In accordance with the Department of Biotechnology (DBT), Government of India, Regional Centre of Biotechnology (RCB) has established an Institutional Biosafety committee (IBSC). The IBSC oversees research with the potentially hazardous biological agents in the Centre.

The primary mission of the IBSC is to ensure the safety of faculty, staff, students and the researchers involved in the biological research at the Centre, and also to protect the general public and the environment from adverse consequences related to that research. The work of the IBSC supports the Centre in meeting all required standards set by the DBT and as necessary, developing specific policies to make any research conducted with hazardous or potentially hazardous biological agents (either naturally occurring or synthetically created) as safe as possible.

The IBSC is composed of nine members so selected that they collectively have experience and expertise in recombinant DNA (rDNA) technology and the capacity to assess the safety of recombinant DNA research and other infectious agents to identify any potential risk to public health or the environment. The IBSC of our Centre is constituted as per the DBT, Govt. of India guidelines and the constitution is as follows:

The IBSC is responsible for reviewing the research with potential hazardous biological agents and rDNA technology in order to assess whether the biosafety containment levels proposed are adequate to protect researchers, the public and the environment from the risk proposed by the work.

The IBSC will determine approval or disapproval of the proposed experiments through review of the application submitted by the principal investigator along with supporting materials as needed. This review and oversight involves independent assessment of the biosafety containment level proposed for the work and through coordination with the biological safety officer, assessment of facilities, procedures, practices, training and expertise of personnel involved in the research.

Specifically, the IBSC is charged with the review and oversight of research with the following potentially hazardous biological agents and experiments:

• Recombinant DNA and synthetic nucleic acid molecules
• Infectious agents
• Biological toxins
• Certain animal-derived tissues, fluids, and cells

The IBSC has to meet at least twice a year to review the status of rDNA projects in the institution. It is important that the Chairman and Member Secretary ensure that regular meetings take place.

More than two meetings may be held as per requirement of the projects. The IBSC members are expected to look into the following during the meetings:

• Action taken on the decisions of the earlier IBSC meetings.
• Characterization of work and approval as per risk category.
• Evaluation of projects and direction to submission for appropriate agencies for approvals.
• Maintaining procedures and other approval requirements.

The role of IBSC assumes major importance in the regulatory framework since it is a Statutory Committee that operates from the premises of the institution and hence is in a position to conduct onsite evaluation, assessment and monitoring of adherence to the biosafety guidelines. The functions in the IBSC of the head of the organization, members, DBT nominees, and Principal Investigator are explained below:

Chairman of IBSC

• The chairman of IBSC has the responsibility to ensure that the biosafety guidelines are followed in his/her institution.
• regular meetings of IBSC are held to review rDNA research and infectious agents-involving projects in the institution.
• open discussion takes place amongst the members in the meetings and the views of external members as well DBT nominee are recorded.
• the facilities at the institution are sufficient to meet the containment levels stipulated for rDNA research and processes.

DBT Nominee

The IBSC has a nominee from DBT who oversees the activities to ensure that safety aspects are being fully adhered to by the organization. The DBT nominee serves as the link between the department and the respective IBSC. In addition to the responsibilities as an IBSC member, the duty of the DBT nominee is to ensure that:

• The committee has been constituted as per the norms of the guidelines.
• The recombinant DNA Safety Guidelines are strictly followed in the institution.
• The IBSC meets regularly, at least twice in a year to review the ongoing activities and provides half yearly reports to RCGM/DBT in the prescribed proforma.
• All the activities are within the perview of the guidelines and in the knowledge of RCGM/DBT.
• The DBT nominee is expected to guide the IBSC on biosafety issues.

Principal Investigator

All recombinant research projects carried out by an organization have a Principal Investigator (PI) and it is the duty of the PI to apprise the IBSC about the nature of the experiments being carried out. Depending upon the risk category, the PI has to inform the IBSC, seek permission of IBSC before starting the experiments or seek permission of the RCGM through its IBSC.

