Recombinant DNA Program - California State Polytechnic ... Program.pdfRecombinant DNA Program (rDNA)...

Mike DeSalvio M.Bt.,

Recombinant DNA Program Biological Containment for Recombinant/ Chimeric DNA and Clones A look at the safe work practices, procedures and requirements for working with Recombinant DNA in research and the Institutional Biosafety Committee.

2013

Michael DeSalvio Environmental Health and Safety

Rev. 10/10/2013

Michael DeSalvio Origination Date: 10/1/2013 Biosafety Specialist Revision Safe: 10/10/2013 Environmental Health & Safety Revision Number: 02

Recombinant DNA Program (rDNA)

Biological Containment for Recombinant/ Chimeric DNA and Clones.

Table of Contents Purpose

Scope

Definitions

Roles and Responsibilities o Administration o Responsibilities of IBC o Institution (EH&S) o Office of Research and Sponsored Programs (ORSP) o Principal Investigator (PI)

Committee Protocols o Review Process

Initial Application and Review Proposal Modifications Site Specific Risk Assessment Suggesting Containment Levels Expiration Renewals

o Determining Risk Groups o Containment Levels

Raising Containment Lowering Containment

o Administrative Action Escalation Procedures

Safe Work Practices and Standard Operating Procedures (SOP)

Regulated Research o Exemptions o Restricted Experiments

Appendix [Enter Appendix]


Purpose

The purpose of this document is to facilitate the functions of the Institutional Biosafety Committee at

California State Polytechnic University, Pomona in the following:

Recognizing and assessing dangers associated with respective research in Biologics, Infectious

Agents, and Recombinant DNA etc;

Assuring federal agencies (NIH and CDC) of compliance in regards to IBC related research;

Assisting faculty researchers to assure safe lab and research practices in the area of Biologics,

Infectious Agents, and Recombinant DNA etc.

Assist the IBC in reviewing research proposals to effectively assess the safety of proposed work.

Review of this document is necessary for Principal Investigators using regulated materials in their

research. The contents of this manual are derived from directions established by Environmental Health

and Safety, existing University policies from other Universities and guidelines from the CDC and NIH. All

Principal Investigators and their staff are expected to comply with the most current version of the

following (where applicable)

All EH&S Policy

o Chemical Hygiene Plan

o Biosafety Plan

o Incident Response Plan

CDC BMBL “Biosafety in Microbiological and Biomedical laboratories”

“NIH Guidelines for Research Involving Recombinant DNA Molecules”

CDC/ USDA Select Agents Program (See Cal Poly Biosafety Plan)

Scope

The Institutional Biosafety Committee, through this document, provides guidance to University

researchers who use Biologics, Infectious Agents and Recombinant DNA (Regulated Materials) within the

scope of their work.


Acronyms and Definitions

BMBL Biosafety in Microbiological and Biomedical Laboratories (CDC/NIH publication) BSO Biological Safety Officer BSP Biosafety Plan (Biosafety Manual) CDC Center for Disease Control and Prevention CPP Cal Poly Pomona EHS Environmental Health & Safety HHS (United States Department of) Health and Human Services IBC Institutional Biosafety Committee IRB Institutional Review Board NIH National Institutes of Health (in HHS) OBA Office of Biotechnology Activities (in NIH) RAC Recombinant DNA Advisory Group (appointed under NIH/HHS procedures) rDNA Recombinant DNA USDA United States Department of Agriculture ORSP Office of Research and Sponsored Programs (Cal Poly Pomona) PI Principal Investigator Regulated Materials: Materials including but not limited to Biologics, Infectious Agents, Select Agents, Recombinant DNA, Controlled Substances and Precursor Chemicals. Controlled Substances: Substances identified by the DEA and DOJ in 21 CFR §1308.11-§1308.15


Roles and Responsibilities

Administration

The Cal Poly administrators, which include the Vice President of Research, the Vice President

for Academic Affairs and all Deans and Department Heads, shall give the IBC the authority to

review all applicable proposals and assess regulatory compliance.

