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Issues in Laboratory Animal Science That Impact Toxicologic Pathology
1
Introduction
As an integral member of the preclinical study team, the toxicologic pathologist needs to have a thorough understanding of the laboratory animal model that serves as a test system for studies with pathology requirements. Genetic, microbial, experimental, and environmental factors greatly affect lesion development in laboratory animals, and an understanding of the interplay of these factors with test article–induced effects is necessary for the pathologist to correctly interpret study findings. In addition to the role of assessing compound-induced tissue findings, the toxicologic pathologist often identifies health-related issues in study animals and must be knowledgeable about the common spontaneous and infectious disease states that arise in long-term preclinical studies. The pathologist must work closely with laboratory animal science professionals who are responsible for oversight of animal colony management and care during the in-life phase of the study.
Toxicology research and testing is becoming increasingly more complex with the advent of new and exciting advances in biotechnology. This is best exemplified by the widespread use of increasingly complex genetically modified mouse and rat models in toxicology testing facilities performing safety assessment bioassays, as well as in basic research laboratories (see Genetically Engineered Animal Models in Toxicologic Research , Vol 1, Chap 23 ) . There is also an increasing sophistication in animal monitoring systems such as telemetric (remote monitoring) assessment of physiological parameters and nuclear and nonnuclear preclinical imaging methods (see In Vivo Small Animal Imaging: A Comparison to Gross and Histopathologic Observations in Animal Models , Vol 1, Chap 13 ). These technologies utilized during in vivo studies contribute to an improved understanding of the pathogenesis of many laboratory animal diseases and lesion states, provide new and useful adjuncts to traditional pathology assessment, and may reduce the number of animals used in studies.
Laboratory animal science is a diverse and complex discipline encompassing many different types of experimental animals. The discipline is a dynamic one and is shaped in part by the regulatory landscape as well as scientific advancements. In the United States, the eighth Edition of the Guide for the Care and Use of Laboratory Animals (NRC, 2011) provides the basis for the care of research animals in the laboratory. There are similar guidance documents elsewhere in the world that govern the highly regulated use of research animals in laboratories ( ). These major guidance documents impact animal care and use programs and influence the conditions under which toxicity studies are conducted.
There are many questions in laboratory animal science that need to be considered during the planning and conduct of a toxicity study ( Table 29.1 ), and the pathologist is an important member of the study team who should be involved in the design phase. The design of toxicity studies often requires professional judgment to balance the needs of the research animal against experimental objectives or other needs such as those of personnel safety, environmental contamination, and/or regulatory requirements (see Practices to Optimize Generation, Interpretation, and Reporting of Pathology Data from Toxicity Studies, Vol 1, Chap 28 and Experimental Design and Statistical Analysis for Toxicologic Pathologists , Vol 1, Chap 16 ). Selected laboratory animal science issues that pertain to the work of pathologists and that influence lesion development will be stressed in this chapter. Remarks in this chapter will be primarily focused on selected issues involving laboratory rodents, given that they are the most commonly utilized animals in toxicity studies. For more in-depth review of specific animal models, the reader is directed to other chapters in this volume (see Part 3—Animal and Alternative Models in Toxicologic Research).
Table 29.1
Laboratory Animal Science Issues to be Addressed by the Toxicology Study Team
Table modified from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology , ed 2, 2002, Academic Press, Table 29.1 , p. 252, with permission.
