Creative Implementation of 3Rs Principles within Industry Programs: Beyond Regulations and Guidelines

Abstract

The industry involved with using animals as an essential part of research has supported the theory and philosophy of the 3Rs for years. However, both the culture and approach surrounding the 3Rs is evolving rapidly, and many institutions are attempting to surpass the regulations and guidelines to implement the 3Rs for improved science and animal welfare. Regulatory documents and guidelines such as the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, the Public Health Service Policy on Humane Care and Use of Laboratory Animals, and the US Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training clearly outline how the IACUC should address the 3Rs, but there are many additional paradigms and resources that an institution can use to promote the 3Rs creatively. We review the legal mandates and guidelines that institutions must or should follow, and we present some creative approaches toward their compliance, including the creation of full-time dedicated 3Rs roles as well as temporary 3Rs-focused positions such as visiting scientist and postdoctoral fellowships and internships. We also discuss how to creatively achieve 3Rs progress through internal committees and working groups, involvement in 3Rs consortia, recognizing 3Rs advances through awards programs, and creating 3Rs volunteer opportunities. Adherence to regulations and guidelines creates a solid foundation for good animal care and science, and creative 3Rs approaches enable the growth of a robust animal welfare culture that enhances the potential for 3Rs benefits to animals and science.

Many institutions support, in numerous ways, the 3Rs of alternatives to animal experimentation, as stated by Russell and Burch in The Principles of Humane Experimental Technique.¹⁷ The concepts of the 3Rs are stated in the Animal Welfare Act regulations, Public Health Service requirements, and institutional policies and standards. Here we examine and highlight several of the mechanisms that can be used to implement the 3Rs in industry programs.

First and foremost in the United States are the responsibilities of the IACUC to apply the principles of the 3Rs to every aspect of animal experimentation. Through the USDA mandate, the IACUC is obliged to ensure the institution is in compliance with the Animal Welfare Act³ and its regulations. Other regulatory guidance such as the Public Health Service Policy on Humane Care and Use of Laboratory Animals (PHS Policy)¹⁶ and its standards as delineated in the Guide for Care and Use of Laboratory Animals (the Guide)¹² are key in shaping the IACUC oversight of the 3Rs and animal welfare oversight in general. Despite the lack of USDA requirements regarding rodents, most facilities meet ethical standards by adhering to PHS Policy requirements or by having an IACUC to oversee welfare of all species. In addition, many institutions have found other oversight mechanisms to promote, provide education about, and ensure vigorous implementation of the 3Rs. These include defined job or committee responsibilities and participation in external associations or consortium.

IACUC Oversight of the 3Rs in the United States

There are 4 main documents that structure how IACUCs address the 3Rs. These are the Animal Welfare Act regulations,⁵ the Guide,¹² the PHS Policy,¹⁶ and the US Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training (US Principles).¹⁹

USDA and the 3Rs

A brief review of the USDA Animal and Plant Health Inspection Service Animal Care Regulations reveals that the term ‘3Rs’ is not used or referenced explicitly. However, the Animal Welfare Act regulations address the consideration of alternatives in section 2.31(d) by stating that:

• (ii) The principal investigator has considered alternatives to procedures that may cause more than momentary or slight pain or distress to the animals, and has provided a written narrative description of the methods and sources, e.g., the Animal Welfare Information Center, used to determine that alternatives were not available.³

Thus there is a mandate to consider alternative methods or procedures to painful procedures. It should be noted that when a study involves multiple painful events, it is necessary to perform an alternative review for each painful procedure.

The USDA has provided the Animal Care Policy Manual² to further clarify the regulations. Although these policies are not considered to be regulations and cannot be used by inspectors to issue a noncompliance citation, they should be carefully reviewed and understood by the IACUC. In policy 12, Consideration of Alternatives to Painful/Distressful Procedures, the USDA first addresses Russell and Burch's principles of the 3Rs—refinement, reduction, and replacement. The expectations regarding consideration of alternatives are clearly stated. They recommend a database search with a narrative that outlines:

• 1) the name(s) of the databases searched (due to the variation in subject coverage and sources used, one database is seldom adequate);

• 2) the date the search was performed;

• 3) the time period covered by the search; and

• 4) the search strategy (including scientifically relevant terminology) used.