Responsibilities of IBC

The mission of the IBC is to ensure that research involving biological agents and/or recombinant DNA is conducted within existing Federal and State laws and guidelines that aim to protect the safety of workers, the general public, and the environment. This objective is achieved through careful planning. Specifically, the IBC is required to:

Review technical and safety-related aspects of the use of all biological agents.

Develop and maintain policies and procedures for use of biological agents.

Ensure compliance with State and Federal reporting requirements for research with

biological agents.

Maintain records of all committee meetings, inspections, protocols, and training.

Review protocols and facilities upon request and periodically as adopted by the

committee.

Classify and (if necessary) inspect facilities with respect to appropriate biosafety levels.

Identify members of the IBC to the NIH Office for Biotechnology Activities (OBA) in

accordance with NIH guidelines.

Adopt Emergency Plans to cover accidental spills and personnel contamination.

Institution (EH&S)

The Institution shall ensure the following:

That the IBC has adequate expertise to evaluate and assess rDNA projects with

the use of ad-hoc consultants if necessary.

If human participants are required, NIH Guidelines appendix M must be

appropriately completed.

No human participants enroll in any studies involving genetic transfer

experiments until IBC and RAC requirements have been met.

File an annual report to the NIH/OBA to include:

o Roster of all IBC members and respective position

o Biographical sketches of all IBC members including community

members.


Ensure no member of the IBC may be involved in the review of approval of a

project in which they are or expect to be engaged or has a direct financial

interest unless asked to provide information for the IBC.

Establish the procedures that the IBC will follow

Make IBC meetings available to the public when possible and consistent with

the protection of privacy and proprietary interests.

Upon request, make available all IBC minutes and any documents submitted to

or received from funding agencies (the latter are required to be made public).

Office of Research and Sponsored Programs (ORSP)

The Office of Research and Sponsored Programs (ORSP) at Cal Poly Pomona assists faculty and

staff in obtaining support for externally funded research. ORSP shall provide the PI with information on

major government agencies, foundations and corporations which support research as well as to provide

assistance in the development, planning and proposal submissions. Additionally, ORSP shall assist the PI

in identifying possible funding sources, developing budgets, completion of applications and negotiating

with funding agencies. The ORSP shall assist the PI by request from the PI or IBC if necessary. The ORSP

is not required to initiate contact with the PI.

Principal Investigator

The Principal Investigator is responsible for ensuring all required documentation is submitted to

the IBC and for determining all required levels of compliance respective to any areas of research. A

detailed description of the Roles and Responsibilities for the Principal Investigator can be found in the

appendices of this document.

On behalf of the institution, the PI is responsible for full compliance with the NIH guidelines in

the conduct of rDNA research. A PI engaged in human gene transfer research may delegate to

another party such as a corporate sponsor to oversee the reporting functions set forth in

appendix M, with written notification to the NIH/OBA of the delegation:

Names

Addresses

Contact information

o PI is responsible for ensuring that the reporting requirements are fulfilled and will be

held accountable for any reporting lapses.

Refrain from Initiation or modification of any research that requires NIH/OBA, RAC and or IBC

approval prior to approval.

Determine whether experiments are covered under Section III-E and ensure the appropriate

procedures are followed.

Report any problems, violations of NIH guidelines or any significant accident or illness to the

BSO, Animal Facility Director (if applicable) etc.

Report new information bearing on the NIH guidelines to the IBC and to NIH/OBA


Be adequately trained in good microbiological techniques

Adhere to IBC approved emergency plans for handling accidental spills and personnel

contamination; and

Comply with shipping requirements for rDNA molecules (appendix H)

PI Must Submit the following information to the NIH:

o Information for certification of new host-vector systems;

o Petition NIH/OBA with notice to the IBC for proposed exemptions to the NIH Guidelines;

o Petition NIH/OBA with concurrence of the IBC for approval to conduct experiments

specified in Sections III-A-1, Major Actions and III-B Experiments that require NIH/OBA

and IBC approval before initiation;

o Petition NIH/OBA for determination of containment for experiments requiring case-by-

case review; and

o Petition NIH/OBA for determination of containment for experiments not covered by the

NIH Guidelines.

o Ensure if using human gene transfer, appendix M is appropriately addressed and

provide a signed letter by the PI on Institutional letterhead to acknowledge the

documentation being submitted to NIH/OBA complies with appendix M requirements.

