Regulations & Guidelines for Recombinant DNA Research (2017) PDF
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2017
S.R. Rao
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This document outlines regulations and guidelines for recombinant DNA research and biocontainment, focusing on biosafety protocols. It details containment procedures, facility requirements, and emergency protocols for various biosafety levels, including risk group assessment. The document was published in 2017 by the Department of Biotechnology, Ministry of Science and Technology, Government of India.
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Storage Import, Disposal & Export Emergency REGULATIONS AND Storage GUIDELINES FOR RECOMBINANT DNA RESEARCH...
Storage Import, Disposal & Export Emergency REGULATIONS AND Storage GUIDELINES FOR RECOMBINANT DNA RESEARCH AND Import, Disposal & BIOCONTAINMENT export Emergency 2017 Manufacture REGULATIONS AND Contained use (Large scale) GUIDELINES FOR (Laboratory) RECOMBINANT DNA RESEARCH AND BIOCONTAINMENT Facility Certification 2017 Manufacture Contained use (Large scale) (laboratory) Facility certification Department of Biotechnology Ministry of Science and Technology Government of India This page is intentionally left blank PREFACE Under the Rules 1989 of Environment (Protection) Act 1986, laboratory biosafety through appropriate containment has been identified as the fundamental part of any biological research. In this direction, DBT had earlier published three guidelines namely “Recombinant DNA safety guidelines, 1990” “Revised Guidelines for Safety in Biotechnology, 1994” and “Revised guidelines for research in transgenic plants, 1998”. During the last two decades, rapid advancement in biology and biotechnology research globally and in India, both in public and private sector institutions, necessitated that the above guidelines are reviewed, updated and harmonised with global best practices and guidelines. Further, research on emerging and re-emerging infections and potential risk associated in handling the pathogenic organisms required to put in place stringent yet practical regulations and guidelines for ensuring biosafety measures for protection of public health and environment. RCGM, the apex body working under Rules 1989 has the mandate to monitor the safety of on-going research projects or activities involving hazardous microorganisms, GE organisms and cells and products thereof. RCGM therefore, has made an extensive effort to update and bring out a consolidated guideline at par with International best practices to prescribe the containment measures for storage, growth, research, manufacture, exchange, import and export of GE and non-GE organisms (microorganisms, animals, plants, arthropods, aquatic organisms) and products of such organisms. As an outcome of efforts involving several deliberations, national and international expert consultations and stakeholder engagements, RCGM is pleased to present “Regulations and Guidelines on Biosafety of Recombinant, DNA Research and Biocontainment, 2017”. The Guidelines cover the regulations on biosafety of rDNA research and handling of hazardous microorganisms and GE organisms or cells in India. It has described stringent and robust facility structures for handling of microorganisms, animals, plants, insects and aquatic organisms and has provided clear instruction on disposal and decontamination of laboratory wastes, emergency procedures etc. The guidelines include a list of risk group agents and determined appropriate containment level for their handling in India. Within the purview of Rules, Institutional Biosafety Committees have been empowered to take adequate precautionary measures for research conducted on risk group 1 and 2 organisms. The approval from RCGM is only required for experiments involving risk group 3 and 4 organisms. In addition, a separate laboratory certification system for handling of risk group 3 and 4 organisms has been developed. Following implementation of these guidelines, it shall be mandatory for all existing high containment facilities at biosafety levels 3 and 4 to acquire this accreditation for working with risk group 3 and 4 organisms. Adoption of these guidelines shall be binding pan India for all public and private organizations involved in research, development and handling of GE organisms (organism includes microorganisms, animals, plants, arthropods, aquatic animals, etc.) and non-GE hazardous microorganisms (microorganism includes parasites, protozoa, algae, fungi, bacteria, virus, prions, etc.) and products produced through exploration of such organisms. I am confident that this document will provide the much needed clarity to all stakeholders on biosafety and biosecurity requirements for manufacture, use, import, export and exchange of hazardous microorganism, GE organisms and cells. I extend my sincere acknowledgements to all expert members, contributions of stakeholders from industry, academia and civil societies for preparing for their inputs in preparation of this document on biosafety in recombinant DNA research and containment yet addressing biosecurity issues. S.R. Rao Senior Advisor & Member Secretary, RCGM Department of Biotechnology Ministry of Science & Technology Government of India This page is intentionally left blank Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 Contents ABBREVIATIONS.......................................................................................................................................v INTRODUCTION...................................................................................................................................... 1 OBJECTIVES.............................................................................................................................................. 1 SCOPE....................................................................................................................................................... 2 CHAPTER 1: REGULATIONS AND COMPETENT AUTHORITIES.......................................................... 3 1.1. SCOPE OF REGULATIONS................................................................................................. 5 1.2. DEFINITION APPLICABLE AS PER RULES 1989................................................................ 5 1.3. COMPETENT AUTHORITIES............................................................................................. 6 1.3.1. Recombinant DNA Advisory Committee (RDAC)............................................. 6 1.3.2. Review Committee on Genetic Manipulation (RCGM)................................... 6 1.3.3. Institutional Biosafety Committee (IBSC).......................................................... 7 1.3.4. Genetic Engineering Appraisal Committee (GEAC).......................................... 7 1.3.5. State Biotechnology Co-ordination Committee (SBCC)................................... 8 1.3.6. District Level Committee (DLC).......................................................................... 8 CHAPTER 2: PRINCIPLES AND COMPONENTS OF CONTAINMENT................................................... 9 2.1. CONTAINMENT............................................................................................................... 11 2.2. PRINCIPLE........................................................................................................................ 11 2.3. FACTORS IN CONTAINMENT.......................................................................................... 13 2.3.1. Physical Containment........................................................................................ 13 2.3.1.1. Procedure.......................................................................................... 13 2.3.1.2. Safety Equipments............................................................................ 14 2.3.1.3. Facility Design.................................................................................... 14 2.3.2. Biological Containment..................................................................................... 15 2.3.3. Laboratory Monitoring..................................................................................... 15 2.3.4. Health and Medical Surveillance..................................................................... 17 2.3.5. Decontamination and Disposal........................................................................ 17 2.3.6. Emergency Procedures..................................................................................... 23 CHAPTER 3: OPERATIONAL GUIDES ON CONTAINMENT................................................................ 25 3.1. MICROBIOLOGICAL BIOSAFETY LEVEL (BSL) FACILITIES.............................................. 27 3.1.1. Purpose.............................................................................................................. 27 3.1.2. Types of Microbiological Biosafety Level Facilities......................................... 27 3.1.2.1. Biosafety Level 1 (BSL-1):.................................................................. 27 3.1.2.2. Biosafety Level 2 (BSL-2):.................................................................. 28 3.1.2.3. Biosafety Level 3 (BSL-3):.................................................................. 29 i Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 3.1.2.4. Biosafety Level 4 (BSL-4):.................................................................. 30 3.1.3. Operational Guide for BSL-1 Facility................................................................ 31 3.1.4. Operational Guide for BSL-2 Facility................................................................ 34 3.1.5. Operational Guide for BSL-3 Facility................................................................ 37 3.1.6. Operational Guide for BSL-4 Facility................................................................ 43 3.2. CONTAINMENT FOR LARGE SCALE OPERATIONS OF GENETICALLY ENGINEERED (GE) MICROORGANISMS........................................... 47 3.2.1. Requirements of BSL-1 Large scale facility...................................................... 48 3.2.2. Requirements of BSL-2 Large scale facility...................................................... 