• The USDA does identify other methodology for researching possible alternatives:

Policy 12—In some circumstances (as in highly specialized fields of study), conferences, colloquia, subject expert consultants, or other sources may provide relevant and up-to-date information regarding alternatives in lieu of, or in addition to, a database search. Sufficient documentation, such as the consultant's name and qualifications and the date and content of the consult, should be provided to the IACUC to demonstrate the expert's knowledge of the availability of alternatives in the specific field of study.¹

However the use of these other routes should be weighed judiciously and carefully by the IACUC. One-line statements regarding alternatives that start with “I am expert” should not be considered to be sufficient.

The guidance in policy 12 addresses the situation when the animal study is prescribed in another federal regulation (for example, toxicology studies) and thus requires the reference of the correct federal regulation in the proposed study. The policy also recommends the review of study requirements with the appropriate agency to determine the availability of acceptable alternatives or animal study refinements.

Policy 12 further stipulates that the principal investigator provide evidence of a reasonable effort to identify alternatives to procedures that elicit more than momentary discomfort. The guidance falls short of requiring the principal investigator to address bona fide alternative methods, it allows the IACUC to query the use of alternatives. The USDA also supports the IACUC in withholding the approval of a study proposal if they are not satisfied with proper assessment of a procedure and its alternatives.

Finally, policy 12 notes the expectation that, when a study modification incorporates a new procedure that involves unrelieved pain, alternatives are considered. The USDA expects that alternatives reviews will be conducted only during triennial de novo reviews and not at each standard annual review.

The PHS Policy, US Principles, and the 3Rs

The PHS Policy¹⁶ and US Principles articulate tenets that scientists are expected to follow when they receive funding from the Public Health Service. Although they do not explicitly identify the 3Rs, the PHS Policy and US Principles include language that is supportive of the 3Rs principles regarding alternatives. Importantly, the PHS Policy states that the research must be performed in accordance with the Animal Welfare Act and must be consistent with the Guide, both of which include the requirement for consideration of alternatives. In addition, the PHS Policy and US Principles stipulate the use of the fewest animals necessary to obtain valid results and therefore require scientific justification of the study size. This emphasis of ‘right-sizing’ the study group reflects the reduction philosophy. Statistical analysis does not always reduce the study group size—it may increase it, in fact—but yielding useful data avoids nonvalid results and the need to repeat studies. Both documents clearly note the need to minimize animal distress and pain, thus indicating the need for consideration refinement or replacement of the procedures to be used, and the PHS Policy directly identifies that nonanimal alternative methods“such as mathematical models, computer simulation, and in vitro biologic systems should be considered.”¹⁶

The Guide and the 3Rs

Institutions accredited by AAALAC¹ must adhere to the 8th edition of the Guide. Among the 4 main documents, The Guide contains the most IACUC guidance on the 3Rs; this information is included in the Key Concepts sections. The Guide covers all species of animals used in research and has a succinct, effective summary of the principles of the 3Rs. The Guide stresses the 3Rs as an important part of decision-making in the design and review of animal research studies and supports the assessment of each study on a case-by-case basis for opportunities for refinement and reduction. The Guide discourages the reuse strategy for reducing the number of animals when reusing an animal compromises its wellbeing—cost reduction alone is not an acceptable justification. In the protocol review section, the Guide defines refinement and replacement asthe “availability or appropriateness of the use of less invasive procedures, other species, isolated organ preparation, cell or tissue culture, or computer simulation.”¹² The 3Rs are not an isolated exercise in performing a literature search but should be incorporated as part of the scientific standards and best practices used for optimal study design. A theme put forth in the Guide is that the principal investigator, IACUC, and institution share the responsibility of the implementing the 3Rs principles. The scientific justification that the Guide requires regarding the number of animals proposed for a study is evidence of the reduction strategy because it encourages appropriate statistical analysis to identify the minimal number of animals needed. Postprocedural care, endpoints, and timely appropriate intervention are important aspects of proposed studies and address the refinement principle. AAALAC endorses the Guide’s view on harm–benefit analysis by the IACUC: “the Committee is obliged to weigh study objectives against animal welfare concerns in accordance with the tenets of the 3Rs.”¹² The Guide further states that particular attention should be placed on opportunities for refinement and replacement in protocols with special considerations, such as unrelieved pain or concerns regarding animal welfare. Appendix A of the Guide has a separate section of references on alternatives.

