Search

The principles of humane experimental technique, first described by Russell and Burch in 1959, focus on minimising suffering to animals used for scientific purposes. Internationally, as these principles became embedded in the various systems of oversight for the use of animals in science, attention focused on how to minimise pain, distress and lasting harm to animals while maximising the benefits to be obtained from the work. Suffering can arise from the experimental procedures, but it can also arise from the manner in which the animals are housed and cared for. Increased attention is therefore being paid to the entire lifetime experience of an animal, in order to afford it as good a quality of life as possible. Russell and Burch were also concerned that animals should not be used if alternatives to such use were available, and that animals were not wasted through poor-quality science. This concept is being revisited through new efforts to ensure that experiments are well designed and properly reported in the literature, that all results--positive, negative or neutral--are made available to ensure a complete research record, and that animal models are properly evaluated through periodic systematic reviews. These efforts should ensure that animal use is truly reduced as far as possible and that the benefits derived through the use of animals truly outweigh the harms.

The ultimate goal of the Three Rs is the full replacement of animals used in biomedical research and testing. However, replacement is unlikely to occur in the near future; therefore the scientific community as a whole must continue to devote considerable effort to ensure optimal animal welfare for the benefit of the science and the animals, i.e., the R of refinement. Laws governing the care and use of laboratory animals have recently been revised in Europe and the US and these place greater emphasis on promoting the well-being of the animals in addition to minimizing pain and distress. Social housing for social species is now the default condition, which can present a challenge in certain experimental settings and for certain species. The practice of positive reinforcement training of laboratory animals, particularly non-human primates, is gathering momentum but is not yet universally employed. Enhanced consideration of refinement extends to rodents, particularly mice, whose use is still increasing as more genetically modified models are generated. The wastage of extraneous mice and the method of their euthanasia are refinement issues that still need to be addressed. An international, concerted effort into defining the needs of laboratory animals is still necessary to improve the quality of the animal models used as well as their welfare.

Simple Summary The issues of openness, transparency and public engagement about animal research have taken focus in several different countries in recent years. This paper gives an account of a two-day-long expert forum that brought together policy experts and academics from Australia, Canada, Germany, New Zealand, Sweden, the United Kingdom and the United States. The aim was to share current governance practices regarding openness and transparency of animal research and to brainstorm ideas for better public engagement. The facilitated conversations were transcribed and analysed to create this report and recommendations that encourage international policy-makers and other stakeholders to engage in genuine dialogue about the use of animals in research. Abstract In November 2013, a group of international experts in animal research policy (n = 11) gathered in Vancouver, Canada, to discuss openness and accountability in animal research. The primary objective was to bring together participants from various jurisdictions (United States, Sweden, Australia, New Zealand, Germany, Canada and the United Kingdom) to share practices regarding the governance of animals used in research, testing and education, with emphasis on the governance process followed, the methods of community engagement, and the balance of openness versus confidentiality. During the forum, participants came to a broad consensus on the need for: (a) evidence-based metrics to allow a “virtuous feedback” system for evaluation and quality assurance of animal research, (b) the need for increased public access to information, together with opportunities for stakeholder dialogue about animal research, (c) a greater diversity of views to be represented on decision-making committees to allow for greater balance and (d) a standardized and robust ethical decision-making process that incorporates some sort of societal input. These recommendations encourage aspirations beyond merely imparting information and towards a genuine dialogue that represents a shared agenda surrounding laboratory animal use.

In the context of the current negotiations between the European Union (EU) and the United States under the Transatlantic Trade Investment Partnership (TTIP), there is the opportunity to look at both legislative frameworks to better pinpoint convergences, synergies, and gaps when it comes to use of laboratory animals for scientific purposes and bring together the best of both worlds. The objectives in this article are to indicate what are the current EU pieces of legislation that are relevant under TTIP regarding the uses of laboratory animals for scientific purposes under the regulations about cosmetics and chemicals, among others. The same approach will be taken to look at the relevant American legal frameworks, that is, the Food and Cosmetics Act and the Toxic Safety Control Act as well as its most recent reauthorization. In conclusion, the authors will identify future frameworks that can contribute to the harmonization of regulatory standards and further steps where TTIP negotiators should strengthen regulatory cooperation. published

Summary Dogs are sometimes referred to as “man’s best friend” and with the increase in urbanization and lifestyle changes, dogs are seen by their owners as family members. Society expresses specific concerns about the experimental use of dogs, as they are sometimes perceived to have a special status for humans. This may appear somewhat conflicting with the idea that the intrinsic value of all animals is the same, and that also several other animal species are used in biomedical research and toxicology. This aspect and many others are discussed in an introductory chapter dealing with ethical considerations on the use of dogs as laboratory animals. The report gives an overview on the use of dogs in biomedical research, safety assessment and the drug developmental process and reflects the discussion on the use of dogs as second (non-rodent)species in toxicity testing. Approximately 20,000 dogs are used in scientific procedures in Europe every year, and their distinct genetic, physiological and behavioral characteristics may support their use as models for e.g. behavioral analysis and genetic research. Advances in the 3Rs (Replacement, Reduction and Refinement of experiments using dogs) are described, potential opportunities are discussed and recommendations for further progress in this area are made.

