Protocol Application for Non-Exempt Activities Involving

UW Oshkosh Office of Sponsored Programs and Faculty Development

Institutional Biosafety Committee PROTOCOL APPLICATION FOR NON-EXEMPT ACTIVITIES INVOLVING rDNA AND SYNTHETIC NUCLEIC ACID MOLECULES

IBC Use Only Protocol Number: Expiration Date:

Please submit one (1) hard copy of this form with required signatures to the Office of Grants and Faculty Development, Dempsey Hall 214. You must also submit one (1) electronic copy to [email protected] ALL individuals listed on this application must provide a copy of their CITI completion report. Instructions for completing CITI training can be found on the last page of this application. Your registration will not be processed until the signed hard copies and CITI training documentation are received.

I. COVER PAGE: Project Identification, Personnel and Signatures PROJECT TITLE: Phone No: PRINCIPAL INVESTIGATOR: E-Mail:

Department:

Phone No:

CO-PRINCIPAL INVESTIGATOR: E-Mail:

Department:

Funding Agency: Grant Identification Number (if federally funded):

New Submission Grant Renewal Revision/Resubmission

Financial Conflict of Interest (FCOI) Statement: Principal Investigators, Co-Principal Investigators, and any other person responsible for the design, conduct, or reporting of the research project who hold significant financial interests related to the project must file a Financial Conflict of Interest (FCOI) Disclosure Form with the Office of Sponsored Programs and Faculty Development and complete appropriate FCOI training through CITI Program. Questions regarding this process can be found on the OSPFD website or by contacting OSPFD at [email protected] or 920-424-3215.

Useful Resources: NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th Edition American Biological Safety Association (ABSA) Risk Group Database American Type Culture Collection (ATCC)

PI Certification: By signing below I am agreeing that all work on this project will maintain compliance with the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. Additionally, these activities will be conducted using biological safety practices described in the CDC/NIH publication titled “Biosafety in Microbiological and Biomedical Laboratories” (BMBL) and by additional stipulations set forth by the University of Wisconsin Oshkosh Institutional Biosafety Committee (IBC). If the IBC approves this application, I agree to execute this research as described; request approval from the IBC for modifications; comply with the guidelines set forth by the IBC, and be responsible for the 1 UW Oshkosh Office of Sponsored Programs and Faculty Development training, supervision and work of my staff. I realize that failure to adhere to the policies related to this research may result in suspension or revocation of permission to perform this research at UWO facilities.

Signature of PI : Date:

Signature of Co-PI: Date:

Signature of Department Chair: Date:

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II. NIH Guidelines Class Determination A. The NIH Guidelines is a regulatory document for research involving recombinant or synthetic DNA or RNA activities. Please select all citations below that apply to your research. Definitions for each section are located in Appendix A: “Definitions for Class Determination”

Experiments utilizing biological infectious agents or biological toxins of RG/BSL 1-2 for purposes other than use or study of recombinant DNA or synthetic nucleic acid molecules. STOP! IF YOU SELECTED THIS CATEGORY YOU SHOULD COMPLETE THE IBC BIOLOGICAL AGENT OR BIOLOGICAL TOXIN FORM. DO NOT PROCEED WITH THIS APPLICATION

III-A. Experiments that Require IBC Approval, RAC review, and NIH Director Approval before Initiation See page 15 of the NIH Guidelines for a full description

Deliberate transfer of drug resistance strain to microorganism that are unknown to acquire the trait naturally, if such acquisition could compromise the use of the drug to control disease agents in humans, veterinary medicine or agriculture.

III-B. Experiments that require NIH/OBA and IBC Approval Prior to Initiation See pages 15-16 of the NIH Guidelines for a full description

Deliberate formation of recombinant or synthetic nucleic acid molecules containing genes for the biosynthesis of toxin molecules lethal for vertebrates at an LD50 of less than 100 nanograms per kilogram body weight (e.g., microbial toxins such as the botulinum toxins, tetanus toxin, diphtheria toxin, and Shigella dysenteriae neurotoxin)

Research equivalent to an experiment that has previously been approved by the NIH Director as a Major Action

III-C. Experiments that Require IBC and IRB Approvals and NIH/OBA Review Before Initiation (and RAC review, as applicable) See pages 16 of the NIH Guidelines for a full description

Deliberate Transfer of any of the following into human research participants:  Recombinant nucleic acid molecules, or DNA or RNA derived from recombinant nucleic acid molecules  Synthetic nucleic acid molecules, or DNA or RNA derived from synthetic nucleic acid molecules, that 1). contain more than 100 nucleotides, possess biological properties that enable integration into the genome, 2). Possess biological properties that enable integration into the genome, 3). have the potential to replicate in a cell or 4). can be translated or transcribed.

