TY - JOUR
AU - Connell,, Nancy
AB - Abstract Research with animals presents a wide array of hazards, some of which overlap those in the in vitro research laboratory. The challenge for environmental health and safety professionals when making their recommendations and performing the risk assessment is to balance worker safety with animal safety/welfare. The care and husbandry of animals require procedures and tasks that create aerosols and involve metabolized chemicals and a variety of physical hazards that must be assessed in addition to the research related risks, all while balancing the biosecurity of the facility and NIH animal care requirements. Detailed communication between health and safety, research, and animal care teams is essential to understand how to mitigate the risks that are present and if modifications need to be made as the experiments and processes progress and change over time. Additionally, the backgrounds and education levels of the persons involved in animal research and husbandry can be quite broad; the training programs created need to reflect this. Active learning and hands-on training are extremely beneficial for all staff involved in this field. Certain areas of research, such as infectious disease research in high- and maximum-containment (biosafety level 3 and 4) facilities, present challenges that are not seen in lower containment or chemical exposure experiments. This paper reviews potential hazards and mitigation strategies and discusses unique challenges for safety at all biosafety levels. animal, biosafety, hazard, risk assessment, biosafety level three Introduction Research with animals presents a wide range of hazards, some of which are present in the in vitro research laboratory and many are unique to vivarium-related operations. Additionally, there are multiple hazards within one experiment, even within one research animal. The challenge for environmental health and safety (EHS) professionals when making their recommendations and performing risk assessment is to balance worker safety with animal safety/welfare. EHS staff need to communicate with veterinary staff to understand the National Institutes of Health (NIH) and United States Department of Agriculture requirements and Association for Assessment and Accreditation of Animal Laboratory Care guidelines for the animals to help inform their recommendations. Animal research can involve chemical, biological, radiological, and physical hazards. In addition, some environments add complexity, such as working in high-containment laboratories, where this balance is even finer and a decision in favor of animal welfare might potentially hinder safety and vice versa. Laboratory safety within animal facilities is not a new topic, but as research technologies change, the protections and reviews must also evolve. Many EHS departments review Institutional Animal Care and Use Committee (IACUC) protocols to provide guidance on worker protection, animal housing, and waste disposal in addition to other procedures involved in the research plan. This paper will cover briefly the general hazards present in animal facilities, both research and husbandry related. The paper will then discuss in more detail how certain hazards are amplified in the setting of high-containment research, where many of the general hazard concerns are present but are supplemented by additional requirements for personal protective equipment (PPE) and understanding of engineering controls. An even finer balance between worker safety and animal welfare must be addressed and implemented in high- and maximum-containment settings. Many of the biosecurity practices that are implemented for the safety of workers and the public also protect the animals. As we discuss these safety measures, we will also examine how these approaches can assist with biosecurity and the prevention of animal infections within facilities. Overview of Hazards and Hazard Mitigation Research with animals typically involves potential risks from certain materials that may be higher due to additional factors that need to be mitigated and harder to control (e.g., movement of animals, expertise levels of workers). These hazards fall into general categories including chemical, biological, physical, and radiological hazards. Many experiments include one or more of these categories, so risk assessments must balance differing risk profiles, presence of multiple hazards, and the best method for worker protection. Use of Chemicals in Research In many animal experimental programs, researchers are studying the effect of chemicals on organ systems, development, and disease treatment, or their ability to cause a certain ailment. Chemical hazards also involve the use of anesthetic gases and drugs used for euthanasia. Many of these drugs are also listed as controlled substances, which require a balance between safety and security. Toxin work is also common. Diphtheria toxin is used in many mouse models to induce a certain phenotype but can cause intoxication in humans if exposed.1 Botulinum toxin and tetrodotoxin are common in physiology laboratories2–4 but are very potent toxins, requiring oversight for safety and proper inactivation, the total quantity in possession by each investigator, and security/access control under the US Federal Select Agent Program.5 In most facilities, animal facility personnel will encounter hazards above and beyond those involved in a research protocol. Cleaning agents and disinfectants used to clean floors, walls, and cages can be very hazardous to workers and require additional precautions such as PPE to protect against splashes and spills. Large disinfectant mixing stations and connections for cage wash detergents are a high hazard area due to the large volume of chemicals used by the cage wash systems; animal facilities may contain hundreds of gallons of chemicals such as acids and bases. Risk assessments and the analysis performed for work with hazardous chemicals will include potential metabolism of these compounds, and the control measures address excretion of neat material, potential toxicity of metabolites, duration of the excretion and hazard presence, and the overall hazardous profile. Once collected, this information is used to compile appropriate guidance for researchers and animal care staff regarding waste disposal for bedding and carcasses, engineering controls needed for animal handling and cage changing, and PPE required for staff. Depending on the chemical hazard, the doses used, the duration of treatments over the life of the animal, and availability of metabolism data, guidance must be implemented for the duration of the study. For some chemicals, the guidance will be active for a certain number of days post treatment (e.g., tamoxifen).6 Use of Biological Materials in Research Biomedical research depends heavily on the use of animal models. Continuing investigation of developmental processes, injury, cancer development, immune response to infection, and development of new drugs and vaccinations is not possible without animal research; some approaches utilize microorganisms to accomplish these goals. Thus, in addition to traditional infectious disease and immunology models involving replication-competent bacteria, viruses, fungi, and parasites, replication-incompetent virus use is universal as a means for treatment delivery in cancer models, genomic editing, and labeling of cells in vivo.7,8 One inherent risk of certain models used in animal research is exposure of the researcher to animal allergens and zoonotic disease. Engineering controls are the best method to protect against allergen exposure and other biological hazards present in research protocols. The most commonly used device is the biological safety cabinet, of which there are many models and types4. In vivarium areas there are also downdraft tables, high efficiency particulate air (HEPA)-filtered