50 3.3. ANIMAL BIOSAFETY LEVEL FACILITIES........................................................................... 51 3.3.1. Purpose.............................................................................................................. 51 3.3.2. Types of Animal Biosafety Level Facilities........................................................ 52 3.3.2.1. Animal Biosafety Level 1 (ABSL-1):.................................................. 52 3.3.2.2. Animal Biosafety Level 2 (ABSL-2):.................................................. 53 3.3.2.3. Animal Biosafety Level 3 (ABSL-3):.................................................. 53 3.3.2.4. Animal Biosafety Level 4 (ABSL-4):.................................................. 54 3.3.3. Operational Guide for ABSL Facilities.............................................................. 56 3.4. PLANT BIOSAFETY LEVEL FACILITIES.............................................................................. 62 3.4.1. Purpose.............................................................................................................. 62 3.4.2. Types of Plant Biosafety Level Facilities........................................................... 62 3.4.2.1. Plant Biosafety Level 1 (PBSL-1):...................................................... 62 3.4.2.2. Plant Biosafety Level 2 (PBSL-2):...................................................... 63 3.4.2.3. Plant Biosafety Level 3 (PBSL-3):...................................................... 64 3.4.2.4. Plant Biosafety Level 4 (PBSL-4):...................................................... 65 3.4.3. Operational Guide for PBSL Facilities.............................................................. 66 3.5. INSECT BIOSAFETY LEVEL FACILITIES............................................................................. 73 3.5.1. Purpose.............................................................................................................. 73 3.5.2. Types of Insect Biosafety Level Facilities.......................................................... 73 3.5.2.1. Insect Biosafety Level 1 (IBSL-1):..................................................... 73 3.5.2.2. Insect Biosafety Level 2 (IBSL-2):..................................................... 73 3.5.2.3. Insect Biosafety Level 3 (IBSL-3):..................................................... 74 3.5.2.4. Insect Biosafety Level 4 (IBSL-4):..................................................... 74 3.5.3. Operational Guide for IBSL Facilities................................................................ 75 3.6. AQUATIC ORGANISM BIOSAFETY LEVEL FACILITIES..................................................... 81 3.6.1. Purpose.............................................................................................................. 81 3.6.2. Types of Aquatic Organism Biosafety Level Facilities..................................... 81 3.6.2.1. Aquatic Organism Biosafety Level 1 (AqBSL-1):............................. 81 3.6.2.2. Aquatic Organism Biosafety Level 2 (AqBSL-2):............................. 81 ii Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 3.6.2.3. Aquatic Organism Biosafety Level 3 (AqBSL-3):............................. 81 3.6.3. Operational Guide for AqBSL Facilities............................................................ 83 CHAPTER 4: CONTAINMENT REQUIREMENT FOR IMPORT, EXPORT AND EXCHANGE............... 87 4.1. INTERNAL HANDLING PROCEDURES............................................................................ 89 4.2. SHIPMENT BY MAIL, AIRFREIGHT OR OTHER COMMON CARRIER............................ 89 4.2.1. Definitions.......................................................................................................... 89 4.2.2. The Packaging Requirements of Biological Materials.................................... 90 4.2.3. Instructions on Packaging................................................................................. 91 4.2.3.1. For Substances that are not subject to Dangerous Goods Regulations............................................................................ 91 4.2.3.2. For Substances Covered under Dangerous Goods Regulations........................................................................................ 92 4.2.4. Additional Requirement for Packaging and Shipment of GE organism or Their Derived Living Products (Propagules, Seeds etc.)................................. 93 4.3. IMPORT AND SHIPMENT............................................................................................... 94 4.4. STORAGE OF GE ORGANISMS AND RELATED MATERIALS.......................................... 95 GLOSSARY 97 OTHER APPLICABLE POLICIES............................................................................................................ 100 REFERRED INTERNATIONAL GUIDELINES........................................................................................ 101 ANNEXURE 103 Annexure 1: List of Infective Microorganisms corresponding to different Risk Groups.................................................................................................................... 105 Annexure 2: Risk Assessment of GE Organisms.........................................................119 Annexure 3: List of Commonly used Host-Vector System..........................................123 Annexure 4: Simplified procedures & Guidelines on Exchange, Import and Export of GE organisms and Products Thereof for R&D purpose.........................................126 Annexure 5: Checklist for Exchange/ Import/ Export of GE organisms and Products Thereof........................................................................................................................... 130 CERTIFICATION OF CONTAINMENT LEVEL 3 AND 4 FACILITIES.....................................................132 ACKNOWLEDGEMENTS..................................................................................................................... 148 iii This page is intentionally left blank Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 ABBREVIATIONS ABSL - Animal Biosafety Level AqBSL - Aquatic Organism Biosafety Level BC - Biological Containment BSC - Biosafety Cabinet BSL - Biosafety Level CDC - Centers for Disease Control and Prevention DBT - Department of Biotechnology DLC - District Level Committee EPA - Environment (Protection) Act GE - Genetically Engineered GEAC - Genetic Engineering Appraisal Committee GLSP - Good Large Scale Practices GOI - Government of India HEPA - High Efficiency Particulate Air IBSC - Institutional Biosafety Committee IBSL - Insect/Arthropod Biosafety Level MoEF&CC - Ministry of Environment, Forest and Climate Change NBPGR - National Bureau of Plant Genetic Resources NIH - National Institute of Health, USA PBSL - Plant Biosafety Level PI - Principal Investigator (R&D/Industry/Others) PPE - Personal Protective Equipment RCGM - Review Committee on Genetic Manipulation RDAC - Recombinant DNA Advisory Committee rDNA - recombinant DNA RG - Risk Group SBCC - State Biotechnology Co-ordination Committee v This page is intentionally left blank Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 INTRODUCTION As mandated in the Rules, 1989 of Environment (Protection) Act, 1986, Review Committee on Genetic Manipulation (RCGM) administered by the Department of Biotechnology, Ministry of Science and Technology has updated “Recombinant DNA safety guidelines, 1990”; “Revised Guidelines for Safety in Biotechnology, 1994” and “Revised guidelines for research in transgenic plants, 1998” and prepared “Regulations and Guidelines on Biosafety of Recombinant DNA Research and Biocontainment, 2017”. These guidelines are based on current scientific information, best practices and from the experience gained while implementing the biosafety frameworks within the country. A series of consultation with researchers, experts, academicians, concerned Ministries/departments and other stakeholders was carried out during preparing this guideline. This document specifies the practices for handling (Manufacture, Use, Import, Export, Exchange and Storage) of hazardous biological material, recombinant nucleic acid molecules and cells, organisms and viruses containing such molecules to ensure an optimal protection of public health and of the environment. The document provides clarity on biosafety requirements and recommendations for laboratory facilities such as facility design, biosafety equipment, personal protective equipment, good laboratory practices and techniques, waste management, etc. OBJECTIVES i. Outline the general principles of containment and establish a minimum standard for laboratories that must be adopted pan India for all handling of genetically engineered (GE) organisms (organism includes microorganisms, animals, plants, arthropods, aquatic animals, etc.) and non-genetically engineered (non-GE) hazardous microorganisms (microorganism includes parasites, protozoa, algae, fungi, bacteria, virus, prions, etc.). ii. Identify the levels of risk(s) associated with GE organisms and non-GE hazardous microorganisms and classification of those organisms into their respective risk groups to select appropriate containment facilities. It also covers certification of containment facilities. iii. Prescribe criteria for Manufacture, Use, Import, Export, Exchange and Storage of any hazardous microorganisms, GE organisms or cells and product(s) produce through exploration of such organisms. iv. Ensure that national authorities, institutions and all other stakeholders involved in research & development are well informed or have access to information on safety thereby facilitating the safe use and handling of hazardous microorganisms, GE organisms or cells and product(s) produce through exploration of such organisms. 