The PI is primarily responsible for ensuring compliance with biosafety standards. The PI functions as a project manager as well as a researcher, communicating with the IBSC and bearing responsibility for training and supervising personnel.

Based on the nature of the GMO or the infectious agent, the PI determines the proper containment level for the project and, in accordance with the DBT Guidelines, develops the necessary experimental protocols. This information is then submitted to IBSC for review.

The responsibilities of PI to IBSC are summarized below:

• To make an initial determination of the required levels of physical and biological containment in accordance with the DBT guidelines.
• To submit the initial research protocol and any subsequent changes (such as changes in the source of DNA or host vector system) to the IBSC for review and approval.
• To ensure that no work is initiated until the research project has been approved by the IBSC and has met all requirements of DBT guidelines.
• Remain in communication with the IBSC throughout the conduct of the project.
• To ensure the safe conduct of the rDNA experiments in his/her laboratory.
• To make available the protocols that describe the potential biohazards and the precautions to be taken to all laboratory staff.
• To instruct laboratory staff about the practices and techniques required to ensure safety, and the procedures for dealing with accidents including the reasons and provisions for any precautionary medical practices advised or requested (e.g. vaccinations or serum collection).
• To supervise the performance of the laboratory staff to ensure that the required safety practices and techniques are employed.
• To undertake corrective measures promptly for any work errors and conditions that may result in the release of recombinant DNA materials or the infectious agent.

Laboratory Personnel (Technician, Technologist, Student, Post-doctorate, Young Investigators)

THE Laboratory personnel must:

Follow all safety guidelines and establish good laboratory practices. They must work within the assigned biological safety containment level and use personal protective equipment as recommended by the PI.

• Immediately notify the PI or Biological Safety Officer (BSO) of any health condition that may be due to their work in the laboratory or any health condition that may be compromised prior to the initiation of a research project (i.e. pregnancy, immunosuppression).
• Follow all practices and procedures as provided by the PI and BSO, and ensure strict compliance with all required biosafety regulations and guidelines.
• Report problems, procedural mistakes, spills, etc. to the PI, and if necessary to the BSO, immediately.
• Report to the PI, BSO or IBSC on non-compliance of biosafety guidelines or policies.

The role of IBSC in rDNA activities within an organization is broadly categorized into research, large-scale experiments/production/field release and import and shipment.

The DBT Guidelines for rDNA Safety Guidelines of DBT (http://www.dbtindia.nic.in) stipulate three categories of research activities i.e. Category I, II and III with increasing level of containment requirements.

• Category I experiments involving self-cloning, using strains and also inter species cloning belonging to organism in the same exchanger group etc. and are exempt for the purpose of intimation and approval.
• Category II experiments falling under containment levels II, III and IV, large scale use of recombinants made of self-cloning in systems belonging to exempt category etc. require prior intimation to IBSC.
• Category III experiments involving toxin gene cloning, cloning of genes for vaccine production, use of infectious animals and plant viruses, self-fusion experiments, field testing and release etc. require review and approval of IBSC before commencement.

Depending upon the category of experiments, IBSC can simply note the information provided by PI, give permission before the start of the experiments or forward it to RCGM for approval.

Large scale trials and production

Although the approval for small scale field trials fall under the purview of RCGM and approval for large-scale trials and production needs to be taken from GEAC, as per the Rules 1989, in all these cases, IBSC has an extremely important role in terms of verifying the information being forwarded to RCGM and GEAC in terms of physical containment conditions, categorization in terms of risk assessment etc. being the statutory body functioning on the premises of the institution. Both RCGM and GEAC depend on the review of the IBSC on the submissions made.

IBSC has to recommend emergency plan in case of large-scale operations, as and when required, which would be then approved by competent authorities. Emergency plan shall include methods and procedures for handling large losses of cultures and organisms.

Import and shipment

The interstate shipment of indigenous etiological agents, diagnostic specimens and biological products need clearance of IBSC and is subject to appropriate packaging, labelling and shipping requirements.

