BIOSAFETY RISK MANAGEMENT

Design and operation of a bioprocessing facility must assure safety of personnel within the facility and those in the surrounding community Protection is achieved through a combination of
engineered facilities, processes, and equipment worker training and education use of personnel protective equipment operational practices validation of machinery and methodologies controlled access to facilities biosafety committee or subcommittee medical and environmental surveillance

 Activities involving LMO are regulated by the Biosafety Act 2007 and Biosafety Regulations 2010, to ensure safe application of biotechnology in a responsible and orderly manner.

BIOSAFETY LEVELS (BSL) FOR CONTAINMENT

 Biosafety containment of the LMO activity is achieved with a combination of facility design, administrative controls, engineering controls or use of Personal Protective Equipment (PPE). There are four BSL for containment based on existing international approaches to pathogenic organisms:
Biosafety Level 1 Biosafety Level 2 Biosafety Level 3 Biosafety Level 4 (BSL1) (BSL2) (BSL3) (BSL4)

 For genetic modification activities, five categories of containment facility are:

Genetic Genetic Genetic Genetic Genetic Modification Modification Modification Modification Modification of of of of of Microorganisms (GM-BSL) Plants (GP-BSL) Animals (GA-BSL) Arthropods (GI-BSL) Aquatic Organisms (GF-BSL)

The genetic modification BSL dictates the minimum level of containment required for carrying out activities with LMO and related materials originating from these organisms.

 If researchers are unsure about the containment level required for their research and development activities, they should consult their respective Institutional Biosafety Committees (IBC). The containment requirements for work with genetically modified (GM) microorganisms are also the fundamental requirement for containment involving plants, animals, arthropods and other aquatic organisms.

BIOSAFETY LEVEL GUIDELINE

Laboratory Facilities

Biosafety Level 1 (BSL-1)

Risk Group 1 Low individual and community risk (BSL1 )
Pathogenity Features A microorganism that is unlikely to cause human disease or animal disease.

Agents include Bacillus subtilis, non-pathogenic E. coli species

BSL1-General
A laboratory classified as GM-BSL1 is appropriate for conducting activities of no known or minimal potential hazard to laboratory personnel, community and environment as listed below:

Research involving the introduction of naked nucleic acids into RG 1 microorganisms Experiments with LMO microorganisms and materials originating from these organisms Experiments involving approved host/vector systems provided

BSL1-Laboratory Facility
a) The laboratory is not separated from the general traffic patterns in the building
b) The laboratory is designed with open benches which can be cleaned easily c) Bench tops should be impervious to water and resistant to acids, alkali, organic solvents and moderate heat d) Laboratory furniture should be sturdy. Space between benches, cabinets and equipment are accessible for cleaning

BSL1-Laboratory Facility
e) Entrances to the laboratory should be posted with an appropriate signage identifying the type of laboratory facility and listing the procedures applicable, including emergency and maintenance procedures. The contact information of the laboratory supervisor or other responsible persons should be listed f) Each laboratory should contain a sink for hand-washing g) The laboratory may be equipped with fumehood h) There should be access to a steam steriliser/autoclave in the same building

BSL1-Work Practices
a) Laboratory personnel working in a GM-BSL1 laboratory should be supervised by a scientist with general training in microbiology, molecular biology or a related science. b) Laboratory coats should be worn during work and removed before leaving the laboratory premises. Personnel may be required to change into footwear specified for the laboratory. Gloves should be worn when handling LMO and material originated from these organisms. c) All persons handling LMO and materials originated from these organisms should wash their hands after removing gloves and before leaving the laboratory.

BSL1-Work Practices
d) Protective eye wear should be worn for anticipated splashes of microorganisms or other hazardous materials to the face. e) All cultures should be clearly labelled and a central logbook kept in the laboratory. f) When experiments are in progress, access to the laboratory should be limited at the discretion of the Laboratory Supervisor. g) Work surfaces should be decontaminated regularly and immediately after any spill of viable material.

BSL1-Work Practices
h) All contaminated liquid or solid wastes should be decontaminated before disposal.
i) Mechanical pipetting devices should be used, mouth pipetting is prohibited. j) All procedures, involving liquids, are performed carefully to minimise the creation of aerosols. k) Eating, drinking, smoking, handling contact lenses and applying cosmetics is prohibited in laboratories.

BSL1-Work Practices
l) Food or drinks for personal consumption should not be brought into the laboratory or stored in laboratory refrigerators. m) An insect and rodent control programme should be implemented.

