Journal of the American Association for Laboratory Animal ScienceVolume 54, Number 2, February 2015

EDITORIAL

Medina et al. Special Issue: Global 3Rs Efforts – Making Progress and Gaining Momentum, pp. 115-118 Domain 3: ResearchDomain 5: Regulatory Responsibilities

SUMMARY: This is an Editorial introduction to this JAALAS issue dedicated to a current update/overview of the 3Rs in international animal research. It introduces the IQ-International Consortium for Innovation and Quality in Pharmaceutical Development and one of its offshoots, the LG-3Rs Leadership Group. The IQ is a 501-3c created by senior leaders from several global pharma and biotech companies in 2010 and its mission is to advance science-based and scientifically driven standard and regulations for the development of pharma products worldwide.

Prior to 2012, there was no global collaborative industry organization to share successes in the 3Rs or to improve the science of the 3Rs, so the LG was created to meet this deficit. The mission of the LG is to promote sharing and integration of high quality scientific practices to advance the 3Rs of animals used in the discovery and development of new medicines, vaccines, medical devices and health care products for humans and animals.

The LG consists of members from 23 IQ member companies and they meet monthly to discuss and guide 3Rs initiatives and ensure the goal progress is being tracked. From this group, a number of Working Groups were formed to focus on various subjects such as Benchmarking, establishing stronger relationships with key decision leaders such as non-governmental organizations, regulatory bodies, and CROs. A new initiative for 2015 is a partnership with AAALAC to implement an annual IQ 3Rs awards program to recognize scientific publications involving the 3Rs.

The article also contains an IQ brochure outlining guidance for creating a successful 3Rs awards program within a facility.

*No questions since this is an editorial introducing the rest of the articles in this issue.

OVERVIEWS

Tannenbaum and Taylor. Russell and Burch's 3Rs Then and Now: The Need for Clarity in Definition and Purpose, pp. 120-132

SUMMARY: The Principles of Humane Experimental Technique was written by Russell and Burch in 1959 to propose a new applied science that would improve the treatment of animals and serve as a foundation for future development of the 3 Rs. To accomplish this goal, it was important for Russell and Burch to provide appropriate definitions of the terms replacement, refinement, and reduction and the concepts of inhumanity. This paper describes and explains their definitions and explores and explains how interpretations have changed and what the implications may be on the Russell and Burch’s original intentions. The authors of this review suggest there is a need for greater clarity in the modern definitions surrounding the 3Rs and through this paper revisit the original work of the Principles for comparison.  THE CONCEPTS OF INHUMANITY AND HUMANITY  Inhumanity – Inhumanity was described by Russell and Burch using a number of unpleasant mental states that can be experienced by research animals, (pain, fear, conflict etc) and overall can be considered as equivalent to distress. Russell and Burch preferred to talk about distress as any single

adverse mental state was not sufficient when considering the minimization of distressful experiences of the animals. Also they wanted to portray the concept of inhumanity to have the potential to apply to a wide range of negative states. They also ensured that distress and therefore inhumanity could be quantified through criteria and measurement sufficiently enough to at least provide a scale with definable extremes of and increments between states of distress (inhumanity) and humanity.

Direct Inhumanity – Russell and Burch define direct inhumanity as the infliction of distress as an unavoidable consequence of the procedure employed even if performed with perfect accuracy and without complications. (ex. – inflicting pain for analgesia research)

Contingent Inhumanity - Infliction of avoidable and unnecessary distress due to factors such as poor techniques and poor husbandry, and can be reduced or eliminated.

Individual vs. Total Inhumanity -  Russell and Burch state that the distress felt while the 3 R’s are ways of reducing inhumanity of an individual, it is just as important that the sum total of inhumanity experienced by all animals in an experiment should also be addressed by the 3 R’s  WELLBEING AND THE 3R’S

The authors of the paper are intent to interpret the use of the term well-being by Russell and Burch. They suggest that Russell and Burch used the term to expand on the scale of humanity vs. inhumanity for evaluating the experiential state of experimental animals by adding another scale between distress and wellbeing (or humanity).  The authors then propose several interpretations of Russell and Burch’s use of the term wellbeing which appears 5 times in Principles. Overall, the lack of appropriate definition of wellbeing is problematic as it can be interpreted to mean an absence of distress or the provision of additional positive states or being that are not easily evaluated.  REPLACEMENT

Russell and Burch define: Replacement - The substitution for conscious, living, higher animals with insentient material. The authors point out that Russell and Burch intended for the ideal of replacement was to use completely insentient material, not animal classes of lower degree of sentience or presumed insentience. They also describe how the current usage of the term indicates that the goal of replacement is separate from the goal of minimizing distress. This is another departure from the Principles, as Russell and Burch saw replacement as the ultimate form of minimizing distress in animals. 

Replacement Technique – A method employing non-sentient material which may replace methods which use conscious living vertebrates.

Relative Replacement - Animals are still required for an experiment, though in the actual experiment they are exposed to no distress.

Absolute Replacement - Animals are not required at all for an experiment.  REDUCTION

Russell and Burch define:

Reduction – Reducing the numbers of animals used to obtain information of a given amount and precision. The authors of the paper defend that it was not intended to mean absolute minimization but simply reduction. References to Russell and Burch should not suggest absolute minimization as being required as it is difficult to know if reduction has been actually been achieved until after firm statistical analysis of an experiment is performed.  Another point made in the paper is that reduction must be described along with the ability to diminish animal distress. By only reducing the number of animals without thorough consideration of the degree to which animal distress is minimized (such as for

economic or perceived public scrutiny reasons) runs the risk of increasing relative distress on animals in the name of exclusively reducing animal numbers.   REFINEMENT

Russell and Burch define:

Refinement – Any decrease in the incidence or severity of inhumane procedures applied to those animals which still have to be used. Russell and Burch also state that the objective is simply to reduce to an absolute minimum the amount of distress imposed on the animal.  The authors point out that a potential deviation from the original intent involves the use of the term pain along with distress. In their opinion, this draws the focus away from the all the possible negative mental states of an animal that would be entirely included if only the word distress was used in the objective.             Documents referencing the Principles exist that add to the definition of refinement to include methods that enhance wellbeing as a result of diminishing distress (or pain and distress). These definitions are problematic for the authors in that wellbeing is not described, and it is unclear if wellbeing can be considered as a positive state of the animal beyond the absence of distress.  It is also an interesting discussion to consider whether wellbeing should be provided to an animal as a method of diminishing distress or if it is something additional that is our due to the animals.  Admittedly ungenerous, they suggest that applying a well-being in addition to relief of distress can have altering effects on the research and must be accounted for.  Overall, providing wellbeing to animals in research is appropriate but one must be careful not to detract from the essence of refinement as described in the Principles, and potentially should be considered as a separate aspect of enhancing the lives of research animals. CONCLUSION

