1) help lay the foundation to build and support a safe and productive research environment:
Figure 1. Four Elements of Research Safety
Leadership
Lead by example, adhere to the rules, and be willing to speak up if you see unsafe practices. Faculty and other supervisor are urged to include safety on the agenda and incorporate it into their group thinking and practices.
Lab members openly discuss safety concerns.
PI/laboratory manager and research group members maintain an environment in which personnel feel free to raise concerns.
Actions confirm safety as a priority that supports and is compatible with good research.
The feedback loop on safety issues (bottom-up and top down) is closed (addressed) at the PI/lab management level.
Design
Take the time to systematically assess risk and plan for the hazards identified. Incorporate safety into laboratory procedures.
PI/lab manager understands the risks of the research being conducted, are actively involved in the laboratory safety program, and integrate safety into the laboratory research culture.
Execution
Take action to control your risks. Make sure you have the right protective equipment, engineering controls are working correctly, and researchers are training to safety perform their work. Principal investigators must enforce the established controls in their lab.
PI/lab manager ensures that the personnel, equipment, tools, procedures, and other resources needed to ensure safety in the academic research laboratory are available.
Lab members identify and manage their own safety environment and are receptive and responsive to queries and suggestions about laboratory safety from their lab colleagues.
Lab members conduct their research using protocols and procedures consistent with best safety practices in the lab.
Adaptability
Research is not a static endeavor; managing safety requires ongoing reassessment, feedback and reinforcement. Encourage reporting by members when identifying and reviewing lessons learned after and using these as teaching opportunities. Involve all lab incidents and near-misses.
PI/lab manager evaluates the laboratory safety status themselves and knows what and how to manage changes to enhance safety in the laboratory.
The PI/lab manager and lab group supports a continuous learning environment in which opportunities to improve safety are sought, communicated and implemented.
Safety discussions become part of regular lab meetings; near misses within the lab are reported in a timely manner and safety information is requested by lab members to prevent future mishaps through understanding HOW and WHY.
2.4Elements to Actions: Research Laboratory Management
Delegation
A PI often delegates responsibilities to a Laboratory Manager or Senior Researcher. While this is an accepted and valuable model of research organization, there are often two potential issues associated with this arrangement: (1) the delegation involves responsibility but may have little or no authority or power to enforce practices, and (2) communication between the PI and Manager can be affected by numerous demands on PI time. Mindfulness of these issues assists in developing and maintaining a strong and healthy research environment. Some key aspects of effective delegation include matching the correct skill level to the task, having firm goals, and providing solid support.
“It’s one of the weirdest aspects of scientific training: You spend years learning how to do the science, hands-on. The natural progression, if you’re successful, is to become head of your own lab and stop spending time at the bench. There just isn’t time or bench space for it anymore: All your time is spent (between teaching gigs and committee meetings) obtaining and managing money and hiring and managing people. You’re no longer a scientist; you’re a manager of scientists and your own scientific enterprise. And what training did you get for that?”
-Jim Austin, Nov. 9, 2007, “Special Feature: Laboratory Management”, Science (https://www.sciencemag.org/careers/2007/11/special-feature-laboratory-management).
Figure 2. Psychological Danger vs Psychological Safety
2.5Risk Assessment for Research
Evaluation and assessment of risk is a key part of designing and conducting an experimental protocol. Not only does a thorough risk assessment allow researchers to systematically identify and control hazards, but it also improves the quality of science through more thorough planning, a better understanding of the variables, and by sparking creative and innovative thinking. It allows one to implement tighter controls which reduces uncertainty and increases the safety and quality of your results/product. Failure to consider risk and hazards from the beginning of experimental design can produce delays, roadblocks, and frustration later in the process.
The Risk Assessment process is broken down into four steps: and by sparking creative and innovative thinking.
1) Explore:
Determine the scope of your work, beginning with research objective. What question(s) are you trying to answer? Conduct a broad review of the literature. Speak with others who have done similar work. Are the risks different for different approaches?
2) Plan:
Outline your procedure/tasks. This may include a deeper dive into specific topics in the literature. Determine hazards associated with each step, and control measures for reducing risk. EH&S can help with more detailed guidance on how to handle certain hazards.
3) Challenge:
What assumptions did you use? Question the importance of each step. Seek advice from others. Ask yourself “what could go wrong?”. Have I missed anything? Consider all possible outcomes, how high is the risk?
4) Assess:
Implement a model, prototype, or trial run. Can you perform a dry run to familiarize yourself with
equipment and procedures? Can you test your experimental design at a smaller scale or with a less hazardous material? Determine if any design changes are needed. Run your experiment and monitor how your controls perform. Assess as you go and make changes as needed.
Important Information
Risk Assessment
It is important to stop, think and plan before doing as well as assess and iterate as you go. Remember, EH&S is always here to assist.
2.6Safety Culture & Biosafety
What is special or unique about Safety Culture for researchers working with biological agents or rDNA? All of the above attributes form the basis for safe research but just like any science specialty, there are unique issues that must be considered when working with these materials, including:
They can be alive and as such, can grow, replicate and sometimes, move.
Their effect on the researcher can be influenced by the health of the researcher
They can spread through numerous mechanisms (droplet, aerosol, mucosal, oral, fecal, blood borne).
They can insert themselves into a genome and have long term effects.
The diagram on the next page illustrates some of the many factors that must be taken into account when planning to work with biologicals and/or rDNA; many of these issues will be discussed in this manual.
Figure 3. Factors to consider when working with biologicals and/or rDNA.
We wish to suggest a structure for the salt of deoxyribose nucleic acid (D.N.A.). This structure has novel features which are of considerable biological interest…It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.
Watson and Crick (1953),Nature
3Recombinant DNA & Synthetic Nucleic Acids: Regulations & Guidelines
3.1NIH Guidelines
The use of recombinant DNA (rDNA) and synthetic nucleic acids (sNA) are regulated by the National Institutes of Health (NIH); the guidelines can be found in the publication NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines) (OBA – NIH Guidelines) (http://osp.od.nih.gov/office-biotechnologyactivities/biosafety/nih-guidelines). These guidelines are the official guide to all rDNA and sNA work done at Stanford. It is important to realize that following these guidelines is the responsibility of all investigators at Stanford University and not solely that of investigators that are funded by NIH.
3.2Exempt rDNA/sNA
The guidelines specify a number of different categories of rDNA/sNA molecules. One of the most important categories is the Exempt category. Experiments that qualify for this category do not need approval by the Stanford University Institutional Biosafety Committee (the Administrative Panel on Biosafety [APB], see Chapter 5). To determine if your experiments are exempt, you can check Section III, Category F in the NIH Guidelines (online); a short reference guide is presented in Table 1 and 2.
3.3Non-Exempt rDNA/sNA
If your experiment does not fall within the exempt categories (Table 2), you must obtain APB approval.
3.4Viral Vectors and Transgenes
All vectors are not the same. More importantly, the class of gene insert can change the Biosafety level of the construct. It is also important to realize that obtaining a cloning/expression vector from a commercial source does not mean it is automatically exempt or a BSL – 1. Table 3 lists many of the more common viral vectors in combination with different classes of inserts and their associated BSL level.
Important Information
Viral Vector Inserts and Envelopes
Some inserts such as oncogenes or toxins will raise the biosafety containment level of the viral vector (See Table 3); the same is true for certain envelopes.
3.5Human Gene Transfer
Protocols involving the use of rDNA/sNA for gene transfer into humans, whether done directly in the subject or in vitro and subsequently put into the subject, must be submitted to both the APB and the Stanford University Institutional Review Board (IRB) for Medical Human Subjects. Federal regulations require the local IBC (at Stanford, the APB), upon receiving submission of a Human Gene Transfer protocol, to review the following aspects to determine if NIH Recombinant Advisory Committee (RAC) review is required:
The protocol uses a new vector, genetic material, or delivery methodology that represents a first-inhuman experience, thus presenting an unknown risk.
The protocol relies on preclinical safety data that were obtained using a new preclinical model system of unknown and unconfirmed value.
The proposed vector, gene construct, or method
of delivery is associated with possible toxicities that are not widely known and that may render it difficult for oversight bodies to evaluate the protocol rigorously.