In October 2010, a group of experts met as part of the transatlantic think tank for toxicology (t4) to exchange ideas about the current status and future of safety testing of nanomaterials. At present, there is no widely accepted path forward to assure appropriate and effective hazard identification for engineered nanomaterials. The group discussed needs for characterization of nanomaterials and identified testing protocols that incorporate the use of innovative alternative whole models such as zebrafish or C. elegans, as well as in vitro or alternative methods to examine specific functional pathways and modes of action. The group proposed elements of a potential testing scheme for nanomaterials that works towards an integrated testing strategy, incorporating the goals of the NRC report Toxicity Testing in the 21st Century: A Vision and a Strategy by focusing on pathways of toxic response, and utilizing an evidence-based strategy for developing the knowledge base for safety assessment. Finally, the group recommended that a reliable, open, curated database be developed that interfaces with existing databases to enable sharing of information.

In 1995, an international group of scientists met in Sheringham, Norfolk, UK, for a workshop entitled The Three Rs: The Way Forward. There, with the participation of William Russell and Rex Burch, the group discussed each of the Three Rs in detail, and made specific recommendations on how to promote them within the scientific community. The Institute for Laboratory Animal Research (ILAR) was established as part of the US National Academy of Sciences, to develop and disseminate information and guidelines for the care and use of laboratory animals. Over the years, the focus of ILAR has been to improve the health, welfare, and psychological well-being of the research animal, using the Three Rs as a foundation. ILAR's programmes include an international component, through which it reaches out to other countries via translations of its report; the ILAR Journal, a quarterly publication that focuses on animal models, animal welfare and protocol review; communications and outreach through the ILAR website and presence at scientific meetings; and special reports in which expert committees make recommendations to improve science and animal welfare. Through the efforts of ILAR and similar organisations, it is now recognised that high standards of humane care lead to better science. However, continued effort is needed to promote the Three Rs in developing countries.

Many countries, professional organizations, and nongovernmental organizations have issued guidelines for adequate veterinary medical care for laboratory animals. They are thus considered internationally to be an essential component of animal care and use programs. In the United States, the Guide for the Care and Use of Laboratory Animals (the Guide; NRC 1996), a publication of the National Research Council through the Institute for Laboratory Animal Research (ILAR), provides guidelines that serve as the basis for the US Public Health Service Policy on Humane Care and Use of Laboratory Animals. Among the critical topics included in the Guide is veterinary medical care for laboratory animals, and the report’s recommendations outline key fundamental issues for institutions. While the Guide primarily provides guidelines for use in

Summary After the publication of the report titled Toxicity Testing in the 21st Century – A Vision and a Strategy, many initiatives started to foster a major paradigm shift for toxicity testing – from apical endpoints in animal-based tests to mechanistic endpoints through delineation of pathways of toxicity (PoT) in human cell based systems. The US EPA has funded an important project to develop new high throughput technologies based on human cell based in vitro technologies. These methods are currently being incorporated into the chemical risk assessment process. In the pharmaceutical industry, the efficacy and toxicity of new drugs are evaluated during preclinical investigations that include drug metabolism, pharmacokinetics, pharmacodynamics and safety toxicology studies. The results of these studies are analyzed and extrapolated to predict efficacy and potential adverse effects in humans. However, due to the high failure rate of drugs during the clinical phases, a new approach for a more predictive assessment of drugs both in terms of efficacy and adverse effects is getting urgent. The food industry faces the challenge of assessing novel foods and food ingredients for the general population, while using animal safety testing for extrapolation purposes is often of limited relevance. The question is whether the latest paradigm shift proposed by the Tox21c report for chemicals may provide a useful tool to improve the risk assessment approach also for drugs and food ingredients.

The TestSmart program was developed in response to the High Production Volume Chemical Challenge, a voluntary initiative under which chemical producers provide basic toxicity data on chemicals produced in greater than one million pounds annually. Specifically, under the Challenge, chemical producers will generate data as needed to complete the Screening Information Data Set (SIDS), as defined by the Organization for Economic Cooperation and Development (OECD). The TestSmart program is a collaborative effort of the Johns Hopkins Center for Alternatives to Animal Testing, the Environmental Defense Fund, Carnegie-Mellon University, and the University of Pittsburgh. The goal of the TestSmart program is to provide a humane and efficient approach to collecting SIDS data. The program has two objectives, one immediate and the other more long term. The immediate objective has been to make recommendations to reduce the number of animals used in collecting SIDS data under the Challenge. This was accomplished, through a group process, by examining the current status of alternative methods for SIDS endpoints and by providing an assessment of the “state of readiness” of current and potential future alternatives. The long-term objective is to provide a model for other programs to follow the TestSmart concept of a more efficient and humane approach to obtain toxicological data of interest to regulators and the public.

Hepatocytes isolated from male Fisher 344VF rats were cultured on two substrates, collagen I and a lactose-derivatized polystyrene (PS-lactose), to compare morphological and functional differences. Hepatocyte morphology changed dramatically depending upon the substrate, shown through actin cytoskeletal staining and scanning electron microscopy. Functional assays performed included albumin secretion, reduced glutathione content, UDP-glucuronosyl transferase, and cytochrome P4501A1 activity. The presence of dexamethasone and dimethylsulfoxide (DMSO) in the media was required for the maintenance of several differentiated functions for cells cultured on collagen. In general, cells cultured on the PS-lactose substrate showed a much slower loss of function over the same period of time. The maintenance of differentiated function of cells on PS-lactose was enhanced with the addition of dexamethasone and DMSO. This is the first report of a culture system in which hepatocytes, cultured on a polymer substrate without additional protein coatings or media additives, have been able to maintain differentiated functions for up to 1 week. (c) 1996 John Wiley & Sons, Inc.