No participants shall be enrolled until approved and reviewed by NIH/OBA, RAC and IBC.

o For clinical trial sites, added after RAC review process, no research participants may be

enrolled at the trial site until the following has been submitted:

IBC approval from the trial site

IRB approval

IRB approved informed consent document

Curriculum vitae of the PI(s) (2 pages or less in biographical sketch format)

NIH grant numbers if applicable.

Submissions by the PI to the IBC

o Make an initial determination of the required levels of physical and biological

containment in accordance with the NIH guidelines;

o Select appropriate microbiological practices and laboratory techniques to be used for

the research;

o Submit the initial research protocol and any subsequent changes to the IBC for review

and approval/disapproval; and

o Remain in communication with the IBC throughout the conduct of the project.

PI responsibilities prior to initiating research

o Make available to all lab staff, the protocols that describe the potential biohazards and

the precautions to be taken;


o Instruct and train lab staff in:

The practices and techniques required to ensure safety,

The procedures for dealing with accidents; and

o Inform the lab staff of the reasons and provisions for any precautionary medical

practices advised or requested (e.g. vaccinations or serum collection).

PI responsibilities during the conduct of the research

o Supervise the safety performance of the lab staff to ensure that the required safety

practices are employed;

o Investigate and report any significant problems pertaining to the operation and the

implementation of the containment practices and procedures in writing to the BSO

when needed and the Animal facility director when applicable. The IBC and NIH/OBA or

other regulatory authorities must be notified when applicable.

o Correct work errors and conditions that may result in the release of recombinant DNA

materials; and

o Ensure the integrity of the physical containment (e.g. Biosafety cabinets) and the

biological containment (e.g. purity and genotypic and phenotypic characteristics.

Procedures

Conflict of Interest

No member of the IBC may be involved in the review or approval of a project in

which he/she serves as PI or has direct financial interest, except to provide information

requested by the IBC.

Review Process

All Principal investigators conducting any research involving Regulated Materials

shall submit an application to the IBC for review. Research involving Regulated Materials

cannot begin before IBC approval has been granted unless approval is not required

based on NIH guidelines. Review Procedures are listed in the appendices.


Initial Application and Review

The IBC is responsible for reviewing, approving, requiring modification (to

secure approval), or withholding approval of those activities related to research

involving biological agents, infectious agents and/or recombinant DNA.

The Biosafety Officer (BSO) has the authority to review protocols for exemptions

in lieu of IBC review. This process will be validated by at least one additional

member of the committee, excluding those classified as non-affiliated members.

Submission Forms:

FORM 1: may require full committee review. This project registration will be valid

for three years from the date of approval. If work is continuing beyond that

time, a renewal project registration form will need to be submitted including

any changes if any.

FORM 2: Requires designated members review. This project registration will be

valid for three years from the date of approval. If work is continuing beyond

that time, a renewal project registration forms will need to be submitted.

FORM 3, 4: Requires designated members review unless the transgenic materials

pose a hazard, then it will be subjected to full review. These forms can be

submitted at any time for approval consideration. These projects once

registered and approved do not require a renewal.

Proposal Modifications

The IBC has the authority to review and approve, require modifications in (to

secure approval), or withhold approval of proposed significant changes in

research using biological agents.

Risk Assessment

A risk assessment matrix is available to determine the level of risk associated

with research. Use of Select Agents will require the use of a Threat Assessment

Matrix. The IBC shall review each research proposal to determine:

If Recombinant DNA (rDNA) is being used, identify all applicable

sections within NIH guidelines. Should no section be determined,

proposals will be designated as exempt under rDNA regulations

(Section III-F).

Use of infectious agents is compliant with blood borne and aerosol/

transmissible disease standards as necessary.

Use of Select Agents & Toxins research is compliant with CDC/ NIH

guidelines.


Suggesting Containment

After determining Risk Groups and applicable sections, the IBC can determine if

an experiment is exempt, requires prior approval or approval from other

outside agencies (e.g. NIH/OBA)

Determining Risk Groups

The IBC shall identify what risk group is associated with the proposed research.