| Institutional Animal Care and Use Committee (IACUC) Considerations: |
| Plan experiment from the standpoint of optimizing animal numbers, assuring proper statistical design, and scientific justification (harm vs. benefit) |
| Review issues of animal pain and distress and consider methods to minimize pain or distress |
| Obtain IACUC review and approval of the experimental protocol |
| Ensure personnel are properly trained with up-to-date documentation |
| Define humane endpoints/criteria for euthanasia |
| Assure dose volumes and blood withdraw volumes comply with IACUC guidelines |
| Study issues: |
| Assure compliance with regulatory testing guidelines |
| Review staffing (e.g., number of trained personnel to cover activities) and associated concerns such as blinding of personnel to allocation groups where appropriate to minimize bias |
| Determine methods of allocation of animals to study without bias (randomization) |
| Establish animal identification and cage identification methods |
| Define acclimation and quarantine periods (e.g., length, location, methods) |
| Review dosing methods and issues related to test compound (e.g., dose schedule, volume, vehicle, special monitoring needs) |
| Review all experimental manipulations of animals, including timing of events |
| Establish procedures for removing animals from study with unexpected morbidity/mortality and determine handling and disposition of animals |
| Animal model selection and use: |
| Determine species and strain and review scientific rationale for model selection |
| Establish age, sex, weight, physiological status, and genetic requirements |
| Choose source or supplier |
| Review genetic and health history of source colony |
| Establish need for any prestudy health or genetic testing of supplied animals |
| Review strain-related causes of morbidity and mortality and institutional experience with chosen animal model |
| Animal environment: |
| Review animal room conditions (e.g., temperature, humidity, lighting, noise, air flow) |
| Establish methods and intervals for environmental monitoring |
| Review housing issues (e.g., group vs. single housing, type of caging, type of bedding, frequency of cage change, room allocation, and methods to prevent bias in housing and handling such as rotation of cage or rack placement) |
| Arrange for special housing as needed (e.g., inhalation or metabolism caging) and methods for acclimation to equipment and special procedures |
| Determine whether other animals (e.g., sentinel animals) or studies (e.g., microbial infections in facility) pose hazard to study animals and institute protective procedures if warranted |
| Establish environmental enrichment methods |
| Diet: |
| Specify type (e.g., open vs. closed formula, natural vs. semipurified ingredients) and form (e.g., pelleted, powder, liquid) of diet |
| Review method of feeding (i.e., ad libitum vs. diet restriction) |
| Determine the need for certified diet or methods for contaminant analysis |
| Review water distribution method (e.g., bottle vs. automatic) and treatment method if any (e.g., autoclaved, filtered, acidified, chlorinated) |
| Determine methods and intervals for monitoring food and water consumption if needed |
| In the case of dosing in feed or water, review stability, mixing, and analytical issues related to the diet, and palatability |
| In-life: |
| Establish clinical parameters to be monitored as well as schedules and methods of monitoring |
| Determine animal health monitoring needs on study |
| Review sentinel monitoring program for study and test facility |
| Review animal manipulation, sampling, and dosing procedures with respect to circadian issues, photoperiodicity, and to minimize interventional stress |
| Review chemical safety issues and cage change procedures |
2
Trends in Global Research Animal Care and Use
Laboratory animal science is a dynamic discipline and there are numerous regulatory changes that occur in response to new scientific knowledge and best practices on how animals should be cared for and used in the research laboratory. Major changes have occurred in how animals are cared for and housed in the toxicology laboratory such as types of caging and social housing arrangements. Work with laboratory animals is a highly regulated endeavor in most parts of the world and those that use in vivo studies must be well versed with the rules and regulations pertaining to their work. In the United States, there are national, state, and local regulations that address many facets of the animal care and use program. In recent years, there has been a trend to examine research animal issues with a global perspective ( ). This is especially true for large pharmaceutical companies that work across numerous facilities located in multiple countries. In some cases, companies have global policies concerning their animal care and use so that they can assure a common best practice approach where warranted.
Over the past decade, one of the major scientific changes in the field of laboratory animal science has been the recognition of the importance of the microbiome in the pathophysiology of research animals ( ). Another important trend in laboratory animal science that has been growing and strengthening over the past several years has been the recognition of the importance of social interactions among research animals ( ). Group housing of social species is the normal default situation for virtually all studies now unless there are well-justified scientific reasons for exception. The ability to socially house animals in the laboratory setting may require changes in standard operating procedures so that food consumption and clinical evaluations can be obtained during the in-life conduct of toxicity studies. An additional trend in the housing of research animals that has gained favor in many laboratory settings is the recognition that cage complexity and enrichment is warranted so that animals can express species-specific behaviors. Many study directors have been reluctant in the past to incorporate these devices, citing differences in responses on study or the possibility for ingestion of contaminants leading to unwanted effects. Environmental enrichment programs are now the norm for the toxicology laboratory as they are for other research animal settings and there are many certified products available to control contaminant levels that might otherwise adversely impact results. In the past, many rodents were individually housed in barren metal wire caging suspended over bedding. Modern rodent housing methods consider the importance of social housing and the need for solid flooring with direct contact bedding that allows for normal behaviors as well as for thermoregulatory needs to be met. There is an increasing realization of the importance of thermoregulation in housing rodents and the role that this factor plays in lesion incidence and development.
