Biological Safety Refresher Training 20067

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Biological SafetyRefresher Training 2006-7

• Stacey Kraemer, Ph.D.
• Biosafety Officer (BSO)
• Environmental Health Safety Division
• CI-1006
• x1-2663
• cell 706-877-8783
• kraemers_at_mcg.edu

rev. 1/29/07
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Administrative Changes
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New Biosafety Protocol (BSP) Applicationshttp//w
ww.mcg.edu/research/ibc/apps.htm

• BSP Primary form (required of all applications)
• Schedules (to be completed to document specific
  research issues, as appropriate)
• A Non-exempt recombinant DNA
• B  Handling and/or culture of mammalian cells,
  tissues, and/or organs requiring gt BSL2
  containment
• C Research involving large-scale (gt10 liter)
  cultures
• D Research involving potentially infectious
  microbial agents (Risk Group gt2) or specimens
  exposed to these infectious microbial agents
  (excluding any material previously described in
  either Schedule A or B)
• E The use of biological materials in association
  with live animals
• F Human gene transfer/therapy
• G HHS/USDA regulated Select Agents and Toxins
• O Use of biological agents /or recombinant DNA
  in applications for sponsored funding or IRB
  approval. Intended to link grant titles and
  HAC/CCRIs to BSP titles for communication and
  verification to DSPA or OHRP.
• Researchers will be asked to verify experiments
  in grants are covered in BSPs (Schedule O, or
  email notification with grant title, funding
  agency, funding period)

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Processing of Applications requireProof of
Biological Safety Training Right-To-Know
Training

• Biological Safety Training
• 1. Initial (didactic) biosafety training
• Required for all research PI and all research
  personnel who will be working with biological
  agents
• Provided once/month
• Contact Ken Erondu (kerondu_at_mcg.edu) sign-up
• 2. Refresher Biosafety Training
• Required annually
• Didactic (as requested)
• Online Biosafety Training (currently contact me
  for email Powerpoint and exam)
• Right-to-Know Training
• 1. Basic RTK (Required once, does not require
  refresher)
• (http//www.usg.edu/ehs/training/rtkbasic/)
• 2. Blood borne pathogen RTK (Required annually
  for all BBP human/NHP workers)
• (http//www.usg.edu/ehs/training/pathogens/table.
  phtml)

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2 Tracks for IBC Reviews

• Expedited Review
• (non rDNA, exempt rDNA)
• After edits, the BSP is submitted to the
  appropriate subcommittee (clinical or
  basic/applied research)
• The subcommittee evaluates the protocol to
  approve/disapprove/hold over for full committee
  review
• Approvals should be received in approximately 3
  days.
• Full-committee Review
• (non-exempt rDNA, higher hazards, recommended by
  subcommittee)
• After edits, the BSP is submits all applications
  received by the 1st day of the month to the full
  IBC for evaluation.
• Discussion/evaluation occurs on the 3rd Wednesday
  of the month.

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Recombinant DNANIH/OBAParticularly concerned
about inadvertent transmission of genetic
material into humans and dangerous application of
rDNA technology (dual use)

• A- IBC approval, RAC review, NIH Director
  Approval
• Deliberate transfer of drug-resistance into
  microorganisms (such that treatment modalities
  for infection would be ineffective).
• B - NIH/OBA and IBC Approval
• Cloning toxic molecules, LD50lt100 ng/kg (OBA
  registration reqd for LD50lt100 ug/kg)
• C- IBC and IRB Approval, RAC Review
• Human gene transfer (Gene Therapy)
• D- IBC pre-approval
• Cloning regions of RG 2-4 agents or restricted
  agents as host and/or vector, includes viral work
  in tissue culture, genetically engineered
  (non-rodent) animals, genetically engineered
  rodents gtBSL2, large scale (gt10 liters) cultures
• E- IBC simultaneous notice
• Work with lt2/3 of eukaryotic viral genome
• Genetically engineered/ transgenic rodents
  (BSL1)
• F- Exempt
• No significant risk to health or the
  environment