Dependent upon the above findings the protocol will be either be submitted for RAC review or the APB will state that RAC review is not required.
Table 1. Recombinant and Synthetic Nucleic Acid Molecules (NIH guidelines).
If your experiment is in an exempt category, APB approval is not necessary. If your experiment does not fall within the exempt categories, you must have current APB approval (also see Table 2).
Is your synthetic nucleic acid designed to: (1) neither replicate nor generate nucleic acids that can replicate in any living cell, and (2) not integrate into DNA, and (3) not produce a toxin that is lethal for vertebrates at an LD50 of less than 100 nanograms per kilogram body weight?
Yes
Exempt (III-F-1)
Is your recombinant or synthetic nucleic acid molecule not in an organism, cell or virus and not been modified or manipulated to render it capable of penetrating cellular membranes?
Yes
Exempt (III-F-2)
Is your recombinant or synthetic nucleic acid molecule solely from a single source that exists contemporaneously in nature?
Yes
Exempt (III-F-3)
Is your recombinant or synthetic nucleic acid molecule solely from a prokaryotic host and propagated in the same host or transferred to another host by naturally occurring means?
Yes
Exempt (III-F-4)
Is your recombinant or synthetic nucleic acid molecule from a eukaryotic host and propagated in the same host?
Yes
Exempt (III-F-5)
Is your recombinant or synthetic nucleic acid molecule from species that naturally exchange DNA?
Yes
Exempt (III-F-6)
Does your genomic DNA contains a transposable element that does not contain any recombinant and/or synthetic nucleic acids?
Yes
Exempt (III-F-7)
Recombinant or synthetic nucleic acid molecule which does not present a significant risk to health or the environment, as determined by the NIH*
Yes
Exempt (III-F-8)
*The NIH has determined that rDNA/sNA from infectious agents of BL-2 (see Appendix A) or above is not exempt and must receive Biosafety approval. Additionally, certain cloning vectors, i.e. Adeno- or Sindbis-based vectors, or amphotropic MMLV based vectors, are some examples of rDNA that are non-exempt.
Table 2. Experiments requiring APB approval.
Approval required for experiments involving:
(Specific NIH Guideline Section)
Further Information and Examples:
Deliberate transfer of a drug resistance trait to microorganisms that are not known to acquire the trait naturally if such acquisition could compromise the ability to control disease agents in humans, veterinary medicine or agriculture. (III-A-1-a)
Transferring a drug resistance trait that is used, had previously been used, may be used (including outside the U.S.), or that is related to other drugs that are used to treat or control disease agents.
Examples include transfer of: erythromycin resistance into Borrelia burgdorferi; pyrimethamine resistance into Toxoplasma gondii; chloramphenicol resistance into Rickettsia conorii; tetracycline resistance into Porphyromonas gingivalis.
Cloning of DNA, RNA or synthetic nucleic acid molecules encoding toxins lethal to vertebrates at an LD50 of <100 ng/kg body weight. (III-B-1)
Cloning toxins (or using plasmids that express toxins with low LD50s).
Transfer of recombinant or synthetic nucleic acid molecules, or DNA or RNA derived from recombinant or synthetic nucleic acid molecules into human research participants. (III-C-1)
Use of recombinant or synthetic nucleic acid molecules, or DNA or RNA derived from recombinant or synthetic nucleic acid molecules, that meet ANY of the following four criteria:
Contain >100nt, or
Possess biological properties that enable genome integration, or
Have the potential to replicate in a cell, or
Can be translated or transcribed.
Examples include: use of a defective adenoviral vector to deliver the CFTR
gene intranasally to patients with Cystic Fibrosis; introduction of an HSVTK
transduced cell line into patients with epithelial ovarian carcinoma;
introduction of a shRNA delivered in a plasmid, bacterial or viral vector.
Risk Group 2, Risk Group 3, Risk Group 4 or Restricted Agents used as Host-Vector Systems. (III-D-1)
The introduction of recombinant or synthetic nucleic acid molecules into Risk
Group 2, 3, 4, or Restricted Agents that meet ANY of the following criteria:
Have the potential to replicate in a cell, or
Possess biological properties that enable genome integration, or
Produce a toxin lethal to vertebrates at an LD50 of <100ug/kg body weight
Examples include: Adenovirus, Herpes virus, Lentivirus, Amphotropic or VSV-g
pseudotyped Murine Retrovirus, Human Retrovirus, Vaccinia virus Vesicular
Stomatitis virus, and Adeno-Associated virus with helper virus.
DNA from Risk Group 2, Risk Group 3, Risk Group 4 or Restricted Agents cloned into nonpathogenic prokaryotic or lower eukaryotic host-vector systems. (III-D-2)
Transfer of DNA from Risk Group 2, 3, 4, or Restricted Agents into
nonpathogenic prokaryotes or lower eukaryotes.
Use of pathogens or defective pathogens as vectors.
Examples include: Adenovirus, Herpes virus, Lentivirus, Amphotropic or VSV-g
pseudotyped Murine Retrovirus, Human Retrovirus, Vaccinia virus Vesicular
Stomatitis virus, and Adeno-Associated virus with helper virus.
Infectious DNA or RNA viruses or defective DNA or RNA viruses in the presence of helper virus in tissue culture systems. (III-D-3)
rDNA experiments involving Risk Group 2, 3, or 4 pathogens.
rDNA experiments involving ≤ 2/3 of the genome from eukaryotic viruses in the
presence of a helper virus.
Examples include: HIV, HTLV-I & II, West Nile Virus, and Lymphocytic
Choriomeningitis Virus
Whole animals, including transgenic animals. (III-D-4)
Experiments utilizing any of the following that may lead to transmissible
infection either directly or indirectly as a result of complementation or
recombination in the animal:
> 2/3 of eukaryotic viral genome, or
Animals containing sequences from viral vectors, or
Stable integration of recombinant or synthetic nucleic acid molecules, or
nucleic acids derived therefrom, into the germline.
Use of viable recombinant or synthetic nucleic acid molecule-modified Risk
Group 2, 3, 4 or Restricted Agent microorganisms tested on whole animals.
Whole plants. (III-D-5)
Experiments involving exotic infectious agents when recombinant or synthetic
nucleic acid molecule techniques are associated with whole plants.
Experiments with plants involving cloned genomes of readily transmissible
exotic infectious agents.
Experiments with plants involving readily transmissible exotic infectious
agents (i.e. soybean rust fungus Phakopsora pachyrhizi, maize streak or other
viruses) in the presence of their specific arthropod vectors.
Experiments involving plants or their associated organisms and the
introduction of sequences encoding potent vertebrate toxins.
Experiments involving microbial pathogens of insects, arthropods or small
animals associated with plants if the recombinant or synthetic nucleic acid
molecule-modified organism can detrimentally impact the ecosystem.
Large-scale DNA work. (III-D-6)
≥ 10 liters of culture combined.
Examples include: Use of ≥10 L fermentor; growing up to five 2 L flasks of rDNA
culture (i.e. E. coli K-12).
Influenza virus. (III-D-7)
Experiments with Influenza virus shall be conducted at the BSL containment
corresponding to the Risk Group of the virus that was the source of the
majority of segments.
Experiments that alter antiviral susceptibility may increase containment level
requirements.
Examples of BSL3 influenza work: 1957-1968 Human H2N2, Highly pathogenic
avian influenza H5N1 strains within the Goose/Guangdong/06-like H5 lineage
(HPAI H5N1), 1918 H1N1.
a Refers to the parental or wild-type virus and some of the common deletions used in viral vectors. MMLV, Moloney murine leukemia virus; SIV, simian immunodeficiency virus. b Refers to ability of vector to infect cells from a range of species. Ecotropic generally means able to infect only cells of the species originally isolated from or identified in. Please note that the ecotropic host for HIV and HSV would be human cells, but the ecotropic host for MMLV would be murine cells. Amphotropic and VSV-G-pseudotyped virus host range includes human cells. c Shown are general categories of cellular genes and functions. Please note that there are differences in the containment level for the same class depending on whether the viral vector integrates into the recipient genome at a high rate. The general categories are as follows: S, structural proteins (actin, myosin, etc.); E, enzymatic proteins (serum proteases, transferases, oxidases, phosphatases, etc.); M, metabolic enzymes (amino acid metabolism, nucleotide synthesis, etc.); G, cell growth, housekeeping; CC, cell cycle, cell division; DR, DNA replication, chromosome segregation, mitosis and meiosis; MP, membrane proteins, ion channels, G-coupled protein receptors, transporters, etc.; T, tracking genes such as those for green fluorescent proteins and luciferases and photoreactive genes; TX, active subunit genes for toxins such as ricin, botulinum toxin and Shiga and Shiga-like toxins; R, regulatory genes for transcription and cell activators such as cytokines, lymphokines and tumor suppressors; Ov and Oc, oncogenes identified via transforming potential of viral and cellular analogs, or mutations in tumor suppressor genes resulting in a protein that inhibits/moderates the normal cellular wildtype proteins.