1 Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 v. Emphasize the need and responsibility of all national authorities institutions and all other stakeholders involved in research to ensure that the public is well informed about the containment strategies followed in India. SCOPE This document covers regulatory scope on rDNA research and handling of hazardous microorganisms and GE organisms or cells in India. Adoption of these guidelines shall be binding pan India for all public and private organizations involved in research, development and handling of GE organisms (organism includes microorganisms, animals, plants, arthropods, aquatic animals, etc.) and non-GE hazardous microorganisms (microorganism includes parasites, protozoa, algae, fungi, bacteria, virus, prions, etc.) and products produced through exploration of such organisms. Note: T hese guidelines do not override any other existing regulations or guidelines, unless specified here. 2 CHAPTER 1 REGULATIONS AND COMPETENT AUTHORITIES This page is intentionally left blank Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 CHAPTER 1 REGULATIONS AND COMPETENT AUTHORITIES 1.1. Scope of Regulations These regulations are to implement the provisions of Rules 1989 of Environment (Protection) Act, 1986 for the manufacture, use, import, export and storage of hazardous microorganisms, GE organisms or cells and products thereof which applies to the whole of India in the following specific cases: i. Sale, offers for sale, storage for the purpose of sale, offers and any kind of handling over with or without a consideration; ii. Exportation and importation; iii. Production, manufacturing, processing, storage, import, drawing off, packaging and repacking of the GE Products; iv. Production, manufacture etc. of drugs and pharmaceuticals, food and food components, distilleries and tanneries, etc. which make use of hazardous micro-organisms or GE organisms one way or the other. 1.2. Definition Applicable as per Rules, 1989 Definitions applicable to this guideline as per Rules, 1989 unless the context requires: i. “Biotechnology” means the application of scientific and engineering principles to the processing of materials by biological agents to produce goods and services; ii. “Cell hybridisation” means the formation of live cells with new combinations of genetic material through the fusion of two or more cells by means of methods which do not occur naturally; iii. “Gene Technology” means the application of the gene technique called genetic engineering, include self-cloning and deletion as well as cell hybridisation; iv. “Genetic engineering” means the technique by which heritable material, which does not usually occur or will not occur naturally in the organism or cell concerned, generated outside the organism or the cell is inserted into said cell or organism. It shall also mean the formation of new combinations of genetic material by incorporation of a cell into a host cell, where they occur naturally (self-cloning) as well as modification of an organism or in a cell by deletion and removal of parts of the heritable material; v. “Microorganisms” shall include all the bacteria, viruses, fungi, mycoplasma, cells lines, algae, protozoans and nematodes indicated in the schedule and those that have not been presently known to exist in the country or not have been discovered so far. 5 Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 1.3. Competent Authorities The Rules 1989 are broad in scope and covers area of research as well as large scale handling of hazardous microorganisms, GE organisms or cells and products thereof. In order to implement the Rules in the entire country, six competent authorities and their roles have been notified (Table 1) for: i. Regulation and control of contained research activities with hazardous microorganisms, and GE organisms. ii. Regulation and control of large scale use of GE organisms in production activity. iii. Import, export and transfer of hazardous microorganisms, GE organisms and products thereof. iv. Release of GE organisms and products thereof in environmental applications under statutory provisions. Table 1: Competent Authorities under Rules, 1989 Competent Authorities Role Recombinant DNA Advisory Committee (RDAC) Advisory Institutional Biosafety Committee (IBSC) Regulatory/ Approval Review Committee on Genetic Manipulation (RCGM) Genetic Engineering Appraisal Committee (GEAC) State Biotechnology Coordination Committee (SBCC) Monitoring District Level Committee (DLC) 1.3.1. Recombinant DNA Advisory Committee (RDAC) This committee functions in the Department of Biotechnology with a role to: i. Review developments in Biotechnology at national and international levels. ii. Shall recommend suitable and appropriate safety regulations for India in recombinant research, use and applications from time to time. iii. Evolve long term policy for research and development in Recombinant DNA research. 1.3.2. Review Committee on Genetic Manipulation (RCGM) This committee functions from the Department of Biotechnology to monitor the safety related aspect in respect of on-going research projects or activities involving hazardous microorganisms, GE organisms and cells and products thereof. The RCGM includes representatives of (a) Department of Biotechnology (b) Indian Council of Medical Research (c) Indian Council of Agricultural Research 6 Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 (d) Council of Scientific and Industrial Research (e) other experts in their individual capacity. RCGM may appoint sub groups to assist RCGM on matters related to risk(s) assessment, in reviewing of existing and preparing new guidelines. RCGM is mandated to bring out manuals of guidelines specifying procedure for regulatory process with respect to activities involving GE organisms in research use as well as industrial and environmental applications with a view to ensure human health and environmental safety. All ongoing research projects involving hazardous microorganisms, GE organisms or cells and products thereof shall be reviewed to ensure that adequate precautions and containment conditions are being met. RCGM lays down procedures restricting or prohibiting production, sale, importation and use of such hazardous microorganisms (Annexure 1), GE organisms or cells. 1.3.3. Institutional Biosafety Committee (IBSC) This committee is constituted by all institutions handling hazardous microorganisms and/or GE The constitution, composition, organisms. The committee is the nodal point for role and functions, information for implementation of the biosafety guidelines and compliance requirements, processes for the interactions within the institution. The to be followed while dealing with committee comprises of the Head of the Institution, hazardous microorganisms, GE Scientists engaged in the recombinant DNA work, organisms or cells and product thereof a medical doctor and a nominee of the Department in line with Rules 1989 is described of Biotechnology. Institutions handling risk-inherent in “Guidelines and Handbook for microorganisms or GE organisms shall prepare, with Institutional Biosafety Committee”. the assistance of the Institutional Biosafety Committee Adherence to the guideline shall be (IBSC), on-site emergency plan and update from time binding for all IBSCs. to time according to the manuals/guidelines of the RCGM and make available as required copies to the District Level Committee/State Biotechnology Co-ordination Committee and the Genetic Engineering Appraisal Committee. 1.3.4. Genetic Engineering Appraisal Committee (GEAC) Genetic Engineering Appraisal Committee (GEAC) [formerly known as Genetic Engineering Approval Committee (GEAC); name changed through Gazette notification, G.S.R No. 613 dated 16th July 2010], has been established under the Ministry of Environment, Forest and Climate Change (MoEF&CC). The major functions of GEAC as prescribed in the Rules 1989 are: i. To appraise activities involving large scale use of hazardous microorganisms GE organisms or cells in research and industrial production from the environmental angle. 7 Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 ii. To appraise proposals relating to release of GE organisms and products into the environment including experimental field trials. iii. The committee or any persons authorized by it has powers to take punitive action under the Environment (Protection) Act, 1986. Submission of applications to GEAC, information on current composition, meeting deliberations etc. is available at http://geacindia.gov.in/index.aspx. 1.3.5. State Biotechnology Co-ordination Committee (SBCC) The State Biotechnology Co-ordination Committee (SBCC) is a monitoring committee at State level. It shall have powers: i. To inspect, investigate and to take punitive action in case of violations of statutory provisions through the State Pollution Control Board (SPCB) or the Directorate of Health etc. ii. To review periodically the safety and control measures established at various institutions handling GE organisms. iii. To act as nodal agency at the State level to assess the damage, if any, due to release of GE organisms and to take on site control measures. 1.3.6. District Level Committee (DLC) There shall be a District Level Biotechnology Committee (DLC) in the districts wherever necessary under the District Collectors to monitor the safety regulations in installations engaged in the use of genetically modified organisms/ hazardous microorganisms and its applications in the environment. The District Level Committee/or any other person/s authorized in this behalf shall visit the installation engaged in activity involving hazardous microorganisms, GE organisms or cells, and , formulate information chart, find out hazards and risk(s) associated with each of these installations and coordinate activities with a view to meeting any emergency. They shall also prepare an off-site emergency plan. The District Level Committee shall regularly submit its report to the SBCC/ GEAC. 8 CHAPTER 2 PRINCIPLES AND COMPONENTS OF CONTAINMENT This page is intentionally left blank Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 CHAPTER 2 Principles and components of containment 2.1. Containment Containment encompasses safe methods (Combination of facilities, practices and procedures) for managing risk-inherent microorganisms, GE organisms or cells in the laboratory environment where they are being handled or maintained. Selection of appropriate containment strategy will ensure safety to laboratory workers, outside people and the environment from hazardous microorganisms, GE organisms or cells by: i. Reducing the exposure, and ii. Preventing their escape and establishment in a natural environment. 