change stations, cage dumping stations, and enclosure devices for aerosol-generating equipment. Risk assessment of protocols involving exposure is used to balance the value of the animal model with the potential hazard of the biological materials invovled.4 In some cases, engineering controls may not be possible or may not be needed due to small volume and low exposure risk, as is the case, for example, with stereotaxic injection methods. Stereotaxic injection involves the use of bulky equipment and often involves the use of microscopes, which precludes the use of biosafety cabinets. Depending on the risk assessment, a researcher can perform work with risk group (RG2) materials in these settings without a primary containment device. Zoonotic disease is a primary concern working with larger animals, such as sheep, cattle, and nonhuman primates. Animals that are otherwise healthy can carry endogenous infections, so an animal bite or scratch in a noninfectious disease model can present an exposure concern. All researchers working with potentially infectious materials require hands-on training that includes training to recognize signs and symptoms of potential infection. Training should include topics such as animal handling, infection models, restraint, use of engineering controls, and necropsy procedures. Husbandry staff should receive detailed training on cage handling, animal handling, and waste processing. High-containment (biosafety level 3 [BSL-3]) and maximum containment (BSL-4) laboratories require detailed understanding of the risks posed by microorganisms in the laboratory and extensive training on animal handling and manipulation of materials within high containment. Additionally, in BSL-3/4 facility work, sterilization is required for all materials leaving the facility, whereas BSL-1/2 locations may collect items to dispose as medical waste or incineration, and some materials may enter the general waste stream.4 Waste-handling requirements may vary and be more flexible at lower biosafety levels. Use of Radiological Materials in Research The use of radiological materials in animal research is not as prevalent as in years past, but the use of irradiators is very common. Irradiators present both safety and security concerns; close oversight is required along with implementation of a personnel reliability program.9 The use of irradiators must consider the time needed for the exposure and animal welfare, as it is not possible to provide additional oxygen into that environment. If there is a problem with opening the irradiator door, for example, the animals might be subjected to oxygen loss. Emergency plans and training are necessary to inform all users of response actions for these types of emergencies. During protocol review, the duration of exposure and number of animals must be evaluated to ensure there are no welfare concerns during the procedures. The use of radiolabeled materials in animal models is less prevalent in recent years, but review by a radiation safety officer must be conducted to ensure proper personnel protection during any experiment with radiolabeled substances, including the animal housing process and waste handling/disposal for bedding, caging, and carcasses. These materials need to decay and cannot be treated/disposed as chemicals and biological hazards, so procedures can be more complicated. Imaging systems used in animal facilities, such as PET or MicroCT systems, involve review and approval by the radiation safety personnel as well to ensure compliance for the radiation present in these systems and protection of the researchers and animals.10 Overview of Physical Hazards Physical hazards are prevalent within the animal research environment both for the researchers and the animal care staff during husbandry and cleaning. Many researchers use animal models to understand the effects of trauma, blast injuries, spinal cord injuries, etc. Depending on the model, there can be noise created, devices used to simulate injury, and other considerations for ergonomics, cleaning, and noise level evaluation for the researchers and animal care staff. Physical hazards also include noises from larger animals, such as dogs and pigs; the barking, snorting, or other sounds will echo in the rooms and can easily exceed Occupational Safety and Health Administration (OSHA) levels. Monitoring of these areas will determine whether persons need to be enrolled in the entity hearing protection program while in the space.11 Cage wash areas also require noise monitoring and may require staff to wear hearing protection if the decibel levels exceed acceptable limits. Not all cage wash or large animal rooms may require hearing protection, and some workers may not be required to use hearing protection based on their tasks and time in the area, based on OSHA requirements.11 It is crucial to engage the department of health and safety for proper hazard evaluation and assessment in this arena to ensure documentation and proper mitigation of the noise hazard. Cage washers and autoclaves can present burn risks to personnel from hot water and hot cages, so workers need access to appropriate footwear, gloves, and other PPE for protection from these hazards. Training is a crucial component to ensure workers are confident in the operation of all equipment to avoid injury during use of large heavy doors, loading apparatuses, and unloading clean cages and autoclaved racks. Finally, ergonomic issues are highly prevalent throughout animal care areas, especially for the husbandry staff, due to repetitive motions; bending, lifting, and other movements are required to handle animal cages and caging racks. In high-containment areas, the PPE can compound potential hazards as it may be difficult to reach animals in higher and lower cages. When possible, devices that push forward the rear wall of the cage to help move the animal closer are preferred, which minimize repetitive bending and reaching. Hazard Mitigation Hazard analysis and the development of mitigation strategies are performed by EHS staff in many industries for a wide range of work requirements. Hazard mitigation strategies are designed specifically for the hazard and the job classification and employ a combination of controls: elimination, substitution, engineering, work practice, administrative controls, and PPE. Animal research is extremely complex, and often experiments will include a combination of chemical, biological, radiological, and physical hazards. The risk assessment must include the hazards presented, the controls mentioned above, and the specifics of the research. Animal husbandry presents additional hazards that are addressed to ensure appropriate protections are in place to prevent exposures or injuries to personnel. Risk assessment is an ongoing and daily process, as new experiments and processes are added into a research portfolio. Engineering controls in an animal facility include chemical fume hoods, biological safety cabinets (BSC), downdraft tables, cage changing, and animal transfer stations. HEPA-filtered enclosure devices for larger equipment (especially in high and maximum containment facilities) and ventilated animal cage racks are also very common and necessary for worker protection. Animal facilities can have a mixture of these cabinets, depending on the original design and the type of research being conducted. For biological work, Class II cabinets are the most common for animal biosafety level (ABSL) 1 and ABSL-2 facilities, including both the Type A2 and B1 or B2 cabinets. Newer facilities tend to favor installation of Class II, Type B cabinets since they can provide researchers simultaneous protection against biological, chemical, and radionuclide hazards and offer product protection, unlike a chemical fume hood.4,12,13 It is important to ensure animal facility personnel receive detailed training on the engineering controls present and