This does not include SV40 T antigen. SV40 T-antigen-containing cells should not be considered more hazardous than the intact virus. SV40 is considered a risk level 1 agent (the lowest level) according to the NIH Guidelines. The prevalence of SV40 infection in the U.S. population due to contaminated polio vaccine does not seem to have caused a statistically significant increase in the rate of cancers. However, the data from the various studies on SV40 association with cancer are equivocal (Strickler et al. 1998; Butel and Lednicky, 1999; Dang-Tan et al., 2004). d This is a general assessment of containment levels for laboratory construction and use of these vectors for nonproduction quantities only based on the 4th edition of BMBL. This table cannot cover every potential use within a research or laboratory settings; as information is gained, risk assessments and containment levels may be changed. Local IBCs should use all available information and their best judgment to determine appropriate containment levels. BSL – 1* refers to the containment level based on parent virus risk group. However, most procedures involving the handling and manipulation of the viral vectors are done at BSL – 2 to protect cell cultures and viral stocks from contamination. e Certain specific strains of poxviruses, such as MVA, NYVAC, ALVAC and TROVAC, are considered low-risk agents and can be handled at BSL – 1 in certain cases.
From Biological Safety Principles and Practices, 4th ed., pg. 524, D.O. Fleming and D.L. Hunt, Ed, ASM Press, 2006
Figure 1. Genome Editing and Gene Drives
Image credit: Kevin Esvelt
Important Information
Human Gene Transfer
Conducting Gene Transfer experiments into human subjects requires both an IRB and an APB protocol.
3.6Genome Editing and Gene Drives
Multiple technologies exist to create permanent genomic modifications in in vitro cell culture and in vivo animal research models (Figure 2). Methodologies include, but are not limited to, Transcription Activator-Like Effector Nucleases (TALENS), Zinc Finger Nuclease mediated DNA repair (ZNF), Meganucleases, and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) (Figure 1). These technologies can be used to create gene drives, a modification of an organism’s genome resulting in a more efficient spread of a trait through the population as compared to Mendelian inheritance. The Vice Provost and Dean of Research, on the recommendation of the Administrative Panel on Biosafety (APB), has established the following policy in order to protect the health of Stanford researchers and the environment.
Figure 2. Glow in the Dark Animals
Example of Gene Editing
Per NIH regulation and as a requirement of Stanford policy, conducting genome-editing experiments on human embryos is prohibited.
Experiments that Require APB Approval
Human Clinical Studies
Study protocols that include either direct gene modification or the administration of donor cells that have been genetically modified must be filed with both the APB and the Administrative Panel on Human Subjects Research (IRB).
Basic Research Studies (In Vitro and In Vivo)
Delivery via viral vectors: Non-exempt viral vectors and Risk Group 1 viral vectors (e.g. AAV) with human target sequences. Genome target scans of the guide RNA (gRNA) sequence is highly recommended to identify the possibility of off-target effects on the human genome. www.rgenome.net/cas-offinder.
Usage of a gene drive (via viral or non-viral delivery methods) with invertebrate and vertebrate animals or on plants: In addition to the description of the planned experiments and safety of the delivery mechanism, the APB protocol must also address the following containment guidelines.
Molecular containment: Will the guide RNA and the nuclease be located in separate loci? Will a synthetic target sequence be used that is absent from the wild type target organism?
Ecological Containment: Will the experiments be performed outside the habitable range of the target organism?
Reproductive Containment: Will a laboratory isolate/organism be utilized that cannot reproduce with wild type organisms?
Barrier Containment: What physical and chemical barriers will be used to contain the target organisms and prevent their release into the environment?
Genome editing tools (delivered via viral or nonviral delivery methods) that:
Modify an infectious agent to increase host range, transmissibility, or pathogenicity of that particular agent.
Modify the host to increase its susceptibility to an infectious agent.
Express a toxin with a low LD50 (≤100 ng/kg) in the genome of both in vitro and in vivo research models.
Figure 3. Plant Research
Exempt Experiments
Genome editing experiments that fall under the exempt category involve the use of nonviral transfection methods (eg. electroporation, lipofection) to create genomic insertions, point mutations and deletions in somatic cells in vitro or in vivo. These insertions include rDNAs that express oncogenes or tumor suppressor genes. However, if the experiment involves the expression of a toxin with an LD50 toxin ≤100 ng/kg, the work becomes non-exempt and an APB protocol submission and approval is required.
Important Information
Using AAV?
Use of AAV with CRISPR or gene editing tools may require APB approval
3.7Transgenic Plants
Experiments to genetically engineer plants by rDNA/sNA methods may require registration with the APB (BL2-P or higher, see OBA – NIH Guidelines and Appendix C for additional information). To prevent release of transgenic plant materials to the environment, the guidelines provide specific plant biosafety containment recommendations for experiments involving the creation and/or use of genetically engineered plants. (Figure 3) Plant Biosafety levels are categorized into BL1-P to BL4-P (Table 4).
Table 3. Viral vectors and transgene containment.
Gene transfer vectora
Host rangeb
Insert or gene functionc
Laboratory containment leveld
MMLV based –
gag, pol, and env deleted
Ecotropic
Amphotropic, VSV-G
pseudotyped
S, E, M, G, CC, T, MP, DR, R, TX,
Ov, Oc
S, E, M, MP, DR, T, G
Ov, Oc, R, CC
TX
BSL – 1*
BSL – 2
BSL2+
BSL – 3
Herpesvirus based –
nonlytic
Broad host range
S, E, M, MP, DR, T, G
Ov, Oc, R, CC
TX
BSL – 2
BSL2+
BSL – 3
Lentivirus based –
HIV, SIV, EIAV, FIV, etc.; gag, pol, env, nef, and vpr deleted
Ecotropic, amphotropic, VSV-G pseudotyped
S, E, M, MP, DR, T, G
Ov, Oc, R, CC
TX
BSL – 2
BSL2+
BSL – 3
Adenovirus based –
serotypes 2, 5 and 7; E1 and E3 or E4 deleted
Broad host range, infective for many cell types
S, E, M, T, MP, DR, R, G, CC
Ov, Oc
TX
BSL – 2
BSL2+
BSL – 3
Alphavirus based –
SFV, SIN
Broad host range
S, E, M, T, MP, DR, R, G, CC
Ov, Oc
TX
BSL – 2
BSL2+
BSL – 3
Baculovirus based
Broad mammalian host cell range
S, E, M, T, MP, DR, R, G, CC
Ov, Oc
TX
BSL – 1*
BSL2
BSL – 2+/BSL – 3
AAV based –
rep, cap defective
Broad host range; infective for many cell types, including neurons
S, E, M, T, MP, DR, G
Ov, Oc, R, CC
TX
BSL – 1*
BSL2
BSL – 2+/BSL – 3
Poxvirus based –
canarypox, Vacciniae
Broad host range
S, E, M, T, DR, MP, CC, R, G
Ov, Oc
TX
BSL – 2
BSL2+
BSL – 3
a Refers to the parental or wild-type virus and some of the common deletions used in viral vectors. MMLV, Moloney murine leukemia virus; SIV, simian immunodeficiency virus. b Refers to ability of vector to infect cells from a range of species. Ecotropic generally means able to infect only cells of the species originally isolated from or identified in. Please note that the ecotropic host for HIV and HSV would be human cells, but the ecotropic host for MMLV would be murine cells. Amphotropic and VSV-G-pseudotyped virus host range includes human cells. c Shown are general categories of cellular genes and functions. Please note that there are differences in the containment level for the same class depending on whether the viral vector integrates into the recipient genome at a high rate. The general categories are as follows: S, structural proteins (actin, myosin, etc.); E, enzymatic proteins (serum proteases, transferases, oxidases, phosphatases, etc.); M, metabolic enzymes (amino acid metabolism, nucleotide synthesis, etc.); G, cell growth, housekeeping; CC, cell cycle, cell division; DR, DNA replication, chromosome segregation, mitosis and meiosis; MP, membrane proteins, ion channels, G-coupled protein receptors, transporters, etc.; T, tracking genes such as those for green fluorescent proteins and luciferases and photoreactive genes; TX, active subunit genes for toxins such as ricin, botulinum toxin and Shiga and Shiga-like toxins; R, regulatory genes for transcription and cell activators such as cytokines, lymphokines and tumor suppressors; Ov and Oc, oncogenes identified via transforming potential of viral and cellular analogs, or mutations in tumor suppressor genes resulting in a protein that inhibits/moderates the normal cellular wildtype proteins. This does not include SV40 T antigen. SV40 T-antigen-containing cells should not be considered more hazardous than the intact virus. SV40 is considered a risk level 1 agent (the lowest level) according to the NIH Guidelines. The prevalence of SV40 infection in the U.S. population due to contaminated polio vaccine does not seem to have caused a statistically significant increase in the rate of cancers. However, the data from the various studies on SV40 association with cancer are equivocal (Strickler et al. 1998; Butel and Lednicky, 1999; Dang-Tan et al., 2004). d This is a general assessment of containment levels for laboratory construction and use of these vectors for nonproduction quantities only based on the 4th edition of BMBL. This table cannot cover every potential use within a research or laboratory settings; as information is gained, risk assessments and containment levels may be changed. Local IBCs should use all available information and their best judgment to determine appropriate containment levels. BSL – 1* refers to the containment level based on parent virus risk group. However, most procedures involving the handling and manipulation of the viral vectors are done at BSL – 2 to protect cell cultures and viral stocks from contamination. e Certain specific strains of poxviruses, such as MVA, NYVAC, ALVAC and TROVAC, are considered low-risk agents and can be handled at BSL – 1 in certain cases.