The minimum PPE required for the BSL-1 laboratory

Biosafety Level 2 (BSL-2)

Risk Group 2 Moderate individual risk, limited community, livestock or environment risk (BSL2) Pathogenicity Features A pathogen that can cause human or animal disease but is unlikely to be a serious hazard to laboratory workers, the community, livestock or the environment Laboratory exposure may cause serious infection. Infectious risk is via direct contact, ingestion or inhalation. Effective treatment, preventive and control measures are readily available and can be implemented to control disease transmission. Risk of spread to a community is limited.

Biosafety Level 2 (BSL-2)

Agents associated with human disease
Generally required for working with any humanderived blood, bodily fluids, tissues in which infectious agent may be unknown

Agents include measles virus, Salmonella species, pathogenic Toxoplasma, Clostridium botulinum, hepatitis B virus

BSL2-General
A laboratory classified as GM-BSL2 is appropriate for conducting activities that has moderate potential hazard to personnel, community and the environment listed below:
Research involving the introduction of naked nucleic acids into RG 2 microorganisms Experiments with LMO and related materials, harboring DNA from a RG 2 microorganism.

BSL2-Laboratory Facility
In addition to the facility design features specified for BSL1, following additional features are essential: a) A hazard warning sign incorporating the universal biohazard symbol and the level of containment together with access restrictions should be displayed on the access door to the laboratory work area.

BSL2-Laboratory Facility
Example of biosafety sign posted outside lab working with infectious agents
Labs biosafety level
Infectious agents under study

Contact information for responsible person and 2 emergency contacts

BSL2-Laboratory Facility
b) The ceilings, walls and floors of the laboratory should be smooth, easy to clean, impermeable to liquids, and resistant to commonly used reagents and disinfectants.

c) A dedicated hand basin of the hands-free operation type should be provided within each laboratory, near each exit.

BSL2-Laboratory Facility

Hand basin facility

BSL2-Laboratory Facility
d) Windows in the laboratory should be closed and sealed. e) An autoclave should be located within the building. Autoclaves should be certified annually by the Department of Occupational Safety and Health/DOSH. f) Freezers, refrigerators or other storage units used for BSL2 microorganisms located outside the designated laboratory should be posted with the appropriate signage.

BSL2-Laboratory ventilation
The laboratory should be well ventilated. If required an inward flow of air should be maintained by extracting room air using mechanical ventilation to ensure directional airflow.

BSL2-Work Practices
In addition to the work practices described in BSL1, the following work practices should be observed: a) The Laboratory Supervisor establishes policies and procedures whereby access is restricted to persons who have been advised of the potential hazard and who meet specific entry requirements (e.g. immunisation). b) Laboratory personnel should receive appropriate training on the potential hazards associated with the work involved and the necessary precautions to prevent exposures. Personnel should receive annual updates, or additional training as necessary for procedural or policy changes.

BSL2-Work Practices
c) Procedures which produce infectious aerosols should be conducted in biological safety cabinets or other physical containment equipment. d) Experiments of lesser biohazard potential can be carried out concurrently in carefully demarcated areas of the same laboratory. e) An institutional biosafety manual is prepared or adopted for use in the laboratories. Personnel are advised on special hazards and are required to read and follow the instructions on practices and procedures.

BSL2-Work Practices
f) Goggles, mask or face shield should be used for anticipated splashes or sprays of infectious LMO and materials originated from these materials to the face, when handling microorganism outside the biological safety cabinet. g) Respiratory protection should be worn when aerosols cannot be safely contained.

h) All solid wastes from GM-BSL2 laboratories, including infectious waste should be decontaminated by autoclaving for at least 30 minutes at a pressure of 15 psi and 121oC.

BSL2-Work Practices
i) Liquid wastes generated during BSL2 activities are to be decontaminated immediately by mixing with a suitable disinfectant j) Extreme caution should be observed when handling needles and syringes to avoid accidental needle-stick injury and generation of aerosols during use and disposal. k) Spills and accidents that may cause over-exposure to LMO and materials originating from these organisms should be immediately reported to the Laboratory Supervisor and the IBC l) A record book should be maintained to provide an up-todate inventory of the procedures performed.

Biosafety Level 3 (BSL-3)

Risk Group 3 High individual, low community risk
Pathogenicity Features Organism, which may be an exotic or indigenous agent with potential to transmit disease mainly via aerosols. Disease caused is severe and may result in death. It could present a risk if spread in the community however effective treatment, preventive and control measures are available

Biosafety Level 3 (BSL-3)

Agents include Mycobacterium tuberculosis, St. Louis encephalitis virus, Francisella tularensis,

Coxiella burnetii

BSL3-General
A laboratory classified as BSL3 is appropriate for conducting activitiesthat has moderate potential hazard to personnel, community and the environment listed below:

Research involving the introduction of naked nucleic acids into RG 3 microorganisms Experiments with LMO and related materials, harboring DNA from a RG 3 microorganism

BSL3- Laboratory Facility

The design, construction and major changes to the GM-BSL3 facility should be certified by a competent authority/organisation endorsed by the NBB before commencement of work. In addition to the facility design features specified for BSL1 and BSL2, following additional features are essential: a) The laboratory should be physically separated from other areas including offices used by laboratory personnel and is not accessible by the general public.