A very important trend in laboratory animal science in toxicity studies has been the advancement of the so-called 3Rs (i.e., Replacement, Reduction, and Refinement) as a culture of animal use. Adherence to working in a 3Rs mode of thinking is now the norm in many countries and has been recently mandated by changes in guidance and regulations in many parts of the world. A section devoted to the 3Rs is incorporated in this chapter due to its importance regarding how animals are used in research and testing in toxicology. The toxicologic pathologist must be aware of 3Rs issues during the design and conduct of animal studies ( ; ). There is strong societal pressure throughout the world for the optimization of animal care and use and many companies put high internal value on the minimization of animal use. The expertise of the toxicologic pathologist is best utilized at the design phase of studies so that animal care and use can be optimized during protocol development. There is great value in close collaboration of the toxicologic pathologist with study directors and principal investigators during the course of the in-life conduct of toxicity studies so that all unexpected findings and events can be understood at the earliest time possible to effect interventions as well as to design modifications to obtain the most data of the highest quality from study endpoints. The use of diagnostic investigation of unscheduled mortality and morbidity can be an important component of optimized animal welfare during the course of the study.
The overwhelming majority of laboratory animals used in biomedical institutions are rodents. Rodent populations are increasing in some institutions primarily due to the burgeoning use of genetically modified mouse models in basic research (see Genetically Engineered Animal Models in Toxicologic Research , Vol 1, Chap 23 ). There has been a diminution in the use of nonrodent species in many areas of toxicology research and testing, and the use of nonhuman primate models is a particularly scrutinized area (see Animal Models in Toxicologic Research: Nonhuman Primate , Vol 1, Chap 21 ). Some pharmaceutical companies require justification for not using other nonrodent preclinical species such as dogs and minipigs prior to utilizing nonhuman primates in drug safety studies (see Animal Models in Toxicologic Research: Dog , Vol 1, Chap 19 and Animal Models in Toxicologic Research: Pig , Vol 1, Chap 20 ). Certain nonhuman primate models, such as chimpanzees, are no longer utilized in safety assessment studies due to regulatory restrictions.
3
Regulatory Issues
3.1
Overview of Rules and Regulations
All scientists who utilize laboratory animals must be knowledgeable about government regulations and guidelines pertaining to the proper care and use of research animals. It should be noted that although regulations differ between regions and countries, there has been a trend toward globalization of animal care and use practices ( ). There are certain aspects of research animal use that are associated with broad societal concerns such as the use of genetically modified animals, humanized animal models, and nonhuman primate models.
Many pharmaceutical and chemical companies are multinational with laboratories in multiple countries and thus scientists must be aware of differing policies that affect animal use. Some companies have developed worldwide practices so that their studies are similar no matter where conducted. In many instances, these practices are clearly delineated in policy statements on company websites so that there is transparency to the public. In the pharmaceutical industry, there has been a trend toward increasingly complex collaborative and contract research interactions, and this has often necessitated development of common approaches to animal care and use issues. Consortia such as the IQ Consortium and the UK's National Centre for the Replacement, Reduction and Refinement of Animals in Research (NC3Rs) are entities that have attempted to address this need ( ; ). The NC3Rs is an independent scientific organization set up by the UK government to find innovative solutions to minimize animal use and improve animal welfare in research.