Table 4. Plant biosafety levels.
From Practical Guide to Containment: Plant Biosafety in Research Greenhouses, Revised Edition, page 13, D. Adair and R. Irwin.
Criteria
Transgenic Plants
Transgenic Microbes: Exotic
Transgenic Microbes: Non-Exotic
Transgenic Insects/Animals/Assoc. Microbes
Not a noxious weed or cannot outcross with one
BL1-P
Not easily disseminated
BL1-P
No detriment to environment
BL2-P or BL1-P+
BL1-P
BL2-P or BL1-P+
Noxious weed or can interbreed with weeds
BL2-P or BL1-P+
Contains complete genome of non-EIA*
BL2-P or BL1-P+
Contains genome of EIA
BL3-P or BL2-P+
Treated with an EIA
BL3-P or BL2-P+
Detriment to environment
BL3-P-4**
BL2-P or BL1-P+
BL3-P or BL2-P+
Involves EIA with detriment to environment
BL3-P or BL2-P+
May reconstitute genome of infectious agent in plants
BL3-P or BL2-P+
Contains Vertebrate Toxin
BL3-P
BL3-P
BL3-P
*EIA—Exotic Infectious Agent **BL4-P containment is recommended only for experiments with readily transmissible exotic infectious agents whether transgenic or not, such as air-borne fungi or viruses in the presence of their arthropod vectors that have the potential of being serious pathogens of major US crops.
Important Information
Plant Research Permits
Additional permits might be required from state and federal agencies before research with plants can be done. Contact Biosafety for information.
4Infectious Agents: Regulations and Guidelines
4.1Tissue Culture, Human and Primate Tissue
The potential laboratory hazards associated with human cells and tissues include the bloodborne pathogens Hepatitis B virus (HBV), Hepatitis C virus (HCV), and Human Immunodeficiency Virus (HIV), as well as agents that may be present in human tissues (e.g. Mycobacterium tuberculosis, Streptococcus, Toxoplasma, etc.) Non-human primate cells and tissues also present risks to laboratory workers (Herpes B virus), as do cells transformed with viral agents such as SV-40, EBV, or HBV, cells carrying viral genomic material and tumorigenic human cells. All are potential hazards due to the possibility of exposure.
Cultured cells which are known to contain or be contaminated with a biohazardous agent (i.e. bacteria or virus) are classified in the same BSL as the agent. Cell lines which do not contain known human or animal pathogens are designated BSL – 1. The following list contains human or primate cells that are to be handled using BSL – 2 practices and containment:
Cells from blood, lymphoid cells, and neural tissue
All primary cell lines
Secondary (immortalized) cell lines
Cell lines exposed to or transformed by a human or primate oncogenic virus
Pathogen deliberately introduced or known endogenous contaminant
Fresh or frozen tissue explants
Note that this list is not conclusive and individual cases will be determined as they occur.
Universal Precautions is the concept of treating all human/primate blood and other body fluids, tissues and cells (including cell lines) as if they were known to be infectious for bloodborne pathogens.
All human blood, blood products, unfixed human tissue and certain body fluids shall be handled with Universal Precautions and BSL – 2 practices.
Table 1. Basis for the classification of biohazardous agents by biosafety level.
BSL 1
Agents that are not associated with disease in healthy adult humans
BSL 2
Agents that are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available
BSL 3
Agents that are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available (high individual risk but low community risk)
BSL 4
Agents that are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available (high individual risk and high community risk)
Table 2. Summary of laboratory facilities for BSL 1 – 4.
BSL
Agents
Practices
Safety Equipment (Primary Barriers)
Facilities (Secondary Barriers)
1
Not associated with disease in healthy adults
Standard Microbiological Practices
As needed to allow for good microbiological practices
Open bench top
Sink required
2
Associated with human disease, hazard = percutaneous injury, ingestion, mucous membrane exposure
BSL – 1 practice plus: Limited access Biohazard warning signs “Sharps” precautions Biosafety manual defining any needed waste decontamination or medical surveillance policies
Primary barriers: Class I or II BSC or other physical containment devices used for all manipulations of agents that cause splashes or aerosols of infectious materials PPE: laboratory coats; gloves; face protection as needed
BSL – 1 plus:
Autoclave available
3
Indigenous or exotic agents with potential for aerosol transmission; disease may have serious or lethal consequences
BSL – 2 practices plus: Controlled access Decontamination of all waste Decontamination of lab clothing before laundering Baseline serum
Primary barriers: Class I or II BSC or other physical containment devices used for all open manipulations of agents PPE: protective lab clothing, gloves, respiratory protection as needed
BSL – 2 plus: Physical separation from access corridors Self-closing, double-door access Exhausted air not recirculated Negative airflow into laboratory
4
Dangerous/exotic agents which pose high risk of lifethreatening disease, aerosoltransmitted lab infections, or related agents with unknown risk of transmission
BSL – 3 practices plus: Clothing change before entering Shower on exit All material decontaminated on exit from facility
Primary barriers: All procedures conducted in a Class III BSC, or Class I or II BSC in combination with full-body, air-supplied, positive pressure personnel suit
BSL – 3 plus: Separate building or isolated zone Dedicated supply and exhaust, vacuum and decon systems Other requirements outlined in the text of the BMBL
Figure 1. BSL – 3 Training
Universal Precautions include frequent hand washing, no mouth pipetting, no food or drink in the lab and proper disposal of biohazardous/medical waste, as well as the use of engineering controls and personal protective equipment (PPE). Engineering controls include biosafety cabinets, ventilation systems, closed top centrifuge rotors, etc.; these are the primary methods to control exposure. PPE such as gloves, lab coats, and eye protection or face shields must be selected and used as appropriate. All material should be treated as medical waste (see Chapter 9).
Important Information
Wash Your Hands
At all Biosafety Levels your last line of protection is the SINK. After finishing all procedures and cleanup, wash your hands with soap and water.