that they understand how the types of biological safety cabinets differ and what materials can be used in them. As a specific example, workers should understand that a Class II Type B2 cabinet should not be turned off if it is the only exhaust route in a room, because it exhausts a large amount of air; turning it off may alter room pressures and balance14,15 The BSC market has many options for engineering controls designed with special features for animal research facilities. It is important when choosing an engineering control to have detailed communication between the department of EHS, research oversight, and animal facility management to ensure that a system is appropriate for the needs of the work and for protection of workers from animal allergens and any hazardous materials. Some BSCs have features to assist with husbandry operations, such as drop-open panels for food and bedding, but these conveniences might not be useful for researchers with respect to cleaning requirements, clutter, or workspace for experimental materials. In an environment with limited funding and/or need for broad programmatic use, simpler approaches may be preferred. Similar challenges are found with caging systems. There are many options, including open, filtered, or ventilated caging. Positive pressure caging should not be used for animals dosed with hazardous chemicals or infected with microorganisms.16,17 Negative pressure caging is typically found in high- and maximum-containment facilities as well as many areas designated as BSL-2. Administrative controls include facility inspections, medical clearance, and training. Training is a crucial aspect for the safety of all persons working with and around animals. The training offered must meet the needs and educational levels of the trainees. Researchers will receive training on the general health and safety, care and use of animals, and the specific methodologies of their research protocols. Training for researchers includes use of specialized PPE (including respiratory protection), agent-specific information, emergency response, and detailed safety training for higher hazard work. Animal care staff must be trained for many additional chemical and physical hazards that the researchers might not encounter. Additionally, members of the animal care staff may not have detailed backgrounds in biology, chemistry, and other sciences, so the training must be designed in a way that is accessible and presents comfortable opportunity for asking questions. Training is always a challenge: there are many demands on the time of all involved, so methods to decrease the length of the training but increase retention are crucial.18,19 Active learning, hands-on training, and mentored training on site are all ways to increase participation and engagement and ensure focus, understanding, and retention of safety-related information.18,20 While signage and access to standard operating procedures remain available as resources and reminders, active and engaged learning has been demonstrated to lead to increased retention and deeper understanding.20 Good work practices are critical for 2 aspects of research: safety and reproducibility. Many aspects of safe research overlap with qualities of good work practices, especially in the biological arena. Improper use of engineering controls can lead to contamination and worker exposure. Researchers will often add new models and procedures but do not indicate how they are learning to perform these experiments; indeed, proper work practices are most often taught by hands-on, mentored training. Depending on the procedure and hazard involved, the IACUC, institutional biosafety committee, and/or veterinarians should be involved in training and oversight to approve and monitor work practices used. This kind of collaboration also helps to create communication among workers at all levels, which leads to a positive culture of safety and compliance. Animal care staff should understand good work practices both for their protection and for animal welfare. Hazard guidance is helpful, but explanation, discussion, and, most importantly, exercising of the application of information to daily work requirements is necessary. Training by senior staff within the animal care program is crucial for proper implementation of evolving or new procedures. There are many different types of PPE used in animal research. Standard garb includes disposable rear-closing gowns or lab coats, booties, hair bonnets, gloves, face masks, and respirators. PPE is also required for all work with animals and will change depending on the experiment and potential hazards involved. Allergen exposure by the aerosol route is a concern with all animal-handling processes and mitigation by use of engineering controls is the first line protection, but respirators should be used as a backup method of protection for certain aerosol-generating activities.21–23 Other PPE (hair bonnets, covering of skin) and policies on laboratory attire (e.g., dedicated uniforms or mandatory clothing changes) protect against skin exposure to allergens.17,21,23 For facilities performing decontamination (discussed in more detail below), it is critical that detailed discussions occur with EHS professionals to assess potential exposures to the chemicals employed in this process to ensure proper PPE is used. Disinfectants used for this process may have low exposure limits, and based on the risk assessment, a respirator with a chemical cartridge may be required. N95 respirators, which are very common in animal facilities for allergen prevention, are not sufficient to protect against chemical inhalation. Any use of respiratory protection must be included in the entity respiratory protection program, and individuals must be medically cleared, trained, and fit-tested. Additionally, staff must ensure they are wearing appropriate gloves, gowns, and other PPE to prevent exposure to skin and mucous membranes during these tasks. Eye protection is critical and should always be used in the animal facility by care staff and researchers. Additionally, face protection in the way of goggles or face shields is used for procedures that could produce splashes or sprays, such as cleaning the floor in a large animal room or working on a downdraft table; for some kinds of fine droplet exposure, such as during the use of high-pressure hosing, more tight-fitting eye protection is appropriate. Other clothing that offers water and chemical resistance is used in larger cleaning procedures, mainly to protect clothing and skin from spray. Proper foot attire is important during cleaning processes in cage wash and other areas with water to prevent slips, trips and falls, and burns.24 Disinfectants, cage wash chemicals, and other cleaning solvents can be hazardous to the worker and may require special gloves or PPE when handling and moving materials.24 As with all hazards, it is crucial for animal care and the department of health and safety to discuss and evaluate each process and to choose the most appropriate clothing on a case-by-case basis. Since there are so many different processes and potential requirements, a matrix or other simplified reference diagram is useful for staff to comply with the requirements in an easy-to-follow mechanism. Special Considerations for Animal Research Animal research relies on close collaboration among the veterinarians, researchers, safety professionals, and the oversight bodies to ensure safe conduct of research with the welfare of the animals in mind. The nature of many animal experiments is to simulate a human condition, and the experiments likely include multiple chemicals, biological, physical, and/or radiological hazards. Additionally, animal husbandry and biosecurity concerns also require cleaning, disinfection, and other processes that are unique to animal research but require hazard assessments of their own. Many IACUCs