III-D. Experiments that Require IBC Approval before Initiation See page 17 of the NIH Guidelines for a full description

rDNA into Risk Group 2 or Risk Group 3 microbes DNA from Risk Group 2 or Risk Group 3 microbes cloned into nonpathogenic prokaryotic or lower eukaryotic host- vector systems other than E. coli K12, S. cerevisiae, K. lactis, B. subtilis, and B. licheniformis DNA from Risk Group 4 microbe cloned into nonpathogenic prokaryotic or lower eukaryotic host-vector systems other than E. coli K12, S. cerevisiae, K. lactis, B. subtilis, B. licheniformis if demonstration that only a fraction of genome is present in recombinant organism. Use of infectious or defective Risk Group 1 or 2 viruses in the presence of helper virus Use of rDNA-modified Risk Group 1, Risk Group 2, or Risk Group 3 microbes in animals Use of recombinant cells in animals Use of viral vectors in animals Experiments involving more than 10 liters of culture in one vessel Experiments with influenza viruses generated by recombinant or synthetic methods Creation of transgenic rodents (including knock-outs) at BSL-2 or BSL-3 Creation, breeding, purchase or transfer of transgenic animals other than rodents Breeding transgenic rodents from one strain for propagation at BSL-2 or BSL-3 3 UW Oshkosh Office of Sponsored Programs and Faculty Development Creation, breeding, purchase or transfer of rDNA-modified arthropods Experiments involving rDNA-modified exotic/detrimental plant species with the potential for serious detrimental effects at BSL-2 or BSL-3 Experiments involving use of rDNA-modified microbes that are exotic or have the potential for serious detrimental impact on plants at BSL-2 or BSL-3 Expression of gene sequences encoding potent vertebrate toxins into plants or associated organisms at BSL-3

III-E. Research Experiments that Require IBC Protocol Application Submission Simultaneous with Initiation See page 21 of the NIH Guidelines for a full description

rDNA into Risk Group 1 microbes not listed in III-F Expression in non-K12 Risk Group 1 E. coli strains. Experiments involving cultured human or primate cells containing less than two-thirds of any eukaryotic viral genome Experiments involving other (non-human, non-primate) cultured cells containing less than two-thirds of any eukaryotic viral genome Creation of transgenic rodents (including knock-outs) Breeding transgenic or knock-out rodents from two parental strains (generating a new strain) Experiments involving rDNA-modified plants that are noxious weeds or can interbreed with noxious weeds Experiments in which DNA that represents the complete genome of a non-exotic infectious agent is introduced into plants Experiments involving rDNA-modified non-exotic microbes with the potential for serious detrimental impact on plants Experiments involving rDNA-modified exotic microorganisms, arthropods, or small animals with no potential for serious detrimental impact on plants rDNA modified whole plants or rDNA-modified organisms associated with whole plants not covered in III-D or III- E-2-b

III-F. Research Experiments that are Exempt from the NIH Guidelines (Still require IBC review) See page 23 of the NIH Guidelines for a full description

Synthetic nucleic acids that: (1) can neither replicate nor generate nucleic acids that can replicate in any living cell, and (2) are not designed to integrate into DNA, and (3) do not produce a toxin that is lethal for vertebrates at an LD50 of less than 100 nanograms per kilogram body weight (i.e. siRNA) Nucleic acids from a prokaryotic or eukaryotic host, including its indigenous plasmids or viruses when propagated only in that host (or a closely related strain of the same species), or when transferred to another host by well- established physiological means Expression in E. coli K12 strains, S. cerevisiae, S. uvarum, K. lactis, B. subtilis or B. licheniformis rDNA in tissue culture containing less than on-half of any eukaryotic viral genome Purchase and/or maintenance of one strain of transgenic rodents at BSL-1 Breeding transgenic rodents from one strain for propagation at BSL-1 Breeding transgenic rodents from two strains (generating a new strain) at BSL-1

III. Research Personnel A. Identify personnel conducting the experiments (including students). Specify degree, applicable CITI training completion dates, and project responsibilities.

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Name Degree Qualifications/Training CITI Training Project Responsibilities Completi on Date

Attach an additional sheet if more space is required.

B. Provide details on the type(s) of project-specific training all personnel listed in III.A. have received. Include hands-on training, training via supervisor, workshops, training duration, number of years/months of experience, etc.