2.2. Principle The principle is the protection of all identified elements from risk(s) posed by organisms (includes risk-inherent; GE and non-GE microorganisms, animal, plants, arthropods, aquatic animals, etc) during their use in laboratory. In practice, it should be achieved in realization of three interrelated steps: i. Identification of elements that should be protected: Containment measures should ensure protection of laboratory worker(s) (Primary elements) who have maximum possibility of exposure to the organisms. In addition, the containment measure should also prevent the escape of organisms and so ensure protection of persons outside the laboratory and the environment (Secondary elements). ii. Identification of potential risk(s) associated with organism(s): It involves assessment of risk(s) associated with the organisms and their classification to appropriate risk groups based on: a. Pathogenicity of the organism towards humans/animals/plants. b. Modes of transmission and host range of the organism. c. Availability of effective preventive treatments or curative medicines. d. Capability to cause epidemics. Based on the above information, infective microorganisms can be classified into four risk groups (Table 1) that allow selection of appropriate biosafety level facilities. In this document, an updated list of infective microorganisms under different risk groups has been provided in Annexure 1. 11 Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 Table 1: Risk Group (RG) classification Risk Group Description RG 1 A microorganism that is unlikely to cause human/ animal/plant disease. RG 2 A microorganism that can cause disease in human /animal/ plant but the laboratory exposures may or may not cause serious infection to individual and risk(s) of spread of infection is limited. RG 3 A microorganism that usually causes serious/lethal human/ animal/ plant disease but does not ordinarily spread from one infected individual to another. RG 4 A microorganism that usually causes serious/lethal human/ animal/ plant disease and that can be readily transmitted from one individual to another, directly or indirectly. iii. Determining appropriate biosafety level facility for safe handling of organisms (includes risk- inherent; GE and non-GE microorganisms, animals, plants, arthropods, aquatic animals, etc.) (Table 2). Table 2. Different biosafety level facilities Organism Facility designation Safety Levels Microorganisms Biosafety Level (BSL) BSL-1 to BSL-4 Animal Animal Biosafety Level (ABSL) ABSL-1 to ABSL-4 Plant Plant Biosafety Level (PBSL) PBSL-1 to PBSL-4 Arthropods/Insects Insect Biosafety Level (IBSL) IBSL-1 to IBSL-4 Aquatic organisms Aquatic Organism Biosafety Level (AqBSL) AqBSL-1 to AqBSL-3 99 T hose agents not listed in Risk Group (RG) 2, 3 and 4 are not implicitly classified as RG 1. For such agents, a risk(s) assessment must be conducted based on the known and potential properties of the agents and their relationship to agents that are listed and then placed in an appropriate Risk Group. 99 enetic engineering can alter/change the overall risk(s) of an organism depending on G the genetic modification. Hence, irrespective of cases, re-evaluation of risk(s) associated with the GE organism will be required to assign requisite containment levels. The risk assessment approaches for GE organisms is presented in Annexure 2. 99 ppropriate containment level for handling of microorganisms, animals, plants, arthropods, A aquatic organisms (both GE and non-GE), is described in this document. 12 Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 2.3. Factors in Containment Depending on the nature of work and organism involved, containment shall be different to ensure optimal protection to the worker as well as environment. The levels of containment shall be determined based upon principle factors as described below: 2.3.1 Physical Containment The strategy is to physically confine the organism under study that can be feasibly adopted to prevent or minimize its exposure to worker and environment ensuring the risk(s) can be prevented or mitigated. It is achieved through the use of three elements of containment i.e. Procedures, Safety equipment(s) and Facility design(s). The protection of personnel(s) and the immediate laboratory environment from exposure to organisms (includes risk-inherent; GE and non-GE microorganisms, animals, plants, arthropods, aquatic animals, etc.) is provided by ‘Procedures’ and the use of appropriate ‘Safety equipment(s)’ (Primary containment). The protection of the environment external to the laboratory from exposure to risk-inherent materials is provided by a combination of ‘facility design’ and operational practices (Secondary containment). The elements are not in hierarchy and should be used with equal priorities in combination to ensure a successful containment. Appropriate combination of these elements lays the foundation for selection of containment facilities for working with organisms/cells pertaining to different risk groups. 2.3.1.1 Procedure These must be followed by workers involved in research and handling of organism in consideration of: i. Strict adherence to standard microbiological practices and techniques. ii. Awareness of potential hazards. iii. Providing/arranging for appropriate training of personnel. iv. Selection of safety practices in addition to standard laboratory practices if required. It is emphasized that good laboratory practice is fundamental to laboratory safety and cannot be replaced by any other means, which can only supplement it. Note: Handling of GE and non-GE organism(s) is the same laboratory will require extra precautionary measures so as to prevent unintentional cross-contamination of non-GE organisms. Means of preventing cross-contamination of other work by GE organisms could include physical separation of the work or separation of the work at different times and ensure proper decontamination prior to commencing work with non-GE organisms. 13 Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 2.3.1.2 Safety Equipments Any equipment that contributes to personnel protection either directly or indirectly from the hazardous biological material is considered for containment. It includes: i. Instruments like biological safety cabinets, autoclave and a variety of enclosed containers (e.g. safety centrifuge cup). The biological safety cabinet (BSC) is one of the principal devices used to provide workers safety from hazardous microorganisms and infectious aerosols. Three types of BSCs (Class I, II, and III) are used in biosafety level facilities. Safety and functionality of each instrument must be monitored monthly for effectiveness and calibrated annually before commencing operations. Equipment such as autoclaves and biological safety cabinets must be validated with appropriate methods (usually by a certified examiner) before being taken into use. The results of the monitoring and calibration must be documented. Recertification should take place at regular intervals, according to the manufacturer’s instructions. If any equipment is found to be defective and the defect has not been corrected, the equipment must be clearly marked to show that it is defective and must not be used for any purpose until the defect has been corrected. ii. Personal protective equipment (PPE) such as gloves, coats, gowns, shoe covers, boots, respirators, face shields and safety glasses, etc. The Head of the laboratory, after consultation with the biosafety officer and IBSC, should ensure that adequate equipment is provided and that it is being used properly. In selecting safe laboratory equipment, the general principles that should be considered include: i. Designed to limit or prevent contact between the operator and the infectious organisms. ii. Constructed of materials that are impermeable to liquids, corrosion-resistant and meet structural strength requirements. iii. Fabricated to be free of burrs and sharp edges. iv. Designed, constructed and installed to facilitate simple operation and to provide ease of maintenance, accessibility for cleaning, and ease of decontamination and certification testing. These are general principles. Detailed performance and construction specifications may be required to ensure that the equipment possess necessary safety features. 2.3.1.3 Facility Design The design of the facility is important in providing a barrier to protect not only to persons working in the facility but also outside of the laboratory and those in the community from infectious organisms which may be accidentally released from the laboratory. Selection of facility is to be determined based on risk(s) associated with the organism. Details of facility design for hazardous microorganisms, GE organisms and cells are mentioned in this guideline. Although, special attention should be paid to conditions like: 14 Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 i. Creation of aerosols. ii. Work with large volumes and/or high concentration of microorganisms. iii. Overcrowded, over equipped laboratories. iv. Infestation with rodents or insects. v. Unauthorized entrance. 2.3.2 Biological Containment Biological containment employs strategies that render an organism used for genetic engineering either incapable of survival in the open or severely reduce its ability to survive or reproduce in the open environment. Such GE organisms would either remain viable only under the selective environmental conditions for which they were designed for or would carry self-contained mechanism(s) that could be induced when need arises to eradicate such GE population. In addition to physical containment, such biological containment hence ensures additional safety while working with GE organisms and provides more flexibility of handling organisms with higher risk(s). It is always advisable to consider biological containment strategies especially if the final aim of the experiment is to release the organisms into the environment. In doing so, it is the responsibility of an investigator to first identify the possible risk(s) associated with the host, vector and modification(s) proposed and select appropriate strategies to reduce or limit: i. The risk(s) associated with host organism. ii. The infectivity of vector to specific hosts. iii. The host-vector survival in the environment. Note: Biological containment must not be considered a standalone containment strategy. 