In the United States, oversight of animal care and use is primarily provided by two national laws, the Animal Welfare Act (7 USC 2131-2157) and the Health Research Extension Act (42 USC 289d). The latter was amended in 1985 by Public Law 99-158 to cover the care and use of animals in research. The regulations that implement the Animal Welfare Act are published in the Code of Federal Regulations (9 CFR 1-3) and are administered by the U.S. Department of Agriculture (USDA). For the purposes of this Act, animals are presently defined as “any live or dead dog, cat, nonhuman primate, guinea pig, hamster, rabbit, or any other warm-blooded animal which is being used, or is intended for use for research, teaching, testing, experimentation, or exhibition purposes, or as a pet” (9 CFR 1.1). Rats and mice bred for use in research are currently exempted from these regulations, although this exemption may be eliminated in the future.
Among the provisions of the Animal Welfare Act are (1) standards to ensure the humane care of animals being transported; (2) standards for care of animals in research facilities, including minimum requirements for housing, feeding, watering, sanitation, handling, adequate veterinary care, and the appropriate use of anesthetic, analgesic, and tranquilizing drugs to ensure the minimization of pain and distress; (3) the reporting of requirements to the USDA showing that professionally acceptable standards for the humane care and use of animals are in effect; (4) establishment of an Institutional Animal Care and Use Committee (IACUC); and (5) establishment of training for all personnel involved in the care and use of animals.
The Health Research Extension Act is implemented by the Public Health Service Policy on Humane Care and Use of Laboratory Animals (PHS Policy) and is administered by the National Institutes of Health, Office for Laboratory Animal Welfare. The policy pertains to all activities conducted and supported by the Public Health Service involving any live vertebrate animal used or intended for use in research, research training, biological testing, or related purposes. The PHS Policy requires compliance with the Animal Welfare Act and use of the Guide for the Care and Use of Laboratory Animals (Guide) (NRC, 2011) as a basis for developing and implementing an institutional program for activities involving animals. No activity supported by the PHS involving animals may be conducted until the institution conducting the activity has provided a written assurance of compliance with PHS Policy.
Many institutions that perform toxicology research and testing are accredited by AAALAC International. AAALAC is a nonregulatory, not-for-profit organization whose mission is to serve as a voluntary accrediting organization that enhances the quality of research, teaching, and testing by promoting humane, responsible animal care and use. Participation in the peer review accreditation program is voluntary and at the initiative of the individual institution. The AAALAC Council evaluates animal programs by conducting site visits and reviewing reports. It relies on performance standards and uses guidance such as the eighth Edition of the Guide for the Care and Use of Laboratory Animals (NRC, 2011) and the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes Council of Europe for the evaluation of laboratory animal care and use programs. AAALAC accreditation demonstrates that a program has achieved a standard of excellence beyond the minimum required by law. It is presently the only accrediting body recognized by the PHS for activities involving animals.
In addition to working under the auspices of the Animal Welfare Act and in some cases PHS Policy, the work of many toxicologic pathologists is conducted under the guidance of the Good Laboratory Practice (GLP) regulations of the Food and Drug Administration or U.S. Environmental Protection Agency ( ). The GLP regulations contain additional provisions for the care and use of laboratory animals. Included among these are certain requirements for animal housing, separation of species, quarantine of new and sick animals, and separation of projects ( ). There are also requirements for monitoring and documenting the health of research animals, as well as requirements for recording any treatments performed on research animals. There is a GLP stipulation that all animals and their enclosures be properly identified. Identification requirements and documentation methods are of particular importance to pathologists who also must track tissues and samples obtained at necropsy.
3.2
Institutional Animal Care and Use Committee
Both PHS Policy and the Animal Welfare Act require organizations using animals for research to designate an institutional official that will be responsible for the animal care and use program, and to mandate the formation of an IACUC. The IACUC is responsible for oversight of the animal care and use program. Specifically, the IACUC must assure that consideration be given to the minimization of pain and distress for all experimental animals. This is particularly important in toxicity testing, where the administration of potentially deleterious test articles can induce animal pain and distress. A specific requirement of the IACUC is to perform a semiannual review of the institution's animal care and use program as well as an inspection of the facilities where animals are housed, using the Guide and the standards of the Animal Welfare Act as a basis for evaluation. Reports of these reviews are sent to the designated institutional official with recommendations for programmatic improvements, if necessary, and corrective actions and timetables if deficiencies are noted. Among its various duties, the primary task of the IACUC is the review of all animal use protocols prior to study initiation to assure compliance with applicable regulations such as the Animal Welfare Act and PHS Policy.
