PSB 474- PHYTOBIOTECHNOLOGICAL ETHICS TOPIC: UNIDO/UNEP/WHO/FAO WORKING GROUP GUIDELINES ON BIOSAFETY Introduction, Biosafety is defined as, “The discipline addressing the safe handling and containment of infectious microorganisms and hazardous biological materials”, It is the prevention of large- scale loss of biological integrity, focusing both on ecology and human health. These prevention mechanisms include conduction of regular reviews of the biosafety in laboratory settings, as well as strict guidelines to follow. Biosafety is used to protect from harmful incidents. Biosafety can also be termed as: The application of knowledge, techniques and equipment to prevent personal, laboratory and environmental exposure to potentially infectious agents or biohazards. Biosafety defines the containment conditions under which infectious agents can be safely manipulated, The objective of containment is to confine biohazards and to reduce the potential exposure of the laboratory worker, persons outside of the laboratory, and the environment to potentially infectious agents. A fundamental objective of any biosafety program is the containment of potentially harmful biological agents. The term "containment" is used in describing safe methods, facilities and equipment for managing infectious materials in the laboratory environment where they are being handled or maintained. The purpose of containment is to reduce or eliminate exposure of laboratory workers, other persons, and the outside environment to potentially hazardous agents. The use of vaccines may provide an increased level of personal protection, The risk assessment of the work to be done with a specific agent will determine the appropriate combination of these elements. Many laboratories handling biohazards employ an ongoing risk management assessment and enforcement process for biosafety. Failures to follow such protocols can lead to increased risk of exposure to biohazards or pathogens. Human error and poor technique contribute to unnecessary exposure and compromise the best safeguards set into place for protection: CONTAINMENT PRINCIPLES Safety in the laboratory is achieved by application of layered, containment principles « in accordance with the risk assessment to prevent exposure of laboratory workers 10 pathogen or the inadvertent escape of a pathogen from the microbiological laboratory Safety layers include primary and secondary containment, Primary containment provides immediate protection to workers in the biological laboratory from exposure to chemical and biological hazards, Primary barriers include biological safety cabinets, fume hoods and other engineering devices used by laboratory technicians while working with a biological hazard. Secondary containment is intended to protect the laboratory worker, the community and the environment from unintended contamination with a biological hazard. Secondary containment consists of architectural and mechanical design elements of a facility that prevent worker contamination and escape of pathogens from the laboratory into the environment. Personal protective equipment (PPE) such as gloves, laboratory coats and Chigor’s cxpy |safety glasses may also be considered a primary containment, however, articles worn on the body are considered a last line of defense and are only used in conjunction with other primary and secoridary containment elements when working with pathogenic organisms. Containment is defined in levels that increase in complexity as the risk associated with the work in the microbiological laboratory increases. All containment levels have defined primary and secondary containment features. These levels are described by a series of working practices, applied technologies and facility design built upon from a common foundation, referred to as biosafety level -1 or BSL-1. One important biosafety element suggested for all 4 biosafety levels is biosafety training Provision of basic training in biosafety is considered a best practice and is usually provided by the Biosafety Officer or Occupational Health Specialist to visitors, students and workers in biological laboratories. Specialized training is also required by the Occupational Safety and Health Administration (OSHA) for work that has the potential to expose laboratory employees to human blood or human blood products and for procedures tha of arespirator. it require the use BIOSAFETY LEVELS Biological Safety Levels (BSL) is a series of protections relegated to autoclave-related activities that take place in particular biological labs. They are individual safeguards designed to protect laboratory personnel, as well as the surrounding environment and community These levels, which are ranked from one to four, are selected based on the agents or organisms that are being researched or worked on in any given laboratory setting. For example, a basic lab setting specializing in the research of non lethal agents that pose a minimal potential threat to lab workers and the environment are generally considered BSL- 1—the lowest biosafety lab level. A specialized research laboratory that deals with potentially deadly infectious agents like Ebola would be designated as BSL-4—the hivhest and most stringent level. The Centers for Disease Control and Prevention (CDC) sets BSL lab levels as a way of exhibiting specific controls for the containment of microbes and biological agents. Each BSL lab level builds upon on the previous level—thereby creating layer upon layer of constraints and barriers. These lab levels are determined by the