IV. Project Abstract A. Provide a summary of the proposed teaching or research activity in such a way that a scientist in another field will understand (layman’s terms). Include methodology that will be used in the project when working with Non-Exempt recombinant or synthetic nucleic acid molecules, touching on: 1). Experimental design, 2). Goals and Purpose, 3). Methods to be used, and 4). Outcomes to be measured.

V. Research Facilities Provide information on where the materials listed in this application will be used, stored, and handled.

AttachBuilding an additional Name sheet if more spaceRoom is required. Number Biosafety Level (BSL-1 or Use of Room (animal BSL-2) housing, research lab, teaching lab, storage room, growth chamber, etc.)

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VI. Animal Use (Vertebrate or Invertebrate)

A. Does the experiment involve the infection and/or use of animals? Yes No (If “No”, skip this section)

Any use of animals for research, teaching or testing purposes must receive approval from the UW Oshkosh Institutional Animal Care and Use Committee (IACUC) prior to Institutional Biosafety Committee (IBC) approval. The IACUC requires that the researcher submit an IACUC Animal Use Application for approval of animal use. Contact [email protected] for more information. IACUC forms can be found at www.uwosh.edu/grants/forms. Please note that the IACUC holds 1-4 meetings per semester and requires the PI to submit protocol applications at minimum two weeks before the next scheduled meeting. IACUC meeting dates can be found at: www.uwosh.edu/grants/iacuc-meeting-dates

B. List animal specie(s) to be used in the procedures outlined in this application. Include animals that are exotic, recombinant, immunocompromised and/or used in combination with pathogens, recombinant materials or organ/tissue/cell cultures. Explain how use of this particular specie(s) is scientifically justified:

C. Disinfection/Inactivation (Animal)

Indicate the procedure(s) used to disinfect or inactivate contaminated surfaces and materials at the completion of work. Animal Species Animal Bedding/Wastes Animal Carcasses Animal Cages/Pens

VII. Plant Use

A. Does the experiment involve the use of exotic, recombinant or pathogen-receiving plants? Yes No (if “No”, skip this section).

Complete this section if plants are exotic, recombinant, and/or grown in association with pathogenic or recombinant microbes and/or pathogenic or recombinant small animals (insects, etc.) List each genus and species of plant used in your research. Include transgenic plants and plants receiving plant pathogens or recombinant organisms.

B. Disinfection/Inactivation (Plant)

Indicate the procedure(s) used to disinfect or inactivate contaminated surfaces and materials at the completion of work.

6 UW Oshkosh Office of Sponsored Programs and Faculty Development Plant Species Plant Materials Soil Pots/Containment

VIII. Recombinant DNA Utilization

A. List genes of interest and marker trails. All genes with antibiotic resistance must be listed. All toxins must be listed. Provide individual details of all higher hazard material. Lower hazard genes such as housekeeping genes may be grouped into categories with representative examples given. Gene Source(s) Gene Name Nature of Insert or Protein Use of Construct (Genus, species, strain) Explain acronyms Expressed (Cloning for sequencing, (Toxin, marker trait, PCR Expression in a virulence factor, DNA repair microbe, tissue culture or gene, oncogene) organism)

Attach an additional sheet if more space is required

IX. Vector Description(s) A. Provide information on the items listed below. Attach a construct map, if available, and indicate any regions that

Gene Transfer Method Vector Backbone Vector Technical Name Risk Attenuation (Conjugation, (Bacterial plasmid, cosmid, Include commercial vendor (Replication defective transformation, phage, virus, synthetic, vector? Ecotropic vector? transduction, liposome, YAC, BAC, etc.) Nontransducing phage? electroporation, viral Temperature-sensitive infection, etc.) growth?)

X. Microbes A. Provide information on all microbes (bacteria, virus, fungi, prions, parasites) which are used to propagate recombinant plasmids and vectors or produce foreign proteins as described in this application. Specify which organisms or cells will receive the microbe and provide strain names or numbers of pathogens when available. Mark “Yes” or “No” in appropriate categories.

7 UW Oshkosh Office of Sponsored Programs and Faculty Development Microbe BSL Human Animal Plant Toxin Large Scale Recipient of Administered (Genus, or Pathogen? Pathogen? Pathogen? Production? Production? rDNA to: species, Risk (Specify (>10 L) Construct? (mice, alfalfa, strain) Group toxin) HeLa cells)

Provide additional information, if needed:

XI. Organ, Tissue or Cell Cultures (OTCC) Provide information on the source of the OTCC, nature of cell lines and whether the OTCC is modified by rDNA. If human tissues or cells are used, the IRB must be involved in the approval process as an IRB application will be required. If transgenic animals are used, indicate if they will be crossed or generated by your lab group. Use of live vertebrate animals will require submission and approval by the IACUC. OTCC Source Technical Passage Description Recipient of Intended Use Potentially (Genus, Name of OTCC (e.g. primary, (oncogenic, DNA (Admin to infectious? species, strain) established) helper/packaging (transient/stable animals, cell (Y/N) , immortalized, ) culture) etc.)