non-exempt rDNA
can start research concurrent with IBC
registration
NIH Guidelines (Section IIIA-F Appendix C)
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Biosafety starts withRisk Assessments
IT DEPENDS!!
Assessment, mitigation and management the risks
associated with working with biological material
in order to reduce those risks to an acceptable
level
Guidelines CDC BMBL http//www.cdc.gov/od/ohs/b
iosfty/bmbl4/bmbl4toc.htm NIH Guidelines
http//www4.od.nih.gov/oba/rac/guidelines/guidelin
es.html ABSA Risk Group Classification Database
http//www.absa.org/XriskgroupsX/index.html Publi
c Health of Canada Biological MSDSs
http//www.phac-aspc.gc.ca/msds-ftss/ Recommendat
ions are based on effect on healthy adults.
Doesnt account for pre-existing diseases,
medications, pregnancy, breast feeding, etc.
Doesnt always account for how the agent will
be utilized. Considerations for lab workers,
support staff, environment, community
Simply because youre aware of the hazards and
you arent concerned for your own safety, does
not mean that the person next to you may not have
legitimate concerns!!
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Risk Assessment Process

• Consideration of risk associated with Agents
  (Risk Group)
• Then Modulate Risk depending on
• Hosts
• Experimental Practices/Applications
• Facilities Proposed
• Personnel Involved
• Environmental factors (e.g. stability)

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Employ Additonal Protection when Aerosol Risk
Increased

• Counter measures include
• Using BSCs
• Additional PPE
• Special equipment
• Safety caps/sealed rotors
• Shielding boxes

• Blowing out pipettes
• Cell sorters (FACS)
• Shaking or Vortexing tubes or Stirring
• Opening lyophilized cultures,
• Opening snap top tubes,
• Breakage of culture containers
• Flaming loops or slides
• Pulling needles out of septums,
• Filling a syringe
• Pouring liquids
• Centrifugation
• Sonication
• Homogenizing
• Blending
• Grinding
• Cell disruption with French press
• Intranasal inoculation of animals
• Cage cleaning, changing animal bedding
• Harvesting infected material from animals, eggs,
  and other virology procedures

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Changes in Regulations/Guidelines
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Who cares?

• National Institutes of Health (NIH)
• Office of Biotechnology Activities (OBA)
• Recombinant DNA Advisory Committee (RAC)
• National Science Advisory Board for Biosecurity
  (NSABB)
• Center for Disease Control (CDC)
• Select Agents http//www.cdc.gov/od/sap
• National Institute of Occupational Safety
  Health (NIOSH)
• Nanotechnology http//www.cdc.gov/niosh/topics/n
  anotech/
• Occupational Safety Health Administration
  (OSHA)
• Environmental Protection Agency (EPA) GA State
  EPA
• US Department of Agriculture (USDA)
• Department of Justice (DOJ)
• World Health Organization (WHO)

/
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Dual-use Research (NSABB) Research with
legitimate scientific purpose, the results of
which may pose a biologic threat to public health
and/or national security
Research that, based on current understanding,
can be reasonably anticipated to provide
knowledge, products, or technologies that could
be directly misapplied by others to pose a threat
to public health, agriculture, plants, animals,
the environment, or materiel.
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• National Research Council of the National
  Academies (2004)
• Biotechnology Research in an Age of Terrorism
  (Fink Report) http//newton.nap.edu/catalog/1082
  7.htmltoc
• Identified Seven Experiments of Concern
• Recommended IBCs given new biosecurity mandate to
  oversee the dual-use research
• Created the National Science Advisory Board for
  Biosecurity (NSABB) to provide guidance.

• NSABB Draft Guidance Documents (2006)
• http//www.biosecurityboard.gov/pdf/NSABB20Draft
  20Guidance20Documents.pdf
• Criteria for Identifying Dual Use Research of
  Concern
• Tools for the Responsible Communication of
  Research with Dual Use Potential
• Set of principles for the responsible
  communication of research with dual use potential
• Points to consider (i.e. a framework) for
  identifying and assessing the risks and benefits
  of communicating research information with dual
  use potential, including options for the
  communication of such research information.
• Considerations for the development of a
  communication plan for research with dual use
  potential
• Considerations in Developing a Code of Conduct
  for Dual Use Research in the Life Sciences.