Russell and Burch emphasized that the definitions of key concepts used in a scientific endeavor express the fundamental aims and priorities of that endeavor. Overall the aim of the Principles, expressed through the definition of the 3Rs, was that the reduction and elimination of animal distress was consistent with sound science. The evolution of the interpretation of the 3Rs to include aspects such as an entitlement of animals to comfort, pleasures, wellbeing or happiness, can be potentially problematic without clear definitions being extended to include these aspects.  QUESTIONS1. What is the proper title of the 1959 book of Russell and Burch where the 3R’s are first presented:

a. The Principles of the 3R’s in Researchb. The Principles of Humane Experimental Techniquec. The Principles of Humane Experimental Researchd. Humane Experimental Research Principles

2. T/F. In Russell and Burch’s 1959 work, the term inhumanity was the equivalent of distress to an animal

3. Infliction of distress as a required, unavoidable and inherent component of particular research was described by Russell and Burch as:a. Direct Inhumanityb. Indirect Inhumanity

4. T/F. Russell and Burch originally considered relative replacement to include the use of animals of lower sentience for distress inducing research.

5. T/F. Russell and Burch defined the refinement as a minimization of pain and distress through appropriate techniques in science.

6. T/F. For Russell and Burch in the Principles, wellbeing was clearly defined as an absence of distress for an animal.

 ANSWERS1. b2. T3. a

4. F. Replacement meant non-sentient materials were used, although they did described the use of degenerate metazoan parasites as non-sentient

5. F. Just a minimization of inhumanity or distress6. F. Well-being has different interpretations  

Bratcher and Reinhard. Creative Implementation of 3Rs Principles within Industry Programs: Beyond Regulations and Guidelines, pp. 133-138

Domain 4: regulatory responsibilities

SUMMARY: This article describes several mechanisms that can be used to implement the 3Rs in industry programs.

1) IACUC Oversight: There are 4 main documents that structure how IACUCs address the 3Rs (the AWA regulations, the Guide the PHS policy and the US Principles). 

*USDA and the 3Rs - the phrase "the 3Rs" is not referenced explicitly, but the AWA Regs state that the PI must consider alternatives to procedures that may cause more than momentary or slight pain or distress to the animals, and must provide a written narrative description of this. The USDA has provided the Animal Care Policy Manual to further clarify the regulations. In policy 12, Consideration of Alternatives to Painful/Distressful Procedures, the USDA first addresses the principles of the 3Rs. Here it is recommended that investigators perform a database search with a narrative that outlines: 1) the name(s) of the databases searched; 2) the date the search was performed; 3) the time period covered by the search; and 4) the search strategy. In some circumstances, conferences, colloquia, expert consultants, or other sources may provide relevant information regarding alternatives. 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. 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 - although they do not explicitly identify the 3Rs, the PHS Policy and US Principles include language that is supportive of the 3Rs. The PHS Policy states that the research must be performed in accordance with the AWA and must be consistent with the Guide. The PHS Policy directly identifies that non-animal alternative methods "such as mathematical models, computer simulation, and in vitro biologic systems should be considered". 

*The Guide   and the 3Rs - institutions accredited by AAALAC must adhere to the 8th edition of the Guide. The Guide contains the most IACUC guidance on the 3Rs of all of the main documents. The Guide stresses the 3Rs as an important part of decision-making in the design and review of animal research studies. The reuse strategy for reducing the number of animals is discouraged when reuse would compromise the animal's well-being. The PI, IACUC, and institution share the responsibility of the implementation of 3Rs principles. AAALAC endorses the Guide's view on harm-benefit analysis by the IACUC.

*Alternatives literature searches - The Animal Welfare Information Center (AWIC) can provide training and online resources for PIs. The Johns Hopkins Center for Alternatives to Animal Testing website, AltWeb, is another site to consider. 

2) Unique Roles And Positions Focused On The 3Rs: Full-time roles dedicated to the 3Rs are becoming a frequent way of advancing the 3Rs in industry. Strategic functions may include developing a 3Rs strategy, effectively communicating the strategy, gaining support from upper management, and facilitating implementation of the strategy. there are other dedicated positions that contribute to a scientific approach to refinement such as full-time behaviorists and enrichment coordinators. 

3) Creatively Achieving 3Rs Impact: The use of internal "alternatives" committees and working groups is often used in industry. Supporting external consortia and participating in consortia-led technical

committees and workshops can help companies achieve 3Rs impact. Creating and hosting an annual internal 3Rs award program, and creating volunteer opportunities can help as well. 

QUESTIONS1. Which document directly identifies that non-animal alternative methods "such as mathematical

models, computer simulation, and in vitro biologic systems should be considered"?a. Animal Welfare Act Regulationsb. US Principlesc. PHS Policyd. The Guide

2. Which documents structure how IACUCs address the 3Rs?a.  AWA regulationsb. The Guidec. PHS policy d.  US Principlese. All of the above

3. How often does the USDA expect that alternatives reviews will be conducted?a. Semiannuallyb. Annuallyc. Biannuallyd. At triennial de novo reviews

ANSWERS1. c2. e3. d

Wickremsinhe and Perkins. Using Dried Blood Spot Sampling to Improve Data Quality and Reduce Animal Use in Mouse Pharmacokinetic Studies, pp. 139-144

Domain 3Primary Species: Mouse (Mus musculus) SUMMARY: Current guidelines and practices limit the maximal total volume of blood that can be collected from a 25 g mouse to approximately 10% to 15% of its circulating blood biweekly, approximately 180 to 270uL. Using DBS technology allows more frequent blood sampling and reduced number of mice used, author experienced a 66% reduction in mice used. Some pharmacokinetic properties differ between plasma concentration and DBS concentrations (Cmax, AUC), while others parameters are similar (Tmax, T1/2). Therefore, it is important to understand drug unbound fraction in plasma and blood, its blood cell affinity, and the anticipated hematocrit range.  Hemocrit Effect refers to bias in the concentrations that arise due to difference between the hematocrit value of the sample and the standard curve used for quantification. However, this variability should be low with inbred animals. Single 20uL spot collected per time point may limit any subsequent reanalysis for sample reanalysis (required for Good Laboratory Practices studies). DBS data should not be equivalent to plasma data since the relationship will be dependent on the blood:plasma partitioning ratio, but both DBS and plasma are equally valid.  QUESTIONS1. In drug pharmacokinetic studies, what term describes the bias in the concentrations that arise due

to difference between the hematocrit value of the sample and the standard curve used for quantification? a. RBC Effectb. Blood: plasma partitioning ratioc.  Washout Periodd. Hematocrit Effect