Risk groups shall be determined by referencing sources including but not limited to,

BMBL 5th ED., NIH Guidelines, Research Precedence, OSHA guidelines etc. Should

multiple Risk Groups apply, the highest respective containment level will be used in the

absence of good reason to downgrade containment levels.

RISK GROUP 1: Not associated with human disease.

RISK GROUP 2: Disease not serious or treatable.

RISK GROUP 3: Serious diseases but likely treatable.

RISK GROUP 4: Serious or lethal diseases and treatment is questionable.


Containment Levels

The IBC has the authority to alter biologic containment levels based on the

associated Risk Group and experiment classification. The highest applicable

containment level shall be used unless sufficient documentation is presented to

justify a lower containment level.

Raising Containment

The IBC is permitted to raise the containment level for certain

experiments if there is significant risk to public health, laboratory/

personnel or campus safety.

Lowering Containment

The IBC is permitted to lower the containment level for certain

experiments as specified in NIH Section III-D-2-a (Experiments in which

DNA from Risk Groups 2, 3 4 or Restricted Agents is Cloned into a non-

pathogenic Prokaryotic or Lower Eukaryotic Host-Vector System)

Administrative Action

The IBC has set the following criteria for investigating the possibility of

withdrawing approval for an ongoing project involving biohazard agents. These are:

1. Noncompliance with the NIH Guidelines for Research with rDNA.

2. Noncompliance with the CDC/ USDA APHIS Select Agent Program.

3. A deviation from the BMBL that was not specified in the IBC application.

4. Failure to comply with the Institution’s IBC Policies and Procedures.

5. Report of concerns involving the unsafe or unapproved use of biological

agents.

Escalation:

1. The PI will be given the opportunity to respond to the allegations in

writing, addressing corrective action and measures of compliance.

2. The Chair will inform ORSP and the PI in writing of the possibility of

approval withdrawal and will call an IBC meeting to review the case. The

PI will be invited to attend and reply to concerns of the committee.

3. The committee will vote whether or not to withdraw approval for the

project. A majority vote of the full committee will be required for

withdrawing approval. The minority view, if any, will be recorded.

The vote of the committee will be reported in writing to the PI and the ORSP. If

the vote is to withdraw approval for the project, a report of the committee investigation

and details of the occurrence, with all supporting documents will be sent to the VP of

Research and Graduate Studies, the Department Chair, the funding agency and the

appropriate government agency with oversight over the specific research area.


General Laboratory Practices

BSL‐1 General Laboratory Practices

1. Access to the laboratory shall be limited. Guests shall be accompanied by laboratory personnel. Doors to the laboratory shall be closed and locked when it is unoccupied. A security access plan and incident response plan will be in place when required to do so.

2. All laboratory personnel must wash their hands with soap and water after working with rDNA, rDNA organisms or biological agents, and prior to exiting the laboratory at all times.

3. No food or drink is permitted in the laboratory at any time, including chewing gum. Application of cosmetics and handling of contact lenses is also prohibited within the laboratory.

4. Mouth pipetting is strictly prohibited in any lab, regardless of rDNA use.

5. Broken glassware must not be handled by hand. Dust pans and brushes or tongs may be used. Broken glassware must be placed in the white boxes designated for this purpose. Plasticware should be substituted for glassware whenever possible. Contaminated glassware shall be disinfected prior to disposal.

6. The use of sharps (needles, razor blades, scalpels) is discouraged. If sharps must be used, the following must be followed. Sharps must be disposed of in appropriate, puncture resistant receptacles designed for this purpose. Needles must not be bent, broken, sheared, recapped, removed from disposable syringes, or otherwise manipulated by hand prior to disposal. Reusable sharps must be stored with all sharp edges covered in block of Styrofoam, or similar material. Contaminated reusable sharps must be decontaminated (chemically or thermally, as appropriate) prior to storage.

7. Gloves must be worn if there is a potential for rDNA, rDNA organisms or biological agents which are hazardous (i.e., infectious) to contact the skin. Gloves shall be removed if contaminated or if the integrity of the protective material has been compromised. Contaminated gloves must be placed in the biohazard bin for autoclaving. Do not reuse gloves. Gloves shall not be worn outside of the laboratory regardless of exposure. Hands must be washed thoroughly after gloves are removed, before leaving the lab.