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Seven Experiments of Concern
Careful consideration should be given to
knowledge, products, or technologies that
1.Would demonstrate how to render a vaccine
ineffective. This would apply to both human and
animal vaccines. Creation of a vaccine resistant
smallpox virus would fall into this class of
experiments. 2. Would confer resistance to
therapeutically useful antibiotics or antiviral
agents. This would apply to therapeutic agents
that are used to control disease agents in
humans, animals, or crops. Introduction of
ciprofloxacin resistance in Bacillus anthracis
would fall in this class. 3.Would enhance the
virulence of a pathogen or render a nonpathogen
virulent. This would apply to plant, animal, and
human pathogens. Introduction of cereolysin toxin
gene into Bacillus anthracis would fall into this
class. 4. Would increase transmissibility of a
pathogen. This would include enhancing
transmission within or between species. Altering
vector competence to enhance disease transmission
would also fall into this class. 5. Would alter
the host range of a pathogen. This would include
making nonzoonotics into zoonotic agents.
Altering the tropism of viruses would fit into
this class. 6.Would enable the evasion of
diagnostic/detection modalities. This could
include microencapsulation to avoid
antibody-based detection and/or the alteration of
gene sequences to avoid detection by established
molecular methods. 7. Would enable the
weaponization of a biological agent or
toxin. This would include the environmental
stabilization of pathogens. Synthesis of smallpox
virus would fall into this class of experiments.
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from NSABB Draft Guidance Documents (2006)
http//www.biosecurityboard.gov/pdf/NSABB20Draf
t20Guidance20Documents.pdf
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Who else is watching?
Private anti-bio warfare weapon community
activist organization
http//www.sunshine-project.org/biodefense/tspibc.
pdf
2003 National Academy of Sciences report
Biotechnology Research in an Age of Terrorism
recommended IBCs given new biosecurity mandate to
oversee the dual-use research Sunshine Project
sent letters to 390 IBCs in the U.S. to survey
their IBC procedures minutes
MCG Failed MCG IBC doesnt do its job of
reviewing research projects to ensure safety- not
even for BSL-3 projects In violation of the NIH
Guidelines, the MCG IBC has effectively abdicated
responsibility for biosafety
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8/15/06 Dukes of Hazards Georgia's Rebel Yell
Against BiosafetyNow Wants to Operate the NBAF
BSL-4 The ongoing failure of the University of
Georgia's Institutional Biosafety Committee (IBC)
to comply with federal safety rules should bring
criticism to the University and to the
ineffective federal overseers of the IBC
system. UGA's continuing noncompliance amounts
to rebellion. The school's scorn for National
Institutes of Health rules is reminiscent of that
of their fellow (fictional) Georgians, the TV
rednecks of the late 1970s who flouted their
disregard for sheriff's authority with a "rebel
yell" ("The Dukes of Hazard"). Except this
sheriff, the National Institutes of Health Office
of Biotechnology Activities, doesn't even bother
to turn on his siren and try to catch UGA to put
it in the biosafety jail that it so richly
deserves. The US Department of Homeland
Security recently named UGA a finalist to receive
a contract to construct a massive new BSL-4
laboratory, the National Bio- and Agro-Defense
Facility (NBAF), a grant with an initial value
estimated at up to 500 million dollars. A more
perverse safety incentive could scarcely be
imagined than to entrust a habitually
noncompliant school with operating such a
facility. UGA remains a leading exhibit of how
little the US government cares about its domestic
rules governing oversight of pathogen research.
6/1/06 The Sunshine Project anticipates that this
will be the first of many complaints filed in
2006, as initial indications are that little has
improved with the IBC system since we last
examined it in detail in 2004. 1 June 2006 Dr.
Amy PattersonOffice of Biotechnology
ActivitiesNational Institutes of
Health COMPLAINT PURSUANT TO THE NIH GUIDELINES
CONCERNING NONCOMPLIANCE BY THE UNIVERSITY OF
SOUTH CAROLINA The Sunshine Project brings to
your attention noncompliance with the NIH
Guidelines by the University of South Carolina.
The Sunshine Project requests that NIH OBA
immediately terminate all NIH funding for
biodefense and recombinant DNA projects at the
University of South Carolina. Rather, USC is
unequivocally in violation of the NIH Guidelines.
This situation requires your action should you
wish the IBC system to be effective and to enjoy
a level of credibility among the public.(5) We
request that relevant funding to the University
of South Carolina be terminated immediately and
that the government seek reimbursement of all
relevant federal funds disbursed to the
University for all research conducted during the
entire period of the institution's noncompliance.
(In this case, this appears to be all such
projects ever funded by NIH at USC.)
http//www.sunshine-project.org/
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Biosafety Reminders!
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Proper Use of Biosafety Cabinets