2.3.3 Laboratory Monitoring Laboratory monitoring is a systematic, regular and preventive activity designed for corrective actions, if required. It is the responsibility of the Head of the Institution to ensure: i. Prevention of any unauthorized entry in the laboratory. Entry and exit procedures must cover: a. Authorization for laboratory staff, visitors, maintenance, A manual of laboratory visiting scholars. monitoring should be b. Entry/exit log or other method to monitor authorized entry. prepared and kept in the c. Required personal protective equipment. facility for information d. Escort requirements. and ready reference to the workers. e. Removal of interfering objects and viable cultures. 15 Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 f. Disinfection procedure prior to entry and maintenance. g. Disinfection procedure for exiting. ii. Allow entry of person having proper training in laboratory safety and immunization, if required (For handling RG 3 and above organisms). Personnel training in biosafety is the key for prevention of laboratory-acquired infections, incidents and accidents ensuring success of any containment strategy. Based on the organism to be handled and the nature of work, training program to be developed and laboratory in charge must play the key role in training of laboratory staff. A training program must include information on safe handling of organisms of different risk groups that are commonly encountered by all laboratory personnel, involving any possible exposure scenarios and decontamination and emergency plan strategies. Training should involve class room work as well as significant one-on-one mentoring in the lab before an individual is allowed to work alone. It may include: a. Inhalation risk(s) (i.e. aerosol production), such as using loops, streaking agar plates, pipetting, making smears, opening cultures, taking blood/serum samples, centrifugation etc. b. Ingestion risks, such as handling specimens, smears and cultures. c. Risk(s) of percutaneous exposures, through the use of syringe and needle techniques. d. Animal handling that may result in bites and scratches. e. Handling of blood and other potentially hazardous pathological materials. f. Decontamination and disposal of infectious material. g. Emergency procedures in case of unwanted breach in containment. iii. Personnel should be advised of special hazards and required to read and follow standard practices and procedures. iv. Persons at increased risk(s) of acquiring infection or for whom infection may have unusually serious consequences (e.g. Immunocompromised, Women during pregnancy, etc.) are informed of their risk(s) and should be restricted from entering the laboratory. Panel of expert scientists of the institute/organization/universities need to review and decide on case by case basis. v. To create an open environment where workers are following proper containment strategies and are fearless to report violations of procedure, identify coworker failings, express concerns and offer suggestions. vi. All safety equipments are working properly and if not, maintenance of the equipment is made immediately. All civil structures are in good condition to ensure proper containment. vii. A regular schedule for housekeeping is maintained. 16 Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 viii. Prevention of diseases in the general or occupational environment. ix. Documentation of daily laboratory activity for immediate consideration of emergency procedures in cases of breach in containment. x. Proper documentation of work involving both non-GE and GE organisms in a same facility should be maintained to ensure that no unintentional cross-contamination of non-GE organisms occurs. Stringency in monitoring procedure(s) must be determined based on biosafety level of the laboratory and should be determined by laboratory supervisors with consultation of scientific experts. 2.3.4 Health and Medical Surveillance The objectives of the health and medical surveillance of laboratory personnel are: i. Prevent individual from acquiring infection during the work ii. Provide for early detection of laboratory-acquired infection. iii. Provide for assessing the efficacy of protective equipment and procedures. iv. Provide for prophylactic vaccinations where needed and monitor booster regimens and assessment of sero conversion, in applicable cases. It is the responsibility of the employing authority and/or the facility in-charge to ensure that health and medical surveillance of laboratory personnel is carried out. 2.3.5 Decontamination and Disposal Decontamination and disposal in laboratories are closely interrelated acts, since disinfection or sterilization constitute the first phase of disposal. All materials and equipment will ultimately be disposed off; however, in terms of daily use, only a portion of these will require actual removal from the laboratory or destruction. These will be referred as biological wastes that need specific treatment to render safe before discard. These include: Steam autoclaving is the preferred method for all decontamination processes. Materials for decontamination and disposal should be placed in containers, e.g. autoclavable plastic bags that are colour-coded according to whether the contents are to be autoclaved and/or incinerated. Alternative methods may be adopted only if they remove and/or kill risk-inherent organisms. The principal questions to be answered prior to disposal of any objects or materials from laboratories dealing with risk-inherent organisms or tissues are: i. Have the objects or materials been effectively disinfected or sterilised by an approved procedure? If not, how the materials will be stored before effective disinfection and/or sterilization? 17 Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 ii. Will the materials be disinfected or sterilised on site or transferred to other laboratory/ area and how it will be transferred? iii. How the disinfected or sterilised material will be disposed off? iv. Does disposal of the disinfected or sterilized objects or materials involve any additional potential hazard, biological or otherwise, to those carrying out the immediate procedure or those who might come into contact with the objects or materials outside the laboratory complex? As part of disposal mechanism: i. All equipment must be decontaminated before being repaired, maintained, or removed from the laboratory. ii. Separate approach of storage and disinfection procedure should be adopted and the same should be well informed to the personnel working in the facility. iii. If on site disinfection/ sterilization is not possible, transport of materials should be carried out by trained staff under appropriate storage container that must be leak proof and tightly sealed. iv. Designated waste disposal area should be available within each facility. v. Wastewater released from laboratories should not be allowed to mix to general public sewage system. Provision should be made to collect such effluents coming from all laboratories and should be treated for proper decontamination before disposal. Microbiological testing may be performed periodically and record should be maintained. vi. All employees who handle biological waste shall be trained regarding the proper segregation, handling, packaging, labelling, storage, and treatment of biological waste. Refresher training is required annually. A training manual must be developed and available in the facility. vii. For facilities that work with RG 3 and above organism and/or perform Category III and above genetic engineering work, it will be mandatory to maintain written records on decontamination and disposal. It should include: Date of treatment, method/conditions of treatment, name of the persons performing the treatment, a written procedure for the operation and testing of any equipment used and a written procedure for the preparation of any chemicals used in treatment. viii. Appropriate disposal mechanisms for chemical or radiological wastes should be developed in discussion with biosafety officer and appropriate local and national competent authorities in conjunction with current mechanism. Unless mentioned in the operational practices in specific containment facility, instructions on disposal of biological materials (classified as Solids, Liquids, Sharps, and Pathological) should be adhered to those mentioned in Table 3. 18 Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 Selection of appropriate decontamination and disinfection strategies for biomedical waste treatment and disposal facilities should be in accordance to those mentioned in the “Revised Guidelines for Common Bio-medical Waste Treatment and Disposal Facilities” (2016) developed by Central Pollution Control Board (CPCB). 19 20 Table 3: Instruction on disposal of laboratory wastes Laboratory waste type Disposal Container (s) Disposal requirements Solid wastes i. The primary container should be i. Keep container and lid clean at all times. E.g. All used gloves, paper leak and puncture proof and must ii. Maintain access to the container - do not put towels, gauze, wipes, have lid. materials on lid. absorbents, disposable ii. A label of “biological waste” should Petri dishes, culture vials, iii. Lid must be in place when waste is not being be visible on container. plastic wares, plants or added to container. iii. It should be lined with a biohazard any parts/ tissues, seeds, iv. No liquid should be discarded along with solid bag of mandated colour that is soil/soil substitute (perlite, waste. leak proof and able to withstand vermiculite, peat mass, autoclave conditions. v. The biohazard bag should be 3/4th fill maximum. etc.) vi. Do not overfill. vii. To transport container(s) outside the facility for decontamination, ensure that the biohazard bag is sealed and the lid is tightly closed. A trolley for transport is preferred. Do not toss. viii. All solid hazardous waste must be autoclaved. ix. Prior to autoclaving, crisscross the bag’s biohazard symbol and/or markings with heat sensitive autoclave tape. x. Ensure the autoclave is set for the appropriate time– selection of time and pressure should Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment ensure proper decontamination. For e.g. Organism containing TSE must be autoclaved at 2017 a higher setting. xi. Once the waste is autoclaved, mark the autoclaved bag with an “Autoclaved/ Decontaminated” sticker. xii. Place the autoclaved bag into an opaque bag of mandated colour and seal it. xiii. Store the bag in a place that could be collected for disposal by cleaning personnel. xiv. As an alternative to autoclaving, other waste disposal methods may be employed as prescribed by the local competent authorities (Pollution Control Board). Liquid Waste i. The container should be leak and i. Liquid waste must be separated from solid E.g. Any media, liquids puncture proof and must have lid. waste. coming from Petri ii. A label of “biological waste” should ii. Liquid waste must be decontaminated on site dishes, culture vials, lab be visible on container. with an appropriate disinfectant/bleach with equipment, recombinant appropriate period of exposure. Nucleic Acids (rNA) in iii. Flush the disinfected material down the sink, all forms (natural and allowing the cold water to run for a period of synthetic e.g., DNA, RNA, time (at least 5 minutes). shRNA, etc.). iv. Do not flush non-aqueous solutions, such as liquefied agarose or unfiltered broths, down the drain as they will clog the drain pipes. Note: Liquid waste generated from higher containment laboratory (BSL-2 and above) should be autoclaved. Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 21 22 Sharps i. The container must be rigid, leak i. All sharps must be placed in appropriate sharps E.g. All needles, syringes, proof, puncture proof and have lid. container. scalpels, razor blades, ii. Keep baffle in place. ii. Once the container is ¾ full, close the top of the pipette tips, Pasteur container. pipettes, glass ware, iii. Line with a biohazard bag of capillary tubes, slides and mandated colour. iii. Sharps contaminated with biological materials cover slips, contaminated must be autoclaved before disposal. iv. Label with “BIOHAZARD SHARPS” broken glassware. sticker. Pathological Must be leak-proof and puncture-proof. Same as ‘solid waste’. E.g. Animal carcasses Lid must be in place when waste is not Incineration of carcasses. suspected to be or being added to container. potentially infected; Line with a red or orange biohazard tissues, organs and any bag. body parts; bedding from Label with “BIOHAZARD” and animal cages, etc. “PATHOLOGICAL WASTE” stickers. Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 2.3.6 Emergency Procedures Emergency contingency plans in consideration of every possible breach in biocontainment should be prepared for each individual laboratory as well as for the institutions. These are best prepared by the individual laboratory supervisor in conjunction with his staff and the biosafety officer. This procedure offers the best prospect of success as it is the immediate staffs that are most familiar with the hazards associated with a particular laboratory. Once the emergency plan is formulated, it should be pasted in conspicuous place in the laboratory for immediate reference. Statutory rules and regulations for each of these will normally be laid down by the competent national or local authorities. Their assistance and guidance should be sought if necessary. Emergency plan should provide for: i. Breakage and spillage. ii. Accidental injection, cuts and abrasions. iii. Accidental ingestion of potentially hazardous material. iv. A potentially hazardous aerosol release (other than in a safety cabinet). v. Breakage of tubes in centrifuges not having safety cups. vi. Chemical, fire, electrical and radiation. vii. Flood and natural disaster. viii. Vandalism. ix. Miscellaneous emergencies including falls due to wet floor, ill health, seizures etc. In addition, emergency plan should also provide: i. Emergency contact numbers, and contact details of other relevant emergency services available. ii. Details of emergency equipment and its location. Note: A part from these, emergency procedures for containment in case of biological disasters should be according to National Disaster Management Guidelines — Management of Biological Disasters, 2008. 23 This page is intentionally left blank CHAPTER 3 OPERATIONAL GUIDES ON CONTAINMENT This page is intentionally left blank Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 CHAPTER 3 OPERATIONAL GUIDES ON CONTAINMENT 3.1. Microbiological Biosafety Level (BSL) Facilities 3.1.1 Purpose BSL facilities are the fundamental laboratory structures for containment purposes. Such facilities will be suitable for: i. Isolation, cultivation and storage and experiments on hazardous microorganisms. ii. Genetic engineering of organisms and their safe handling. iii. Handling of toxins, tissues, etc. 3.1.2 Types of Microbiological Biosafety Level Facilities 3.1.2.1 Biosafety Level 1 (BSL-1): BSL-1 will be applicable for: i. Isolation, cultivation and storage of Risk Group (RG) 1 microorganisms those are abundant in natural environment. ii. Experiments on RG 1 microorganisms provided that the experiments will not increase environmental fitness and virulence of the microorganisms. iii. Category I genetic engineering experiments on microorganism: This category includes experiments which generally do not pose significant risk(s) to laboratory workers, community or the environment and the modifications have no effect on safety concerns. Examples are: a. Insertions of gene into RG 1 microorganism from any source, deletions, or rearrangements that have no adverse health, phenotypic or genotypic consequence. Modification should be well characterized and that the gene functions and effects are adequately understood to predict safety. b. Experiments involving approved host-vector systems (refer to Annexure 3) provided that the donor DNA is originated from RG 1 microorganism, not derived from pathogens. The DNA to be introduced should be characterized fully and will not increase host or vector virulence. c. Experiments involving the fusion of mammalian cells which generate a non-viable organism, for example, the construction of hybridomas to generate monoclonal antibodies. 27 Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 d. Any experiments involving microorganism belonging to RG 1. For e.g. self-cloning, fusion of protoplasts between non-pathogenic RG 1 organism. Before commencement of Category I GE experiments, the investigator should intimate the IBSC about the objective and experimental design of the study along with organisms involved. IBSC should review the same as and when convened for record or action if any. It is desirable to designate a separate area in the facility with proper labelling for Category I GE experiments to avoid any chances of contamination. 3.1.2.2. Biosafety Level 2 (BSL-2): BSL-2 will be applicable for: i. Isolation, cultivation and storage of RG 2 microorganisms. ii. Handling of environmental samples collected from environment that is unlikely to contain pathogens. Isolation of microorganisms from those samples and subsequent experiments. iii. Experiments on RG 2 microorganisms or isolates from environment mentioned above, provided that the experiments will not increase environmental fitness and virulence of the microorganisms. iv. Category II genetic engineering experiments on microorganism: These experiments may pose low-level risk(s) to laboratory workers, community or the environment. Examples are: a. Experiments involving the use of infectious or defective RG 2 viruses in the presence of helper virus. b. Work with non-approved host/vector systems where the host or vector either: does not cause disease in plants, humans or animals; and/ or is able to cause disease in plants, humans or animals but the introduced DNA is completely characterized and will not cause an increase in the virulence of the host or vector. experiments using replication defective viruses as host or vector. c. Experiments with approved host/vector systems, in which the gene inserted is: a pathogenic determinant; not fully characterized from microorganisms which are able to cause disease in humans, animals or plants; or an oncogene. 28 Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 d. Modification leading to persistent transient disruption of expression of gene(s) that are involved directly or indirectly in inducing pathogenicity, toxicity, survival, or fitness. Modification should be well characterized and the gene functions and effects are adequately understood to predict safety. e. Work involving fragments of Transmissible Spongiform Encephalopathy (TSEs) proteins or modified TSEs proteins that are not pathogenic and is not producing any harmful biological activity. f. Experiments in which DNA from RG 2 or 3 organisms are transferred into non-pathogenic prokaryotes or lower eukaryotes. However, handling of live cultures of RG 3 organism should be performed in BSL-3 laboratory. All category II GE experiments require prior authorization from IBSC before the commencement of the experiments through submission of information in the prescribed proforma. It is desirable to designate a separate area in the facility with proper labelling for Category II GE experiments to avoid any chances of contamination. 3.1.2.3 Biosafety Level 3 (BSL-3): BSL-3 will be applicable for: i. Isolation, cultivation and storage of RG 3 microorganisms. ii. Handling of environmental samples collected from environment that is likely to contain pathogens of potential disease consequences. Isolation of microorganisms from those samples and subsequent experiments. iii. Experiments on RG 3 microorganisms or isolates from environment mentioned above provided that the experiments will not increase environmental fitness and virulence of the microorganisms. iv. Category III and above genetic engineering experiments on microorganism: These kinds of experiments pose moderate to high risk(s) to laboratory workers, community or the environment. Examples are: a. Experiments on RG 2 and RG 3 microorganisms where insertion of gene directly involved in production of toxin or allergen or antimicrobial compounds. b. Insertions of gene into RG 3 microorganisms from any source, deletions, or rearrangements that affect the expression of genes, whose functions or effects are not sufficiently understood to determine with reasonable certainty if the engineered organism poses greater risk(s) than the parental organism. 