The import of regulated materials for research (e.g. toxin genes, hybridomas, cell cultures, organelle) and specifying conditions under which the agent or vector is shipped, handled and use are issued by RCGM. In case of plants, the import is routed through the Director, National Bureau of Plant Genetic Resources on the basis of the import permit issued by the DBT, based on recommendations of the RCGM. However, all these proposals need to be submitted by the PIs through thei IBSC.

Assignment of Biosafety Levels (BL) - It is the responsibility of the PI to initially assign the BL to his/her protocol. This level may be changed either upward or downward during review by the IBSC. All parties involved in assigning BLs will follow the standard levels of 1, 2 or 3 as outlined in DBT’s Guidelines for Research Involving Recombinant DNA Molecules. These are summarized below.

The details of microorganisms falling into each category are given in the Recombinant DNA Biosafety Guidelines, 1990.

Containment facilities

In general, biosafety begins with ensuring the workplace, whether it is a laboratory or open fields, is safe for the working staff, the general population and the environment by proper containment.

The containment could be physical, where there are real barriers to prevent escape, or biological where the organism is designed not to be able to survive in any environment other than that of the laboratory. The containment facilities and biosafety practices have been defined in detail in “Recombinant DNA Safety Guidelines, 1990” of DBT. A summary of recommended biosafety levels for infectious agents is given below.

Table: Recommended Biosafety Levels for Infectious Agents

• Biohazard symbol should be placed at the entrance of recombinant DNA work area.
• Access to the laboratory should be limited or restricted only to the workers.
• Eating, drinking, smoking, storing food, and applying cosmetics should not be permitted in the laboratory work area.
• Laboratory coats, gown or uniforms should be worn while in the laboratory. The protective clothing should be removed before leaving laboratory for non-laboratory areas.
• Gloves should be worn while handling the rDNA materials and infectious agents to avoid skin contamination.
• All contaminated liquid or wastes should be decontaminated by ethanol treatment or autoclaving before disposal.
• Contaminated materials should be stored in leak-proof containers till decontamination.
• Mechanical pipetting devices should be used; mouth pipetting is prohibited.
• Work surfaces are decontaminated at least once a day.
• Persons should wash their hands after handling recombinant DNA materials and infectious agents.
• Creation of aerosols should be minimized.
• Biological Safety Cabinets should be used for routine work with rDNA materials and infectious agents.
• Control measures should be taken to avoid entry of insects and rodents.
• Spills and accidents which result in rDNA exposures, or exposure to infectious organisms should be immediately reported to laboratory in-charge and IBSC.

The code of practice for a basic laboratory applies as follows except where modified:

• The two-person rule should apply, whereby no individual works alone within the laboratory.
• A hazard warning sign should be displayed on laboratory doors, identifying the agent, the name of the laboratory supervisor and other responsible person(s) and indicating any special conditions of entry into the area (immunizations, etc.).
• Laboratory clothing that protects street clothing (i.e. solid front or wrap-around gowns, scrub suits, coveralls, etc.) must be worn in the laboratory. Laboratory clothing must not be worn outside the laboratory and must be decontaminated before being laundered.
• When appropriate, respiratory protective equipment should be worn in rooms containing infected animals.

• If adjustable, the window should be lowered to 8 inches, with a >75 ft/min air velocity.
• Keep the amount of equipment used or stored in the cabinet to a minimum.
• Before work is started, everything needed for the procedures should be placed in the cabinet, and let cabinet air exhaust run for a few minutes.
• Nothing should be placed on or blocking the front or rear grills.
• Contaminated items should be segregated from clean ones and located so that they never have to pass over clean items.
• Avoid disrupting the air barrier in a safety cabinet by frequent and rapid arm movements and bringing hands in and out of the cabinet.
• Waste containers should be placed inside the cabinet to avoid breaking the air barrier and bringing contaminated items out into the room.
• Do not use a burner inside a Class II BSC because the air currents induced counter to the normal air flow, can cause contamination of the work surface and ignite ethanol and other materials in the cabinet and damage the HEPA filter.
• When working with biohazards, keep absorbent towels and decontaminating solutions in the cabinet and wipe down work surface with ethanol prior to and at the completion of each session, and after any small spills.
• Decontaminate all equipment removed from the cabinet. Pipetting aids and tools that are used repeatedly should remain in the cabinet.
• Decontamination of the entire cabinet (filter, plenums, work surfaces and the fan) is achieved by exposing these areas to a paraformaldehyde vapor. This type of decontamination must be performed only by a trained professional.
• If the power to the unit fail during use, stop work with biohazardous agents immediately, seal all cultures securely, and decontaminate the work area with a suitable disinfectant. BSC should be put on UPS for continuous power backup in case of power failure.
• Horizontal Laminar Flow Hoods provide a very clean environment but must be used only for the manipulation of non-hazardous materials. Since the operator sits in the downstream exhaust from the clean bench, this equipment must never be used for the  handling of toxic, infectious, or sensitizing materials, including volatile chemicals, cell culture materials (except plant cell cultures), or drug formulations.