• Serves to ensure biosafety as well as the
  sterility of the science!

• Do not block your grates in the front or the
  back.
• Do not over-crowd your BSCs
• Allow the unit to run for approximately 15
  minutes BEFORE and 15 minutes AFTER use. (Class
  IIA2 cabinets exhaust 30 air, re-circulate 70)
• Clean all work surfaces, interior vertical
  surfaces and face shields BEFORE use (to protect
  the science)
• Clean all work surfaces, interior vertical
  surfaces and face shields AFTER use (to protect
  the scientists)
• Avoid moving hands in and out of the BSC and
  sweeping side-to-side motions
• Do not use open flames inside biosafety cabinets

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Biological Safety Cabinets

• Open flames in BSCs can
• Interfere with laminar airflow of BSC required
  for containment sterility.
• Damage the HEPA filters or adhesives in the
  filter units or other components of the BSC
• Can impact warranty issues or UL ratings
• Fire safety risk, esp. when used with alcohol
  surface decontamination

• Do not use Class IIA2 (unducted) BSCs for
• Volatile chemicals
• Radiological materials

Biosafety Cabinet Fire from Ignition of Alcohol
Spray
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Good Tissue Culture Techniques

• To ensure biosafety and the science!

• Place all reagents and supplies inside BSC before
  experiment.
• Work from clean to dirty. Consider using
  decontamination trays, filled w/ disinfectant.
• Collect all liquid wastes in vacuum aspirators in
  which disinfectant has been placed. Make sure
  flasks are placed in secondary containment.
• Do not allow vacuum traps to become overfull. Do
  not contaminate vacuum line and decontaminate
  liquids before disposal.
• Consider HEPA filters in the vacuum lines
• Do not leave pipettes on aspirator hoses after
  use
• Dispense pipettes gently along the sides of tubes
  to avoid aerosols

aerosol generated from pipette
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Other safe practices to use

• Wash your hands after glove removal!
• Dont touch your face or unprotected areas with
  contaminated hands.
• Remove gloves DISPOSE before leaving lab. Do
  NOT re-use gloves!!
• Remove gloves without touching outsides of the
  gloves (which may be contaminated).
• Cover any open wounds with occlusive bandage.
• Anyone with skin lesions or dermatitis on hands
  shouldnt work with infectious agents!
• Cover pressurized vials with gauze soaked in
  alcohol
• Standard PPE lab coat, gloves, eye protection

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Other practices to keep in mind

• Transport of biological agents outside of
  laboratory areas should be in
• Closed, leakproof, primary container
• Inside a well-labeled closed, leak-proof, durable
  secondary container
• All overt exposures, large or public spills
  should be reported to PI and BSO.
• Sharps containers should be placed near work
  areas. Do not leave sharps unprotected.
• Label any waste containers with biohazard
  stickers.
• Decontaminate surfaces at the end of the work day
  or after any spill
• Taped-down benchkote paper or diapers are not
  recommended

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Factors that Effect Decontamination
IT DEPENDS!!