include representative(s) from their health and safety programs to review the applications and provide guidance on waste disposal, experimental procedures, and other safety requirements. It is common for animal facilities to have a 1-page hazard sign on the outer door to a procedure room to provide information regarding the PPE requirements, engineering controls, and waste-handling procedures. Communication is important since experiments are fluid, equipment may fail, and alternatives need to be in place. Additionally, it is important to foster open communication so that in the event someone does not follow the proper hazard guidance, follow-up and additional training can be performed. The next section will cover, in more detail, some special issues that arise during the day-to-day operations of an animal research program. Carcass and Waste Disposal Hazard review and waste management are everyday concerns for those working in an animal facility and are common topics of conversation among safety professionals. Hazardous waste production is commonplace during these experiments, including hazardous carcasses, which require special disposal methods. Mixed hazardous wastes (e.g., chemical/biological) are a commonplace occurrence for an in vitro setting, but a mixed hazard carcass is not as easily disposed, since many hazardous waste vendors do not accept them for removal. The most important factor when determining how to handle hazardous wastes and mixed wastes is communication. There are multiple people within an institution, and even within the health and safety department, that will be knowledgeable about how to handle this situation. Depending on state laws and Environmental Protection Agency-listed wastes, medical waste disposal is an option, which greatly simplifies matters, as items are placed into medical waste containers instead of having to be segregated into multiple containers. It is important to know how the animal facility segregates and disposes of carcasses, and other options should be available if a new experiment is proposed that will not fit into the existing framework. In addition to carcasses, chemicals are often administered in drinking water, and that waste cannot be poured down the drain; appropriate signage provides disposal guidance and procedures for how to clean the drinking water bottles. Frequently, this kind of material is collected as liquid hazardous waste. Some materials are not always deemed hazardous and have a time period in which they are treated as hazardous, but subsequently, the experiment/cages and animals are downgraded and treated as nonhazardous. A good example of this approach is when animals are exposed to a viral vector for gene delivery or cell labeling. Most viral vectors are replication-incompetent: they enter the target cell but do initiate a new round of replication to produce new virus particles. Each viral expression system must be evaluated to determine an appropriate timeframe during which all viruses will have infected the target cells and have integrated or expressed the genes, as required by the particular experiment. Furthermore, virus shedding can vary according to route of administration and animal model. After a designated time period, carefully determined by experimentation and approved by the Institutional Biosafety Committee, when no further shedding of virus will occur, the animals are moved to a new clean cage and are deemed no longer potentially hazardous.25,26 Depending on the entity and processes in place, some may choose to base the guidance on the individual hazard, but this kind of ad hoc approach can be confusing to the animal care technicians, and, in practice, the animals are not moved to new cages until normal cage change time to keep costs lower. In many cases, entities choose to streamline the guidance for hazard identification, providing uniform guidance for downgrading a particular hazard (e.g., all viral vector cages are hazardous for 7 days post infection).25,26 For many types of research models, especially those involving genomic editing and the use of transgenic animals, the risk to persons is diminished during cage change or is not present at any time; however, the carcass will require disposal as medical waste since there was a genetic modification to the animal. This consideration is determined on an ad hoc basis and will be provided in hazard assessment to the animal care staff. Many animal programs dispose of all animal carcasses as medical waste as a general precaution. Several approaches are used in studies involving tumor models. Human cell lines are classified by OSHA as potentially infectious, unless formally characterized to be free from bloodborne pathogens and include documentation from a biosafety professional or scientific expert documenting a risk assessment; they are traditionally handled according to BSL-2 guidelines. Some tumor cell lines have been well characterized, and such documentation is available including data indicating that cells are shed by the animal (e.g., nonmetastatic tumor studies). In these cases, institutions may downgrade the animal, caging, and overall experiment to BSL-1 guidelines based on risk assessment. Other entities will maintain BSL-2 practices for the duration of the experiment to provide consistency to the husbandry staff. These carcasses will require medical waste disposal, and sometimes autoclaving, to ensure that no risks are present upon disposal. Caging Considerations: Clean Rooms vs. Infectious Materials Biosecurity is a primary concern for both the animal care personnel and the animals in their care. Veterinarians, investigators, and the IACUC are concerned with the risk of animal pathogen infection in a breeding colony or any research animal, as such an event can greatly affect the health and well-being of the animals in their care as well as experimental data. Animal research must balance biosecurity concerns with potential worker exposure concerns. The primary methods to prevent infection from outside pathogens after quarantine is positive pressure ventilation for animal housing, the use of PPE, and a dedicated room entry order. However, a potential concern with these practices is that animals in barrier housing may be exposed to a chemical, or potentially infectious material, and positive pressure caging (depending on the specific style) may not be appropriate because poor seals or improperly fitted lids may allow escape from the cage. For cages with infected animals that are actively shedding pathogens or with animals excreting hazardous chemicals, the potential for release out of the cage might not be allowable based on risk assessment. There are mechanisms to decrease the positive pressure and there are specific cages designed for containment, but these are not options are not always available at an entity.16 Some facilities may not have a cage ventilation system that is integrated with their HVAC system and instead rely on static or stand-alone caging or disposable cage racks for animals that require a higher level of containment. Older facilities may not have ventilated racks, so disposable or static caging is used when needed. Use of disposable caging depends on several factors. Cages must be cleaned after use and, depending on the hazard review, may require autoclaving before cleaning. Some facilities may not have autoclaves on site, and alternatives must be in place, which can include chemical disinfection or disposable caging with medical waste disposal. Finally, some experiments will produce cages where highly hazardous chemicals are excreted and thus cages cannot be placed in a cage wash and/or the facilities may lack autoclaves, cage washers, and/or animal transfer stations for cage changing. In these cases, disposable caging is often used, so the entire cage and all bedding can be collected and disposed as hazardous waste. These cages should be placed