BSL3- Laboratory Facility

b) Physical separation of the high containment laboratory from access corridors or other laboratories or activities is achieved by a double door system where entry to the laboratory facility is gained through an airlock.
c) The laboratory and airlock should be sealable to permit safe decontamination with gases. d) All room penetrations should be sealed to ensure they are air tight

BSL3- Laboratory Facility

e) Access doors to the laboratory or containment module should be self-closing and open outwards.
f) One side of the laboratory wall should have a glass panel or observation window to view laboratory occupants from the outside. g) An autoclave for decontaminating laboratory waste should be available within the laboratory, preferably located within the barrier wall of the GM-BSL3 laboratory, but not situated in the airlock.

BSL3- Laboratory Facility

h) Liquid effluents should be discharged in a manner appropriate to the type of waste. i) A telephone and fax machine or other means of outside communication should be provided inside the laboratory unit. j) An audible emergency alarm should be provided within the laboratory to indicate a loss of negative pressure and a visible alarm is provided outside the laboratory to indicate the same. k) The BSL3 facility should be tested and certified annually at least every 12 months.

BSL3- Laboratory ventilation

The laboratory ventilation should incorporate the following features: a) A ducted exhaust air-ventilation system is provided b) Air supply and exhaust from Class II biological safety cabinets should be connected in a manner that avoids any interference with the air balance of the cabinets or buildings exhaust system. c) The proper directional airflow into the laboratory is verified by airflow tests.

Wash hands after removing PPE at the hands-free sink

Eye/face wash: understand how to use, flush at least weekly, and identify closest station outside the BSL-3 lab (in event of release outside of containment)

BSL3- Work Practices

In addition to the work practices described in BSL1 and BSL2, the following work practices should be observed: a) The laboratory senior management is required to establish policies and procedures whereby access to the laboratory is restricted to authorised persons who have been advised of the biohazard and who meet any medical requirements. b) All personnel should have specific training in handling pathogenic organisms and in the use of safety equipment and controls.

BSL3- Work Practices

c) The laboratory door should be locked when the room is unoccupied.
d) If experiments involving other organisms that require lower levels of containment are to be conducted in the same laboratory concurrently with work requiring BSL3 level physical containment, such experiments should be conducted in accordance with all BSL3 level practices.

BSL3- Work Practices

e) Work surfaces of biological safety cabinets and other containment equipment should be decontaminated
f) PPE that protects street clothing (e.g. tyvex suit and boots). In addition, appropriate respiratory protection should be made available. g) Liquid wastes generated during BSL3 activities should be decontaminated immediately with suitable disinfectant. All solid wastes are decontaminated by autoclaving before disposal for incineration by a government authorised service provider

BSL3- Work Practices

Tyvek suit

BSL3- Work Practices

i) A medical surveillance programme should be instituted for all persons entering a BSL3 facility
j) An Emergency Response Plan should be documented and personnel should be trained in these procedures which include spill management, emergency evacuation if necessary, as well as incident reporting system.

Biosafety Level 4 (BSL-4)

Risk Group 4 High individual and community risk (BSL4) Pathogenicity Features Organism, which may be an exotic agent or new agent usually able to cause life-threatening human disease. The infectious disease is readily transmissible from one individual to another. Infectious disease may be transmitted via aerosol or via an unknown route. Effective treatment, preventive and control measures are not readily available.

Biosafety Level 4 (BSL-4)

Agents (all viruses) include Marburg virus, Ebola virus, viruses that cause CongoCrimean hemorrhagic fever, Lassa fever

(transmission electron micrograph of Ebola virus)

BSL4-General
A laboratory classified as BSL2 is appropriate for conducting activities that may cause serious harm to the laboratory personnel, community and environment if accidentally released listed below:
Research involving the introduction of naked nucleic acids into RG 4 or exotic agents Experiments with LMO and related materials, harboring DNA from a RG 4 microorganism

BSL4- Laboratory Facility

The design, construction and major changes to the BSL4 facility should be certified by a competent authority/organisation endorsed by the NBB before commencement of work. In addition to the facility design features specified for BSL1, BSL2 and BSL3 the following additional features are essential: a) A BSL4 facility is housed in a separate building. An outer and inner change-room separated by a walk-through double door shower airlock should be provided for personnel entering and leaving the facility. The outer door of the facility should be lockable.