This is the report of the thirty-third of aseries of workshops organised by the Euro-pean Centre for the Validation of AlternativeMethods (ECVAM). ECVAM™s main goal, asdefined in 1993 by its Scientific AdvisoryCommittee, is to promote the scientific andregulatory acceptance of alternative methodswhich are of importance to the biosciencesand which reduce, refine or replace the use oflaboratory animals. One of the first prioritiesset by ECVAM was the implementation ofprocedures which would enable it to becomewell-informed about the state-of-the-art ofnon-animal test development and validation,and the potential for the possible incorpora-tion of alternative tests into regulatory pro-cedures. It was decided that this would bebest achieved by the organisation of ECVAMworkshops on specific topics, at which smallgroups of invited experts would review thecurrent status of various types of

A problem traditionally encountered with primary hepatocyte cultures is their rapid dedifferentiation, which is reflected not only in decreased liver-specific functions, but also in dedifferentiated morphology: the cells flatten, depolarize, and lose many of the surface characteristics of normal hepatocytes in vivo. However, culture conditions that maintain primary rat hepatocytes in a healthy and highly differentiated state were recently developed: the hepatocytes are cultured in Chee's Medium supplemented with dexamethasone and dimethyl sulfoxide (DMSO) on collagen-coated Permanox dishes. In addition to retaining labile hepatocyte-specific functions (e.g., P450 activity and albumin synthesis), these hepatocytes also have a differentiated morphology. They have numerous microvilli and are cuboidal and cluster into cords reminiscent of hepatic trabeculae. Their subcellular organelles have normal morphology, and specialized junctions and bile canaliculi form within the membranes of adjacent cells. Actin fibers cluster at these canalicular surfaces. These hepatocytes also synthesize blood clotting factors, which aggregate into fibrin meshworks between cells. Taken together, these morphological data suggest that these hepatocytes are polarized and generally have an appearance very similar to parenchymal cells in the liver, and that the same culture conditions that promote retention of liver-specific functions are also critical to the maintenance of physiological morphology. In contrast to other hepatocyte cultures, this differentiated morphology, including the polarized nature of the cells, is established without the use of serum or flexible or complex extracellular matrices and shows a close link between cellular architecture and tissue-specific function.

The desire to develop and evaluate drugs as potential countermeasures for biological and chemical threats requires test systems that can also substitute for the clinical trials normally crucial for drug development. Animal models have limited predictivity for drug efficacy, as is well known from many disappointments in clinical trials. Traditional in vitro and in silico approaches are not really game changers here, but the substantial investment into novel tools now underway might bring about a second generation of alternative approaches. The avenue pursued focuses primarily on the development of a Human on a Chip, i.e., the combination of different three-dimensional (stem) cell-based organ equivalents combined with microfluidics. The prospects of such approaches, their impact on the field of alternative approaches, and necessary complementary activities are discussed. The need to adapt quality assurance measures and experiences from validation is stressed.

The use of mice in research is governed by a range of laws, guidelines and policies that differ among countries and continents. In a modern regulatory system, these instruments generally aim to facilitate good quality science while safeguarding animal welfare and so promote public confidence in this highly sensitive area of animal use.

Systemic toxicity testing forms the cornerstone for the safety evaluation of substances. Pressures to move from traditional animal models to novel technologies arise from various concerns, including: the need to evaluate large numbers of previously untested chemicals and new products (such as nanoparticles or cell therapies), the limited predictivity of traditional tests for human health effects, duration and costs of current approaches, and animal welfare considerations. The latter holds especially true in the context of the scheduled 2013 marketing ban on cosmetic ingredients tested for systemic toxicity. Based on a major analysis of the status of alternative methods (Adler et al., 2011) and its independent review (Hartung et al., 2011), the present report proposes a roadmap for how to overcome the acknowledged scientific gaps for the full replacement of systemic toxicity testing using animals. Five whitepapers were commissioned addressing toxicokinetics, skin sensitization, repeated-dose toxicity, carcinogenicity, and reproductive toxicity testing. An expert workshop of 35 participants from Europe and the US discussed and refined these whitepapers, which were subsequently compiled to form the present report. By prioritizing the many options to move the field forward, the expert group hopes to advance regulatory science.

In October 2010, a group of experts met as part of the transatlantic think tank for toxicology (t4) to exchange ideas about the current status and future of safety testing of nanomaterials. At present, there is no widely accepted path forward to assure appropriate and effective hazard identification for engineered nanomaterials. The group discussed needs for characterization of nanomaterials and identified testing protocols that incorporate the use of innovative alternative whole models such as zebrafish or C. elegans, as well as in vitro or alternative methods to examine specific functional pathways and modes of action. The group proposed elements of a potential testing scheme for nanomaterials that works towards an integrated testing strategy, incorporating the goals of the NRC report Toxicity Testing in the 21st Century: A Vision and a Strategy by focusing on pathways of toxic response, and utilizing an evidence-based strategy for developing the knowledge base for safety assessment. Finally, the group recommended that a reliable, open, curated database be developed that interfaces with existing databases to enable sharing of information.