The IACUC review of protocols requires an assurance that the animal model is appropriate and that the proposed experimental design uses a suitable number of animals in a manner likely to achieve the scientific objectives of the study. There is a requirement that investigators consider alternatives to the use of animals where possible and that every effort be made to minimize pain and distress. IACUC review also includes review of the proposed method of euthanasia. Toxicologic pathologists may be called upon to serve as members of the IACUC due to their familiarity with study design and animal care and use issues.
4
Euthanasia of Research Animals
The toxicologic pathologist should be involved in all facets of preclinical study design and protocol development, and special attention should be directed to laboratory science issues that affect the necropsy (see Basic Approaches to Anatomic Toxicologic Pathology , Vol 1, Chap 9 ). A very important part of the necropsy is performance of the euthanasia procedure. The pathologist needs to determine the proper method of euthanasia and to assure oversight of the procedure. In general, the euthanasia method selected should be in accordance with the recommendations of the American Veterinary Medical Association (AVMA) Panel on Euthanasia ( ). Euthanasia in the necropsy laboratory can present special challenges when it is associated with terminal surgical procedures, such as during the collection of certain samples and during the conduct of organ perfusion methods. These methods require a proper understanding of anesthetic management and monitoring procedures and involve careful description of the procedures to the IACUC for approval.
The method of euthanasia chosen should (1) have the ability to produce death without causing pain or distress to the animal; (2) be reliable; (3) be safe to personnel; (4) be nonreversible; (5) cause minimal emotional distress to necropsy personnel; and (6) be compatible with the scientific objectives of the experiment. There are many examples of untoward effects of the euthanasia method on research endpoints. Anesthetic overdose using injectable or inhalant anesthetic agents followed by exsanguination is a commonly utilized method favored by many toxicologic pathologists. This methodology is compatible with excellent tissue preservation and can be carried out efficiently. A variety of commonly used methods for rodent and nonrodent species euthanasia are associated with tissue artifacts ( ; ). These include pulmonary hemorrhage associated with carbon dioxide (CO 2 ) inhalation and tissue trauma associated with decapitation and cervical dislocation methods. Histopathologic assessment of tissues is often only one of several study endpoints, and thus the pathologist must understand the needs of the entire experiment (e.g., clinical biochemistry, hematology, body fluid analysis, immunologic assessments) so that the most appropriate euthanasia method can be chosen to minimize untoward effects on the study (see Clinical Pathology in Nonclinical Toxicity Testing, Vol 1, Chap 10 ). Clinical pathology parameters may be affected by rapid acid–base shifts induced by CO 2 or terminal hypoxia. Other toxicologic endpoints such as hormone levels, neurotransmitter activity, liver metabolism, and immune function parameters can be affected by certain anesthetic regimens and euthanasia methods ( ).
Because euthanasia of research animals is so critical to the work of the pathologist, it is imperative that controversies concerning these methods be well understood. Guidance often changes over time. Even the most commonly used method of euthanasia in rodents, inhalation of CO 2 , has had changes and conflicting methodology (e.g., rate of administration) during the past 2 decades and there have been changes in the AVMA guidance concerning recommendations for how to conduct the technique to minimize the occurrence of pain and distress. The latest guidance from the AVMA recommends introducing the gas into the chamber at a rate of 30%–70% of the chamber volume per minute in order to reduce distress to the rodent. There have also been controversies concerning the use of decapitation and cervical dislocation in animals. In all instances, the selection of the technique for euthanasia should be reviewed by pathologists involved with the study and discussed with the study director and institutional veterinarian to assure compatibility with research endpoints. The method must be approved by the relevant institutional animal care and use and ethical review committees. Pathologists must consider the possibility that variance in euthanasia administration, prior to necropsy and exsanguination, between animals may also contribute to biologic variation observed in research results.