2. In drug pharmacokinetic studies, what is a method that can reduce the number of animals used, reduce variability due to collection of pharmacokinetic and pharmacodynamics data from the same mouse, and reduce stress to mice due to reduced blood collection. a.  Mandibular bleedingb.  Dried Blood Spot technologyc. Tail snip bleedingd. ELISA plasma concentrations

 ANSWERS 1. d2. b

Caron et al. Miniaturized Blood Sampling Techniques to Benefit Reduction in Mice and Refinement in Nonhuman Primates: Applications to Bioanalysis in Toxicity Studies with Antibody–Drug Conjugates, pp. 145-152

SUMMARY: Minimizing the number of animals in regulatory toxicity studies while achieving study objectives to support the development of future medicines contributes to good scientific and ethical practices. Toxicokinetic determinations evaluate the relationship between the systemic exposure of a drug and its toxicity. In rodent species, the number of animals specifically dedicated to toxicokinetic determinations in a toxicity study mainly depends on the number of sampling time points, sample volume, sampling techniques, and physiologic limitations that may affect animal welfare and consequently influence the overall toxicological assessment. Study demonstrated the feasibility, applicability, and relevance of miniaturized blood sampling procedures in the context of the toxicologic assessment in mice and NHP. Figures are useful in this article.

Procedure for blood sampling in lightly restrained cynomolgus monkeys. (A) The lateral side of the heel is visualized. (B) After local disinfection and puncture with a 21-gauge needle, blood (50 μL) is collected in a microtube precoated with lithium heparin.

Procedure for blood sampling in lightly restrained mice. (A) The lateral saphenous vein is visualized after local application of lubricant. (B) A puncture is performed by using a 25-gauge needle. (C) The blood volume (25 μL) is collected into a glass capillary tube precoated with lithium heparin.

Figure 5. Procedure for blood sampling in lightly restrained cynomolgus monkeys. (A) The lateral side of the heel is visualized. (B) After local disinfection and puncture with a 21-gauge needle, blood (50 μL) is collected in a microtube precoated with lithium heparin.

Avery et al. The Use of Systematic Reviews and Reporting Guidelines to Advance the Implementation of the 3Rs, pp. 153-162

SUMMARY: The 3 Rs (replacement, reduction, refinement) have become the guiding principles for ethical use of animals in biomedical science. The number of published reports in scientific journals using animals has increased in an almost exponential manner over the previous decades. In spite of the increase in published reports efforts to enhance the application of the 3 Rs has been hampered by shortcomings in identifying, collecting, and implementing published information, publication bias wherein negative results are less likely to be published, incomplete reporting of relevant details in published reports, and selection bias in literature searches.

Systematic review is a method to “review a clearly formulated question that uses systematic and explicitly methods to identify, select, and critically appraise relevant research, and to collect and analyze data from the studies that are included in the review. Statistical methods (meta-analysis) may or may not be used to analyze and summarize the results of the included studies.”

A systematic review of reviews concerning the 3Rs was performed. It found only 10% of reviews evaluated explicitly reported on the 3Rs, and those exclusive mentioned reduction. None of the evaluated reviews mentioned reduction or refinement. Systematic review of animal studies is a relatively new endeavor. If the 3Rs are considered during the initial planning stages of a review it may assist in assembling and implementation of the principles.

QUESTION1. Identify components of systematic review:

ANSWER1. Systematic review includes:

a. Clearly formulated questionb. Explicit methods to identify, select, and critically appraise relevant researchc. Statistical methods (meta-analyses) d. Summary of results

Bourcier et al. Improving Prediction of Carcinogenicity to Reduce, Refine, and Replace the Use of Experimental Animals, pp. 163-169

SUMMARY: Because pharmaceutical are designed to have biological effects, and because they are often taken for long periods of time, potential for toxicities are of concern.  Many toxicities are discovered during clinical trials, but because of the time frame required for carcinogenicity, teratogenicity, and mutagenicity, often these effects are assessed in non-clinical studies.  The current gold standard for assess carcinogenicity is the 2-year rodent bioassay. This assay is known to detect most of the known human carcinogens, however the assay is flawed. This system is insensitive because of small animal numbers, compared to large numbers of people exposed to the drug for extended periods. These studies are also lengthy, expensive,  and animals are exposed to doses that often far exceed doses likely to be used clinically. Exposing animals to these extreme doses can alter biologic processes that are not relevant in clinical exposures. This system can also result in drug induced tumors, with questionable relevance to humans.

In the 1970’s it was thought that a series of in vitro tests could successfully identify potential carcinogens and thereby substitute for the 2 year rodent bioassay, however a study in 1987 showed that genotoxic chemicals were often carcinogens, there are many nongenotoxic carcinogens.

Another system developed to supplant the rodent bioassay was the Syrian hamster embryo transformation assay. Because of mechanisms by which drugs s induce cancer, which are often related to pharmacology, hormonal imbalance, and immune suppression, that are unlikely to be replicated in this assay, this assay has overall been rejected as being useful in drug development.

With consideration of the 3 R’s, in silico models have become more important in risk assessments because they offer early insight into potential safety issues for new agents. QSAR (Quantitative structure-activity relationship) models provide rapid, cost effective assessments of the toxicity of molecules based on chemical structures.  This system again, has its limitations, given the complexity and longevity of many medications in clinical use. These methods will require many modifications, but provide the ability to replace animals, and give sight to much of the potential for carcinogenicity.

Based on ICH guidelines a single long term rodent carcinogenicity study with the addition of one other supplementary study to provide information that is not readily available from the long term assay is recommended. Rats are recommended as the model for the long term assay, unless there is evidence favoring another species. When in vivo tests are used, transgenic mice may be used as a secondary carcinogenicity assay, however it has been found that transgenic mice studies miss some known and probable human carcinogens.

It is also recommended that a WOE (weight of evidence) approach for biotechnology-derived products are used with a carcinogenicity assessment is warranted, however produce specific assessments may be needed depending on the treatment duration, patient population, or biologic activity of the proposed product. In specific cases, a single rodent species may be considered.