IACUC oversight of the 3Rs principles should include oversight of training and assurances during the semiannual program review. The 3Rs principles should be a component of the training of the IACUC, scientific personnel, and vivarium staff. In addition, the IACUC training program should include understanding the expectations regarding the search for alternatives. The IACUC should also evaluate the training program and resources available for PIs.

Alternatives literature searches

Alternatives literature searches are an important part of applying the 3Rs and complying with the regulations but may be considered one of the most onerous aspects of protocol writing. Noncompliance with the requirements for consideration of alternatives can lead to issues with regulatory and accreditation organizations. As part of the IACUC's general oversight of animal welfare, a strong program should be in place to help the principal investigator understand the alternatives search requirements, make resources available to aid in the search, and to provide guidelines regarding assembly of the narrative. Many institutions provide internal guidance documents that summarize the process and include helpful references. The Animal Welfare Information Center (AWIC)⁴ of the Agriculture Library can provide training and online resources for principal investigators. The AWIC runs training courses several times a year, these courses are very helpful for Information and Library Specialists at institutions. Another online resource to consider is the AltWeb site sponsored by the Johns Hopkins Center for Alternatives to Animal Testing.¹³ How to conduct a literature search is beyond the scope of this paper, but AWIC has authored a brochure that contains helpful tips for principal investigators, including:

• 1) Complete and review the search before completing the protocol;

• 2) Assess and evaluate the alternative possibilities and be prepared to support their use, or nonuse, in writing;

• 3) Make sure the terminology, search strategy, database(s) searched, and dates of search are provided; and

• 4) Keep a copy of the search strategy, database(s) searched, and year of search for future use.

Furthermore, when a PI needs to update a previously completed search for de novo review of a protocol without significant changes, the search can be updated by using the same terminology and search strategy; the dates to be used are that of the original protocol to the present.

To give member of United States IACUC a European perspective, we here provide a brief review of the 3Rs in a recent legislation that has guided national research welfare laws in the European Union. Directive 2010/63/EU of the European Parliament and of the Council on the Protection of Animals Used for Scientific Purposes (the Directive)¹⁸ was adopted in September 2010. The Directive serves as a centralized standard, and all member countries must adopt this centralized standard into their national laws on animal research by January 2013. The Directive has numerous direct and indirect references to animal alternatives and the 3Rs. As the Directive states, “The care and use of live animals for scientific purposes is governed by internationally established principles of replacement, reduction and refinement.”¹⁸ Principles of the 3Rs are to be considered systematically in project design, animal breeding, and animal care. Several levels of 3Rs oversight responsibility include an institution's animal welfare body, which is to advise the staff on 3Rs application as well as the national project review process, and periodic reviews of animal use and 3Rs status relative to those throughout the European Union.

The IACUC should not be the only bastion of 3Rs principles. As we stated earlier, a true institutional effort is more holistic and includes the IACUC, scientists, attending veterinarian and institutional policy. We now address additional paradigms or resources that an institution can use to promote the 3Rs in a creative manner.