on a negatively ventilated rack or maintained in static mode. Disposable caging is not considered an environmentally sustainable option and is expensive, but the ability to dispose the lid and cage, without removing the bedding, and to eliminate the washing step for reuse can be a huge benefit for certain research programs. Facility Decontamination Traditional animal facilities require routine facility decontamination due to concerns with infectious materials such as murine norovirus, pinworms, and mouse hepatitis virus; floods and other facility-related contamination concerns; and/or a generalized cleaning/room turnover process. In high- and maximum-containment laboratories, facility decontamination is performed when changing a room over between studies, when switching from one infectious agent to another, or before a maintenance shutdown.27,28 Depending on the size of the facility and the type of decontamination needed, the process can be performed by the facility users or an outside vendor can be employed. High- and maximum-containment facilities routinely include this type of work in their maintenance budgets and perform the decontaminations more frequently; personnel may possess in-depth understanding of the risks and of mitigation processes. Facility decontamination processes marry chemical safety and industrial hygiene safety with biosafety and require coordination of multiple safety professionals to provide comprehensive guidance for the workers. It is crucial to have communication between EHS and animal care about equipment purchased, disinfectants used, and also the final goal, as this will greatly inform EHS recommendations and the overall risk assessment, discussed further below. The chemicals most commonly used for facility decontamination are sterilants. It is important first to distinguish between disinfectants and sterilants. Sterilants are chemicals that when used at appropriate levels can destroy all forms of microbiological life; disinfectants by definition remove many or all of the microbes targeted, with the exception of spores.29 Not all facility decontamination procedures require sterilants, but the use of disinfectants, even for general room cleaning, requires review and risk assessment by EHS to provide guidance on worker protection for these processes. Both groups of chemicals are hazardous to the micro-organisms they are designed to terminate but also have properties that can cause harm to humans. The effectiveness of the decontamination is confirmed by the use of biological indicators, most commonly the use of Geobacillus sterothermophilus or Bacillus atrophaeus spores.30,31 It is important to consider the method of decontamination to ensure the indicator chosen is appropriate for the chemical and the method. Historically, formaldehyde was used in most facilities, but recently chlorine dioxide and vaporized hydrogen peroxide (VHP) have gained favor and are widely used. These agents leave small amounts of residue and can be used with sensitive equipment such as imagers, mass spectrophotometers, cell sorters, and microscopes.30,32,33 Despite opinions about VHP use causing equipment damage, VHP has been widely used for many years. Sterilants are strong chemicals that can lead to harmful exposure to humans and animals at the concentrations used for facility decontamination. The concentrations used for facility decontamination can reach above the level that NIOSH has determined to be immediately dangerous to life and health in spaces during decontamination procedures. In our experience, reported levels of VHPs in BSL-3 reach approximately 500 hundred parts per million (ppm): the permissible exposure limit is 1 ppm, and the Immediately Dangerous to Life and Health level is 75 ppm.34 Thus, meters should be used to monitor concentrations in the facility and surrounding areas, to ensure that the chemical agent is not leaching, and to determine when it is safe to reenter the area. Each chemical has requirements for how it is used in terms of concentration and contact time; specific requirements for PPE need to be followed by all personnel. A standard operating procedure should be generated for decontamination processes, including instructions for the use of the specific equipment, disinfectant (concentration, dwell time, and preparation), use of chemical-monitoring devices, PPE, chemical and biological indicators, and documentation for the process and results. The standard operating procedure should also include the method of facility aeration or neutralization of the chemical, which is the method by which the concentration decreases to the point at which it is safe to reenter the facility. The NIOSH guide must be consulted to determine this level.34 This process must include persons from the animal facility, EHS, and facilities oversight to ensure an understanding of the facility capabilities and coordination of HVAC shutdown during this process. Note that full coordination of the facilities oversight is required in the instance of HVAC shutdown, as consequences may extend to other departments and sections of the building. The best practices for decontamination are to use a combination of chemical and biological indicators for efficacy of the process. The choice of chemical disinfectant and the concentration used to ensure killing of the target microorganism(s) must be coordinated with the use of a biological indicator to confirm killing (as appropriate). Biological indicators are placed throughout the room as well as in hard-to-reach areas, typically next to the chemical indicators. Once the decontamination process is finished, the indicators are collected, added to culture media, and incubated according to manufacturer’s instructions. Positive and negative controls are included for comparison. Most of these produce colorimetric change to indicate growth of the bacteria in the media. Absence of color change means the decontamination was successful. The compilation of all data produces a comprehensive report for persons requiring reentry to that space whether they are researchers looking for assurance that a pathogen has been killed or maintenance staff entering for work in a high-containment facility. Animal Transport-Infected/Exposed Animals Safe transfer of animals is necessary for any research program. Common reasons for transport include the need for a designated breeding colony in one location with animal use in another facility, creation of transgenic animals and the movement to the housing/breeding facility for additional experiments, the use of specialized equipment located in only one facility, biosecurity issues within a facility that necessitate movement for cleaning, and others unique to specific programs. Uninfected animals can be transported by the usual means, but if the animals are infected there are additional concerns to protect the workers and vehicle. Animal transport should be performed using methods acknowledging the potential for animal allergens and/or potential exposure to any present hazard but also in a way that balances health and safety for the animal (e.g., temperature, humidity, caging types, including HEPA-filtered transportation devices). Animal facilities guided by their IACUCs have policies for the vehicles and temperature requirements to meet animal care requirements and proper caging. It is important to note that animals infected with pathogens are not permitted to be transported by air according to International Air Transport Association (IATA) regulations.35 US Department of Transportation regulations are less restrictive for road transport, but one must consider and determine the risk of transporting an animal that is infected with an infectious agent and the risk of exposure or contamination. Transporting animals infected with a microorganism classified as risk group 3 or 4 is generally not performed since it is difficult to maintain proper