It is suspected that more flexibility in the ICH guidelines may complement the 2 year rodent assay. As it is recognized that the 2 year rodent assay has many key shortcomings, it is theorized that efforts to develop other meth methods for clinical monitoring of the potential for cancer risks from pharmaceuticals may be more fruitful than building a better rodent bioassay QUESTIONS1. According to the Blue Book, how much food do mice consume per day (after weaning)?2. According to the Blue Book, mice consume how much water? (Pg 41 D2 L 3)

ANSWERS1. 3-5 grams2. 6-7 mL/day

 Casey et al. A New Path Forward: The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and National Toxicology Program's Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM), pp. 170-173

Domain 3: Research (Replacement, Reduction and Refinement techniques) SUMMARY: In 2013, ICCVAM decided to redirect their focus towards fostering more productive communication with stakeholders and international collaborators, while simultaneously improving the relevancy and coordination of ICCVAM recommendations. ICCVAM was created in 2000 to facilitate the development, validation, and regulatory acceptance of new and revised regulatory test methods that employ the 3 R’s while maintaining scientific integrity. NICEATM provides scientific and operational support for ICCVAM activities. Three areas of priority of ICCVAM through 2017 are 1) reducing the number of hamsters used in Leptospira vaccine potency testing through use of an ELISA test, 2) evaluating the contribution of LD50 tests for hazard labeling and PPE and investigating in vitro assays for predicting oral LD50 values, and 3) developing a plan for evaluation of alternative skin sensitization test methods and strategies. To increase federal agencies’ awareness of international 3R efforts, ICCVAM will communicate with the Organization for Economic Cooperation and Development (OECD), which has guidelines that are internationally agreed-upon for use in the 34 OECD member countries to determine safety of chemicals. Additionally, in 2009 the International Cooperation on Alternative Test Methods (ICATM) was established to, in part, promote timely international adoption of alternative test methods. Current participating organizations are from the US, Europe, Japan, Korea, and Canada. Finally, Tox21 is a multiagency collaboration that aims to shift chemical toxicity assessment toward target-specific, mechanism-based observations obtained through in vitro high-throughput and high-content screening assays to test 10,000 substances to determine “adverse outcome” pathways to toxicity, in hopes to eventually use in vitro assay data to predict adverse effects in vivo.   QUESTIONS1.  How many federal agencies are involved in the ICCVAM committee?2.  The ICCVAM Authorization Act formally established ICCVAM as a permanent committee at which

institution? ANSWERS1.  15, including Departments of Ag, Defense, Energy, and Transportation, the EPA, FDA, NCI,

NIOSH, NIEHS, NIH, and OSHA.2.   National Institute of Environmental Health Sciences (NIEHS)  Turner et al. Roles of the International Council for Laboratory Animal Science (ICLAS) and International Association of Colleges of Laboratory Animal Medicine (IACLAM) in the Global Organization and Support of 3Rs Advances in Laboratory Animal Science, pp. 174-180

Domain 4: Regulatory responsibilities

SUMMARY: Practical implementation of the 3Rs at national and regional levels around the world requires long-term commitment, backing, and coordinated efforts by international associations for laboratory animal medicine and science, including the International Association of Colleges of Laboratory Animal Medicine (IACLAM) and the International Council for Laboratory Animal Science (ICLAS). Together these organizations support the efforts of regional organization and communities of laboratory animal science professionals as well as the development of local associations and professional colleges that promote the training and continuing education of research facility personnel and veterinary specialists. The recent formation of a World Organization for Animal Health (OIE) Collaborating Center for Laboratory Animal Science and Welfare emphasizes the need for research into initiatives promoting laboratory animal welfare, particularly in emerging economies and regions with nascent associations of laboratory animal science. 

The mission of ICLAS is to promote and coordinate the development of laboratory animal science throughout the world, particularly in developing countries. The council fosters international collaboration, quality definition and monitoring of laboratory animals, the collection and dissemination of information on laboratory animal science, and harmonization in the care and use of laboratory animals. ICLAS encourages humane use of animals in research by recognizing ethical principles and by promoting 3Rs tenets. The International Association of Colleges of Laboratory Animal Medicine (IACLAM; www.iaclam.org) constituted in 2006.

It is global organization that brings together national and regional CLAM with the goal of providing a common platform at the global level for communication among and representation of these colleges and their diplomats in various activities.

IACLAM currently consists of 4 member colleges, the American College of Laboratory Medicine; European College of Laboratory Animal Medicine; Japanese College of Laboratory Animal Medicine; and Korean College of Laboratory Animal Medicine Currently, the membership of the 4 colleges totals approximately 1155 active diplomats in 24 countries IACLAM, as an international organization, provides a readily accessible source of expertise from credentialed specialists in laboratory animal medicine Veterinarians undertaking the practice of laboratory animal medicine can demonstrate specific competency by becoming certified by an established group of specialists (also known as a College of Laboratory Animal Medicine [CLAM]) who have defined a set of skills and knowledge that each member, typically called a ‘Diplomat’, should possess. QUESTIONS1.  T\F?  The ICLAS committee’s goal is standardization of animal welfare and use all over the world.2. When, and by whom the 3Rs' published.3.  What established between OIE ILAR and IACLAM in 2012 and what its aim? ANSWERS 1. F. Each country should be able to maintain an animal welfare oversight system that reflects its

cultures, traditions, religions, laws, and regulations.2. In 1959, the report by Russell and Burch was published as 'The Principles of Humane Experimental

Techniques'3. In 2012, the first OIE collaborating center for laboratory animal science and welfare was established

in partnership with ILAR with the support of IACLAM representatives The long-term aim of this laboratory animal science and welfare center is to promote research and educational initiatives to enhance the acceptance and adoption of 3Rs tenets around the world.

Bayne et al. The Evolution of Animal Welfare and the 3Rs in Brazil, China, and India, pp. 181-191

Domain 4: Regulatory responsibilities  SUMMARY: More research is being done across country borders increasing the need for assurance that the quality of animal welfare and caliber of animal research conducted are equivalent among research partners around the globe. Global harmonization of animal care and use standards and practices is needed. One way to achieve this is the successful integration of the 3Rs in animal care and use programs. Here we review status of implementation of 3Rs worldwide by focusing on 3 countries with large, fast-growing economies.  CIOMS and ICLAS push incorporation of the 3 Rs (Replacement, Reduction, and Refinement) in the “International Guiding Principles for Biomedical Research Involving Animals”. Many nations now include 3Rs in regulations, codes, or guidelines that govern the use of animals.