8. All procedures must be performed such that the generation of aerosols and/or splashes is minimized. Centrifuge tubes containing hazardous (i.e., infectious) materials must be capped prior to centrifuging. Sonication of such solutions must be conducted in a BSC, chemical fume hood, or the solution must be in a loosely covered container during the sonicating procedure. Chemical splash goggles must be worn while performing procedures likely to generate splashes or aerosols. Lab coats must be worn when splashes are likely.

9. All work surfaces must be decontaminated after the completion of work and after any spills. Solutions of 70% ethanol or 10% bleach may be used, as appropriate. All equipment and instruments must be decontaminated prior to service or removal from the laboratory.


10. All cultures, stocks, plates or other items containing non‐NIH exempt rDNA, rDNA organisms or biological agents must be disinfected prior to disposal. Bulk quantities of NIH exempt material must also be treated prior to disposal. Liquid phase materials must be brought to a concentration of 10% bleach and allowed to stand for 20 min. (or until the indicator changes color for media) prior to disposal. Material which cannot be chemically disinfected must be autoclaved at 120⁰C for one hour prior to disposal. Contact the Biology Safety Coordinator for details on autoclaving.

11. Each laboratory P.I. shall provide training to laboratory employees regarding their duties, necessary precautions to prevent exposures and exposure evaluation processes. This shall include risk factors that increase susceptibility to infection. At risk individuals are encouraged to self‐identify to health services for counseling and guidance. Training is required at least annually or when procedures change such that additional training is justified.

BSL‐2 General Laboratory Practices All BSL-1 practices shall be followed.

1. Access to the laboratory shall be restricted. The doors to the laboratory will remain locked when unoccupied. Only authorized personnel (i.e., those individuals working in the lab) may enter the area. A security access plan and incident response plan will be present if required to do so.

2. All laboratory personnel must wash their hands with soap and water after working with rDNA, rDNA organisms or biological agents, and prior to exiting the laboratory.

3. No food or drink is permitted in the laboratory at any time, including gum. Application of cosmetics and handling of contact lenses is also prohibited within the laboratory.

4. Mouth pipetting is strictly prohibited.

5. Broken glassware must not be handled by hand. Dust pans and brushes or tongs may be used. Broken glassware must be placed in the white boxes designated for this purpose. Plasticware should be substituted for glassware when possible. Contaminated glassware shall be disinfected prior to disposal.

6. The use of sharps (needles, razor blades, scalpels) is discouraged. If sharps must be used, the follow must be followed. Sharps must be disposed of in appropriate, puncture resistant receptacles designed for this purpose. Needles must not be bent, broken, sheared, recapped, removed from disposable syringes, or otherwise manipulated by hand prior to disposal. Reusable sharps must be stored with all sharp edges covered in block of Styrofoam, or similar material. Contaminated reusable sharps must be decontaminated (chemically or thermally, as appropriate) prior to storage.

7. In addition to item 7 under BSL‐1 General Laboratory Practices above, gloves, chemical splash goggles (and face masks or shields, if necessary) and lab coats must be worn. Contaminated protective equipment (goggles, face shields and lab coats) must be properly disinfected (either thermally or chemically) prior to donning again or laundering. Contaminated face masks should


be disposed of in the biohazard bin. Used protective equipment must not be stored in non‐laboratory areas, such as offices.

8. In addition to item 8 under BSL‐1 General Laboratory Practices above, all work with live cultures of rDNA organisms or biological agents must be performed inside of a Class II BSC.

9. All work surfaces must be decontaminated after the completion of work and after any spills. Solutions of 70% ethanol or 10% bleach may be used, as appropriate. All equipment and instruments must be decontaminated prior to service or removal from the laboratory.

10. All cultures, stocks, plates or other items containing non‐NIH exempt rDNA, rDNA organisms or biological agents must be disinfected prior to disposal. Bulk quantities of NIH exempt material must also be treated prior to disposal. Liquid phase materials must be brought to a concentration of 10% bleach and allowed to stand for 20 min. (or until the indicator changes color for media) prior to disposal. Material which cannot be chemically disinfected must be autoclaved at 120⁰C for one hour prior to disposal. Contact the Biology Safety Coordinator for details on autoclaving.