Incubators can become the inadvertent and undesired repository of microorganisms. Although they may present a hazard to laboratory workers, most often they are a source of contamination of laboratory cultures. Besides the moist surfaces, rubber gaskets, the humidity trough if present, and the fan mechanism are areas in which contaminating microorganisms concentrate.

• It is recommended that an anti-microbial agent such as Zepharin chloride be added to the humidity source water (do not use sodium azide).
• Panels, trays and other removable parts should be autoclaved and the gaskets and non-removable parts wiped thoroughly with 70% ethanol every 2 months.

Use this type of hood only for the removal of vapors released or generated by chemical reactions involving mildly toxic materials, acids (not heated), and organic solvents. In this type of hood, do not use hot perchloric acid, hot concentrated acids, highly toxic materials, unstable chemicals or explosive.

• Do not use large pieces of equipment in the hood.
• Close doors and windows when hoods are in operation.
• Avoid foot traffic and rapid arm/body movement.
• Place chemical sources and equipment at least 6 inches behind the face of the hood.
• Do not extend your head inside of the hood while experiments are being performed.
• Perform work with the sash height as low as possible (at most 10-12 inches).
• Keep fume hoods and adjacent work areas clean since solid debris can enter the hood’s exhaust duct work.
• Protect spark sources from flammable vapors. Permanent electrical receptacles are not permitted in the hood.
• Do not cut holes into the hood or its duct work.
• Do not store chemicals in a fume hood unless storage is the sole use of the hood. Only those chemicals necessary to perform the experiment should be left in the hood.
• Do not use hood for evaporation as a means of chemical disposal.

Use this type of hood only for the removal of vapors released or generated by chemical reactions involving mildly toxic

PI should maintain a document accessible to all lab personnel, with a list of procedures to be followed in case of an accident such as a spill, injection, ingestion, aerosolization, splash, etc. The document should have the information on agent specific actions, known first aid procedures, effective disinfectants / neutralizing agents (include location in lab), and known symptoms associated with exposures to the agent(s). In case of emergency, please call Principal Investigator, Security, emergency medical personnel etc., and inform the IBSC.

It is the responsibility of the PI to make appropriate notifications in the wake of an occupational exposure, laboratory accident, loss of potential containment, etc. The IBSC should be notified of incidents or adverse events involving biological agents or toxins.

In case of accidental exposure to infectious material e.g., infected body fluid, pathogenic strains etc., prophylaxis measures should be initiated as follows:

• Immediate repeated washing with soap, water, antiseptic solution (where applicable)
• Report to respective PI and the Biosafety Officer
• Initiation of post-exposure counselling and testing and treatment (as applicable)

Records of appropriate training (technical, safety, procedural, etc.) of lab personnel  should be maintained. PI should ensure enrolment of new lab personnel to biosafety training and notify the IBSC on December 1st of each year about training status of all lab personnel.

The Biosafety Officer will assess requirement of vaccination for students and staff working in various laboratories depending on the micro-organism used and the level of exposure. A regular regime will be followed with documentation of baseline serum values and subsequent values for different antibodies as decided by the Biosafety Officer.