One study found that despite doubling numbers of articles published annually, the proportion of studies using animals fell by 30%, with a 50% drop in the average number animals used per published paper. Another study found no large fluctuations in number of experiments based on increasing ethical concern for lab animals published in Nature Medicine 1998-2003. It did find a dramatic increase in the number of articles including statement about “the relevant laws and institutional guidelines with which each experiment should comply, and the institutional committees which approved each experiment, as requested by Nature Medicine”. Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines – intended to improve detail published literature – include #, characteristics of animals used, randomization or blinding to decrease bias in study, and well-described statistical methods applied to the data. >300 journals endorsed ARRIVE guidelines but may not have resulted in actual improvement in quality of information contained in preclinical research publications. Canadian study participants felt they were already practicing reduction in numbers and were still reluctant to use analgesics. Netherlands studies indicated scientists had problems with searching for alternatives and had limited knowledge of specialized databases. There is room for greater understanding and implementation of the 3Rs. Global progress possible due to institutions participating in AALAC accreditation. AALAC adopted OIE’s Terrestrial Animal Health Code as a reference resource. AAALAC also requires conformance with 1) Guide for the Care and Use of Laboratory Animals the 2) Guide for the Care and Use of Agricultural Animals in Research and Teaching, and the 3) European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (ETS 123). Both the lab animal guide and the European resource encourage use of the 3Rs.  PUBLIC AWARENESS OF THE 3RS

Brazil: Lay public was largely unaware of the 3Rs. After an activist group broke into a research facility, the scientific community wanted to inform the public about how research is regulated in Brazil. Laboratory animal scientists gave lectures to students and television and newspapers interviewed people in the field.

China: Chinese culture has long-standing traditions of Confucianism, Taoism, and Buddhism that advocate concern for living things. Many multinational pharmaceutical companies and contract research organizations have had a positive influence on animal welfare in China, including tangible evolution of the acceptance of the 3Rs.

India: Ethical concern about the use of animals in research is gaining traction due to strict guidelines by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). However, the general public is mostly unaware of the 3Rs.  AWARENESS OF THE 3RS IN THE SCIENTIFIC AND VETERINARY COMMUNITIES

Brazil: Scientists have become increasingly aware of the 3Rs, mainly since 2000, due to international collaborations in which adherence to the 3Rs was expected. An Ethical Committee has also been established in almost every university. Scientists who work with animals must be conscious of the 3Rs and apply them in their day-to-day work. However, only a small number of veterinarians are aware of the 3Rs – primarily those who take part in research at or work for universities.

China: Dr. Ruishan Liu is now considered a pioneer in modern Chinese laboratory animal science. He studied lab animal medicine at the University of Maryland and lectured on animal welfare, the 3Rs, and animal rights activities when he returned to China in the late 1980s. Lab animal science has been developing quickly since then. Currently, the broader veterinary and scientific communities in China are very well aware of the 3Rs and animal welfare, although there is uneven implementation of these principles in different regions of the country.

India: Scientists who conduct animal-based research and lab animal vets who support them are familiar with the 3Rs.

 INCORPORATION OF THE 3RS INTO LAW OR PREVAILING GUIDELINES

Brazil: In 2008, the Arouca Law was approved which addressed aspects of the 3Rs. Specifics, including the use of alternatives, use of minimal possible animals, analgesia or anesthesia should be used for painful procedures, and if adjunctive procedures will be performed on an animal it should be during the same anesthetic period and the animal should be euthanized before recovering. This law does not actually discuss implementation. A Brazilian Guide is currently being written.

China: In 2012, a grant was given by the Ministry of Science and Technology (MOST) to establish a Chinese laboratory animal care and use accreditation process similar to AAALAC. The Chinese Association of Laboratory Animal Science (CALAS) recently established a committee to develop Chinese standards for lab animal welfare and ethics. There is a chapter on animal welfare added to the Regulations on the Management of Experimental Animals in 2004. This chapter represents the first time that animal welfare is to be regulated in China. The Guideline on Humane Treatment of Laboratory Animals by MOST in 2006 is considered the country’s first broad animal welfare regulation. They require each institution to establish an IACUC.

India: Experimentation on animals in India is regulated by the CPCSEA, a statutory body under the Prevention of Cruelty to Animals Act, 1960. The committee is to ensure animals are not subjected to unnecessary pain or suffering before, during, or after the performance of experiments on them. Institutions are also required to have an Institutional Animal Ethics Committee (IAEC) to evaluate projects for their validity and examine the effective implementation of the 3Rs. The aftercare and rehabilitation (a fourth R) of NHP and dogs after use in scientific experiments is mandatory. Rehabilitation of an animal extends until the animal resumes a normal existence, and investigators discharge their duty by providing a lump-sum amount as costs for rehabilitation and care to cover its expected lifespan.  IMPLEMENTING THE 3RS IN THE CONDUCT OF RESEARCH

Brazil: The investigator must address the 3Rs in the protocol form of a proposed study. The principle investigator must document consideration of non-animal alternatives or a “lower” species. The Ethical Committee also advises the researcher to consult with a statistician to help reduce the number of animals used. The Ethical Committee must be provided the curricula vitae of project personnel. In 2012, the Brazilian Society of Alternative Methods was formed. The Ministry of Science, Technology and Innovation established a Brazilian National Network of Alternative Methods to be coordinated by the Brazilian Center for Validation of Alternative Methods and INMETRO (Brazilian accreditation body for the accreditation of laboratories).

China: 3Rs are required to be considered when developing and reviewing an animal study protocol. The Chinese government has supported animal replacement studies. Animal test information databases have been funded by MOST in order to share research results and hopefully reduce the number of animals used. The 3Rs are strongly advocated and promoted in the animal research community in China.

India: CPCSEA appoints a representative to every registered institute or organization to oversee the welfare of the animals housed or used for experiments or breeding. The CPCSEA nominee and IAEC monitor the effective implementation of the 3Rs in animal research.  TRAINING IN THE 3RS

Brazil: Training of personnel provided by the Ethical Committees of the institutions and by the Brazilian Laboratory Animal Science Association.

China: Seminars are frequently held covering the 3Rs. Since 2003, a Beijing international symposium on animal welfare has been held regularly. In 2001, MOST issued the Regulation on the Management of the Laboratory Animal License System. Under this regulation, a person who works with laboratory

animals must receive training and a license from the local administrative office of laboratory animals. To obtain an animal use license, a person needs to attend five days of government-provided training courses and take a daylong test that includes animal welfare and the 3Rs.