BSL4- Laboratory Facility

b) Fumigation chamber or ventilated air-lock is provided for passage of materials, supplies or equipment that is not brought into the facility through the change room.
c) As much valve and control equipment as possible should be located outside the laboratory boundary to minimise the need for service personnel to enter the laboratory.

BSL4- Laboratory Facility

d) Walls, floors and ceilings of the facility should be constructed to form a sealed internal shell that facilitates fumigation and is animal and insect-proof. The internal surfaces of this shell should be resistant to liquids and chemicals, thus facilitating cleaning and decontamination of the area. All penetrations in these structures and surfaces should be sealed. e) If there is a central vacuum system, it should not serve areas outside the facility. In-line HEPA filters should be placed as near as practicable to each point of use. Filters are installed to permit in-place decontamination and replacement. Other liquid and gas services to the facility are protected by devices that prevent backflow.

BSL4- Laboratory Facility

f) A double-door autoclave is provided for decontaminating materials from the facility and from the inner clothing change room. g) A pass through dunk tank or decontamination chamber should be provided so that materials that cannot be autoclaved can be rendered safe for removal from the facility. h) An automatic changeover emergency power source, emergency lighting and communication system should be provided to ensure continuing operation of the ventilation systems, biological safety cabinets, room access and controls.

BSL4- Laboratory Facility

i) For certain requirements,
i) A specially designed suit area may be provided within the facility. ii) Personnel who enter this area should wear a onepiece positive pressure suit that is ventilated by a life support system that includes an alarm and emergency back-up breathing air system. iii) Entry should be via an airlock fitted with a personal body shower into an anteroom leading to a second airlock fitted with a chemical disinfectant shower provided to decontaminate the surface of the suit before the personnel leaves the area.

BSL4- Laboratory Facility

i) A double door autoclave with doors at each end is provided for decontaminating waste materials to be removed from the suit area. ii) An air supply for connection to the positive pressure suit should be provided in the anteroom. iii) The exhaust air should be filtered through two sets of HEPA filters installed in series. iv) The air pressure within the suit area should be lower than that of the adjacent entry, exit and nonsuit areas. v) A duplicate ventilation system, exhaust fan and an automatically-starting emergency power source should be provided to automatically re-establish laboratory ventilation and pressure conditions in event of equipment failure.

BSL4- Laboratory Ventilation

The laboratory ventilation should have the following features:
a) A separate supply and exhaust non re-circulating air ventilation system should be provided. b) Both supply and exhaust air should be filtered through HEPA filters. c) The filtered air from Class III biological safety cabinets is discharged through the facility exhaust system in a manner that avoids any interference with the air balance of the cabinets or the facility exhaust air system.

BSL4- Laboratory Ventilation

d) The ventilation control system should raise an audible alarm within the laboratory and at an attended location when room differential air pressures depart from set points.

BSL4-Work Practices
In addition to the work practices described in BSL1, BSL2 and BSL3, the following work practices should be observed: a) Accessibility is managed by the Laboratory Supervisor, Biological Safety Officer (BSO) or other persons responsible for the physical security of the facility.

b) A facility operations manual and SOP should be prepared by the management. Practical and effective protocols for emergency situations should be documented in the SOP.

BSL4-Work Practices
c) Authorised persons should comply with the instructions and all other applicable entry and exit procedures in the manual and SOP d) All personnel should be trained in the specific working aspects of the containment laboratory, including handling of the relevant pathogens, cleanup of infectious spills and use of safety equipment.

BSL4-Work Practices
e) A risk assessment of the working environment should be undertaken by management, taking into consideration all matters influencing the safety of personnel.
f) Personnel should enter and leave the facility through the clothing change and shower rooms, except in cases of emergency, for example fire or explosion inside the containment laboratory, personnel can use the air-locks to leave the laboratory.

BSL4-Work Practices
g) Supplies, materials and specimens should only be brought into the facility through the change and shower rooms, the double-door pressure steam steriliser, the fumigation chamber, the airlock or the dunk tank.
h) A primary container holding viable or intact biological material should only be opened in the maximum containment laboratory.

BSL4-Work Practices
i) No material should be removed from GM-BSL4 laboratory unless it has been autoclaved or decontaminated..
j) RG 4 biological materials should be stored only within the facility. If RG 4 biological materials are to be removed from the maximum containment laboratory in a viable or intact state, they should be transferred in a nonbreakable, sealed primary container, and then enclosed in a non-breakable, sealed secondary container, which is removed from the facility through a disinfectant dunk tank, fumigation chamber or an air-lock designed for this purpose.