Unique Roles and Positions Focused on the 3Rs

Although not yet mainstream, full-time roles dedicated to the 3Rs are becoming a frequent way of advancing the 3Rs in industry. Responsibilities of such roles vary and can be administrative, strategic, or scientific. The approaches may differ, but all are valuable and useful aspects of promoting the 3Rs and enhancing a culture of ethical animal research. Administrative functions include coordinating and managing all aspects of either a local or global 3Rs program to achieve consistent expectations, thus maximizing the effect of the program. Strategic functions may include developing a 3Rs strategy, effectively communicating the strategy, gaining support from upper management, and facilitating implementation of the strategy. Having support from senior management is an essential component of a successful 3Rs strategy. In addition, having a central liaison to share 3Rs information and practices across the company can be remarkably effective, especially in larger institutions or global companies with multiple sites, where replication of efforts can be a common and counterproductive problem. The 3Rs liaison can be a well-known resource who is well-informed about the various 3Rs initiatives, pulling together common efforts for collaboration for optimal resource management and effect. Scientific functions could include coordinating and conducting innovative 3Rs research projects to address ethical issues and to provide scientific data to support decision-making.⁸ 3Rs research is essential to assess how refinements or other 3Rs efforts, such as enrichment, may affect study outcomes and to optimize adoption of 3Rs initiatives that enhance and don't hinder the research. Dedicated resources are especially valuable in a time when research and animal care staff are fully occupied by their primary responsibilities with little time to volunteer to 3Rs research efforts. A 3Rs scientist can lead these studies but must get input from scientists who have expertise with the animal model in question as well as statisticians to ensure the best study design. This team approach ensures that all concerns are addressed in the design of the 3Rs study and paves the way for rapid adoption of the 3Rs if the scientific data support the change. This working group approach will be discussed in further detail but has been an effective way of bringing together staff whose primary responsibility is not 3Rs-related to achieve a positive outcome.

Beyond full-time 3Rs roles, there are other dedicated positions that contribute to a scientific approach to refinement specifically, such as full-time behaviorists and enrichment coordinators. Through expertise and experience, behaviorists and enrichment coordinators can develop strategic behavioral management and enrichment assessment programs. Such program enhancements are especially valuable for global programs that manage enrichment at multiple sites and programs with species that benefit from positive reinforcement training, such as dogs and nonhuman primates. In addition to the positive outcome on wellbeing through allowing animals increased control over their environment, positive reinforcement training can have the added benefit of increasing the efficiency of research activities, enabling changes such as decreasing staff requirements.¹¹ With heightened attention to the value of assessing both the welfare and scientific effects of enrichment, these positions allow a strategic approach and can both enhance animal welfare and contribute research data to guide the use of enrichment across animal research programs.²⁰

In addition to fulltime, dedicated roles, various temporary positions have been used to contribute to the influence of 3Rs activities, such as visiting scientist programs, postdoctoral positions, and internships. An internal, cross-departmental, visiting scientist program is one approach to using and sharing internal resources while allocating dedicated time to focus on work necessary for 3Rs implementation.⁶ The goal of such a program is to foster a more innovative culture through the cross-fertilization of ideas, information, and skills and to provide scientists with unique development opportunities that can be tailored to their needs and interests—in this case, the 3Rs. In addition to enabling the participating scientist to acquire new skills and expertise, this program helps to develop and empower employees to return to their home departments as 3Rs leaders.¹⁴ Although postdoctoral positions and internships may be more difficult to plan because of logistics and the need for a dedicated mentor, they also can be effective in achieving internal benefit with decreased resource requirements. These temporary roles can contribute to developing a balanced animal care staff and to training scientists who are focused on ethical research and the 3Rs and who in turn can enrich the field themselves. The disadvantages of temporary positions include the need to outline and approve projects ahead of time and the intrinsic interim nature of the position, which limits further work or development when the work timeline is complete. Rather, temporary positions often don't afford sufficient flexibility or time to achieve a noteworthy effect. Conversely, all of these positions offer numerous advantages that go beyond achieving 3Rs changes.

Having dedicated staff (full-time or temporary) broadly contributes to a robust culture of animal welfare.¹⁵ In addition to improvements in animal welfare, 3Rs initiatives often help to decrease costs, promote the efficient use of resources, and shape scientific research, such as through improved compound selection. In addition, the work performed by dedicated staff can serve as a catalyst to stimulate similar research by other members of the institution. The benefits of creating a central 3Rs liaison who is responsible for communicating, networking and facilitating sharing of 3Rs efforts and positive effects across the program cannot be overemphasized. Providing dedicated staff to advance the 3Rs not only improves communication and implementation of these principles but enhances employee awareness of and sensitivity toward ethical topics within an animal research program.