following Risks related to containment Severity of infection Transmissibility Nature of the work conducted Origin of the microbe Agent in question Route of exposureThe reason biosafety levels are so important is because they di i : ey dictate the type of work practices that are allowed to take place in a lab setting. They also heavily influence the over design of the facility in question, as well as the type of specialized safety equipment used within it. The following is an explanation of each biosafety level—what they mean and how they diffe in safety measures and best practices. z: ei hon a citer BSL-1 As the lowest of the four, biosafety level 1 applies to laboratory settings in which personnel work with low-risk microbes that pose little to no threat of infection in healthy adults. An este a microbe that is typically worked with at a BSL-1 is a non-pathogenic strain of E. coli. This laboratory setting typically consists of research taking place on benches without the use of special contaminant equipment. A BSL-1 lab, which is not required to be isolated from surrounding facilities, houses activities that require only standard microbial practices, such as: Mechanical pipetting only (no mouth pipetting allowed) Safe sharps handling Avoidance of splashes or aerosols Daily decontamination of all work surfaces when work is complete Hand washing Prohibition of food, drink and smoking materials in lab setting Personal protective equipment, such as; eye protection, gloves and a lab coat or gown Biohazard signs BSL-1 labs also require immediate decontamination after spills. Infection materials are also decontaminated prior to disposal, generally through the use of an autoclave. BSL-2 ‘This biosafety level covers laboratories that work with agents associated with human diseases (ie. pathogenic or infections organisms) that pose a moderate health hazard. Examples of agents typically worked with in a BSL-2 include equine encephalitis viruses, as well as Staphylococcus aureus (staph infections). Hepatitis B virus, HIV, the salmonellae, and Toxoplasma spp. are representative of microorganisms assigned to this containment level. BSL-2 is appropriate when work is done with any human-derived blood, body iluids, tissues, or primary human cell lines where the presence of an infectious agent may be unknown, BSL-2 laboratories maintain the same standard microbial practices as BSL-I labs, but also include enhanced measures due to the potential risk of the aforementioned microbes Personnel working in BSL-2 labs are expected to take even greater care to prevent injuries such as cuts and other breaches of the skin, as well as ingestion and mucous membrane exposures. In addition to BSL 1 expectation, the following practices are required in a BSL 2 lab setting 3Appropriate personal protective equipment (PPE) must be worn, including lab coats and sloves. Eye protection and face shields can also be worn, as needed. All procedures that can cause infection from aerosols or splashes are performed within a biological safety cabinet (BSC), An autoclave or an alternative method of decontamination is available for proper disposa The laboratory has self-closing, lockable doors. A sink and eyewash station should be readily available. Biohazard warning signs Access to a BSL-2 lab is far mor I e restrictive than a BSL-1 lab. Outside personnel, or those with an increased risk of cont: ‘amination, are often restricted from entering when work is being conducted. BSL-3 Again building upon the two prior biosafety levels, a BSL-3 laboratory typically include work on microbes that are either indigenous or exotic, and can cause serious or potentially lethal disease through inhalation. Examples of microbes worked with in a BSL.3 includes; yellow fever, West Nile virus, and the bacteria that causes tuberculosis, Mycobacterium tuberculosis, The microbes are so serious that the work is often strictly controlled and registered with the appropriate government agencies. Laboratory personnel are also under medical surveillance and could receive immunizations for microbes they work with. Common requirements in a BSL-3 laboratory include: Standard personal protective equipment must be wom, and respirators might be required Solid-front wraparound gowns, scrub suits or coveralls ate often required All work with microbes must be performed within an appropriate BSC A Sustained directional airflow to draw air into the laboratory from clean areas towards potentially contaminated areas (Exhaust air cannot be re-circulated) 'ss hands-free sink and eyewash are available near the exit A self closing set of locking doors with access away from general building corridors Access to a BSL-3 laboratory is restricted and controlled at all BSL-4 BSL-4 labs are rare. However some do exist in a small number of places in the US and around the world. As the highest level of biological safety, a BSL-4 lab consists of work with highly dangerous and exotic microbes. Infections caused by these 'ypes of nee a frequently fatal, and come without treatment or vaccines, Two examples of such mic include Ebola and Marburg viruses following container In addition to BSL-3 considerations, BSL-4 laboratories have the following requirements: abPersonnel are required to change clothin, 8 before entering, shower upon exiting Decontamination of all materials before exiting Personnel must wear appropriate personal protective e: ‘well as a full body, air-supplied, positive pressure suit A Class III biotogical safety cabinet -quipment from prior BSL levels, as A BSL-4 