• Nature of the agents being disinfected
• Some disinfectants are appropriate for some
  agents, and not others!
• Disinfectant used and its concentration
• Exposure time and temperature
• Nature of surface being disinfected
• Pourous surfaces are more difficult to
  decontaminate
• Organic material can interfere with disinfectant
  functions
• Amount of microorganisms present
• The need to preserve the matrix
• Can we destroy the object which needs
  decontamination?
• Chemical Safety (most disinfectants change
  biohazards into chem hazards!)

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Decontamination

• Steam autoclaving
• Autoclaves must be certified regularly (monthly)
  to ensure decontamination!
• Containers must have opening to allow for steam
  penetration for decontamination and
  sterilization!
• Chemical Decontamination
• includes Fixation- paraformaldehyde, formalin,
  glutaraldehyde
• Physical methods
• Incineration, irradiation, filtration

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Chemical Decontamination
Use appropriate method to match the agents used
in the laboratory (should be detailed on
Biosafety protocols!
Bleach Effective against veg. bacteria,
mycobacteria, viruses, fungal spores, and some
bacterial spores FRESH (lt24 h) 10 bleach (i.e.
0.5 sodium hypochlorite) Undiluted bleach
concentration reliable only 1 year after
purchase 30 minute exposure time Avoid on
stainless steel surfaces (unless rinsed
immediately) Iodine/Iodophors Effective against
lipid-enveloped viruses, veg. bacteria, fungi
Variable effect against mycobacteria,
non-enveloped viruses and bacterial spores
25-1600 ppm available halogen eg. Wescodyne,
Povidone-Iodine Can stain and can corrosive to
stainless surfaces (unless rinsed)
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Chemical Decontamination (continued)
Alcohols Effective against lipid-enveloped
viruses and veg. bacteria Not effective against
Adenovirus, blood-borne pathogens, bacterial
spores Ethanol or Isopropanol (70) up to 10
minute contact time for some agents Evaporation
may require re-application for increased contact
time Flammable! Phenolics Effective against
lipid enveloped viruses and veg. bacteria,
Variable activity agains fungi and mycobacteria,
depending on product. Limited activity against
non-enveloped viruses 5-10 phenol, depending on
product eg. Vesphene, Biozide Quaternary
Ammonium Compounds (Quats) Effective against
Gram positive bacteria Limited activity against
enveloped viruses, fungi, and Gram neg.
bacteria eg. Roccal, Germex, Zephiran, Coverage
Plus
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Chemical Decontamination (continued)
Aldehydes (glutaraldehyde, formaldehyde) Effective
against most agents, depending on contact
time Non-enveloped viruses and mycobacteria (20
min.) Bacterial spores (3 hours) 2-4 in aqueous
solution Requires adequate ventillation, health
risks eg. Cidex Peroxygens Effective against
veg. bacteria, mycobacteria, fungi, viruses and
spores (depending on formulation) 6 working
solution (fresh) Vapor-phase hydrogen peroxide
(VHP) vs. surface decon Incompatible with
aluminum, copper, zinc, brass, natural rubber and
some plastics eg. Virkon
Good Resource for disinfection guidance Public
Health of Canada Biological MSDSs
http//www.phac-aspc.gc.ca/msds-ftss/
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You will need Biosafety Protocols (BSPs)
submitted to the IBC for research involving

• Any potentially infectious agent
• Infectious or potentially infectious agents
  (viruses, viral vectors)
• Human or non-human primate tissues, blood, or
  other materials
• Cell cultures (gtBSL2, eg. human or non-human
  primate origin, infected cells, some genetically
  modified cells)
• Any recombinant DNA
• Exempt and Non-exempt rDNA protocols
• Shipping of dangerous goods
• Dry ice, liquid nitrogen
• Clinical, diagnostic specimens
• Infectious material
• Tissues
• Cells/cultures
• Toxins of Biological origin
• Genetically modified organisms (GMOs) which could
  transmit the rDNA
• Microbial agents (bacteria, yeast, fungi, prions,
  parasites)