5
Selection of Animal Models
5.1
Overview
The use of laboratory animal models will continue to play a critical role in toxicologic pathology. They have proven to be useful in toxicology testing because they share many similarities with humans. Broad translation of cellular, tissue, organ, and system similarities of xenobiotic-induced toxicologic pathology findings in animals is the long-standing premise for nonclinical in vivo testing of test articles intended for administration in humans. Relative comparisons of toxicities in nonclinical species with those in humans support this continued use. There are metabolic, anatomic, and physiologic similarities that allow for comparisons in absorption, distribution, and excretion of xenobiotics, but differences in these attributes also must be appreciated that potentially impact translation (see ADME Principles in Small Molecule Drug Discovery and Development—An Industr ial Perspective , Vol 1, Chap 3 and Biotherapeutic ADME and PK/PD Principles , Vol 1, Chap 4 ). The small size, docile nature, short life spans and gestation periods, and large litter size make rodents very economical animal models to maintain, breed, and use in the conduct of toxicology studies, including for life-long studies used to assess carcinogenicity. Historical databases and publications on spontaneous disease characteristics of mice and rats and other laboratory animal species make them invaluable animal models for toxicology studies ( ; , ).
For the proper interpretation of toxicology and pathology data, the age, sex, physiologic status, microbiologic status, nutrition, and genotype of the test animals must be considered. It is also necessary to consider the environment in which they are bred, maintained, and observed. Most modern toxicology testing facilities use barrier-reared rodents with defined microbial flora, perform health surveillance on the colony, and order animals from reliable suppliers. These sources should be able to provide information concerning diet and feeding methods, breeding procedures, genetic control, caging, and husbandry as part of the source history.
5.2
Issues of Translation in Animal Model Selection
Translation of animal model toxicity to humans is a controversial topic, but nonclinical testing is codified and required by harmonized international regulatory expectations. These regulations are predicated on the known value of hazard characterization (e.g., tissue, organ, or system injury caused by the test article as evident in histopathologic evaluation, and correlative clinical signs, clinical pathology, organ weight changes, or other allied and appropriate evaluations of injury or functional perturbation). Hazard characterization is used to screen out drugs in development considered to have high risk for causing similar human toxicity. Risk assessment is used after hazard characterization to establish a risk–benefit ratio for a specific disease indication, and nonclinical safety testing is essential to establish a safe starting dose in clinical trial subjects (e.g., based on a No Observed Adverse Effect Level, or NOAEL, in animals) and to define predicted safety margins based on comparison of estimates of the test article exposure needed to provide efficacy for the disease and estimates of therapeutic index from toxicokinetic assessment of exposures in animals (see Interpreting Adverse Effects , Vol 2, Chap 15 ] and Risk Assessment , Vol 2, Chap 16).
Retrospective evaluation of toxicity in animal studies compared to human toxicity to determine translatability must take into account that true positives are typically not identified in both animals and humans ( ; ). Drugs showing greater toxicity in animals are typically “screened out” and not often advanced to clinical trials if the risk–benefit ratio is deemed too high for continued development. Further, toxicity in animals is often manifest at a high dose that is not pursued in clinical trials, so genuine positive concordance to human outcomes in the case of animal toxicity will generally remain undetermined unless patients are more sensitive to toxicity than nonclinical species or have contributing concomitant conditions or therapies not tested for in animals. True negatives may also not be easily determined since it would be considered unethical to escalate the human dose beyond the efficacious range to specifically test for toxicity. Instead, only drugs with low order or manageable toxicity will advance to human testing and development. Manageable toxicities are those that have suitable benefit to risk ratio for the specific indication; identifiable safety biomarkers that can measure toxicity early in the course of injury; readily reversible toxicity; a reasonable margin of safety; and/or a reaction in animals already known to be species specific. Translational knowledge for drug toxicity has grown over time and continues to be readily applied so that unnecessary toxicity testing can be avoided or minimized in animals, with cautious assessment of potential human risk, the constant overarching requirement in pharmaceutical research and development.