355 Critical Evaluation of the Use of Dogs in Biomedical Research and Testing Joanne Zurlo 1, Kathryn A. Bayne 2, Dorothy Cimino Brown 3, Tanya H. Burkholder 4, Vicki L. Dellarco 5, Amy Ellis 6, Laura Garrett 7, Robert Hubrecht 8, Erik R. Janus 9, Lewis B. Kinter 10, Elizabeth Luddy 11, LaVonne D. Meunier 12, Diana G. Scorpio 13, James A. Serpell 14, and Rory J. Todhunter 15 Center for Alternatives to Animal testing, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA; AAAlAC International, Frederick, MD, USA; University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA, USA; Division of Veterinary Resources, Office of the Director, National Institutes of Health, Bethesda, MD, USA; Office of Pesticides, US environmental Protection Agency, Washington, DC, USA; Center for Drug evaluation and Research, US Food and Drug Administration, Silver Spring, MD, USA; University of Illinois, College of Veterinary Medicine, UrbanaChampaign, Il, USA; Universities Federation for Animal Welfare, Wheathampstead, Hertfordshire, UK; Steptoe & Johnson, llP, Washington, DC, USA; AstraZeneca Pharmaceuticals lP, Wilmington, De, USA; Center for Veterinary Medicine, US Food and Drug Administration, Rockville, MD, USA; GlaxoSmithKline, King of Prussia, PA, USA; Department of Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Baltimore, MD, USA (current address: California National Primate Research Center, University of California, Davis); University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA, USA; Cornell University College of Veterinary Medicine, Ithaca, NY, USA

We examined the pattern of expression of several proto-oncogenes during nonneoplastic growth and in acinar cell neoplasms in the rat pancreas. The levels of c-myc, c-raf-1, and c-Ki-ras mRNAs were increased in regenerating pancreata following surgical partial pancreatectomy and following administration of camostat. We also investigated proto-oncogene expression associated with the progression of pancreatic cancers in azaserine-treated rats. Injection of a single dose (30 mg/kg) of azaserine (O-diazoacetyl-L-serine) to 14-d-old rats leads to a variety of neoplastic lesions in the rat pancreas. Total RNA was isolated from lesions in various stages of tumor progression, including adenomas, carcinomas in situ, and invasive carcinomas. We observed increased expression of c-myc, c-raf-1, and c-Ki-ras in azaserine-induced adenomas and carcinomas. Actin expression was also increased in these tissues, whereas amylase expression was variable. However, when compared to the normal growing pancreas, the level of proto-oncogene expression in the adenomas and carcinomas was disproportionate to the degree of cellular division in those tissues. Thus, the alterations induced by azaserine apparently caused a deregulated increase in expression of cellular oncogenes associated with growth regulation.

Laws that mandate replacement alternatives, reduction alternatives, and refinement alternatives (the Three Rs) in scientific research have been passed in the United Kingdom, Germany, the Netherlands, the United States, and the European Union over the past decade. Full implementation of this newly developed legislation depends upon scientists' ability to understand animal welfare issues and to accept the legitimacy of the public's interest in the conduct of science. The European Centre for the Validation of Alternative Methods (ECVAM), established by the European Commission in 1991 to promote the scientific and regulatory acceptance of alternative methods, recently sponsored a workshop to discuss the current status of the Three Rs and to make recommendations aimed at

The National Academies continue the activities of the Institute for Laboratory Animal Research (ILAR), the recognized leader in developing and disseminating guidelines for laboratory animal care, breeding, and use, (including the internationally accepted standard Guide for the Care and Use of Laboratory Animals); resources for identifying animal models for human diseases and physiological processes; and reports on the humane and scientific use of laboratory animals (including education and training of scientists and technicians in the care and use of laboratory animals; reduction of pain and distress in research animals; occupational safety and health of employees; and nomenclature of inbred, transgenic, and other genetically defined animals). The program goal is to improve the humane and scientifically valid use of laboratory animals as well as the availability, quality and care of laboratory animals. ILAR accomplishes this goal through a core program, carried out by staff, and a special-project program, carried out by National Academies-appointed experts. Both programs are guided by a 16-member advisory council (the Council). The Council meets three times a year to provide program direction and strategic planning; to oversee the communication and information programs (consisting of the ILAR web site and the ILAR Journal); to oversee special projects; and to direct ILAR's international programs (including participation as the US national member of the International Council of Laboratory Animal Sciences).

The Institute for Laboratory Animal Research (ILAR) is a component of the Division on Earth and Life Studies (DELS) at The National Academies. The Department of the Army ?PRIVATE! has provided partial support for ILAR for many years to enable to ILAR fulfill its mission. ILAR was founded in 1952 and is a national and international leader in developing guidelines for animal care, breeding, and use; descriptions of animal models for human diseases and physiological processes; and reports on specific issues of humane care and use of laboratory animals. ILAR's mission is to help improve the availability, quality, care, and humane and scientifically valid use of laboratory animals and to disseminate its reports and other relevant information to the biomedical and laboratory animal science communities. ILAR accomplishes its goals through its core program, which is carried out by the staff, and its special-project program, which is carried out by NRC-appointed committees with staff assistance. A 1 5-member Council composed of experts in laboratory animal medicine, zoology, genetics, medicine, ethics, and related biomedical sciences directs both programs. The Army finds partially support general office opera- tions, the Animal Models and Genetic Stocks Information Program, publication of ILAR Journal, and work of the Council.