Areas subject to Universal Precautions must have appropriate signs posted on doors and equipment; these signs can be obtained from EH&S (723.0448). Additional information on Universal Precautions is presented in Figure 2.
Biological agents are classified by Risk Group (RG); RG 1 being the least pathogenic to RG 4 being the most. The RG, together with the work to be done (experiments) is assessed to determine the Biosafety Level (BSL).
Important Information
Biosafety Levels
Risk Group (RG) 1 – 4 + Work (research) = Biosafety Level (BSL) 1 – 4
Historically agents are often referred to as BSL – 1-4 (vs RG1). Although this is not technically correct this manual stays with the norm and uses BSL regarding agents.
4.3Biosafety Level Classification
Stanford University follows the categorizing of infectious agents into levels as described in Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th edition (https://www.cdc.gov/labs/BMBL.html), written and published by the Centers for Disease Control (CDC) and NIH. The BMBL describes combinations of microbiological practices, laboratory facilities, and safety equipment in combination with four biosafety levels for various agents infectious to humans. The descriptions of biosafety levels (BSL) 1 – 4 parallel those in the NIH Guidelines for research involving recombinant DNA. Biosafety levels are also described for infectious disease activities that involve laboratory animals or plants. It is important to note that the guidelines presented in the BMBL are considered minimal for containment, and will be customized as needed. The BSL categories are divided up by risk of disease combined with availability of preventive and therapeutic treatments. The four groups are shown in Table 1. For the list of agents and their categories, see Appendix A or go to https://my.absa.org/Riskgroups.
Table 3. Biosafety level work practice requirements.
Limit access to lab while BSL – 2 work is being conducted
Restricted
Not permitted
Bench-top work
Permitted
Permitted only for lowrisk procedures
Not permitted for biohazardous materials
Not permitted for biohazardous materials
Decontamination
Daily and following any spill
Daily and following any spill
Daily; immediately following work with biohazardous materials, and following any spill
Daily; immediately following work with biohazardous materials, and following any spill
Eating, drinking applying lip balm, etc.
Permitted only in designated clean areas
Permitted only in approved and designated clean areas
Not permitted at any time; Food or drink may not be brought into or through lab
Not permitted at any time; Food or drink may not be brought into or through lab
Lab coats
Recommended
Required when work with BSL – 2 is being conducted
Required (wraparound disposable preferable)
Required (wraparound disposable required)
Personal Protective Equipment
Based on risk assessment
Required: Wear appropriate combinations of special protective clothing for all activities with biohazardous materials
Required: Wear appropriate combinations of special protective clothing for all activities with biohazardous materials
Required: Wear appropriate combinations of special protective clothing plus NIOSH N95 respirators or equivalent for all activities with biohazardous materials
Biological Safety Cabinet (BSC)
Not required
Required for all aerosol generated processes*
Required for all work with biohazardous agents
Required for all work with biohazardous agents
Storage Equipment
No Biohazard signs required
Biohazard signs required, all equipment must be labeled with contents
Biohazard signs required, all equipment must be labeled with contents
Biohazard signs required, all equipment must be labeled with contents
Physical containment
Decontaminate equipment immediately after use
Use physical containment devices during procedures that have a high potential to create aerosols* when using biohazardous material; Decontaminate immediately after use
Use physical containment devices (centrifuge safety cup, sealed centrifuge rotor) for all activities using biohazardous material; Open containers in a BSC; Decontaminate immediately after use
Use physical containment devices (centrifuge safety cup, sealed centrifuge rotor) for all activities using biohazardous material; Open containers in a BSC; Decontaminate immediately after use
Hand-washing facilities
Required
Required
Required (foot, elbow, or electronic activation preferable)
Required (foot, elbow, or electronic activation required)
Pipetting
Only mechanical device
Only mechanical device
Only mechanical device
Only mechanical device
HEPA-filtered vacuum lines
Recommended
Required
Required
Required
*Procedures include but not limited to: centrifuging, grinding, blending, vigorous shaking or mixing, sonic disruption, opening containers of biohazardous materials after above procedures.
4.4Laboratory facility requirements
Each BSL has its own corresponding requirements for the laboratory facilities.
T0he physical requirements described in Table 2 will be used in conjunction with additional protective mechanisms (see Chapter 7) to achieve personnel and environmental safety.
Important Information
Aerosols
Did you know aerosols can be generated by pipettes? Vortexing an infectious agent? Do it in a biosafety cabinet.
4.5Biosafety Level Work Practice Requirements
Each BSL is associated with work practices that address the potential risks. Along with practices for BSL1, 2 and 3, is a list of practices labeled as BSL – 2+. This category is used for BSL – 2 agents that are worked with using BSL – 3 practices. (Table 3)
4.6Biosafety Level 3 Laboratories
Biosafety Level 3 (BSL3) laboratories involve research using agents that are associated with serious or lethal human disease for which preventative or therapeutic treatments may be available. These laboratories are designed to protect individuals and the public through the containment of the agents used by both engineering and administrative controls. Any waste generated from these facilities must be sterilized before disposal outside of the facility. Access to these laboratories are tightly controlled and involve a rigorous mentored training process designed by the laboratory to ensure that individuals are both capable and highly knowledgeable in all procedures involving the agent and facility in use. (Figure 1)
The primary hazards to personnel working with BSL – 3 agents involve autoinoculation, exposure to aerosols and ingestion. In order to prevent infection through these modes of transmission, containment devices such as a biosafety cabinet should be used along with procedures and practices that are thoroughly vetted before work begins. In addition to primary engineering and administrative controls, the facility itself must meet strict design guidelines to insure the environment and public are protected from any accidental release inside the facility.
Due to the intrinsic risk associated with work at BSL – 3, each researcher’s knowledge and ability must be evaluated individually, with a gradient of experience and proficiency expected. All researchers must demonstrate competency with a minimum base skill set along with being secure in their potential to ask questions and express concerns. All labs should have an internal expert knowledge source to serve as a mentor to ensure specific skills are passed on.
To ensure the safety of all researchers and the environment, the APB requires BSL – 3 researchers to demonstrate appropriate theoretical knowledge and practical skillsets in the following areas:
Adhering to general lab safety procedures, including donning and doffing PPE.
Setting up, cleaning out, and properly using the biosafety cabinets.
Bringing materials into and out of the biosafety cabinet.
Growing and manipulating cultures safely, with emphasis on the importance of avoiding aerosol generation during all operations.
Performing the essential procedures required of most protocols, such as centrifugation, plating and incubating.
Using the autoclave, disposing of waste.
Emergency management and procedures.
Training should be thoroughly documented and consist of multiple sessions that culminate in a practical test to assess the skill of the researcher. These records should be kept in the lab and sent to the Biosafety group for evaluation. The results for these tests will also serve as the basis for access to the BSL3 facility.
Formal training is strongly encouraged. There are a number of intensive BSL – 3 training courses offered across the country; please contact the Stanford Biosafety group to discuss what is best suited for individual needs.
Contact Biosafety if you are considering doing research with a BSL3 agent.
Figure 2. Stanford Universal Precautions.
4.7Poliovirus Eradication and Containment
Due to the success of worldwide efforts to contain and eliminate polio, the CDC and WHO are moving towards the eventual eradication of all poliovirus. Currently, all poliovirus type 2 (PV2) materials, including WPV2, vaccine-derived poliovirus type 2 (VDPV2), and Sabin type 2-related poliovirus, are subject to containment. This includes both laboratory strains and isolates and other potentially infectious materials, such as stool or respiratory specimens that originate from areas with a high prevalence of poliovirus. The CDC and WHO plans to move towards the containment of all poliovirus types.
Users of these materials will eventually be asked to register with the CDC as a designated poliovirus essential facility. The criteria for these facilities are similar to Biosafety Level 3 laboratories with additional biosecurity elements. Please refer to the CDC webpage (http://bit.ly/2zjJGmA) and the WHO GAPIII document (http://bit.ly/2yOzPBX) for more information on the upcoming requirements and conditions for poliovirus research.
Laboratories using poliovirus or other materials potentially containing poliovirus are encouraged to re-evaluate their use of these materials and destroy any unneeded samples. If you are currently working with poliovirus or materials that may contain poliovirus contact the Biosafety group.