BSL4-Work Practices
k) All practices within the facility involving agents assigned to RG 4 should be conducted in Class III biological safety cabinets or alternatively Class II biological safety cabinets used in conjunction with one-piece positive pressure personnel suits ventilated by a life-support system.
l) Precautions should always be taken with any contaminated sharp items including needles and syringes, slides, pipettes, capillary tubes and scalpels..

BSL4-Work Practices
m) A system should be set up for reporting accidents and exposures to microorganisms and for the medical surveillance of illnesses that are potentially laboratory associated.

Scientists working in a BSL-4

BL4 Lab - Max Containment

Containment Levelof Laboratories

CL1:-Ill just do it on the bench over there.. CL2:-OK, so Ill have to use the hood. CL3:-Thats that room along the corridor that noone goes in? CL4:-Spacesuits?

BIOSAFETY CABINET

Biosafety Cabinet
Biological safety cabinets are the primary means of containment in process support laboratories and during early stages of culture development Based on design and the protection afforded, biological safety cabinets are designated as Class I, II, and III Must be properly maintained and regularly serviced Must be certified annually and when new, moved, or repaired

Biosafety Cabinet
Must be used in conjunction with good microbiological techniques Provide protection for personnel, product, and environment The cabinets should be decontaminated with formaldehyde gas or an equivalent disinfectant before testing when there have been used for handling RG 2, 3 or 4 microorganisms. Penetration of the decontaminants throughout all the sections of the cabinets is essential.

BSC Class I
A Class I cabinet is a ventilated cabinet for personnel protection. Air flows inward, away from the operator. The exhaust air from this cabinet filters through a High Efficiency Particulate Air (HEPA) filter.

 This cabinet is used in three operational models: i. with full-width open front ii. with an installed front-closures panel (having four 12-cm diameter openings) without gloves iii. with an installed front-closure panel equipped with arm-length rubber gloves. The face velocity of the inward flow of air through the full-width open front is 30 m per minute or greater.

BSC Class I

BSC Class II
A Class II cabinet is a ventilated cabinet for personnel and product protection. It has an open front with inward airflow for personnel protection and HEPA filtered mass airflow for product protection. The cabinet exhaust air is filtered through a HEPA filter. The face velocity of the inward flow of air through the full-width open front is 30 m per minute or greater.

BSC Class II
While HEPA filters are effective for trapping particulates and infectious agents, these filters will not capture volatile chemicals or gases As general, no BSC suited for handling volatile toxic substances

BSC Class II (A & B)

BSC Class III

A Class III cabinet is a closed-front ventilated cabinet of gas-tight construction, which provides the highest level of personnel protection among biological safety cabinets. The interior of the cabinet is protected from contaminants outside of the cabinet. The cabinet is fitted with arm-length rubber gloves and is operated under a negative pressure of at least 1.25 cm water gauge. All air supply is filtered through HEPA filters. Exhaust air is filtered through two HEPA filters before being discharged to the outside environment.

BSC Class III

HEPA FILTER

 Control of airborne particulates in indoor environments is critical to develop quality products, protect employees from contact with hazardous materials, or prevent health problems from prolonged exposure to allergens HEPA filters in biological safety cabinet
particle sizes efficiency and penetration filter standards and performance testing

 HEPA filter are rated to remove particles down to 0.3micrometer with efficiency of 99.97% Filters are susceptible to shock-induced mechanical damage Biosafety cabinet must be certified after initial installation, after relocation, after repair and at yearly interval

 The certification test depend on the type of cabinet

Downflow velocity and volume testing (I and II) Inflow velocity test (I and II) Negative pressure testing (II and III) Air flow smoke pattern test (I and II) HEPA filter leak test (I-III) Cabinet leak test (II and III) Alarm and interlocks (III)

BSC SUMMARY
Biohazard level BL1-3 BL1-3 BL4 Protection provided Personnel Yes Yes Yes Product No Yes Yes Environme nt Yes Yes Yes Cabinet class I II III

LAYOUT

 The layout of the facility affects

efficiency of operations the potential for containment prevention of cross-contamination

Contained, clean, and dirty areas should be identified on the process flow sheets and the building layout drawings Movement of personnel, equipment, process streams, and air across containment boundary must assure integrity of containment

 Flows must be unidirectional (clean to dirty areas)should not cross No back-tracking The biohazard containment areas and the aseptic product filling areas should be located in different wings of the facility, with no sharing of common hallways or direct access Adequate space No overcrowded