The increasing complexity of different drug modalities (drug tools) that are available in current pharmaceutical discovery to target human disease has brought evolution in toxicity testing approaches, along with slowly adapted regulatory expectations and guidelines for these respective modalities. Drug toxicity translation from animals to humans should no longer be collectively considered. Modality-based approaches now recognize the diversity of toxicity expression and translatability. These approaches are science driven, and in select cases, animal testing is not scientifically valid ( ). Small-molecule drug modalities are typically tested in animals for hazard characterization, and this can manifest as target mediated (i.e., exaggerated pharmacologic effect), off-target toxicity (i.e., receptor-like activity in a tissue, organ, or system, not expected in the desired disease pathway or expected pharmacologic target), and chemically mediated toxicity (i.e., nonpharmacologic or indirect mechanisms that are chemically induced and independent of the drug target, and often related to absorption, distribution, metabolism, and excretion). Concordance of small-molecule toxicity to human toxicity identified in subsequent clinical trials has been studied and found to have high correlation in most organ systems, particularly where comparison of rat and nonrodent testing is combined ( ). This concordance is also most likely to be characterized in toxicity studies of 1-month duration or less. Biopharmaceutical compounds such as monoclonal antibodies and peptides are singularly developed for efficacy on human-specific biologic systems; their toxicity expression in animals is essentially divided into translationally relevant exaggerated pharmacologic activity in a pharmacologically relevant species (e.g., typically nonhuman primate) and less relevant antihuman drug reaction (e.g., neutralizing antibodies, immune complex disease) (see Protein Pharmaceutical Agents , Vol 2, Chap 6). Antisense oligonucleotides present a different challenge as these drug modalities create toxicity in rodent and nonrodents that have low translational relevance to human; however, worldwide regulatory agencies are increasingly aware of these issues through case-by-case learning, and through publications that advance collective understanding of risk translation (see Nucleic Acid Pharmaceutical Agents , Vol 2 , Chap 7). Cellular therapies (for example, stem cell reengineering or lymphocyte effector cell reengineering) are entirely designed as human specific, and animal testing for these modalities has very limited purpose (see Stem Cells and Regenerative Medicine , Vol 2, Chap 10).
5.3
Genetic Considerations
There are three main classes of laboratory rodents used in research laboratories: isogenic strains (i.e., all individuals are genetically identical), outbred stocks (i.e., from parents that are not closely related), and mutants (e.g., spontaneous and genetically modified). All three groups of rodents have been extensively used in toxicology research and testing, although critical evaluation of how rodent models were historically chosen has not been well elucidated in the literature. More recently, humanized mouse models have entered the arena of therapy development to examine human tissue responses and to create surrogate human disease models ( ). In some humanized mice, a mouse gene is replaced by either a human gene, genomic sequence, or regulatory element. In other models used in therapy discovery and development, immunodeficient mice are engrafted with functional human biological systems.
There has been a continuing debate concerning the choice of rodent stocks and strains most suitable for toxicity studies ( , ). This debate has intensified recently because of apparent genetic drift in certain outbred stocks of rats, leading to significant declines in life span, increased background of spontaneous disease, and changes in other parameters that impact historical data ( ; ). Scientific debate in favor of using multiple inbred strains within the same toxicity study to more closely mimic heterogeneity of human populations has not led to changes in how rodents are used in safety assessment testing in the pharmaceutical industry. Outbred sources of rodents are still in broad use.
Outbred stocks of rodents are often known by generic names such as Sprague–Dawley rats or Swiss mice but should always be designated by their proper genetic nomenclature. As a result of genetic assortment, inbreeding, and selection, different colonies of outbred stocks will be genetically different from each other within a few years. It is important for scientists to understand that commercial rodent producers maintain multiple colonies of many of the important outbred stocks; therefore, the relationship between the various colonies is dependent on the breeding practices utilized. Control of these breeding practices is critical to assure uniformity, even among single suppliers. Although genetic drift can be minimized using large colony sizes and specific breeding schemes, genetic quality control of outbred stocks is difficult and expensive and requires significant supplier effort.