The Institute for Laboratory Animal Research (ILAR) is a component of the Division on Earth and Life Studies (DELS) at The National Academies. The Department of the Army has provided partial support for ILAR for many years to enable to ILAR fulfill its mission. ILAR was founded in 1952 and is a national and international leader in developing guidelines for animal care, breeding, and use; descriptions of animal models for human diseases and physiological processes; and reports on specific issues of humane care and use of laboratory animals. ILAR's mission is to help improve the availability, quality, care, and humane and scientifically valid use of laboratory animals and to disseminate its reports and other relevant information to the biomedical and laboratory animal science communities. ILAR accomplishes its goals through its core program, which is carried out by the staff and its special-project program, which is carried out by NRC-appointed committees with staff assistance. A 15-member Council composed of experts in laboratory animal medicine, zoology, genetics, medicine, ethics, and related biomedical sciences directs both programs. The Army funds partially support general office operations, the Animal I Models and Genetic Stocks Information Program, publication of ILAR Journal, and work of the Council.

This article examines the status and application of alternatives defined as replacements, refinements, and reduction for screening high production volume (HPV) chemicals. It specifically focuses on the Screening Information Data Set (SIDS), a series of toxicological tests recommended by the Organization for Economic Cooperation and Development to screen such chemicals. Alternative tests associated with acute, repeat-dose, genetic, and reproductive and developmental toxicity were examined at 2 meetings of academic, industry, and regulatory scientists and their status determined. Tests were placed in 1 of 3 categories: ready for immediate use, in need of or currently undergoing validation, or needing research/developmental work. With respect to traditional acute toxicity testing, the basal cytotoxicity approach was placed in the category of research with the up-and-down, fixed-dose, limit test, and the acute toxic class categorized as available for immediate use and the neutral red assay under validation. Cell culture methods that could provide information on acute target organ toxicity were all categorized in the research stage. Studies of the Ah receptor were placed under validation. All alternative tests for repeat-dose toxicity were placed in the category of research. With regard to genetic toxicity, the Ames, mouse lymphoma, and Chinese hamster ovary methods were considered ready for immediate use, while the in vitro micronucleus and Syrian hamster ovary assays were placed in the validation category. All alternatives for developmental toxicity, with the exception of gene chip technology, were placed in the category of validation. Gene chip technology is considered to be in the research stage. For reproductive toxicity, sperm motility and morphology were considered as ready for immediate use, with the other assays categorized as needing validation or in the research stage. Follow-up to these results is obvious. Work needs to be conducted to move those tests from the research stage to the validation and use stage. This is one approach to the development of alternatives to SIDS. Progress along these lines would apply not only to SIDS but also to toxicology in general.

The Institute for Laboratory Animal Research (ILAR), formerly a component of the Commission on Life Sciences (CLS) within the National Research Council (NRC), is now part of the Division on Earth and Life Studies (DELS) following a reorganization of the NRC that involved consolidation of various components. Partial support for ILAR has been provided for many years from the Department of the Army to enable ILAR to fulfill its mission. ILAR was founded in 1952 and has become a national and international leader in developing guidelines for animal care, breeding, and use; descriptions of animal models for human diseases and physiological processes; and reports on specific issues of humane care and use of laboratory animals. ILAR's mission is to help improve the availability, quality, care, and humane and scientifically valid use of laboratory animals and to disseminate its reports and other relevant information to the biomedical and laboratory animal science communities. ILAR accomplishes its goals through its core program, which is carried out by the staff, and its special-project program, which is carried out by NRC-appointed committees with staff assistance. The number of committees and size of the staff are dependent on the number of special projects and available funding. A 15-member Council composed of experts in laboratory animal medicine, zoology, genetics, medicine, ethics, and related biomedical sciences directs both programs. The Army funds partially support general office operations, the Animal Models and Genetic Stocks Information Program, publication of ILAR Journal, and work of the Council.

The publication in 1959 of Russell and Burch's The Principles of Humane Experimental Technique initiated a new era in the history of the debate between science and animal protection, the era of alternatives. Although Russell and Burch never used the word “alternatives” (speaking instead of the “three Rs”: replacement, reduction, and refinement), and although the animal protection movement was somewhat moribund at the time in both England and the United States, within a decade of the book's publication a number of signs indicating a resurgence of public interest in the issue began to appear. At the same time, increased knowledge and understanding of neurophysiology and ethology heightened scientific awareness of the significance of humane treatment in the laboratory and professionalized laboratory animal science.

An hepatocyte culture system was developed for potential use in toxicological studies in vitro. Rat hepatocytes were isolated by two-step collagenase perfusion and cultured on Vitrogen-coated Permanox dishes in a modified Chee's medium containing 1 microM dexamethasone and 1% dimethylsulfoxide. The cells remained highly viable for at least 10 d as determined by lactate dehydrogenase release and total protein levels. Albumin secretion into the medium, as a measure of differentiated function, was maintained at elevated levels over the course of 10 d in culture. A number of CYP activities were determined by the analysis of testosterone metabolism in freeze-thawed cells, diazepam metabolism in live cells, and specific assays for CYP 1A1/2, 2B1/2, 2E1, and 3A. Results of these assays indicated that a wide range of CYP isozymes were maintained, some activities were enhanced under the conditions of culture and some activities were inducible. Activities of the phase II enzymes, glutathione S-transferase and UDP-glucuronosyltransferase, and glutathione levels were also maintained in the cultured hepatocytes for at least 6 d. These results strongly support the use of this hepatocyte culture system for in vitro toxicological studies.