4.8Select Agents and Toxins
Select Agents and Toxins are a collection of designated infectious agents and toxins that, by their nature, have the potential to pose a severe threat to public health and safety; this threat has resulted in the creation of a number of legislative acts.
Initiated with the Antiterrorism and Effective Death Penalty Act of 1996 (http://bit.ly/2k9cMgJ), and bolstered by the USA Patriot Act of 2001 (http://bit.ly/2f7oyYP) and the Public Health Security and Bioterrorism Preparedness and Response Act of 2002 (http://bit.ly/2k8sGrD), the Select Agents and Toxins program oversees the transfer, possession, and use of biological agents (viruses, bacteria) and toxins that have the potential to be a severe threat to public or environmental health. Possession of the specified agents or toxins without registration carries severe civil and criminal penalties. Possession of Select Agents or Toxins over exempt amounts is not allowed at Stanford at this time and would require prior approval from the Vice Provost and Dean of Research and registration with the FSAR Program. The application and further information may be found on the Federal Select Agents Registry (FSAR) website: https://www.selectagents.gov/SelectAgentsandToxins.html
Stanford University is currently not registered for possession of viable select agents. For use of any biological select agent, contact the Biosafety Program.
4.9Stanford University Select Toxins Program
Possession of small quantities of select toxins may be exempt from registration with the NSAR program. The Stanford Select Toxins Program summarizes the University’s requirements for possession of NSAR Select Toxins under the exempt quantities. For additional information please go to the EH&S web site (http://bit.ly/2j7NAWW).
Figure 3. Overview of the Process for Institutional DURC Oversight
Important Information
Prions and Prion-like Proteins
Prions and prion-like proteins are defined as proteins (human or animal) that fall into one of the below categories:
Superoxide dismutase 1 (SOD1); transactivations response element (TAR) DNA-binding protein-43 (TDP43); RNA-binding proteing Fused in Sarcoma (FUS); Ubiquilin (ALS/Lou Gehrig’s disease)Proteins that are highly associated with proteinopathies, including, but not limited to:
Proteins that confer a disease state that is transmissible from cell to cell.
Proteins that have a fibrillar or aggregated form that has
been shown to “seed” a pathology associated with a disease.
4.10Requirements for Research with Prions and Prion-Like Proteins
Per NIH guidelines (http://osp.od.nih.gov/officebiotechnology-activities/biosafety/nih-guidelines) and the Stanford University Administrative Panel on Biosafety policy (see Chapter 5, Charge to the APB), the APB requires researchers to have an approved APB protocol and follow specific guidelines for working with prions and prion-like proteins.
For APB oversight, prions and prion-like proteins are defined as proteins (human or animal) that fall into one of the below categories:
Proteins that are highly associated with proteinopathies, including, but not limited to
Proteins that confer a disease state that is transmissible from cell to cell.
Proteins that have a fibrillar or aggregated form that has been shown to “seed” a pathology associated with a disease.
Specific in vitro or in vivo work with such proteins is classified as BSL2 or ABSL2 and requires an APB approved protocol. This includes, but is not limited to, the following types of work:
Synthesis, use or production of protein in high concentration
Generation or use of mutated proteins
Generation or use of fibrillar or misfolded forms of proteins
APB protocols must include established prion disinfection/decontamination and destruction / disposal protocols, or specific Standard Operating Procedures (SOPs). These SOPs must be provided for review by the APB. If necessary, contact Biosafety for appropriate methods. Refer to the following references for established infection control guidelines for disinfection /decontamination:
Biosafety Manual (see Chapter 11, Waste and Decontamination)
4.11Dual Use Research of Concern (DURC)
A subset of research, as defined by the Federal government, that has the greatest potential for generating information that could be readily misused to threaten public health and national security has been termed “dual use research of concern” or DURC. (Figures 3 & 4)
The United States Government (USG) is presently limiting the scope of DURC policies to a subset of 15 biological agents and toxins that are considered Select Agents and are regulated by the US Department of Health and Human Services and the U.S. Department of Agriculture. Additionally there are 7 categories of experiments that come under DURC.
01. Enhances the harmful consequences of the agent or toxin;
02. Disrupts immunity or the effectiveness of an immunization against the agent or toxin without clinical or agricultural justification;
03. Confers to the agent or toxin resistance to clinically or agriculturally useful prophylactic or therapeutic interventions against that agent or Figure 4. Research Subject to DURC Policies Does research directly involve one or more of 15 agents or toxins listed in the policy? Does research aim to or reasonably predict to produce one of seven listed experimental effects? Does research meet definition of DURC? Requires additional federal and local oversight and risk mitigation strategies to address dual use concerns Yes Yes Yes 42 Stanford University Biosafety Manual toxin or facilitates their ability to evade detection methodologies;
04. Increases the stability, transmissibility, or the ability to disseminate the agent or toxin;
05. Alters the host range or tropism of the agent or toxin;
06. Enhances the susceptibility of a host population to the agent or toxin; or
07. Generates or reconstitutes an eradicated or extinct agent or toxin.
Work with these agents/toxins under these circumstances requires additional review (Figure 4). This review is multi-layered (Figure 3) and extensive. Contact Biosafety for further information or if you are planning any work that could be considered to fall under DURC.
5Administrative Panel on Biosafety
5.1Administrative Panels on Research Compliance at Stanford
The Administrative Panels on Research Compliance assure the institution’s compliance with federal, state and local regulation of research and teaching activities by reviewing those activities which involve the use of human subjects, laboratory animals, biohazardous agents, recombinant DNA, or radiological hazards. In addition, the Administrative Panels are responsible for assessing current research policy and helping formulate new policy governing the conduct of research and training at Stanford with respect to the subjects or agents under the jurisdiction of each panel.
Important Information
APB Oversight
Biohazardous materials include any organism that can cause
disease in humans, or cause significant environmental or
agricultural impact, such as:
Bacteria
Viruses
Parasites
Prions and Prion-like proteins
Fungi
Human or primate tissues, fluids, cells, or cell cultures/lines that are known to or are likely to contain infectious organisms
Human or animal tissues, fluids, cells, or cell cultures/ lines that have been exposed to infectious organisms
Animals known to be reservoirs of zoonotic diseases
The Biosafety programs oversees the use of the recombinant and synthetic nucleic acid molecules. This includes:
Recombinant and synthetic nucleic acid molecules
Transgenic animals
Trangenic plants
Human gene transfer or studies using recombinant DNA
Administrative Panel on Biosafety (APB)
The NIH has mandated the presence of an Institutional Biosafety Committee for all organizations that come under NIH regulations. At Stanford University, the Administrative Panel on Biosafety (APB) is an established Institutional Biosafety Committee (IBC) that reviews projects involving infectious agents, recombinant DNA (rDNA), and synthetic nucleic acid molecules (sNA). APB approval is required for all work that uses biological agents classified as BSL – 2 or above, or rDNA classified as non-exempt by the NIH. Per NIH guidelines (http://bit.ly/2B2yUDX) and the Stanford University Administrative Panel on Biosafety Charge (see below) the APB also requires researchers to have an approved APB protocol and follow specific guidelines for working with prions and prion-like proteins; additional information can be found in Chapter 4.
All research personnel using BSL 2 or 3 biohazardous agents must be appropriately trained and familiar with the safety procedures in handling these materials. The PI/Laboratory Director is responsible for training and ensuring that all biohazardous agents are used at the appropriate level of biological containment.
APB approval is not required for experiments which involve the use of BSL 1 agents exclusively (without the use of recombinant DNA molecules). However, any investigator working with human blood, clinical specimens, human tissues/tissue culture, or other potentially infectious materials must still meet the compliance requirements of the OSHA Bloodborne Pathogen Standard.
5.2Compliance Panel Oversight
Human Clinical Research Protocols (IRB/APB)
Protocols involving the use of infectious agents and/or rDNA/sNA for gene transfer into humans, whether done directly in the subject or in vitro and subsequently put into the subject, must be submitted to both the APB and the Stanford University Institutional Review Board (IRB) for Medical Human Subjects prior to initiation of protocol. The APB usually reviews the human clinical research protocols prior to the IRB review. Like the APB, the IRB uses eProtocol for its reviews.