containment of the agent and also to provide appropriate animal care. Short-term movement can be performed but usually within a building. A typical transport box may not be sufficient and a more suitable cage with appropriate filters and containment is required. Persons transporting the animals must have training and an understanding of the requirements for institutional policies as well as applicable laws. Containment Challenges The primary and secondary containment requirements for high- and maximum-containment work differ between BSL-3 and 4 and are much more specific and detailed compared with BSL-1/2 to protect the workers and the environment from these pathogens4. For instance, BSL-1/2 facilities do not require the facility to contain materials more than a generalized negative pressure and in fact, negative pressure at BSL-1 and BSL-2 is only a recommendation. The regulations and guidance differ when discussing BSL-3, BSL-3-Ag (large animal), and BSL-4 research and differ if select agents or certain viruses, such as risk group 3 influenza, are used in animals.5,36,37 The federal entities and agencies that regulate or have published guidelines are listed in Table 1. Table 1 List of Federal Entities and Agencies With Oversight of High-Containment Work Regulation or Guideline . Federal Entity/Agency . Purview . NIH Guidelines for the Use of Recombinant or Synthetic Nucleic Acids NIH-OSP Recombinant and synthetic nucleic acids BMBL 5th Edition CDC, NIH Biosafety guidelines, risk assessment Respiratory Protection Standard OSHA Respiratory protection requirements Bloodborne Pathogens Standard OSHA Human or nonhuman primate blood or other potentially infectious material 9 CFR 122 USDA and Health and Human Services Importation and transfer permit requirements Importation permit requirements 7 CFR Part 331, 9 CFR Part 121 42 CFR Part 73 United States Department of Agriculture and Health and Human Services Select agent regulations and import of infectious agents The Policy for Government Oversight of Dual Use Research of Concern NIH-OSP Review by institutional review entity and during grant submission for subset of select agents The Guide for the Care and Use of Animals AAALAC Institutional assurance for animal research Health Research Extension Act of 1985 NIH Office of Laboratory Animal Welfare Health and Welfare of Research Animals Covered by NIH Funded Research 7 USC Chapter 54 and 9 CFR Part 1, Subchapter A USDA Animal Welfare Act Regulation or Guideline . Federal Entity/Agency . Purview . NIH Guidelines for the Use of Recombinant or Synthetic Nucleic Acids NIH-OSP Recombinant and synthetic nucleic acids BMBL 5th Edition CDC, NIH Biosafety guidelines, risk assessment Respiratory Protection Standard OSHA Respiratory protection requirements Bloodborne Pathogens Standard OSHA Human or nonhuman primate blood or other potentially infectious material 9 CFR 122 USDA and Health and Human Services Importation and transfer permit requirements Importation permit requirements 7 CFR Part 331, 9 CFR Part 121 42 CFR Part 73 United States Department of Agriculture and Health and Human Services Select agent regulations and import of infectious agents The Policy for Government Oversight of Dual Use Research of Concern NIH-OSP Review by institutional review entity and during grant submission for subset of select agents The Guide for the Care and Use of Animals AAALAC Institutional assurance for animal research Health Research Extension Act of 1985 NIH Office of Laboratory Animal Welfare Health and Welfare of Research Animals Covered by NIH Funded Research 7 USC Chapter 54 and 9 CFR Part 1, Subchapter A USDA Animal Welfare Act AAALAC, Association for Assessment and Accreditation of Animal Laboratory Care; CDC, Centers for Disease Control and Prevention; NIH, National Institutes of Health; NIH-OSP, National Institutes of Health Office of Science Policy; OSHA, Occupational Safety and Health Administration; USDA, United States Department of Agriculture. Open in new tab Table 1 List of Federal Entities and Agencies With Oversight of High-Containment Work Regulation or Guideline . Federal Entity/Agency . Purview . NIH Guidelines for the Use of Recombinant or Synthetic Nucleic Acids NIH-OSP Recombinant and synthetic nucleic acids BMBL 5th Edition CDC, NIH Biosafety guidelines, risk assessment Respiratory Protection Standard OSHA Respiratory protection requirements Bloodborne Pathogens Standard OSHA Human or nonhuman primate blood or other potentially infectious material 9 CFR 122 USDA and Health and Human Services Importation and transfer permit requirements Importation permit requirements 7 CFR Part 331, 9 CFR Part 121 42 CFR Part 73 United States Department of Agriculture and Health and Human Services Select agent regulations and import of infectious agents The Policy for Government Oversight of Dual Use Research of Concern NIH-OSP Review by institutional review entity and during grant submission for subset of select agents The Guide for the Care and Use of Animals AAALAC Institutional assurance for animal research Health Research Extension Act of 1985 NIH Office of Laboratory Animal Welfare Health and Welfare of Research Animals Covered by NIH Funded Research 7 USC Chapter 54 and 9 CFR Part 1, Subchapter A USDA Animal Welfare Act Regulation or Guideline . Federal Entity/Agency . Purview . NIH Guidelines for the Use of Recombinant or Synthetic Nucleic Acids NIH-OSP Recombinant and synthetic nucleic acids BMBL 5th Edition CDC, NIH Biosafety guidelines, risk assessment Respiratory Protection Standard OSHA Respiratory protection requirements Bloodborne Pathogens Standard OSHA Human or nonhuman primate blood or other potentially infectious material 9 CFR 122 USDA and Health and Human Services Importation and transfer permit requirements Importation permit requirements 7 CFR Part 331, 9 CFR Part 121 42 CFR Part 73 United States Department of Agriculture and Health and Human Services Select agent regulations and import of infectious agents The Policy for Government Oversight of Dual Use Research of Concern NIH-OSP Review by institutional review entity and during grant submission for subset of select agents The Guide for the Care and Use of Animals AAALAC Institutional assurance for animal research Health Research Extension Act of 1985 NIH Office of Laboratory Animal Welfare Health and Welfare of Research Animals Covered by NIH Funded Research 7 USC Chapter 54 and 9 CFR Part 1, Subchapter A USDA Animal Welfare Act AAALAC, Association for Assessment and Accreditation of Animal Laboratory Care; CDC, Centers for Disease Control and Prevention; NIH, National Institutes of Health; NIH-OSP, National Institutes of Health Office of Science Policy; OSHA, Occupational Safety and Health Administration; USDA, United States Department of Agriculture. Open in new tab It is beyond the purview of this article to delineate the specifics of all the regulations and requirements and the nuances of all applicable animal models, but we will discuss some important, general challenges that can apply in multiple ways across high-containment laboratories. The current regulations and guidance documents overlap with respect to engineering controls and work practices for high-containment laboratories. They are promulgated by the National Institutes of Health, Centers for Disease Control and Prevention, and the United States Department of Agriculture Animal and Plant Health Inspection Service, and these agencies inspect laboratories often to ensure compliance.5,37 The requirements complement each other by discussing work practices, facility requirements, and other containment practices for specific pathogens and delineate requirements for human, animal, and plant pathogens. It is important to cross-check all applicable Centers for Disease Control and Prevention, United States Department of Agriculture, and National Institutes of Health regulations to ensure