Lactose and heparin were covalently coupled to poly(chloromethyl styrene) and the modified polymer was used as a substrate for rat hepatocyte culture. Lactose and heparin are recognized by rat hepatocytes and can be used to mediate cell attachment to the substrate. Rat hepatocytes cultured in serum-free media on these substrates were able to maintain enzymes and peptides important in the detoxification functions of hepatocytes, without the addition of hormones such as dexamethasone, media additives such as DMSO, or complex biological extracellular factors.The growing interest in large-scale cell culture and in tissue engineering requires substrates of different geometries. Therefore, we have fabricated the derivatized polymers into microcarriers and, most recently, foams. These three-dimensional structures, combined with the chemistries of the polymers, provided the hepatocytes with more cell-cell interactions and in vivo-like geometries than conventional flat-dish culture.

Epidemiological and experimental data strongly support a causal relationship between exposure to excessive levels of estrogens and the development of cancer in various tissues. In this paper, we have presented background information that shows a correlation between the prolonged use of oral contraceptives and the development of liver cancer. The clinical data supported the hypothesis that the estrogenic components of oral contraceptives were promoters of hepatocarcinogenesis, and the experimental evidence in support of this hypothesis and bearing on the mechanisms involved are also reviewed. The effects of estrogens on liver neoplasia and growth are: (i) synthetic steroidal estrogens are potent promoters of hepatocarcinogenesis in female rats; (ii) these estrogens stimulate liver growth at doses that are not hepatotoxic; (iii) the mechanisms by which the estrogens stimulate liver growth are indirect and include the enhancement of a serum/plasma growth factor, co-mitogenic effects which result in enhanced responsiveness of cultured hepatocytes to epidermal growth factor and decreased sensitivity of hepatocytes to growth inhibition by transforming growth factor-beta; (iv) the co-mitogenic effects of synthetic estrogens extend to endogenous estrogens and natural product estrogens; and (v) the co-mitogenic effects of estrogens for epidermal growth factor are associated with increased epidermal growth factor receptor protein levels caused by an increase in the half-life of the receptor protein. The synthetic estrogens also have weak "complete" carcinogenic activity in rat liver and strong complete carcinogenic activity in Syrian hamster kidney and Armenian hamster liver. Evidence from the literature is presented in support of a hypothesis that this process may involve indirect genotoxicity mediated through redox cycling and the formation of hydroxylated DNA bases. This process, together with the potent promoting activity of these estrogenic chemicals, may account for their complete carcinogenicity.

The objective of this study was to determine whether activation of c-Ki-ras occurs in carcinogen-induced rat pancreatic tumors. DNAs from 27 samples, which included adenomas, carcinomas in situ, and adenocarcinomas arising in azaserine-treated rats and corn oil-gavaged rats along with a nafenopin-induced rat pancreatic adenocarcinoma were examined for mutation of c-Ki-ras at codons 12, 13, and 61 by using the polymerase chain reaction. Our results indicate that activation of c-Ki-ras is not a common event during pancreatic carcinogenesis in the rat.

The authors are at the Johns Hopkins Center for Alternatives to Animal Testing (CAAT), Johns Hopkins School of Public Health, 111 Market Place, Suite 840, Baltimore, MD 21202-6709, USA. E-mail: caat@caat.spharbor.jhu.edu In 1959, British scientists William M. S. Russell and Rex L. Burch wrote that scientific excellence and humane use of laboratory animals are inextricably linked. In The Principles of Humane Experimental Technique,* they described the “three Rs”—reduction, refinement, and replacement—of animal use in the life sciences, concepts now known as “alternatives.” Legislation mandating the incorporation of the three Rs into animal research and testing has been passed in the United States and Europe. Because some scientists view discussion of alternatives as driven exclusively by political and social forces rather than by scientific considerations, an international group of scientists prominent in the alternatives field met at a May 1995 workshop to reaffirm the scientific basis of the three Rs and to discuss how these concepts can best be disseminated to the scientific community.† Published surveys of the scientific literature have found inefficient use of animals due to poor experimental design or inappropriate statistical analysis of results or both. Reduction alternatives allow comparable amounts of data to be obtained from fewer animals or more information to be obtained from a given number of animals. The number of animals used should be the minimum necessary to test the experimental hypothesis and give statistically usable results. Refinement alternatives are methods that eliminate or minimize pain and distress or enhance animal well-being. Assessments of animal pain and distress are currently based on subjective evaluation of abnormal behavior and appearance. Because proper evaluation of pain relies largely on the ability to understand the behavior and needs of each species of laboratory animal, it is best for investigators to assume that a procedure that inflicts pain and distress on humans will inflict pain and distress on animals. Much pain and distress can be diminished or eliminated with the proper use of anesthetics and analgesics. Researchers can enhance animals9 well-being by using environmental enrichment techniques, such as proper handling, appropriately sized cages, and group housing of social species. Replacement alternatives are methods that use organisms with limited sentience or that do not use whole animals. They include improved information exchange to avoid unnecessary repetition of animal experiments; physicochemical techniques and structure-activity relations; mathematical and computer models; use of invertebrates, plants, and microorganisms; in vitro methods; and human studies, including the use of human volunteers, postmarketing surveys, and epidemiology. In the biomedical sciences, in vitro methods are increasingly being used, not because they provide precisely the same information as do animal studies but because they offer the best scientific approach. Such methods often use results from past animal studies as a basis for cellular and molecular investigations. Successful implementation of the three Rs requires that scientists and technicians be formally trained to scientifically and ethically evaluate the use of laboratory animals and to perform animal experiments that meet the highest scientific and animal welfare standards. Coursework should be included in graduate training programs and should focus on strengthening the principles of experimental design and teaching competence in animal handling, how to make ethical decisions about using animals in experiments, and how to find alternative methods. As the participants at the workshop concluded, present and future scientists should be encouraged to view the three Rs as an intellectual challenge and an opportunity to enhance the scientific, economic, and ethical value of their research.