Stem Cell Research (SCRO/APB)
All research involving the use of Stem Cells come under the Institutional Review Board/Stem Cell Research Oversight Panel (IRB/SCRO Panel) within the Research Compliance Office (RCO). As mandated by State Law, all University research projects involving human stem cells must be reviewed and approved by the IRB/SCRO Panel. The IRB/SCRO Panel is responsible for providing scientific and ethical review of all proposed research projects involving all human stem cells. This review is in addition to other compliance panel reviews that may be required such as Animal Care and Use, or Biosafety. The IRB/SCRO uses eProtocol for its reviews.
Research involving infectious agents and/or rDNA/sNA and Animals (APLAC/APB)
Research involving the use of infectious agents and/or non-exempt rDNA/sNA with animals (in vivo) requires both APB and Administrative Panel on Laboratory Animal Care (APLAC) approval. These approvals are not required to be obtained in any specific sequence but both must be approved prior to any research being done. APLAC uses eProtocol for its reviews.
Important Information
Research Compliance Panels
at Stanford
Panel
Oversight
APB
Biologicals BSL 2/3
rDNA/synthetic nucleic acids
Prion/Prion-like proteins
Use of biologicals/rDNA in humans
Use of biologicals/rDNA in animals
APLAC
Animals
IRB
Human Subjects
SCRO
Human Stem Cells
The APB Review Process
At Stanford University, research involving rDNA and/or Biohazardous Agents is regulated by the NIH and comes under review of the Stanford University Administrative Panel on Biosafety (APB).
Research that involves the use of the above materials (excluding exempt rDNA (see Chapter 2) and clinical research that requires Institutional Biosafety Committee approval MUST be reviewed and approved by the APB prior to work being done. The APB, along with the Stanford IRBs, APLAC and SCRO, uses eProtocol, a web-based system that coordinates new protocols, updates, renewals, and reminders.
The flow chart in Figure 1 illustrates the eProtocol Biosafety workflow.
The APB meets the third Wednesday of each month; protocol applications that require APB review, if submitted by the first of the month, will normally be reviewed by the panel at that month’s meeting.
Protocol Actions
Renewal: an update is required on the anniversary of the protocol approval; this is an opportunity to ensure that all personnel, project information, locations, etc. are current. The eProtocol system will send the PI, research coordinator and administrator (if designated) automatic email reminders prior to the due date. If an annual update is not submitted the eProtocol system will close the protocol.
Revision: any approved protocol can be revised at any time during its approval cycle to update personnel, project information, locations, etc. Revisions will be reviewed on the same panel schedule as noted above.
Protocol Terminations
Duration of approval: BSL – 2 projects are normally approved for three years, with a requirement for renewals (annual updates); BSL – 3 and human clinical projects are approved for one year. Annual updates and revisions of projects are done through eProtocol Biosafety.
Violations and Termination of APB Approval: An approved user who willfully or negligently violates the University, federal, or state rules and regulations governing the use of biohazardous agents/rDNA may have his/her APB approval suspended or revoked by the Biosafety Officer pending review by the APB. The Biosafety Officer will prepare a report which will describe the violations in detail and will discuss the matter with the Chair of the APB who will then determine the final course of action.
5.3Charge to the Administrative Panel on Biosafety (Revised August 2015)
General Charge
Policy for ensuring the safe use and handling of biohazardous agents and recombinant DNA (r-DNA) at Stanford is provided in the Charge to the Administrative Panel on Biosafety. The Administrative Panel on Biosafety reviews all University research and teaching activities involving the use of biohazardous agents, recombinant DNA molecules and synthetic nucleic acid molecules that require approval (“biosafety activities”), as defined below. Through these reviews, the Panel ensures that the activities described in the previous sentence and the related facilities are in compliance with applicable University policies and external regulations. The Panel is also responsible for review of biological agents as they relate to Biosecurity, identifying risks associated with the potential misuse of information, technologies, or products that may be generated.
The Panel advises the University and recommends policies to guide investigators and the Department of Environmental Health & Safety (EH&S) in carrying out the University’s Biosafety Program in the acquisition, use, training, transfer, storage, disposal, and emergency response procedures for all biosafety activities. The Panel’s objective shall be to ensure that such activities meet standards of good practices consistent with safety of personnel and the general public in ways that best facilitate relevant research or teaching activities of the University.
The Panel is responsible for reviewing all University projects conducted by Stanford faculty, staff, students, and/or visiting scientists which involve biosafety activities at Stanford facilities. In addition, the Panel may be asked by the University administration to review research protocols on behalf of other institutions with which Stanford has formal affiliation agreements. Under Stanford’s current “Institutional Biosafety Committee” agreement with the Veterans Affairs Palo Alto Health Care System (VAPAHCS), the Panel shall review all biosafety protocols from Stanford researchers located at the VAPAHCS and from VAPAHCS researchers not otherwise affiliated with Stanford University.
The Panel shall function so as to discharge the University’s obligations placed upon the Panel by current governmental requirements, including those described in the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines), the Centers for Disease Control and Prevention (CDC) Guidelines, the U.S. Department of Health and Human Services (HHS), and Occupational Health & Safety Administration (OSHA) Regulations. To this end, the Panel shall assist protocol directors in meeting their responsibilities.
All biosafety activities involving the use of Biosafety Level 2 or 3 agents AND/OR non-exempt recombinant DNA AND/OR synthetic nucleic acid molecules, as defined by the National Institutes of Health (NIH), AND/OR agents identified as Dual Use Research of Concern shall be reviewed by the Panel regardless of the source of funding for the project. The Panel may approve research protocols with or without modifications, or withhold approval of all or any portion of a protocol. The Panel may delegate review and approval of protocols that meet specific requirements to a voting member of the panel. This subset of protocols must be agreed upon by the full Panel and approved by the Dean of Research.
All human subject protocols involving gene transfer, as defined in the NIH Guidelines, shall be reviewed by the Panel in coordination with the Administrative Panel on Human Subjects in Medical Research (see Review Process for Biosafety and Human Subjects Gene Transfer Experiments).
The Panel shall assess suspected or alleged violations of protocols, external regulations, or University policies which involve biosafety activities. Activities in which serious or continuing violations occur may be suspended by the Panel or the Institutional Biosafety Officer. In such cases, the Panel will immediately notify the affected investigator(s), the relevant school dean, the Vice Provost and Dean of Research, appropriate University officers and others as required by University policies and external regulations.
Upon request, the Panel shall review and comment on proposed external regulations dealing with biosafety. When appropriate, the Panel will formulate draft policies and procedures for approval by the appropriate University bodies and promulgation by the Vice Provost and Dean of Research.
Important Information
Definitions of Recombinant DNA and Infectious Agents that must be approved by the APB prior to use.
Biohazardous Agents
Infectious/pathogenic agents classified in the following categories: Biosafety level 2, 3, and 4, or
Other agents that have the potential for causing disease in healthy individuals, animals, or plants.
Recombinant DNA Molecules
Molecules which are constructed outside living cells by joining natural or synthetic DNA segments to DNA molecules that can replicate in a living cell, or
DNA molecules that result from the replication of those described above.
Synthetic Nucleic Acid Molecules
Can replicate or generate nucleic acids that can replicate in a living cell, or
Are designed to integrate into DNA, or
Produce a toxin with a LD50<100ng/kg body weight.
Synthetic nucleic acids that are deliberately transferred into one or more human subjects and
Are >100 nucleotides, or
Can integrate into the genome, or
Can replicate in a cell, or
Can be transcribed or translated.
Gene Transfer
Delivery of exogenous genetic material (DNA or RNA) to somatic cells for the purpose of modifying those cells.
Dual Use Research of Concern
A subset of research, as defined by the Federal government, that has the greatest potential for generating information that could be readily misused to threaten public health and national security has been termed “dual use research of concern” or DURC.
Guidelines
All biosafety protocols shall be available for review by any member of the Panel. The Panel shall maintain records of research protocol reviews and minutes of meetings, including records of attendance and Panel deliberations. The activities of this Panel are subject to the Guidelines on Confidentiality of Administrative Panel Proceedings.