XII. Toxic Molecules A. Do the DNA clones contain genes for the biosynthesis of toxic molecules lethal for vertebrates?

No (Skip this section) Yes

B. If ‘Yes”, Provide LD50 information below: 8 UW Oshkosh Office of Sponsored Programs and Faculty Development

XII. Source A. Explain how the material(s) will be acquired: Laboratory Stock Off-campus Collection Off-campus Researcher Directly from animal/plant tissues

Other (describe):

XIV. Transportation A. Will recombinant or synthetic nucleic acid molecules be transported between locations (i.e. between labs in the same building, between different buildings on campus, between campus and satellite off-campus locations)? Guidelines for appropriate transportation can be found in the BMBL, 5 th Edition

No (Skip this section) Yes

B. If “Yes”, please explain transportation procedures. What precautions are used during transport?

XV. Storage and Security A. Explain how the rDNA materials will be stored and maintained while at UW Oshkosh facilities. Include information on security to ensure unauthorized people cannot access the rDNA materials: Lyophilized Chemical-fixation Refrigerator (Provide location: ) Freezer (Provide location: ) Storage cabinet (Provide location: ) Incubator (Provide location: ) In vivo (Also complete Appendix A: “Animals”)

Other (describe):

B. Will storage space(s) be secured and/or locked? Provide explanation including who will have access to the materials:

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XVI. Disposal/Disinfection/Inactivation (General) A. Explain how rDNA materials are inactivated and disposed. If a chemical disinfectant is used, state the kind and concentration. Include information on how surfaces (lab benches, sharps, plastic or glassware) are disinfected:

XVII. Personal Protective Equipment A. Please select the personal protective equipment (PPE) to be worn for the procedures described on this form. Questions regarding PPE requirements should be directed to [email protected]

10 UW Oshkosh Office of Sponsored Programs and Faculty Development Hand Protection Examination gloves Heat resistant gloves Cold resistant gloves

Eye Protection Safety glasses Goggles Face shield Goggles + Face shield

Body Laboratory coat (white) Scrubs Blue smock/coveralls Tyvek body suit Waterproof boots

Hair Hair cover/hat

Respiratory Guidance on respirator selection can be found at: http://www.cdc.gov/niosh/docs/2005-100/pdfs/2005- 100.pdf N-95 mask Air purifying respirator (half mask/particulate) Air-supplied respirator

Other (Explain):

B. Provide additional information, if needed, on PPE use for the proposed work:

XVIII. Equipment A. Select the type of equipment you will use with the materials listed on this proposal. Biosafety Cabinet: Autoclave: Centrifuge Containment: Will your work require use of a Will your work require use of an Will your work require use of a biosafety cabinet? autoclave? centrifuge? Yes No Yes No Yes No

Cabinet Class: I II III

Model: Autoclave Location: Sealed buckets/rotors? Yes No

Cabinet Location: Autoclave Testing Method: Rotor opened in BSC? Yes No

Certification Date**: Autoclave Testing Frequency: Centrifuge inside BSC? Yes No11

Other Equipment (Explain): UW Oshkosh Office of Sponsored Programs and Faculty Development

Class I cabinets are ventilated for personnel protection and have an inward flow of air away from operator. Exhaust air is filtered through a HEPA filter. Class II cabinets are ventilated for personnel and product protection having an open front with inward air flow for personnel protection, and HEPA filtered mass recirculated air flor for product protection. Exhaust air is filtered through a HEPA filter. Class III cabinets are closed-front ventilated cabinets with gas tight construction which provide the highest level of personnel protection. The cabinet is fitted with arm-length rubber gloves and is operated under negative pressure. All supply air is filtered through HEPA filters. Exhaust air is filtered through two HEPA filters or one HEPA filter and one incinerator.

Autoclave testing: Autoclave efficacy testing must occur on a regular basis. Monthly testing is generally the acceptable frequency but this should be confirmed with your department stockroom manager or department chairperson. Testing method can include spore vials, spore strips, or chemical strips.

Emergency Preparedness This section requests a summary of all emergency response plans that are in place for your laboratory or work space.