The major objective of this study was to investigate the expression of members of the raf family of proto-oncogenes during rat liver regeneration. The steady-state level of expression of both c-raf-1 and A-raf-1 increased three- to fivefold 18-24 h following partial hepatectomy, and it returned to basal levels by 72 h. Expression of c-myc and Ha-ras mRNA was increased at 3 and 18-24 h, respectively, confirming previous reports. Increased steady-state levels of c-raf-1, A-raf-1, and Ha-ras mRNA were also detected in hepatocytes isolated from rat liver 24 h after partial hepatectomy. Thus, elevated expression of the raf genes closely correlated with that of Ha-ras, beginning at 12 h and reaching maximal levels during the first peak of DNA synthesis following partial hepatectomy.

The ability of the D-isomer of azaserine to induce atypical acinar cell nodules (AACN) in pancreas and to cause DNA damage in pancreas and liver was evaluated. Rats were injected with equivalent doses of D- or L-azaserine and numbers of AACN were counted after 4 months. DNA damage in pancreas and liver of rats treated in vivo, and in pancreatic acinar cells treated in vitro with D- or L-azaserine was determined by alkaline elution. Results show that D-azaserine does not significantly induced AACN in pancreas, nor does it cause extensive DNA damage in comparison with L-azaserine, suggesting that the differential effect of the 2 isomers is related to stereospecificity in either transport or metabolism.

The objective of this study was to compare the ability of heat stock (HS) with that of another type of cellular stress, UV irradiation, to cause the induction of enhanced viral reactivation, a process that may represent an SOS-type repair process in mammalian cells. Studies performed to evaluate the effect of HS on growth of Vero cells revealed that HS at 45°C for 45 min caused inhibition of cell growth similar to that caused by UV irradiation at 12 J/m 2 , but this inhibition was not observed at HS treatment for 5–15 min, or at a UV fluence of 2 J/m 2 . Enhanced reactivation of UV-irradiated Herpesvierus was observed in cells which had been pretreated by HS for > 30 min or UV at 12 J/m 2 . The synthesis of new proteins following HS for 15 and 45 min and UV at 12 J/m 2 was examined by [ 35 S]methionine-labeling experiments. The new synthesis of two HS proteins with molecular weights of 46 000 and 78 000 was induced by both levels of HS, but to a much greater extent at the high dose. These proteins were not detected in response to UV irradiation. These results indicate that, like UV irradiation, HS at levels inhibitory to cell growth induced enhanced viral reactivation in Vero cells. The results also suggest that at least two proteins in the HS protein family are not necessary for this response to occur.

Previously, the induction of pancreatic carcinogenesis in the rat using azaserine has involved a multiple-dose treatment protocol. The objective of the present study was to determine the effect of multiple azaserine treatments on pancreatic DNA synthesis and to develop a protocol for a single-dose initiation of pancreatic carcinogenesis by azaserine in the rat. Pancreatic DNA synthesis in young rats, which was determined by measuring the amount of [3H]-thymidine incorporation into DNA, was found to be elevated at 4.3 weeks of age and to decrease to a baseline level by 6.3 weeks. Treatment of 4-week-old rats with azaserine resulted in a dose-dependent inhibition of [3H]-thymidine incorporation into both pancreatic and liver DNA. Maximum inhibition was seen at 10 mg/kg body weight. This inhibition was followed by a gradual return of incorporation to normal values over a 48 h period. One week following pretreatment with four weekly injections of azaserine at 30 mg/kg, [3H]-thymidine incorporation into pancreatic and liver DNA was significantly elevated, suggesting that multiple injection protocols caused enhanced DNA synthesis which could have a co-carcinogenic and/or promotional effect. Single-doses of azaserine (10, 30 and 60 mg/kg) given at 7 weeks of age caused the appearance of more atypical acinar cell nodules (AACN) than when given at 5 weeks of age. The most effective dose was 30 mg/kg. Using alkaline elution, we determined that this response was due to the occurrence of more DNA damage in the 7-week-old animals. Thus, these results demonstrate a rationale for the use of single-dose initiation protocols in the pancreas. An effective single-dose protocol for induction of AACN in azaserine-treated rats fed semi-synthetic diet is presented.