The following guidelines are established to aid the Panel in the exercise of its responsibilities:
Biohazardous Agents
Protocols involving Biosafety level 2 and 3 biohazardous agents must be reviewed and approved by the Panel prior to the initiation of use of agent. Approval of Biosafety level 3 agents may be granted for no more than one year after review at a convened meeting of a quorum of the Panel (i.e., a majority of the voting members) with the affirmative vote of a majority of those present. Biosafety level 2 protocols are approved for 3 years.
Protocols involving Biosafety level 1 agents that do not involve recombinant DNA are not reviewed by the Panel.
Research using Biosafety level 4 agents are not currently being carried out at Stanford.
Toxins and Select Agents
The routine use of most toxins will not require APB review and approval. However, the Panel will notify the Department of Environmental Health & Safety (EH&S) if any experiments involve the isolation and production of certain toxins (from live biological organisms) that are listed in the U.S. Departments of Health and Human Services (HHS) and Agriculture (USDA) (https://www.selectagents.gov/SelectAgentsandToxinsList.html) USA PATRIOT Act and Public Health Security and Bioterrorism Preparedness and Response Act of 2002 and the select agent regulations (7 CFR Part 331, 9 CFR Part 121, and 42 CFR Part 73).
Recombinant DNA
Recombinant DNA experiments involving certain Risk Group 1 agents and all Risk Group 2 and 3 agents require Panel approval before initiation. In addition, Panel approval is required prior to the commencement of any proposed recombinant DNA project which involves pathogenic agents, human subjects, live animals, plants, and/or planned release of recombinant DNA organisms into the environment. Protocols are approved for 3 years.
Synthetic Nucleic Acid Molecules
Any work using synthetic nucleic acid molecules that are deemed by the NIH to be non-exempt from the NIH Guidelines must have APB approval prior to commencing work. Protocols are approved for 3 years.
Gene Transfer
Human Subject protocols involving gene transfer must be reviewed and approved by the Panel prior to initiation of protocol. Approval may be granted for no more than one year after review at a convened meeting. RAC review occurs before final Panel approval in order to inform that Panel of the RAC’s recommendations before the Biosafety Panel makes its final determination.
Experiments classified as “Exempt” in the NIH Guidelines do not require Panel review.
Dual Use Research of Concern
Potential DURC must be reviewed by the Panel prior to initiation of work. All Federal requirements must be met prior to and for the duration of work. Approval may be granted for no more than one year after review at a convened meeting. The appropriate Federal agency must approve the work and risk mitigation plan prior to final Panel approval.
Conflict of Interest
In accordance with the NIH Guidelines, no member of tge APB may be involved (except to provide information requested by the APB) in the review or approval of a project in which he/she has been or expects to be engaged or has a direct financial interest.
Decisions of the APB
If an investigator has concerns with respect to procedures or decisions of the APB, the investigator may discuss his/her concerns with the Vice Provost and Dean of Research. Neither the Vice Provost and Dean of Research, nor the Provost, nor any other Stanford official or committee may approve a Chapter 5: Administrative Panel on Biosafety (APB) 51 protocol that has not been approved by the decision of the Panel, nor apply undue pressure on the Panel to reverse a decision.
Membership
The Panel is appointed by the Vice Provost and Dean of Research and shall be made up of at least five members with expertise in general issues of laboratory biosafety, use of infectious materials, and recombinant DNA technology. Individuals on the Panel include faculty and staff, one student nominated by the ASSU Committee on Nominations who is either an upperclassman or preferably a graduate student with previous biosafety experience, two members from the local community not otherwise affiliated with the University, and any others who may be invited to serve when their expertise is required.
Voting ex officio members shall include representatives of the: Department of Environmental Health & Safety (Biosafety Officer) and Department of Comparative Medicine (a veterinarian). Non-voting ex officio members shall include representatives of the: Department of Environmental Health & Safety (Associate Vice Provost), Office of Vice Provost and Dean of Research and Office of General Counsel (consultation basis).
The term of membership on the Panel is a 12-month renewable period beginning October 1 through September 30.
Reporting on Obligations
The Panel reports to the Vice Provost and Dean of Research. The Biosafety Officer is the institutional official responsible for the day-to-day operation of the Biosafety Program and reports to the Associate Vice Provost for Environmental Health & Safety.
Panel Meetings
The Panel shall meet as necessary to conduct its business but no less than bi-monthly. The Chair shall submit an annual report of Panel activities and deliberations to the Vice Provost and Dean of Research.
Staff Support
EH&S and the Office of the Vice Provost and Dean of Research shall provide the necessary staffing and administrative assistance. EH&S shall provide technical expertise and advice as necessary for the Panel to fulfill its duties.
6Training
6.1Base-Level Tier I Training
Stanford University offers numerous training courses and materials for employees of all levels and backgrounds. A basic list of required trainings for laboratory workers is shown in Figure 1 (Note: a hard copy of this poster is available through EH&S).
The course entitled Biosafety (EHS—1500—WEB) available through Stanford University Axess) provides the basic, Tier I level training in Biosafety. Laboratory workers in the Stanford University School of Medicine are required to complete Biosafety (EHS—1500—WEB), Chemical Safety for Laboratories (EHS—1900—WEB), and Compressed Gas Safety (EHS—2200—WEB). Alternatively, the course entitled Life Sciences Research Laboratory Safety Training (EHS—4875—WEB), which covers Biosafety, Chemical Safety and Compressed Gas Safety, can be completed.
6.2Tier II Bloodborne Pathogens (BBP)
In 1993, CAL/OSHA published the Bloodborne Pathogens Rule (Title 8 CCR GISO 5193); the fundamental premise of this rule is an approach to infection control termed Universal Precautions.
Important Information
Universal Precautions
Universal Precautions assumes that all human cells, cell lines, human blood, blood products, and certain body fluids are contaminated with HIV, HBV, HCV, or other bloodborne pathogens and that these materials be handled accordingly.
The Bloodborne Pathogens Standard (29 CFR, Bloodborne Pathogens. – 1910.1030) applies to all occupational exposure to blood or other potentially infectious materials. Blood means human blood, human blood components, and products made from human blood. Bloodborne Pathogens means pathogenic microorganisms that are present in human blood and can cause disease in humans. These pathogens include, but are not limited to, hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV). Additionally, “Other Potentially Infectious Materials” (OPIM) are included under this standard. OPIM means (1) The following 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, and all body fluids in situations where it is difficult or impossible to differentiate between body fluids; (2) any unfixed tissue or organ, including cells and cell lines, (other than intact skin), from a human (living or dead); and (3) HIV-containing cell or tissue cultures, organ cultures, and HIV, HBC, or HCV (or other) containing culture medium or other solutions, and blood, organs, or other tissues from experimental
animals infected with HIV, HBV or HCV. The above additionally applies to non-human primate materials.
In accordance with the above information, BBP training (considered a Tier II level training) is mandatory and is available under Bloodborne Pathogens (EHS—1600—PROG). This course is entirely web based and requires annual updates (EHS—1601—PROG), also available on the web. To help determine if a worker is at risk for contact with BBP, please use the questions listed below:
Important Information
Is Bloodborne Pathogen training required? If a single box can be checked as yes, BBP training is required.
Will the person:
Work with human blood, blood products or body fluids?
Work with unfixed human cells (including tissue culture cells and cell lines), human tissues or organs?
Work with non-human primates (NHP) or NHP blood, blood products or body fluids?
Work with unfixed NHP cells (including tissue culture cells and cell lines), NHP tissues or organs?
Work with bloodborne pathogens (e.g. HIV, HBV, HCV or other infectious agents able to be spread via blood)?
Work with animals or animal tissues that have been infected with a BBP?
Perform tasks which may potentially result in exposure to human or animal blood, body fluids, organs, or tissues which are infected with the hepatitis B virus or other bloodborne pathogens?
Handle sharp instruments such as knives, needles, scalpels, or scissors which have been used by others working with human blood or other potentially infectious materials to include human organs, tissue or body fluids or used by others working with similar body parts and fluids from animals infected with the hepatitis B virus or other bloodborne pathogens?
If the answer to any of the above questions is yes, then the worker is consider
Important Information
Important Information
Important Information
Important Information
Important Information
Important Information