Creatively Achieving 3Rs Impact

Creatively achieving 3Rs impact can be accomplished through a variety of programs and in numerous animal research settings. Because there is no one-size-fits-all approach, creative implementation must be tailored to the needs of the institution, interests of staff, and availability of resources. Approaches include the use of internal committees or working groups, awards programs, and volunteer opportunities. Each of these approaches is not limited to application in industry but can be adapted to any research setting to advance 3Rs practices.

Internal committees and working groups.

The pharmaceutical industry is a recognized leader in the application of 3Rs practices, but having dedicated staff lead these alternatives efforts may not meet the needs of a particular institution for various reasons. A growing trend in the industry is to develop ‘alternatives committees’ to help promote and implement a robust 3Rs strategy.⁷ These committees bring together expertise from across scientific and animal care areas, potentially globally, with varied influence and experience from across the animal research program. The main functions of an alternatives committee might include assisting in scientific evaluation, prior to the implementation of alternative methods within and across global sites, and suggesting new opportunities for 3Rs improvements. Another positive outcome that can result is the ability to provide support to research investigators who wish to pursue a 3Rs project but have insufficient time, resources, or personnel. Rather, with the premise of ‘power in numbers,’ a project promoted as an alternatives committee initiative may be more likely to be supported by management as part of the 3Rs strategy. These committees can be formal, where annual goals are developed and then shared with the company's management and research staff, or informal, where they simply serve as a central source of communication. Either way, an alternatives committee can make a significant contribution by allowing a cross-functional team of experts to develop a cohesive and collaborative company-wide approach to the 3Rs.

Beyond the creation of a formal committee, another effective means of achieving 3Rs impactis to form project-specific working groups by bringing together all who have some stake in the target project. This diversity allows consideration of all aspects of a project including obstacles to implementation, differences in levels of support, and potential concerns with implementation (for example, scientific, financial, resource-related). By integrating everyone involved into a working group, all potential problems or concerns associated with pursuing a specific project can be balanced with the potential benefits, and a collective decision can be made regarding whether a project should move forward. This collaboration allows for a cohesive and harmonized approach. When the decision to pursue a project is made, everyone likely to be involved has already become invested in the project, a situation that likely will help it to succeed. Although potentially a slow process that presents challenges regarding scheduling, with appropriate planning and setting of timelines, a working group approach can be successful. One obvious issue is that such an approach requires a responsible leader to move the working group forward. In addition, top-down management support is often needed to get the working group started, followed by bottom-up support to see the project through to completion. With sound research hypotheses, clear expectations, and reasonable distribution of responsibilities, most staff likely will be happy to participate in work that promotes a growing commitment to ethical animal research and alternatives.

3Rs consortia.

Another way that companies achieve 3Rs impact is by supporting external consortia and by participating in consortia-led technical committees and workshops. Involvement in consortia can contribute to the 3Rs either directly or indirectly. Examples of indirect contributions include improved predictive ability and thus more efficient use of animals, improved ethical justification of animal use, and increased use of in vitro assays, which often lead to replacements or reductions in in vivo animal studies. Some consortia are geared specifically toward more explicit scientific aims, for example, finding improved models or assays for the prediction of drug-induced liver injury (MIP-DILI), cardiac safety (ILSI HESI–Cardiac Safety Technical Committee), and developmental and reproductive toxicology (ILSI HESI–DART Technical Committee and the Zebrafish Consortium). Other, more broadly focused partnerships support working groups and consortia as goals change, such as the International Consortium for Innovation and Quality (IQ) in Pharmaceutical Development's 3Rs Leadership Group as discussed in the editoral on page ; the European Union Reference Laboratory for Alternatives to Animal Testing (EURL–ECVAM); the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM); the National Center for the Reduction, Refinement, and Replacement of Animals in Research (NC3Rs), and the European Partnership for Alternative Approaches to Animal Testing (EPAA). Challenges to working with consortia include the time required to make a significant contribution. In addition, the time to obtain meaningful results is often slow, given that results rely on the involvement of many colleagues from multiple institutions. Most consortia rely on financial support for involvement, so management must be supportive and believe that financial investment will benefit the company before any results are achieved. Finally, although many topics discussed fall within a precompetitive space, legal agreements regarding involvement and those required to provide useful data to such consortia can be quite cumbersome and at times unachievable. Alternatively, there is a greater probability of success when multiple experts across industry work together toward a common goal. In addition, a group's influence typically increases as its membership increases, so that once a general consensus about a 3Rs topic is achieved through these consortia, it is often at a stage where implementation or adoption of changes happens quite naturally.^9,10

Awards programs.