29 Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 c. Insertions of nucleic acid from any source, deletions, or rearrangements that have known or predictable phenotypic or genotypic consequence in the accessible environment that are likely to result in additional adverse effects on human and/or animal health or on managed or natural ecosystems, e.g., those which result in the production of certain toxins. d. Research involving the introduction of nucleic acids (recombinant or synthetic) into RG 3 organisms or organisms listed in SCOMET items (http://dgft.gov.in). e. Genetic engineering of organisms isolated from environment where there are reported cases of disease prevalence and possibility of presence of infectious microorganisms. All category III and above GE experiments require prior authorization from IBSC and subsequent approval from RCGM before commencement of the experiments through submission of information in the prescribed proforma. 3.1.2.4. Biosafety Level 4 (BSL-4): BSL-4 laboratory is the maximum containment laboratory. Strict training, strictly restricted access and supervision are required and the work must be done under stringent safety conditions and positive pressure personnel suits. BSL-4 will be suitable for: i. Isolation, cultivation and storage of RG 4 microorganisms. ii. Handling of samples collected from environment/patients that are likely infected with RG 4 organisms with serious/fatal health effects. iii. Experiments on RG 4 microorganisms or isolates from environment/patients mentioned above to find remedial measures. iv. Category III and above genetic engineering experiments on microorganisms involving introduction of nucleic acids (recombinant or synthetic) into RG 4 microorganisms or exotic agents. Note: i. BSL facilities are not meant for: a. Permanently housing/keeping/rearing of any animals, arthropods or aquatic organisms for longer than the time required to complete laboratory procedures on them. b. The growing of any plants, except those in tissue culture bottles or fully contained in a plant growth chamber. ii. Genetic engineering experiments not covered under any of the above four categories will require case by case evaluation for selection of appropriate containment strategies. Prior 30 Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 approval and/or permission from the IBSC and/or the RCGM shall be required to initiate such experiment. Few examples are: a. Clubbing of experiments pertaining to different categories. b. Any experiments involving primates, dogs, large animals, and human participants within the laboratory. c. Experiments involving the use of infectious or defective RG 3 and above viruses in the presence of helper virus. d. Experiments using DNA which encodes a vertebrate toxin having an LD50 of less than 100 μg/kg. e. Experiments with genes that alter the growth status of cells such as oncogenes, cytokines and growth factors. f. Experiments aimed at controlling natural populations. iii. All existing BSL-3 and 4 facilities must be certified by RCGM. A format for certification is available in this guideline. iv. The new BSL-3 and 4 facilities shall require certification at the time of commissioning operations as per the format. 3.1.3. Operational Guide for BSL-1 Facility A) Facility design i. Facility should be a fully enclosed space bounded by walls, doors, windows, floors and ceilings. ii. Ample space must be provided for the safe conduct of laboratory procedures. iii. Walls, ceiling, and floors should be smooth, easily cleanable, impermeable to liquids, and resistant to the chemicals and disinfectants normally used in the laboratory. Floors should be slip resistant. Exposed pipes and ducting should stand clear of walls. Horizontal runs should be avoided to prevent dust collection. iv. Adequate illumination should be ensured for carrying out all activities. Undesirable reflection is to be avoided. v. Bench tops should be impervious to water and resistant to disinfectants, acids, alkalis, organic solvents and moderate heat. vi. Laboratory furniture should be sturdy and open spaces between and under benches, cabinets and equipment should be accessible for cleaning. vii. Storage space must be adequate to hold supplies for immediate use and thus prevent clutter on bench tops and in the aisles. Additional long-term storage space, conveniently located outside and working areas, should also be provided. 31 Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 viii. Wash-basins, with running water, should be provided in each laboratory room, preferably near the exit. ix. Doors should have appropriate fire ratings, be self-closing, and have vision panels. x. There are no specific ventilation requirements. In planning new facilities, consideration should be given for providing a mechanical ventilation system that provides an inward air flow and exhaust without recirculation. If there is no mechanical ventilation, windows should be openable, preferably having fly proof screens. Skylights should be avoided. xi. Drainage exits should be fitted with barriers to prevent entry of arthropods and rodents. xii. Space and facilities should be provided for the safe handling and storage of solvents, radioactive materials and compressed gases. xiii. Safety systems should cover fire, electrical emergencies, emergency shower and eyewash facilities. xiv. First-aid areas or rooms suitably equipped and readily accessible should be available. xv. A good-quality and dependable water supply is essential. There should be no cross-connections between sources for laboratory purposes and the drinking water supply. The public water system must be protected by a back-flow preventer. xvi. A reliable electricity supply with adequate capacity should be available. There should be emergency lighting to permit safe exit. A standby generator with automatic cut-off is desirable for the support of essential equipment-incubators, freezers, etc. xvii. There should be an insect and rodent control measures. xviii. Facilities for storing outer garments and personal items and for eating and drinking should be provided outside the working areas. xix. “No Smoking” “No Eating” and “No Drinking” signs should be displayed clearly inside and outside the laboratory. xx. Access to the laboratory area should be designed to prevent entrance of free-living arthropods and other vermin. B) Safety Equipments i. Pipetting aids-to replace mouth pipetting. ii. Screw-cap tubes and bottles - to provide positive specimen containment. iii. Disposable Pasteur pipettes, whenever available, to avoid glass. iv. Sterile plastic disposable transfer loops and spreader etc. to avoid incineration of regular loops, glass spreader etc. 32 Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 C) Personal Protective Equipment Working in BSL-1 laboratory do not require any Personal Protective Equipment (PPE), although care should be made to avoid spillage of biological material on street clothing for which use of apron is recommended. D) Procedures i. Mouth pipetting should be prohibited. ii. Eating, drinking, storing food, and applying cosmetics should not be permitted in the laboratory work area. iii. Avoid touching various body parts while handling the microorganisms. iv. Wash hand after entering, post work and before leaving the laboratory with sanitizing agents. v. The laboratory should be kept neat, clean and free of materials not pertinent to the work. vi. Work surfaces should be decontaminated at least once a day and after any spill of potentially dangerous material. vii. Members of the staff should wash their hands after working before leaving the laboratory. viii. All technical procedures should be performed in a way that minimizes the creation of aerosols. ix. Laboratory doors would be kept closed when work is in progress. x. Children are not permitted in laboratory working areas. E) Laboratory monitoring i. There should be no unauthorized entry in the laboratory. ii. Only the trained personnel to enter the laboratory. iii. Entry and exit should be limited when work is in progress. iv. Immediately after work, the workplace and the used instruments should be cleaned with a disinfectant and the materials used in work should be placed back to its position. v. No viable cultures are left unattended and either stored or incubated as per need. vi. Record of work should be duly registered in the register available. F) Waste management There is no specific requirement on waste management in BSL-1 facility. However, waste disposal procedure must meet the pollution control requirements. Any effluents from laboratories should be pre-treated and microbiological testing of treated effluents along with record should be available. 33 Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 G) Health and Medical Surveillance These microorganisms are unlikely to cause human or animal diseases of veterinary importance. Ideally, however, staff members should be subjected to a pre-employment health surveillance procedure regarding medical history. Prompt reporting of illness or laboratory accident is desirable and all staff members should be made aware of the importance of maintaining good laboratory safety practice. H) Emergency procedures All spills, accidents and overt or potential exposures to infectious materials should be reported immediately to the laboratory supervisor. A written record should be prepared and maintained. Appropriate medical evaluation, surveillance and treatment should be provided. 3.1.4. Operational Guide for BSL-2 Facility The operational program for BSL-1 laboratory will also apply to Biosafety Level 2 laboratory, with additional modifications as follows: A) Facility design i. An autoclave for decontamination of potentially hazardous laboratory wastes should be available in the same building as the laboratory. ii. Biological safety cabinets for handling of risk-inherent microorganisms of RG 2 should be used. iii. Laboratory may be kept under constant CCTV surveillance. iv. The biohazard warning symbol and sign (Fig. 1) must be displayed on the door(s) of the rooms where microorganisms of RG 2 are handled. Fig. 1 B) Safety instruments i. Autoclaves - to sterilize contaminated material. ii. Biological safety cabinets to be used whenever: a. Procedures with a high potential for creating hazardous aerosols. These may include centrifugation, grinding, blending, vigorous shaking or mixing, sonic disruption, opening containers harbouring hazardous materials whose internal pressure may be different from the ambient pressure, intranasal inoculation of animals, and harvesting infected tissues from animals or eggs. b. High concentrations or large volumes of hazardous microorganisms are handled. Such materials may be centrifuged in the open laboratory if sealed heads or centrifuge safety cups are used and if they are opened only in a biological safety cabinet. 