11. In addition to item 11 under BSL‐1 General Laboratory Practices above, the P.I. shall ensure that no laboratory personnel conduct work involving rDNA, rDNA organisms, or biological agents of a BSL‐2 categorization until proficiency in standard and special microbiological practices has been demonstrated.

12. A sign incorporating the universal biohazard symbol and name of the organism in use must be posted on the laboratory door, along with emergency contact information.

Infectious Material Spill and Exposure Procedures

The following detail the procedures that must be followed in the event of a spill of material containing infectious organisms or biological agents at BSL‐2 containment. All laboratory personnel must be very familiar with these procedures so that successful execution of the protocols can be carried out during emergency situations. Any spill or accident involving rDNA that leads to personal injury (i.e. needle stick), illness or a breach of containment must be reported to the Office of Biotechnology Activities (OBA) at NIH.

Spill Procedure

1. Alert everyone in the lab to the spill and evacuate the immediate area.

2. Wear the appropriate personal protective equipment to clean up the spill. At a minimum this must include gloves, chemical splash goggles, a face mask or shield, and a lab coat. If the spill clean‐up requires additional respiratory protection, do not attempt to clean up the spill. Instead, evacuate the lab, closing the door behind you, and report the incident to the campus police at x3070 or 911 from an on campus telephone only, or dial (909) 869-3070 from a cell phone. Note that dialing 911 from a cell phone or otherwise off-campus telephone will direct your emergency call to a CHP dispatch far from campus. 911 from an on campus line will go


directly to University Police. Identify the organism, the need for respiratory protection and any other hazardous conditions present (eg. hazardous chemicals involved in the spill).

3. Remove broken glass, if any, with tongs or some other mechanical device. Do not use your hands. If a dust pan and brush must be used, attempt to minimize the spread of the contaminated liquid. The collection apparatus and glass should then be decontaminated by soaking in a 10% bleach (or 70% ethanol, if appropriate) disinfectant solution for 20 min., followed by rinsing with water. The glass can then be disposed of in the glass waste box.

4. Place an absorbing towel or spill pillow, as appropriate to the spill size, on the liquid. Carefully disinfect the area with the disinfectant solution. Ensure that all of the liquid is contained by the absorbing material, and let stand for 20 minutes.

5. After the elapsed time, transfer the absorbing material to a biohazard bag.

6. Apply more disinfectant solution to the spill area and contain with absorbing material. After all of the liquid is absorbed, transfer the absorbing material to the biohazard bag.

7. After disinfecting, rinse the spill area with water and paper towels, or mop.

8. Contaminated gloves and face masks should be placed in the biohazard bag. Other contaminated personal protective equipment can be disinfected as appropriate. Lab coats should be autoclaved prior to laundering. The Biology Safety Coordinator should be contacted for disposal of the biohazard bag.

9. Wash hands thoroughly with soap and water before exiting laboratory.

10. Report the spill to EH&S x4697. Exposure Procedure

1. Small spills to the hands or lower arms should be washed thoroughly with soap and water.

2. If a person is splashed in the eyes, assist the person to the eyewash station, and flush the eyes for 15 minutes. Wear gloves, lab coat and chemical splash goggles while assisting the victim.

3. Contaminated clothing should be removed for spills to the body, and placed in a biohazard bag or bin. Wear gloves, lab coat and chemical splash goggles while assisting the victim. Wash the affected area thoroughly with soap and water.

4. Contact the campus police at x3070 to report the incident and request medical evaluation. Report the identity of the organism and type of exposure (i.e, splash to the skin or eye, inhalation, etc.)

5. Reports of all exposures must be made to the P.I. and Institutional Biosafety Committee (IBC). Spills of non‐infectious materials handled at BSL‐1 containment levels do not require special clean‐

up procedures. Exposures to non‐infectious materials handled at BSL‐1 containment levels should


be treated by washing with soap and water for skin contact, or flushing the eyes with water for 15

minutes for eye contact. If irritation develops, seek medical evaluation.

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