XIX. Emergency Contact Information A. Provide emergency contact information for the following: i. PI and Research Staff ii. Program Staff iii. Student Health Center, personal physician, physician familiar with the materials, Environmental Health and Safety, biohazards disposal company, department, or contact, etc. as appropriate

XX. Associated Health Risks A. Describe the health risks, as applicable, associated with the biological materials you will use. Include information on available treatments and their recommended use for persons handling or in the presence of these materials.

XXI. Potential Exposure Hazards A. Identify potential exposure hazards (e.g., aerosol generation when transferring, mixing, or centrifuging, accidental inoculation, ingestion, use of needles/sharps, etc.). If rDNA or synthetic nucleic acids will be used in animals, be sure to specify additional risks related to animal handling, husbandry or by-products

B. Explain the procedure for mitigation of an exposure were to occur. How will personnel health/safety be protected?

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XXII. Laboratory Acquired Infections A. Identify potential hazards and risk for laboratory acquired infections (LAI)

XXIII. Accidental Spill and Containment Breach Response Procedure A. Provide a spill response and/or containment breach procedure, as applicable, that will be utilized in the event of a spill or breach. What steps will be taken to ensure the spill or breach is contained?

XXIV. Aerosol Generating Activities Certain laboratory procedures are at risk for generating aerosols from solid or liquid materials. If not properly contained, these activities can result in a laboratory-acquired infection for staff. A. Do you perform any of the following activities with the materials listed in this protocol?

Vortexing/Blending/Sonicating/Homogenizing Opening lyophilized cultures, culture plates, ampoules, tubes and bottles Flaming inoculating needles, slides or loops Animal inoculations Animal cage changes Necropsy Other (Explain below)

B. Are you working with any of the materials mentioned in this application outside of containment (on a lab bench without closed containers)? 13 UW Oshkosh Office of Sponsored Programs and Faculty Development Yes No

If “Yes”, explain below. Specify which biological materials are used outside of containment. Include the procedure used with the materials.

XXV. Violations to Safety Protocol A. Explain what steps will be taken if personnel are found to be performing work out of compliance with established laboratory safety protocols or procedures. Please note that any incidents such as exposures, personal injury (needle stick), accidental spills, or containment breaches where recombinant DNA or synthetic nucleic acid molecules are utilized must be reported to the Institutional Biosafety Committee within 24 hours by completing the IBC Incident Report form and e-mailing to [email protected]

FOR IBC USE ONLY:

Reviewer Recommendations: Approval Approve as submitted

Require Revisions Modifications deemed to be minor in nature can be reviewed through expedited review by the IBC Chair or his/her designee(s) Significant Modifications will be reviewed by the Full Committee at a convened meeting

Table Table ( Ex: IBC needs additional information from PI in order to make a determination, did not have time to review, need additional expertise, or loss of quorum)

Disapproval Unable to meet requirements for approval under NIH Guidelines. (Ex: Risks significantly outweigh benefits or value of the knowledge to be gained OR there are significant ethical concerns)

Risk Group and Biosafety Level Determination

14 UW Oshkosh Office of Sponsored Programs and Faculty Development Risk group determined by convened IBC: Risk Group 1 (RG 1) Risk Group 2 (RG 2) Risk Group 3 (RG 3) Risk Group 4 (RG 4) Biosafety Containment Level determined Biosafety Containment Level 1 (BSL 1) by convened IBC: Biosafety Containment Level 2 (BSL 2) Biosafety Containment Level 3 (BSL 3) Biosafety Containment Level 4 (BSL 4) IBC Approval Date:

Instructions for CITI Training for Biosafety and Security Program (BSS): 1. Enter the training at www.citiprogram.org a. Select: New Users Register Here b. Create your profile and affiliate with University of Wisconsin Oshkosh. c. You will be prompted by response driven questions that will determine what course and modules you will be required to complete based on your role in the Biosafety and Security Program. a. Example: If you are a committee member and a faculty researcher on campus, answer the questions according to both of your roles. d. The program will provide you with a customized list of courses to complete. The course list will appear on your Main Menu page. e. You can enter the training by clicking on any of the courses listed in your Main Menu. Each course is compiled of numerous modules. There is typically a short quiz after each module. You must complete the quizzes to earn credit for the course. 2. Training may take 1-3 hours to complete depending on your role(s) in the program, so please plan accordingly. 3. At the completion of the training, a completion report is issued verifying the curriculum completed. A hard copy of the Training Certificate must be attached to the protocol application or registration form that you submit to the IBC. 4. The CITI Program training is valid for three (3) years. A refresher course will be required at the time of expiration for continuing projects. 5. For more information or questions about the CITI training requirement, please contact: IBC Administration: [email protected]

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