One objective of this study was to determine the effects of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) treatment on DNA synthesis in regenerating rat liver. Rats were subjected to a two-thirds hepatectomy followed 20 h later by i.p. injection of N-OH-AAF. 4 h after carcinogen injection, it was found that N-OH-AAF caused a dose-dependent inhibition of [3H]thymidine incorporation into liver DNA. This inhibition was followed by a gradual, but incomplete recovery beginning 28 h after carcinogen treatment. Radioimmunoassay of deoxyguanine-C8 adducts remaining in liver DNA indicated that the recovery began prior to detection of adduct removal. The second objective of the study was to determine the effects of DNA damage on the size distribution and elongation of nascent hepatocyte DNA. Hepatocytes, which have been shown to demonstrate a pattern of inhibition and subsequent recovery of DNA synthesis following UV irradiation similar to that seen in vivo upon treatment with N-OH-AAF (Zurlo and Yager, 1984), were cultured under conditions that promote replicative DNA synthesis. The size distribution of nascent DNA after UV irradiation was determined by pH step gradient alkaline elution analysis. [3H]Thymidine pulse times and subsequent chase times were adjusted to equalize amounts of DNA synthesis in control and UV-irradiated cells. The results show that UV irradiation caused a dose-dependent decrease in the size distribution of nascent DNA suggesting an inhibition of elongation. Pulse-chase studies revealed that subsequent joining of nascent chains in UV-irradiated hepatocytes occurred at a rate comparable to or faster than controls and that this could be inhibited by caffeine. The results obtained from both the in vivo and in vitro studies show that resumption of DNA synthesis and nascent strand elongation occur on damaged templates. These observations along with our previous studies demonstrating the ability of UV-irradiated hepatocytes to carry out enhanced reactivation of UV-irradiated herpes virus lend support to the idea that DNA damage leading to inhibition of DNA synthesis may induce SOS-type processes which if mutagenic may play a role in the initiation of carcinogenesis.

Carcinogen treatment of cultured mammalian cells prior to infection with u.v.-irradiated virus results in enhanced virus survival and mutagenesis suggesting the induction of SOS-type processes. In this paper, we report the development of a primary rat hepatocyte culture system to investigate cellular responses to DNA damage which may be relevant to hepatocarcinogenesis in vivo. We have obtained data demonstrating that enhanced reactivation of u.v.-irradiated Herpes simplex virus type 1 (HSV-1) occurs in hepatocytes irradiated with u.v. Cultured hepatocytes were pretreated with u.v. at the time of enhanced DNA synthesis. These treatments caused an inhibition followed by a recovery of DNA synthesis. At various times after pretreatment, the hepatocytes were infected with control or u.v.-irradiated HSV-1 at low multiplicity, and virus survival was measured by direct plaque assay. U.v.-irradiated HSV-1 exhibited the expected two-component survival curve in control or u.v. pretreated hepatocytes. The magnitude of enhanced reactivation of HSV-1 was dependent on the u.v. dose to the hepatocytes, the time of infection following u.v. pretreatment, and the level of DNA synthesis at the time of pretreatment. These results suggest that u.v. treatment of rat hepatocytes causes the induction of SOS-type functions that may have a role in the initiation of hepatocarcinogenesis.

Knowledge of the carcinogenic process has been dramatically increased by the application of recombinant DNA technology and the discovery that mutated cellular genes capable of causing neoplastic transformation can be isolated from spontaneously appearing and carcinogen-induced human and experimental animal tumors. That single genes can cause neoplastic transformation was first demonstrated in studies conducted on acutely transforming retroviruses.1,2 Such viruses, the first being the Rous sarcoma virus, were shown to carry single genes, termed oncogenes (see Chapter 13), that were responsible for their transforming ability. It was also clearly shown that these retroviral oncogenes had homology to cellular genes and were in fact initially derived from cellular genes by retroviral transduction. The cellular homologs of the retroviral oncogenes are referred to as cellular oncogenes or proto-oncogenes. To date, about 20 retroviral oncogenes, each originating from a cellular precursor proto-oncogene, have been identified.3

The binding of [14C] azaserine or its metabolites to DNA and protein in the organs of rats and hamsters was determined at various time after treatment with [14C] azaserine. The specific activity of 14C labelling of DNA and protein was determined. Rat liver DNA and protein were most extensively labelled at 90 min post-injection, but by 24 h the specific activity decreased to the levels found in pancreas and kidney. Thymus contained negligible amounts of radioactivity at all time-points. DNA and protein from hamster pancreas contained more label than did DNA and protein from rat pancreas. The results suggest that factors other than DNA binding play a role in determining the species and organ specificity of azaserine.

N-nitrosobis(2-oxopropyl)amine (BOP), a potent pancreatic carcinogen in hamsters that has failed to induce pancreatic tumors in rats, was studied for its effects on the DNA of both rat and hamster pancreas in order to relate DNA damage (as measured by alkaline elution) to carcinogenicity in vivo. At doses of 10, 20, and 40 mg BOP/kg, extensive DNA damage was detected in male Syrian golden hamster pancreas but Lewis rat pancreatic DNA was not affected. Only at doses of 100 mg BOP/kg or greater could pancreatic DNA damage in the rat be detected. DNA damage was also observed in both rat and hamster livers at 10, 20, and 40 mg BOP/kg. Alkaline elution analysis of DNA from isolated rat and hamster acinar cells treated in vitro with BOP revealed that only hamster acinar cell DNA was damaged. Rat acinar cell DNA was unaffected at all doses examined, up to 200 micrograms BOP/ml medium. Unscheduled DNA synthesis studies in cultured acinar cells confirmed the observations that BOP is genotoxic to hamster but not to rat acinar cells. The results strongly suggested that rat pancreas did not have the ability to metabolically activate BOP, which accounted for lack of both BOP-induced DNA damage and carcinogenicity in the rat.