Another way of indirectly achieving 3Rs impact is by creating and hosting an annual internal 3Rs awards program or by providing funding to external awards and grant programs. With the appropriate resources, internal awards programs can be quite effective and can provide the additional incentive and support needed for internal staff to either work toward 3Rs implementation or to share their work across the company. Research scientists often implement the 3Rs on their own, but without appropriate communication and recognition, most members of the company remain uninformed about these new methods or approaches. An internal awards program allows for the recognition of employees who go above and beyond to make a significant contribution by the adoption of alternatives (3Rs) into the program of animal research. In addition, this type of program contributes to building a culture supportive and conducive to ongoing 3Rs research. Some companies even make an annual event of the awards program by sponsoring 3Rs-related symposia showcasing both internal and external speakers as well as presentations through which the annual awards recipients to share their work. This type of program can start small and be expanded as the internal culture and support for such a program grows. Funding external 3Rs awards or grants programs can be a bit more challenging, given that a company must have existing financial resources and support to fund specific external programs. In addition, because many in the veterinary or animal welfare offices of companies are involved in the same organizations that fund annual 3Rs-related grants, conflicts of interest can become problematic, in view of the fact that members of the funding company should not have direct involvement in the recipient selection process. However, although limited, external grants are excellent ways to sponsor research supportive of the 3Rs and to empower persons wanting to conduct 3Rs research.

Volunteer opportunities.

Finally, a unique way of growing a culture of animal care and thus indirectly promoting 3Rs advancement is by making available volunteer opportunities in which employees can participate. Examples of volunteer opportunities include developing and presenting both internal and external programs that lead to dialogue and discussion about humane animal research and the 3Rs. These types of presentations are most beneficial when they present a balanced overview about animal research, including sharing information about what efforts and impact are being made in the 3Rs arena. Another example is encouraging involvement in refinements such as dog play and socialization programs, dog tooth-brushing programs, and behavioral modification programs. Allowing employees, both those directly involved in animal research as well as those that work in other areas of the company, an opportunity to bring about such refinements automatically contributes to intrainstitutional support and education. This hands-on, direct interaction offers a firsthand experience of the animal program, fosters a sense of responsibility for and pride in contributing to a refined approach, and provides a basis on which to form an opinion and to further support a well-rounded culture of care within the company. Although these programs come with some risk due to of the increased number of people who have access to the animal facilities, appropriate screening and education can mitigate this risk, which is far outweighed by the benefits of having more people involved in supporting 3Rs efforts.

In conclusion, although adhering to the regulations and guidelines ensures a sound animal program with regard to the adoption of the 3Rs, institutions are increasingly looking for creative ways to actively promote the 3Rs as part of a robust culture of care that goes beyond minimal requirements or standard practices. The regulations and guidelines help programs to build a foundation of animal care and use that is based on the 3Rs, after which each institution can enhance their 3Rs culture through a variety of different approaches that are based on specific program needs and resource availability. Centralization of 3Rs information and program oversight provides a valuable internal resource that can help to gauge advances. Involvement in external 3Rs consortia facilitates collaborative learning in a precompetitive space and improved alignment with industry peers. These and other mechanisms for promoting robust institutional incorporation of 3Rs principals and practices can be highly effective in promoting a stronger culture of animal welfare and growing the level of support from a broad base of scientists, veterinarians, animal care technicians, and the IACUC. A broad-based, team approach to the 3Rs, with at least one key leader to coordinate efforts (when feasible), ensures that multiple champions of alternatives are empowered to advance ethical science and animal welfare. This is a mutually beneficial and sustainable paradigm to consider.