Listed below are the materials necessary to develop a fairly inexpensive general spill kit for a laboratory. This kit should be able to handle <1 litre of most acids, bases, and solvents found in a typical laboratory. Do not attempt to clean up a spill of greater volume. The following materials shall constitute a general laboratory spill kit.

• Large plastic box to hold the contents of the kit (3-5 gallons)
• Plastic dust pan and brush (non-sparking)
• Chemical safety goggles and face shield
• Appropriate chemical resistant gloves (ex. neoprene)
• 3-5 waste disposal bags that can be sealed/closed 4-5 absorbent pads Note: brown paper towels and other combustible tissue may ignite when brought into contact with certain chemicals
• pH paper
• Bleach (if biohazards are present)
• Absorbent paper towel for liquid spills
• Sodium bicarbonate for acids
• Citric acid for bases
• “Powersorb Universal Absorbent Pads” (3M) or other “universal” pads

This general chemical spill kit is not meant for use with mercury, hydrofluoric acid, sodium metal, cytotoxic drugs, and numerous other chemicals. It is the responsibility of all laboratory personnel to evaluate a potential spill and develop spill response procedures for the specific hazards present. This kit can be modified to meet the needs of your laboratory. Refer to the MSDSs for the chemicals handled in the lab to identify if special spill materials are needed.

• Skin exposure: Vigorously wash affected skin with plenty of soap and water while removing contaminated clothing and shoes.
• Eye exposure: Wash eyes for at least 10 minutes with plenty amounts of water, lifting the upper and lower eyelids occasionally.
• Seek follow-up medical attention by contacting your supervisor for referral to the medical facilities or for medical assistance call IBSC.

Follow these procedures for cleaning-up spills of blood and blood products. The same procedures can be used for cleaning up other body fluids. For any assistance call IBSC. Prior to beginning the clean-up, wear personal protecting equipment (PPE) i.e. rubber, latex, PVC or similar type gloves, lab coat etc.

• The following items may be needed in handling the spill.
  
  • 10% bleach solution or Lysol.
  • Gloves
  • Clear plastic bags
  • Biohazard labels (available from IBSC)
  • Leak-proof sharps containers.
  • Brush &amp; dustpan, or tongs or forceps for picking up sharps material.
  • Disinfectant wipes
  • Gauze piece
• Cover all the spill area with a paper towel/blotting sheet and then pour freshly prepared mixed 10% bleach and water solution. Allow solution to soak into the contaminated material for 20-30 minute.
• Any glass, needles, or other sharp objects that may puncture the skin will should not be picked up by hand. Only brush and dustpan, or forceps are allowed to pick the sharp material.
• If you do not have such equipment available, contact IBSC for assistance. Wipe up bleached material with paper towels or absorbent pads. It may be necessary to use a scrub brush to remove the material and clean the area.
• Wash hands properly or use disinfectant wipes and then wash your hands as soon as possible
• Place bleached material, gloves and other disposable materials into a labeled biohazard bag and place into either another labeled biohazard bag or container. Ensure lids are firmly sealed on all waste containers when spill clean-up is complete and keep biohazard waste container in a biohazard waste room until picked by biohazard waste agency.

• Familiarize yourself with the potential hazards of chemicals. Keep record of MSDSs of the hazardous chemicals used in the laboratory.
• Evaluate the type of toxicity of the hazardous chemical (i.e. corrosive, irritant, sensitizer, and carcinogen), the possible routes of exposure (i.e. inhalation, skin absorption, ingestion,ninjection) and hazards of flammable and explosive chemicals.
• Appropriate eye protection and protective apparel should be used during the chemical handling and follow the appropriate procedure to minimize exposure.
• Smallest container or the amount of hazardous material that will be used within a reasonable period should be purchased.
•  First aid is always the top priority. If you spill a hazardous material on yourself, remove any potentially contaminated clothing immediately. Seek appropriate medical treatment.
• If material spills in your eye, flush for at least 15 minutes (for corrosive materials, you may need to flush for up to 60 minutes – review the (MSDS). Seek appropriate medical treatment.
• Use leak-proof containers, metal cans, or plastic-coated bottles for storing and transporting hazardous material.
• Do not place glass containers of chemicals on the floor and avoid fallen or leaking gas cylinders.