compliance across the spectrum and how they are applicable to animal welfare requirements. Most of the experiments performed in high and maximum containment are broken down into sections, each of which may require a specialized engineering control for worker and environmental protection; infection, observation, and additional treatments; and necropsy. Thus, a detailed risk assessment is performed analyzing each of these processes to determine the risks and mitigation strategies. For example, sample processing follows the necropsy or treatment timepoints, and at this stage samples are commonly moved to another suite or facility for analysis (ABSL-3 to BSL3); if transfer is to a lower biosafety level (e.g., ABSL-3 to BSL2), the sample must be inactivated by validated methods, and in the case of select agents, in line with current regulatory requirements. Primary containment includes biological safety cabinets and ventilated housing racks, whereas secondary containment includes the room design and the HVAC system. BSL-3-Ag and BSL-4 use the rooms as primary containment due to additional requirements for room seals and testing, which can provide some flexibility to the required containment equipment, caging types, and required PPE. Research facilities working with large animals (Ag facilities) have additional challenges and requirements for housing, facility design, and personal protection, mainly due to the size of the animals, the complexity of moving them (gates, runs lifts, etc.), and housing requirements. Staff working in these areas receive detailed training on the containment equipment located in the laboratory, the similarities and differences, how to determine if the equipment is not working properly, and the required reporting procedures. Since containment laboratories have many different devices, a good portion of staff training, especially for the animal husbandry staff, will involve explanations of the design, use, and function of engineering controls. In addition to facility design, work practices and safe processes are integral for worker protection in a high-containment facility. Researchers and animal care staff must have detailed training on the facility, engineering controls, their specific animal model, the techniques performed, sharps safety, aerosol prevention, and detailed pathogen handling when working in these environments, especially for injecting animals for sedation, infection, and surgeries.18 The use of parenteral injections at high containment can lead to serious incidents resulting in grave illness, so mitigating this risk is highly important. Training for researchers in these facilities is usually a step-wise process, combining didactic training with hands-on training.18 Each part of the experiment requires different engineering controls, PPE, and animal care requirements (e.g., sedation or restraint). Experiments performed at BSL-1 and BSL-2, and sometimes chemical exposures, require some type of restraint of the animal for the infection. At BSL-1 and BSL-2, it is customary to use a strong grip, sometimes a mechanical restrainer and, rarely, sedation at the time of infection; an exception is exposure during a surgical procedure, which always requires sedation. In high and maximum containment, chemical restraint (sedation) should be the primary method used. However, some procedures cannot be performed under sedation, as some animals are overly sensitive to these drugs, leading to a decrease in overall well-being and/or illness; additionally, experimental design may preclude sedation. The use of anesthetics or restraints requires communication and decision-making across animal care, research, EHS, and the high-containment administrations. When working at high-containment facilities, researchers will implement different methods of restraint to provide as much protection to the worker from accidental movement of the animal, which can result in an increased risk for auto-inoculation, bites, scratches, and other potential exposures to the pathogen. Additional PPE (e.g., Kevlar or puncture-resistant gloves/sleeves) should be required whenever possible for all procedures using sharps at high- or maximum-containment facilities; this PPE is also highly recommended (although not required) for nonhuman primate and other large animal models to prevent bites or scratches. Training must include how to use this PPE, initially in a lower containment setting, until the staff member is comfortable. Finally, the balance between sedation of the animals during procedures/transport and animal welfare and personnel protection is an important challenge. Transport of infected animals from a housing room to a procedure room or between facilities is a common event. Not all animals require sedation, but some larger animals, such as rabbits, are easily stressed; moving them between facilities is less stressful with low levels of sedation, requiring special caging systems. Similarly, procedures should be developed to move the animals safely in the event a facility shutdown or another emergency when the animals must be relocated. In the case of animal research, many models require aerosolization of bacteria/viruses for infection. Research programs with a large aerobiology program have aerobiology cores, which are invaluable in planning experiments for these facilities. Aerosol exposure equipment can be quite large and typically only rodent systems will fit into a standard Class II BSC; for rodents and tuberculosis, whole-body aerosolization is becoming more common. For systems of slightly larger research animals, the equipment rarely fits into a standard BSC and requires a specialized enclosure device with HEPA filtration to ensure worker safety during the exposure. BSL-3-Ag research requires additional specialization due to the size of the animal and the fact that the facility room acts as primary containment.4 The HEPA filters on these units are subject to annual leak testing and certification—just like BSCs. It is recommended for select agent aerosolization to be performed in a Class III BSC, which is costly in terms of initial install, and maintenance of the cabinet and the dedicated HVAC system.12 Even though a microorganism causes respiratory disease, aerosolization may not be needed, so it is important to fully understand and discuss research methodology requirements. Intranasal infection will provide this infection model but will not require the large aerosol equipment or the specialized cabinet. Intranasal infection is usually performed as an alternative to aerosol exposure for many different infectious disease models, and most animal models will fit into a BSC; a downdraft table might be used for the procedure but this approach increases exposure risk to personnel, as a downdraft table is not a primary containment device. Both infection models typically require sedation of the animals to ensure their safety in the aerosol exposure device and/or to allow the liquid placed into the nostrils to diffuse. Finally, whole-body aerosol exposure (e.g., using a Madison aerosol chamber) does not require sedation of small animals. Housing of animals is typically within negative pressure cage racks, but housing larger animals in ventilated cages may prove challenging. BSL-3 Ag facilities will use containment enclosure devices, open caging, and in some cases larger pens for the animals. Negative pressure-ventilated caging systems are crucial for maintaining containment, especially for animals that can spread infection by coughing or sneezing, but they can be bulky, which creates concern for animal welfare such as the requirement for light, social housing, sight, smell, and other interactions. Regulators have required containment curtains over these cages, which can be damaged by the animals and create a physical issue for workers when trying to observe/remove animals from the cages. Petitions and discussions with