laboratory is extremely isolated—ofien located in a separate building or in an 'solated and restricted zone of the building. The laboratory also features a dedicated supply and exhaust air, as well as vacuum lines and decontamination systems Knowing the difference in biosafety lab levels and their corresponding safety requirements is imperative for anyone working with microbes in a lab setting, The laboratory director is specifically and primarily responsible for the safe operation of the laboratory. His/her knowledge and judgment are critical in assessing risks and appropriately applying these recommendations. The recommended biosafety level represents those conditions under whieh the agent can ordinarily be safely handled, Special characteristics of the agents used, the training and experience of personnel, procedures being conducted and the nature or function of the laboratory may further influence the director in applying these recommendations. No Containment: tfoxconainmene Defined organisms > It gives countries that do-not yet have domestic biosafety regulations in place a legal basis and a methodology to make informed decisions on import of Living Modified Organisms (LMOs). It contributes to intemational harmonisation of national biosafety regulations by international agreement on some of the key elements of domestic biosafety regulations such as definitions, information requirements, principles and methodology on risk assessment, treatment of confidential information etc > It contains a crucially important mechanism for international information exchange on biosafety through its Biosafety Clearing House (BCH3). Inth , this Manual provides practical guidance for national authorities that are in the process of developing national biosafety regulations: to is | contains an overview of the broader international context relevant to biosafety ParPart Tl contains Guidance on what is needed to. establi is capacities for the development and/or trade of LMOs, ee ne I contains guidance on some’ practical aspects when applying the procedures of the B import of LMOs for intentional releases: This version of the Manual, which is curently ¥eing used-in UNIO the distant leaming biosafety programme, contains Part I and Part Ill. In the Autumn of 2010 Part II will be s mpleted and be published on the UNIDO website together with updates of Part I and Part The Food and Agriculture Organization (PAO) The Food and Agriculture Organization (FAO) mandate was created in 1945 to lead intemational efforts to defeat hunger. Serving both developed and developing countries, FAQ acts as a neutral forum whete all-nations meetias equals to-negotiate-agreements and debate policy. FAO also assists developing countries and countries in transition fo modernize and improve agriculture, forestry and fisheries practices énd-ensure, good nutrition for all. Since its founding, FAO has focused special'dttention on developing rural areas, home to 70 percent of the world's poor and hungry people. FAO's ioverall is to-raise levels of nutrition, improve agricultural produetivity, better the lives of rural populations and contribute to the growth of the world economy, The Committee on. Agriculture (COAG) in-1999-recommended FAO to develop a strategic approach in biotechnology-and biosafety. The resulting FAO Statement on Biotechnology, which was published in March 2000, iclides, among others, the following points Biotechnology provides -powerfil tools. for ‘the: sustainable development of agriculture, ficheries and forestry, as well'as the food industry. When appropristely integrated with other technologies for the production of food, agricultural products and services, biotechnology can te of significant assistance in meeting the needs of an expanding and increasingly urbanized population in the next millennium. Genetic engineering could lead to higher yields on imaginal lands in countries that tday cannot grow enough food to feed their people. FAO is also aware of the ¢ottcern aboutthe potential risks posed by certain aspects of biotechnology. FAO supports science-based evaluation:system that would objectively determine the benefits and risks of each individual LMO, This'calls for a cautious case-by-case approach to address legitimate concerns for the biosafety'of each product or process prior 10 ts release PAO considers that efforts:should be made to ensire that developing countries, in general, and resource-poot farmers, in particular, benefit moze from biotechnological research, while continuing to have access to @ diversity of sources of genetic material.WHO Biosafety Guidelines WI orld: Health i WHO (World Health Organisation) has long recognized that safety and, in particular, ological safety are i > H ; gical ‘ure important international issues. WHO. published ‘the first edition of the Aboratory biose! ety ‘manual in 1983. The manval’ encouraged countries to accept and implement basic concepts in biological safety and to develop national codes of practice for the safe handling of pathogenic microorganisms in laboratories within their geographical Dorders. Since 1983, many countries have used the-expert guidance provided in the manual to develop such codes of practice, A second edition ofithe mantial swas published in 1993. ues to provide international leadership in biosafety through this third edition of nal by addressing biological ‘safety and. security. issues facing us in the current millennium. The third edition stresses throughout the importatice of personal responsibility Recent world events have revealed new threats to,public:health through deliberate misuse and release of microbiological agents, and’ toxins;:“The ‘third. edition’ therefore also introduces (biosecurity concepts ~ the protect which n of fiierobiglogical assets from theft; loss or divers uld lead to the inappropriate use of these agents to cause public health harm. The third edition of the WHO Laboratory biosafety matual is a kelpful reference and guide to nations that accept the challenge to develop and establish national codes of practice for sécuring microbiological assets, yet ensuring their availability for clinical, research and ft epidemiological purposes. f Few of WHO Biosafety guidelines: ! 