• All staff and students in the biological laboratory/Chemical Laboratory who handle hazardous chemicals should be familiar with spill response procedures.
• The possibility of a spill and preparation for handling it should be anticipated in setting up your experiment. Appropriate precautions will alleviate many associated complications
• Ensure there are no ignition sources in the area. If you feel there is risk of fire or explosion, evacuate the lab and treat as a major spill.
• Alert people in the lab that a spill has occurred and ask them to evacuate; close doors to the affected area.
• If spilled material is flammable, turn off ignition and heat sources if it can be done safely.
• If it can be done safely, block off any drains where the spill may enter. Acids typically have a sour taste and a pH less than 7. Neutralize spill with Sodium bicarbonate, sodium carbonate/baking soda and wait until bubbling/fizzing has stopped.
• Bases have a bitter or a stringent taste and a pH greater than 7. Bases are also called alkaline compounds and are neutralized by using a weak acid. Common bases are sodium hydroxide, potassium hydroxide and ammonia. Neutralize spill with citric acid, sodium bisulfate.
• All spill residue and spill clean-up material needs to be placed in a high-density polyethylene or polypropylene bag with attached hazardous waste tag.

• Remove jewellery because it can form bonds with the mercury (amalgamate).
• Change into old clothes that can be discarded.
• Put on rubber gloves and safety glasses.
• Carefully clean up any broken glass. Wear rubber gloves to avoid contact with mercury and to prevent cuts. Place the glass in a rigid, wide mouth container that can be sealed with a lid.
• Work from the outside of the spill area towards the center. Using stiff paper or a mercury vacuum, slide any droplets of liquid mercury onto a plastic dustpan, and away from any carpet or other porous material.
• Use a flashlight to illuminate the mercury spill and to help spot small droplets. An eyedropper or an adhesive strip can be used to pick up small droplets.
• Sprinkle sulfur powder over the contaminated area and rub it gently all over the surface and into the cracks with a paper towel. Sulfur powder binds with mercury. Use a paper towel dampened with water followed by wiping with another damp paper towel to clean up the sulfur and mercury. Label the container with the mercury spillage tag and send it to Biohazard waste agency.

• Employees and students who have been through the biosafety training are designated to clean up blood or potentially infectious material spills that occur in their work areas.
• Blood or potentially infectious material spills which occur inside buildings, in an area outside of a trained person’s work or research area, shall be cleaned up by the designated housekeeping person (trained employee) who is on duty at that time in consultation with housekeeping in-charge.
• Blood or potentially infectious material spills which occur outside of a building, but on the Centre property shall be cleaned up by a trained person in consultation with housekeeping in-charge.
• Blood or potentially infectious materials that are on a person’s body shall only be cleaned up by a trained first responder, who is trained.
•  Only approved cleaning products may be used to clean up blood or potentially infectious materials. Specific products and instruction will be provided for housekeeping personnel and other designated spill clean-up employees during training provided by IBSC.

13.0 WASTE DISPOSAL

As per Section 6, 8 and 25 of the Environment (Protection) Act, 1986 of the Central Government the waste must be disposed-off in the following manner.

@@ Chemicals treatment using at least 1% hypochlorite solution or any other equivalent chemical reagent. It must be ensured that chemical treatment ensures disinfection.

##Mutilation/shredding must be such so as to prevent unauthorized reuse.

@There will be no chemical pretreatment before incineration. Chlorinated plastics shall not be incinerated.

* Deep burial shall be an option available only in towns with population less than five lakhs and in rural areas.

Colour coding and type of container for disposal of bio-medical wastes

• Colour coding of waste categories with multiple treatment options, shall be selected depending on treatment option chosen.
• Waste collection bags for waste types needing incineration shall not be made of chlorinated plastics.
• Categories 8 and 10 (liquid) do not require containers/bags.
• Category 3 if disinfected locally need not be put in containers/bags.