the NIH have allowed for open caging to be used at BSL-4, with additional facility requirements.5,37 Depending on how the room is designed, and the type of cage used, the bottom row of cages can be very dark, which is not good for the animal but also creates a challenge for the worker who needs to handle the animal in a manner to ensure proper care and to minimize any exposure. These containment curtains also affect light penetration to the cages, which can create AAALAC-related animal welfare concerns. Ergonomic issues also arise when working with animals in containment, especially in containment caging. The cages are low to the ground, and to reach the cages while wearing cumbersome PPE is very challenging. Not all cages come with “squeezeback” mechanisms, which assist in reaching for the animal when needed. For larger animals and animals that require loose housing or open caging, many facilities utilize containment equipment such as plastic enclosures, in addition to HEPA filters, to allow easier cleaning. PPE may also be modified and increased since a worker may need additional protection from body fluids during procedures and necropsy. High-containment (BSL-3-4) laboratories have strict requirements for carcass and waste disposal, requiring sterilization of all materials before leaving the laboratory space and treatment of liquid effluents, including water from personnel showers. These requirements differ slightly depending on the microorganisms and the specific regulations cited, but in general, liquid, solid, and carcass waste must have specific procedures for disposal.4,5,37 Mixed waste streams can create complications as some radionuclides cannot be autoclaved, nor can certain chemicals. Waste disposal can be complicated, as BSL-3-Ag, BSL-4, and some BSL-3 research requires effluent waste systems and tissue digesters and therefor require additional training of staff and oversight by safety. Materials leaving the facilities need to be sterilized, and the equipment used must be validated and each cycle used must be validated to the particular waste material. General laboratory waste will likely require a different autoclave cycle than animal cages and animal carcasses. Different animal carcasses will require different sterilization times and if they are frozen, the cycle will be longer, since they will need to thaw and then sterilize. Each cycle must be validated with biological indicators in representative waste, which can be a long process full of troubleshooting to optimize just the right timing. It is not uncommon for carcasses to need 4 hours of sterilization time, and the autoclave sizes vary on the facility size, animal caging, and other materials that require sterilization. Persons working in this environment require training on how to validate an autoclave, the general use and how to determine if a cycle did not pass, and reporting for issues. Additionally, training needs to include potential hazards of removing the extremely hot materials, the proper PPE, and how to properly use a larger autoclave to ensure people do not enter it or get hurt by the large doors. Liquid effluent systems can be as simple as adding disinfectant, or a system connected to the drains in the building that sterilizes the water before releasing into the sanitary sewer system. Work with a subset of risk group 3 influenza viruses and BSL-3-Ag and BSL-4 facilities requires separate effluent waste systems that are ultimately connected to the building drainage system.4,5,37 These systems utilize chemicals to treat the water or high temperatures. The water will collect and then when the tank is full, a cycle for sterilization is run. Biological indicators are used to ensure full sterilization of the liquids, which are released into the sewer systems once the cycle completion and indicators confirm killing. Since waste decontamination is crucial for protecting the public and the environment, lack of autoclaves or effluent waste systems can halt operations or lead to a buildup of waste that must be stored. Tissue digesters are common and come in all sizes for ease of use; they can provide an alternative method for carcass disposal. These also must be validated to ensure proper function. Entities must have detailed plans in place so all understand the process if a system is out of order. Usually a facility can go a day or so, but sometimes, it means a full halt in operations. For critical systems, backup equipment is usually present since ceasing operations is not usually an option when animals are involved. Technology has allowed for the influx of equipment into containment laboratories, but there is much that is too burdensome or expensive to house inside a containment facility. Additionally, many samples can be inactivated for further analysis in a BSL-1/2 environment. Tissues are commonly fixed in formalin for histopathology, and samples for DNA, RNA, and protein analysis may also be removed. Since the materials will be handled at a lower biocontainment level, often without an engineering control, it is imperative that they be free of pathogen. Solvent-based extraction methods are best practice for nucleic acid and protein purification from samples, as most solvents render pathogens inactive. Some—but not all—kit-based materials will also inactivate materials. The United States Federal Select Agent Program has strict requirements for validated inactivation of materials that fit the definition of select agent materials.5 There are many other microorganisms studied at high containment that also must be inactivated. To date, there are no regulatory requirements as detailed as the select agent requirements, but those are being used as a framework. Historically, high-containment laboratories validate their inactivation procedures for all materials leaving containment. Most procedures utilize chemicals for fixation or extraction of nucleic acids. Some procedures cannot utilize formalin for tissue fixation and require the use of an irradiator. Unfortunately, the dosage of radiation required to kill the infectious materials is large, and not all irradiators have the capability to kill the pathogen without severely damaging the tissue samples. BSL-3 samples are often sent from the laboratory as infectious materials to another entity for irradiation and can then be sent back for manipulation outside of a containment facility. Summary Animal research is a broad and complex arena with many different potential hazards to the workers in that area. It is important for EHS professionals to engage with animal care professionals and researchers to discuss and understand the research components, the equipment utilized, and the potentially hazardous aspects of this work. Open communication and detailed training is crucial, especially for animal care staff to understand and discuss concerns about the hazards they encounter on a daily basis. Animal facility operations and safety are always evolving, changing, and improving. These research programs excel when relationships are established and all players are brought in to evaluate how programs will change, new technologies and procedures, facility renovation, and hazard assessment as it applies across the board. The participation of EHS staff in semi-annual IACUC inspections is crucial to observe the facilities and help identify areas for improvement or concern from the animal care staff. 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In:April 2019 . © The Author(s) 2019. Published by Oxford University Press on behalf of the National Academy of Sciences. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Laboratory Safety, Biosecurity, and Responsible Animal Use
JF - ILAR Journal
DO - 10.1093/ilar/ilz012
DA - 2019-12-31
UR - https://www.deepdyve.com/lp/oxford-university-press/laboratory-safety-biosecurity-and-responsible-animal-use-J0BurEGFY6
SP - 24
VL - 60
IS - 1
DP - DeepDyve
ER -