India: Indian Council of Medical Research established the Laboratory Animal Information Service at the Indian Cancer Research Centre. This facility performed the first-ever survey of the conditions of animal facilities in the country and started regular courses at the junior level for animal handling and experimentation. The government of India established the National Institute of Animal Welfare in Ballabhgarh to promote awareness and disseminate information about animal welfare to the public.  EVOLUTION OF THE AWARENESS AND IMPLEMENTATION OF THE 3RS

Brazil: The majority of scientists have embraced the 3Rs. One of the most important outcomes in Brazil regarding the implementation of the 3Rs has been its effect on education. The majority of university classes use alternatives and the number of animals used in education in Brazil has decreased dramatically.

China: Modern lab animal science has undergone rapid development since the early 1980s. Since 1994, the China State Bureau of Technical Supervision has issued over 100 national standards (GB) that address quality control for several lab animal topics. The new GB is 95% similar to the 8 th edition of the Guide, including references to the 3Rs, indicating that China aims to align with international practices and standards in lab animal research.

India: CPCSEA has incorporated the 3Rs as a foundation for formulating guidelines and for evaluating animal research protocols. Ever since the implementation of CPCSEA guidelines, awareness among Indian scientists is steadily increasing, and most of the scientists who are using animals in biomedical research in India are aware of the guidelines and the 3Rs.  QUESTIONS1. What 3 primary standards does AAALAC utilize in its global assessments?2.  What is the regulatory authority in India regarding animal use?3. What is the 4th R that has been made mandatory for dogs and NHP in India?             ANSWERS:1. Guide for the Care and Use of Laboratory Animals

Guide for the Care and Use of Agricultural Animals in Research and TeachingEuropean Convention for the Protection of Vertebrate Animals 

2. Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) 3. Rehabilitation        

Fleetwood et al. Making Progress and Gaining Momentum in Global 3Rs Efforts: How the European Pharmaceutical Industry Is Contributing, pp. 192-197

Domain 5: Regulatory Responsibilities; TT5.1. Laws, regulations, policies and standards SUMMARY: The European Federation of Pharmaceutical Industries and Associations (EFPIA) represents the pharmaceutical industry operating in Europe, and includes 40 companies and 33 national associations. The EFPIA and member companies recognize the ethical dilemma of using animals in research, and take efforts to not only comply with existing regulations, but also go further by supporting the 3Rs and promoting higher animal welfare standards. This paper illustrates some of the EFPIA’s recent activities in support of the 3Rs principles and animal welfare. 

Contributing to Global Discussion on the 3Rs and Animal Welfare: Pharmaceutical industry experts played a leading role in the drafting and legislative process of Directive 2010/63, which updated and ensured common high standards regarding animal welfare across the whole of Europe. The Directive imposed many new or improved standards such as mandatory ethical review and authorization of

studies involving animals, a new evaluation scale which categorizes procedures by degree of pain, suffering distress or lasting harm expected, and new housing and caging standards. Another new regulation states that NHPs should be sourced from second-generation (F2) born-in-captivity animals. The EFPIA has helped with implementation of these new standards and work out the feasibility of these new policies.

Measuring 3Rs Progress: the Research and Animal Welfare (RAW) working group of the EFPIA selected 5 key performance indicators based on a survey with which to assess the effect of investments in 3Rs. They are as follows: 1) examples in reduction of severity, 2) evidence of senior executive ownership for 3Rs, 3) existence of internal 3Rs structures, 4) number, subject and impact of internal 3Rs awards, and 5) involvement in external 3Rs initiatives

Transport: Global 3Rs Opportunities and Risks: High quality, reliable animal transport is very important to both research and animal welfare. The RAW group is contributing to discussions about regulations and processes to further improve the quality of animal transport.

The Era of Global Communication: The RAW group is driving several initiatives regarding improving communication. These include a published report illustrating the industry’s efforts and commitment to the 3Rs, an “Animal Testing Perspectives” blog, and an “Animal Research for Life” website.

Solving the challenges of modern animal research will require multi-stakeholder and international collaborations. Some examples of these types of initiatives include the Innovative Medicines Initiative (IMI) and the European Partnership for Alternative Approaches to Animal Testing (EPAA). In addition, individual member companies of the EFPIA also work together as part of other initiatives that have 3Rs effects.

QUESTIONS1.  According to the Directive 2010/63, where should NHPs for research be sourced from?

a.  Wild-caught animalsb.   F1 born-in-captivity animalsc.  F2 born-in-captivity animalsd.  F3 or more born-in-captivity animals

2.  Which of the following is NOT an example of how the RAW group is using the internet to help improve communication regarding animal research and welfare?a.  A blogb.  A Twitter accountc.  A website

 ANSWERS1.  c. F2 born-in-captivity animals2.  b. A Twitter account

Burden et al. Pioneering Better Science through the 3Rs: An Introduction to the National Centre for the Replacement, Refinement, and Reduction of Animals in Research (NC3Rs), pp. 198-208

Domain 3: Research Domain 5: Regulatory Responsibilities SUMMARY: The National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) is an independent scientific organization based in the UK.  The purpose of this organization is to use the 3R’s to accelerate advances in science and business practice, apply new technologies, minimize the use of animals in research and improve animal welfare.  In addition to focusing on the importance of the 3Rs for social and ethical reasons, the NC3Rs was formed to promote the use of the 3Rs for improving the quality of science.

The NC3Rs uses different tools and strategies to accomplish its goals. 

1. Funding research training and early career development2. Supporting open innovation

a.  CRACK IT is an open-innovation platform that supports research and development using the 3Rs.  An annual competition is held to solve a Challenge and promote collaboration and innovation.

3. Influencing policy, practice and regulatory changea. Shaping the academic research landscapeb.  Influencing practices within the pharmaceutical and chemical industriesc. Improving the science of animal experimentsd. Improving animal welfare

4. Providing information resources CASE STUDIESApplying The 3Rs In Asthma: Research: Asthma is a complex disorder  that is considered one of the most common respiratory diseases, affecting 300 million people currently undergoing treatment globally, and has a significant economic burden.  Despite significant research focus, very few novel treatments have been developed in the past 50 years.