34 Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 C) Personal Protective Equipment i. The use of laboratory coats, gowns or uniforms is required to prevent contamination of street clothing. ii. Goggles and face protection must be used when there is a potential for splashes of microorganisms or other hazardous materials. iii. Face mask and appropriate gloves may be worn as protection while handling animals. iv. Appropriate gloves should be worn for all procedures that may involve accidental direct contact with blood, infectious materials, or infected animals. Gloves should be removed aseptically and autoclaved with other laboratory wastes before disposal. When disposable gloves are not available, re-usable gloves should be used. Upon removal they should be cleaned and disinfected before re-use. v. All PPE should be removed so that the transfer of infectious materials to areas beyond where they or animals are being handled is minimized. vi. Used disposable PPE should be disposed off with other contaminated waste and reusable PPE (i.e., goggles) should be appropriately decontaminated before reuse. vii. Reusable protective clothing should be laundered through laboratory laundry facility only and it must not be taken home. If visibly contaminated, laundry should be placed in a biohazard bag before being placed with other items to go to laundry. D) Procedures i. All contaminated liquid or solid materials should be decontaminated before disposal or reuse; contaminated materials that are to be autoclaved or incinerated at a site away from the laboratory should be placed in durable leak-proof containers, which are closed before being removed from the laboratory. ii. Containers used to collect, handle, process, store, or transport within a facility, potentially infectious materials must be durable, leak-proof and have a lid. The containers must be properly labelled with the contents and a biohazard symbol. iii. Laboratory coats, gowns, or uniforms should be worn in the laboratory; laboratory clothing should not be worn in non-laboratory areas; contaminated clothing should be disinfected by appropriate means. iv. Safety glasses, face shields and other protective devices should be worn to protect eyes and face from splashes and impacting objects. v. Only persons who have been advised of the potential hazards and meet any specific entry requirements (e.g. immunization) should be allowed to enter the laboratory working areas. 35 Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 vi. Hypodermic needles and syringes should not be used as a substitute for automatic pipetting devices in the manipulation of infectious fluids. Cannulas should be used instead of sharp needles wherever possible. vii. Never wear contact lenses when working with infectious microorganisms. viii. Add disinfectant to water baths to contain spread of infectious substances. ix. Use sealed rotors, sealed buckets, or a guard bowl cover complete with gasket as well as safety centrifuge tubes (tube or bottle carrier with sealable cap or “O” ring cap) for potentially infectious samples/otherwise hazardous samples. Before use, tubes should be checked for cracks. x. All technical procedures should be performed to minimize the formation of aerosols and droplets. Whenever there is an increased risk(s) of aerosolization, work should be conducted in a biological safety cabinet. xi. Always use secondary leak-proof containers when transporting samples, cultures, inoculated Petri dishes, and other containers of hazardous microorganisms. Packages containing viable microorganisms must be opened in a facility having an equivalent or higher level of physical containment unless the microorganism is biologically inactivated or incapable of reproduction. E) Laboratory monitoring Monitoring should ensure that: i. Only highly trained personnel are entering in the facility. ii. Person working in the facility are not transporting the laboratory materials including hazardous organism outside the laboratory environment either without permission or without proper transport strategy with prior approval from competent authority. iii. Person working in the laboratory are well aware about the microorganism(s) to be handled and its associated risks. iv. Accidental spill or splashes are cleaned immediately, reported and recorded. F) Waste management Decontamination and disposal mechanism should be in strict adherence to those mentioned in “Decontamination and disposal”. i. Autoclaves and sterilizers for treatment of solid wastes need specially designed accommodation and services. ii. Incinerators may need to be of special design and equipped with after burners and smoke- consuming devices. 36 Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 G) Health and Medical Surveillance i. Pre-employment health surveillance is necessary. This screening should include the past medical history. A clinical examination and the collection of a baseline serum sample would be advantageous and, in some cases, may be necessary. ii. Records of illness and absence should be kept by the facility in-charge and it is the responsibility of the laboratory worker and his own medical officer to keep the facility in-charge informed of all absences due to illness. iii. Women of child-bearing age should be made aware, in unequivocal terms, of the risk(s) to the unborn child of occupational exposures to hazardous microorganisms, such as Rubella, Cytomegalovirus, etc. The precise steps taken to protect the foetus will vary, depending on the microorganisms to which exposure may occur. H) Emergency Procedures Same as BSL-1 3.1.5. Operational Guide for BSL-3 Facility This level of containment requires strengthening of BSL-2 laboratory operational and safety programmes as well as the provision of added structural safeguards and the mandatory use of biological safety cabinets. Therefore, the facility in-charge must first comply with the BSL-2 guidelines and additionally have those specific for BSL-3 facility. The major changes are in: Procedures, Facility design and Health and medical surveillance. Laboratories in this category should be registered or listed with the appropriate national authority(ies). A) Facility design i. The laboratory should be separated from areas that are open to unrestricted traffic flow within the building. Additional separation may be achieved by using a laboratory at the blind end of a corridor, a partition and door, a double-door system where entry to the laboratory should be through an ante-room or airlock. Airlock doors must be self-closing and fitted with seals at the top, bottom and both sides of the door. Airlock doors must contain a viewing panel unless the airlock functions as a shower airlock. Physical mechanisms (e.g., interlocking or alarm system) must be in place to ensure that only one door can be opened at any time. ii. The surfaces of walls, floors, and ceilings should be water resistant and easy to clean. Openings in these surfaces should be sealed to facilitate decontaminating the area. iii. A foot or elbow-operated wash-hand basin should be provided near each laboratory exit door. iv. Windows in the laboratory should be closed and sealed. 37 Regulations and Guidelines on Biosafety of Recombinant DNA Research & Biocontainment 2017 v. Access doors to the laboratory should be self-closing and lockable. vi. An autoclave for decontamination of laboratory wastes should be available within the laboratory. If infectious wastes have to be removed to another area in the same building for disinfection, they should hold and transported in a covered, leak-proof container. vii. There should be a ventilation system that establishes a negative pressure into the laboratory. Personnel must verify that proper direction air flow (into the laboratory) is achieved. viii. The work area must be maintained at an air pressure of at least 50 Pa below the pressure of adjacent areas outside the facility when both doors of the airlock are closed. When either door of the airlock is open, the work area pressure must remain at least 25 Pa below that of adjacent areas outside of the BSL-3 containment barrier. ix. The work area must be equipped to measure and display the pressure difference between the facility and the areas adjacent to the facility. The display must be located so that it can be read immediately before entering the facility. x. The facility must be equipped with an alarm that will alert relevant persons both inside and outside the facility and be immediately activated when the pressure in the facility is more than 25 Pa above the set point. xi. The facility must have an emergency stop button for the ventilation system, which is easily accessible in case of an emergency. The emergency stop button must operate independently of the main ventilation control and main facility pressure control system such that emergency isolation of the ventilation can be implemented in the event of central control malfunction. xii. Supply or replacement air to the facility must have HEPA filtered. xiii. The exhaust air from the facility must pass through a HEPA filter and must be tested by qualified person. The exhaust HEPA filter must be mounted in a gas-tight housing, with sealed access doors and the ductwork between the facility and the HEPA filter housing must also be gas-tight. The design and location of the filter housing must allow for access to and integrity testing of the HEPA filter. xiv. The building exhaust system can be used for this purpose if the exhaust air is not recirculated to other areas of the building. Air within the laboratory can be recirculated. xv. In laboratories that have supply air systems, the supply air and exhaust air systems are interlocked to ensure inward air flow at all times. xvi. The HEPA-filtered exhaust air from Class I and Class II biological safety