1, Waste handling f Waste is anything that is to be discardéd. In laboratories; decontamination of wastes and their ultimate disposal are closely’ isterrélated. In terms of daily use, few if any contaminated materials will require actual removal from the laboratory or destruction, Most glassware, instruments and laboratory clothing will be reused or recycled, The overriding principle is that all infectious materials should be decontaminated, autoclaved or incinerated within the laboratory. The principal questions to be asked before discharge of any objects or from laboratories that deal with-potentially infectious microorganisms or animal tissues are: 1. Have the objects or materials been effectively decontaminated or disinfected by approved procedure? 2, If not, have they been packaged in an approved manner for | immediate on-site incineration or transfer to another facility with incineration capacity? 3. j Does the disposal of the decontéminated objects or materials involve any additional potential hazards, biological or otherwise, to those who carry out the immediate disposal procedures or who might come into contact with discarded items outside the facility? rials —2. Decontamination: Steam autoclaving is the preferred method for all decontamination (processes. Materials for “scontamination and disposal should be placed in containers, e.g. aut6clavable plastic bags, that are colour-coded according to whether, the ‘contents ate to be autoclaved and/or Alternative methods may be envisaged only if they remove and/or kill jctoorganisms inciner; 3. Biosafety and recombinant DNA technology Recombinant DNA technology involves combining genetic material from different sources thereby creating genetically modified organisms. (GMOs) that may have never existed in nature before Initially there was concer among molecula biologists that such organisms might have unpredictable and undesirable properties that could represent.a:biohazard if they escaped from:the laboratory. This concern became the focus.of a scientific conferghice-held in Asilomar, CA, USA, in 1975. At that meeting, safety issues were discussed and the first-guidelines for recombinant DNA technology were proposed, The subsequent 25+ years. of research experionce ‘have’ deitionstrated that genetic engineering may be conducted in a’safe manner when an appropriate risk asdessmient is performed and adequate safety measiires are ised, Recombitiant-DNA technology or genetic engineering was first used to clone DNA segments in bacterial hosts in orde? to overexpress specific gene products for further studies. Recombinant DNA molecules have also beet ised to‘ereate GMOs sueh as transgenic and “knock out” animals end transgenic plants, Recombinant DNA technology has already had an enormous impact on biology and medicine, and will probably have an even greater influence now that the nucleotide sequence of the entire human genome is available, Tens of thousands of genes of yet unknown functions will-be studied:using recombinanDNA technology, Gene therapy may become a routine treatment for certdin diseases, andtew Veot0rS for ene transfer ate likely to be devised using genetic engineering techniques: Also, transgenic plants: produced ‘by recombinant DNA technology may play an increasingly important role in modem agricultuie. Experiments involving the construction or use of GMOs should be coniduéted after performing a biosafety risk assessment, The pathogenic properties and’aniy potential hazards associated with such organisms may be novel and not ‘well-characterized, The properties of the: donor organism, the nature of the DNA sequences that will be transferred, the properties of the recipient onganism, and the properties of the environment should be evaluated, These factors should help determine the biosafety level thet is required forthe safe handling of the resulting GMO, and identify the biological and physical containment systems that should be used.Transgenic and “knock-out” animals Animals carrying foreign genetic material (transgenic animals) should be handled in containment levels appropriate to the characteristics of the products of the ‘oreign genes. Animals with targeted deletions of specific genes (“knock-out” animals) do not Senerally present particular biological hazards, Examples of transgenic animals include animals “eptors for viruses normally unable to infect that species. If such animals escaped from he laboratory and it i i the laboratory and transmitted the transgene to the wild animal population, an animal reservoir for that “ius could theoretically be generated. This: possibility has béen discussed for poliovirus “sscularly relevant in the context of poliomyelitis eradication, Tyansgenie mice expressing the Poliovirus * 103 + receptor generated in different laboratories were susceptible to poliovirus ‘isct'cn Sy various inoculation routes and