Initiation Of The NC3Rs Program In Asthma:  In 2009, the NC3Rs brought together participants from academia and industry to identify opportunities for improving animal models of asthma.  The findings of the workshop directed future investment in this type of research.  The NC3Rs investment in asthma research now stands at $2.3 million and has resulted in several peer-reviewed publications.   CRACK IT Challenge: Rodent Big Brother: One of the first CRACK IT Challenges was based on an idea by AstraZeneca scientists who wished to obtain more information from animals that were already being used in drug development.  Much data from rodent studies is lost because it is not recorded or observed.  The system developed during this challenge utilizes video recording and software that automatically recognizes and distinguishes the location and behavior of individual animals when animals are housed together. CRACK IT Solution: An early screen for emetic liability of novel chemical entities by using the amoeba Dictyostelium. This project allowed the submission of a model for emetic testing that could be utilized in the compound development process. Within one month of the Solution being posted on the CRACK IT website, GSK proposed a collaboration to utilize this model for testing the palatability of bitter compounds. Developing practical guidance to minimize the use of NHPs in the development of monoclonal antibodies

The large number of mAbs in research and development programs are driving an increase in the use of NHPs.  In an effort to work on reduction, a project was initiated in 2006 with a workshop project to imagine alternatives if NHPs were unavailable for use.  This resulted in the publication of an article outlining the strategies that were discussed.  After the workshop, an expert working group was established to determine the best strategy and implement and integrate potential alternatives into current practice and study design. An data-sharing exercise showed that in some cases, rodents would be an acceptable alternative to NHPs, as well as the use of fewer dose and recovery groups. Strategic CRACK IT Challenge:  Improving the predictive capacity of in vitro cytokine release assays to reduce animal use and drug attrition.

In 2011 a project was started to develop in vitro human cell-based models for the testing of protein-based biopharmaceuticals (including mAbs) to allow the prediction of human cytokine release and to develop a parallel assay that uses NHP cells to predict cytokine release in preclinical safety assessment. Looking To The Future: The NC3Rs identifies 5 interrelated areas as challenges for the future: practice, procedures, people, places and policy. The future strategy will build on the previous 10 years of

experience based on collaboration, international outreach, and back-to-basics approach.  The NC3Rs will continue to provide new resources to scientists and institutions as well as relevant training of how to use them. QUESTION1. The 3R’s consist of :

a.  Replacement, refinement, reductionb.  Replacement, refinement, revisionc.  Replacement, revision, reductiond.  Replacement, reduction, review

 ANSWER1. a

Cozigou et al. The European Partnership for Alternative Approaches to Animal Testing (EPAA): Promoting Alternative Methods in Europe and Beyond, pp. 209-213

Domain 3: Research; K12. Replacement, Reduction and Refinement techniques SUMMARY: The European Partnership for Alternative Approaches to Animal Testing (EPAA) was launched in 2006 as a unique and innovative public-private partnership, with the goal of promoting the 3Rs to meet regulatory requirements based on better and more predictive science. EPAA was developed by the European Commission (EC) in collaboration with industry stakeholders. Sectors represented include agrochemicals, animal health, chemicals, cosmetics, fragrances, pharmaceutical, and soaps and detergents. EPAA’s existence and success relies on transparency for decision-making, consensus among members, and mutual trust for each side of the public-private partnership. Cross-sector dialogue between EPAA members focuses on both the scientific aspect of research project dedicated to the application of the 3Rs and the regulatory aspect to better understand and facilitate the implementation of 3Rs principles. This article highlighted a growing need for international collaboration and implementation of non-animal alternatives, pointing to stem cell safety testing and vaccine batch release testing as case examples. QUESTIONS1. T/F: In 1986 the European Council adopted Directive 86/609/EEC to regulate animal welfare.2.  EPAA’s organizational structure does NOT include which of the following?

a.  The Steering Committeeb.  The Mirror Groupc.  European Union Reference Laboratory for Validation of Alternatives to Animal Testing (EURL-

ECVAM)d.   A private-public partnership

 ANSWERS1.  T2.  c. EURL-ECVAM was created by the European Commission in 1991.

Settivari et al. Predicting the Future: Opportunities and Challenges for the Chemical Industry to Apply 21st-Century Toxicity Testing, pp. 214-223

Domain 3: Research (Replacement, Reduction and Refinement techniques) SUMMARY: Whereas conventional toxicology uses mainly animal-based, descriptive methods, a paradigm shift emerging in which computational approaches, systems biology, high-throughput in vitro toxicity assays, and high-throughput exposure assessments are beginning to be applied to mechanism-based risk assessments in a time and resource-efficient fashion.

In systems biology, computational biology, and high-throughput assay development have led to an enhanced understanding of toxicology pathways

As a result a new paradigm often referred to as ‘Toxicity Testing in the 21st Century,’ or simply ‘ Predictive Safety Assessment ’ has emerged.

In predictive safety assessment, an integrated suite of in vitro, molecular, and biochemical assays is combined with cheminformatics approaches to predict the likelihood of adverse effects occurring under various exposure scenarios.

Cheminformatics refers to mathematical, chemical, structural, and statistical modeling tools to predict chemical effects on human health or the environment. This approach integrates all relevant information on a compound and its structural or substructural analogs to make preliminary assessments of potential toxicity. It also involves qualitative identification of structural alerts , read-across through the identification and assessment of closely related analogs of the target chemical, and advanced data mining techniques to identify putative modes of action (MoA)

The structure-based (quantitative structure activity relationship [QSAR]) prediction approach is among the first developed and most commonly used cheminformatics tools for safety assessment In vitro biologic profiling includes the use of 2 or 3D cell models that comprise relevant morphologic and biochemical signaling processes and are designed as either one-to-one replacements of existing in vivo assays or as tools to specifically evaluate mechanistic processes, which underlie adverse effects

Given the reductionist nature of in vitro assays, these models are less suitable for addressing more complex biologic endpoints (for example, developmental toxicity, repeat-dose toxicity). To address this limitation, integrated testing strategies are applied, where a series of computational, biochemical, and in vitro tools are applied to predict perturbations in key events leading to an adverse outcome

In the case of complex endpoints for which the mechanism is not well characterized (for example, reproductive toxicity), a combination of in silico, high-content ‘omics,’ and bioinformatics approaches are applied to delineate broader mechanistic pathways

“Omics” profiling has been applied to classify chemicals into different toxicity classes for read-across purposes

High-throughput screening is another common predictive tool, in which large numbers of chemicals are tested by using simple biochemical or in vitro assays to query their effects on a single biologic response (for example, the US Environmental Protection Agency’s ToxCast program)The millions of data points generated by these approaches are modeled by using high-end computational tools and network models for efficient safety characterization and for prioritizing chemicals for more comprehensive testing During the early stages of new chemical development, the need is to conduct a preliminary evaluation of new candidates from which 1 or 2 are selected for further development. Testing strategies at such early stages usually involves a tiered testing approach, with interim decision points or ‘stage gates’ for further advancement of the molecule.