the resulting disease. was clinically and histopathologically simi to human poliomyelitis, However, the mouse: ‘model differs from humans in that alimentary ‘act replication of orally administered poliovirus'is eiter'inefficient or’doesinot occur. It s therefore very unlikely that escape of such transgenic mice to the wild would result in the establishment of a new animal reservoir for poliovirus. Nevertheless; this‘example indicates that, for each new line of ‘transgenic animal, detailed studies should be conducted to-determine the routes by which the animals can be infected, the inoculum size required for infection, and the extent.of virus shedding by the infected animals. In addition, all measures should'be:tiken to assure, strict containment of receptor transgenic mice. Transgenic plants: Transgenic plants expressing genes that confer tolerance to herbicides or resistance {0 inseots are currently a matter’ of considerable controversy in many parts of the world. The discussions focus on the food-safety of such plants, and gn the long-term ecological consequences of their cultivation. Transgenic plants expressing genes of animal or human origin are used to develop medicinal and nutritional products. A tisk assessnient should determine.the appropriate biosafety level for the production of these plants. Risk assessments for genetically modified organisms Risk assessments for work with GMOs should consider the characteristics of donor and recipient/host organisms. Examples of characteristics for consideration include the following. Hazards erising directly from the inserted gene (donor organism) Assessment is necessary in situations where the product of the inserted gene has known biologically or pharmacologically active properties that may give rise to harm, for example: 1, Toxins 2. Cytokines 3. Hormones 4. Gene expression regulators 5 ‘Virulence factors or enhaneers’6, Oncogeni¢ gene sequences 7, Antibiotic resistance 8. Allergens. The consideration of such cases should include an estimation of the level of expression required to achieve biological or pharmacological activity. Hazards associated with the recipient/host 1. Susceptibility of the host 2. Pathogenicity of the host strain, including virulence, infectivity and toxin production 3 Modification of the host range 4. Recipient immune status 5, Consequences of exposure. Hazards arising from the alteration of existing pathogenic traits Many modifications do not involve genes whose products are inherently harmful, but adverse effects may arise as the result of alteration ofexisting | i ing non-pathogenic or pathogenic traits, Modification ‘of n ‘o identify these potential hazards, ‘not exhaustive). Sue ‘ormal genes may alter pathogenicity, ae : the following points. may be considered (the list is 18 Nate an inerease in infectivity or pathogenicity? 2. Could any disabling Within the recipie "e ecipient be overcome as a result of the insertion ofthe foreign gene? 3. Does the © evleode a ps mutation ‘thogenicity determinant from another organism? 4, Ifthe foreign DNA does enicity determinant, is it foreseeable that this gene could contribute to the ‘y of the GMO? 5. Is treatment available? ‘6, Will the susceptibility of the GMO to ics Or other * forms of therapy be affected as a consequence of the genetic modification? 7. Is ' GMO achievable? Further considerations The use of whole animals or plants for experimental purposes also requires careful consideration, Investigators must comply with the ctions and requirements for the conduct of work with GMOs in host countries and institutions, Countries may have national authorities that establish guidelines for work with GMOs, regulations, re and may help scientists classify their work at the appropriate ‘biosafety level. In some cases classification may differ between countries, or countries may decide to classify work at @ lower or higher level when new information on/a particular-vector/: host system becomes available. Risk assessment is a dynamic process that takes into account new developments and the progress of science. The performance of appropriate risk assessments‘ will assure that the benefits of recombinant DNA technology remain available to humankind in the years to come. 4. Chemical spills Most manufacturers of laboratory chemicals issue charts describing methods for dealing with spills. Spillage charts and spillage Kits are also available commereially. Appropriate charts should be displayed in a prominent position in the laboratory. The following equipment should also be provided: 1. Chemical spill kits 2. Protective clothing, e.g. heavy-duty rubber gloves, overshoes or rubber boots, respirators 3. Scoops and dustpans 4. Forceps for picking up broken glass 5. Mops, cloths and paper towels 6. Buckets + 109 + 7. Soda ash (sodium carbonate, Na2 COs) or sodium bicarbonate (NaHCOs) for neutralizing acids and corrosive chemicals 8. Sand (to cover alkali spills) 9. Non-flammable detergent. The following actions should be taken in the event of a significant chemical spill. 1. Notify the appropriate safety officer. 2. Evacuate non-essential personnel from the area, 3. Attend to persons who may have been contaminated. 4. If the spilled material is flammable, extinguish all open flames, tum off gas in the room and adjacent areas, open windows (if possible), and' switch off electrical equipment that may spark. 5. Avoid breathing vapour from spilled material. 6. Establish exhaust ventilation if it is safe to do so. 7. Secure the necessary items (see above) to clean up the spi