The process (tier 1) begins with cheminformatics, which includes the collection and evaluation of any existing toxicity information for the chemical being evaluated as well as toxicity data on structurally related analogs. This effort is accompanied by QSAR predictions (global or local prediction models) to identify structural alerts and to generate hypotheses on toxicity and putative MoA The subsequent tier in the testing strategy may involve targeted in vitro assays, which are selected based on the putative MoA and alerts identified in the first tier

This assessment may include either a single or a battery of in vitro assays, depending on the complexity of the hypothesized endpoint.

Chemicals advancing through the first 2 tiers usually move on to regulatory-guideline–required in vivo studies, which can be further customized based on the information gleaned from earlier tiers. Another major challenge that the chemical industry faces regards pressures to address the so-called ‘toxics information gap.’

Regulatory agencies as well as external stakeholders are looking toward predictive toxicology approaches, such as the use of cheminformatics, validated in vitro assays, and the grouping of substances into categories to support the use of read-across to improve the quantity and quality of information on these chemicals and thereby to fill the data gaps.

Of these approaches, the grouping of substances into categories and the use of read-across offers significant opportunities to reduce the need for new animal-based toxicologic studies.

The acceptance of the use of read-across by regulatory agencies therefore depends on the strength of the justification underpinning it. As such, perhaps the greatest challenge faced when using read across is providing robust scientific justification for why read-across between analogs or within a category is acceptable and likely to be associated with minimal uncertainty in the predictionThe issue of uncertainty in the use of read-across may be addressed by the use of uncertainty factors allowing the use of read across to be accepted and a subsequent risk assessment performed even in cases where there are minimal data supporting the read-across justification.

Another possibility for using these tools to reduce the uncertainty is to build them into an integrated testing strategy and to make use of Bayesian networks in a ‘Bayesian Network Integrated Testing Strategy.’

An alternative approach to strengthening read-across justifications and reducing uncertainty is to consider the use of read-across in the context of the broader 21st-century toxicity testing paradigm. It is apparent that the formation of categories or groups of analogs based on structural similarities complements the use of the broad array of predictive toxicology tools and that these can provide support for the justification that underpins the use of read-across within the category. Regarding in vivo chemical safety assessments, a recent approach has been the implementation of integrated testing strategies in line with 3R principles, which allow for analysis of these endpoints, along with toxicokinetics and MoA, within a single toxicity study design.

When considering 3R principles, further application of the integration of toxicokinetics into toxicity studies supports read-across arguments to waive additional in vivo testing; this approach could be strengthened dramatically by the addition of toxicokinetic data to provide information regarding systemic exposure and metabolism of the chemical(s) in question and to determine the bioequivalence of related chemicals.

Although in vivo integrated testing strategies reduce animal use, time, and cost for results, efforts are ongoing to evaluate the compatibility of these endpoints within a single study design and to ensure that each of the endpoints can be evaluated and interpreted appropriately. In addition, fulfilling the individual guideline(s) for added endpoints such as genetic toxicity and immunotoxicity can be challenging, considering that these guidelines specify different durations of exposure, route of administration, and criteria for achieving an acceptably high dose level. QUESTIONS1. For toxicological testing of new chemicals, in an tiered testing approach the phases are (number

each group chronologically)a. Cheminformaticsb. In vitro biological profiling

c. Standard regulatory toxicology (in vivo)2. T/F. The use of toxic genomics and/or metabolomics are useful to refine the structure activity

relationship (SAR) based predictions and the use of read-across3. Speaking about toxic genomics techniques, which one/s of the following sentences is/are false?

a. Assessment of gene expression by using in vitro models provides comparable results to in vivo models

b. They provide support for the grouping of substances without resorting to extensive in vivo studies

c. Are not a predictive toxicology toold. Have been extensively used in the characterization of modes of toxic action for chemicals

 4. Which of the following strategies are used to reduce read-across uncertainty?a. Use of uncertainty factorsb. Integrated testing strategy and Bayesian networksc. Forñation of categories complemented by predictive toxicology toolsd. All of the above

 ANSWERS1. 1) Cheminformatics; 2) In vitro biological profiling; 3) Standard regulatory toxicology2. True3. d. is false4. All of the above 

Landi et al. Consideration and Checkboxes: Incorporating Ethics and Science into the 3Rs, pp. 224-230

Domain 3: Research Domain 5: Regulatory Responsibilities SUMMARY: The primary means of producing high-quality research and ensure that harm is minimized in animals is the 3Rs. This is a framework of replacement, reduction and refinement. This group has noted that some approaches to the 3Rs may result in a 'check box mentality' in which IACUC members, researchers, administrators and caretakers check off a list of tasks to evaluate a protocol. This group has provided reasons for thinking that the 3Rs approach could be enhanced by more explicit discussion of the ethical assumptions used to arrive at an approved research protocol during IACUC review. They suggest that moral considerability and all of the related issues it gives rise to should be incorporated into the IACUC discussions of 3Rs. 

Problems with the 'check box approach' explored in this paper include the following:  The approach can limit critical thinking about reviews by making the process more about following a

set of instructions and about holistically considering how the procedures might affect the welfare of animals

The approach can lead to a bias in favor of the status quo The approach may make it possible for members to conduct reviews without seriously considering

all ethical and scientific assumptions underlying different approaches

This group provides an alternative means for IACUC to utilize the 3Rs. They offer the discussion that the IACUC would be well served by apply the notion of moral considerability to evaluations of specific protocols in laboratories. In order to achieve the additional dimensions to the 3Rs discussions, a working knowledge of different ethical views would enhance the effectiveness of the process. In order to illustrate this the group has provided two examples where they contrast what they term the ‘check box approach’ with the ‘considerability approach’.

Utilizing a ‘check box approach’ the IACUC would confirm that the PI has conducted a search for alternatives, the number of animals and number and size of groups would be noted and scientific justification and study design would be discussed for possible modifications. By incorporating moral

considerability it allows recognition of the animal and its needs independent of the study design and therefore expands the breadth of thought and discussion.  QUESTION1.  What are the criteria used to determine sentience?  ANSWERS1.   Capable of experiencing pleasure, pain and other feelings