In general, toxicologic pathologists feel most comfortable using animal models for which they have historical data on the incidence of spontaneous and test article–induced lesions. The lesion database for any animal stock or strain is best utilized when it is based on in-house studies with animals that have a similar genetic history and are maintained under identical environmental conditions to the study being conducted. Thus, many institutions become reluctant to change the design of studies or to use new animal models if they feel that the changes will negate their ability to utilize historical data. This is particularly true when studies need to be reported to regulatory agencies. Historical data can provide an extremely valuable tool for study interpretation, but sometimes the value of these databases is overstated. Genotypic qualities of rodent models change over time due to selection procedures, alterations in breeding schemes, and genetic drift. These genetic changes have led to changes in the incidences of many neoplasms and nonneoplastic findings in both isogenic strains and outbred stocks. In addition to alterations in genetic qualities of rodents, animal husbandry practices change over time, contributing to the need to view historical databases as living documents subject to change. Animal housing, trends in stocking density, and many other aspects of animal husbandry such as diets and feeding practices contribute to significant changes in historical databases of both neoplastic and nonneoplastic lesions.
It is important to understand individual stock and strain characteristics when choosing rodent models for toxicology experiments. In some instances, anatomical, biochemical, or physiological attributes may be used to select a model for use. The longevity of the selected model is an important characteristic to consider for certain long-term studies. Regulatory requirements dictate certain survival criteria, and the decreasing longevity in certain stocks of ad libitum–fed rats in recent years has led to difficulty in some laboratories. Various approaches have been used in efforts to reverse the declining trends in survival. These efforts have included changes in breeding schemes and husbandry procedures (such as types of diets and the way in which rodents are fed) ( ). In a number of instances, institutions have changed their routine stocks and strains for chronic bioassays based on longevity issues.
A key point in properly selecting a rodent model is to understand the impact of strain-related spontaneous lesions on the proposed study endpoints. For instance, if the lung is a suspected target organ of toxicity, the Fischer 344 rat strain is probably not the animal model of choice for a long-term study. This strain has a very high incidence of leukemia, with pulmonary vascular infiltrates that would compromise the ability of the pathologist to diagnose interstitial lung disease. Similarly, if a nose-only method was the proposed route of exposure in a long-term inhalation study, one might not wish to choose a rat strain with a high incidence of spontaneous mammary tumors that would mechanically interfere with animal placement in the nose-only apparatus. In virtually all rodent stocks and strains, examples can be found of target organ compromise by a genetic predisposition to a high spontaneous background of lesions. There is no ideal rodent model for all types of toxicity studies, and one must critically evaluate model selection for each experiment. This is an area where active discussion between the study director, laboratory animal science professionals, and the toxicologic pathologist is warranted before decisions are made on specific models.
5.4
Issues to Consider When Sourcing Animals
In addition to considerations of stock and strain, it is critically important to consider the source colony of the animal model selected for toxicity studies and not just take into account the vendor from which a model is procured. Animals derived from different source colonies can have significant differences in response to xenobiotics for a variety of reasons related to differences in housing and husbandry, diet, microbiome factors, pathogens, and other nongenetic reasons ( ; ). Animals derived from differing source colonies can have varying spontaneous as well as induced lesion incidence. It is important for toxicology professionals to monitor the health and conditions of source colonies and to consider what happens to animals prior to their arrival in test facilities. The importance of procurement issues such as transport stress, quarantine procedures, and animal training and acclimation methods should not be underestimated. Animal stress is associated with both biochemical changes as well as morphological alterations in a number of organ systems, most notably the immune and endocrine systems ( ).
The size of source colonies and husbandry methods used by animal vendors can have practical implications for toxicity studies. For instance, there can be issues associated with litter effects in long-term rodent bioassays used for carcinogenicity assessment. It takes many age, weight, and sex-matched animals to set up a carcinogenicity study and some vendors include entire litters in the shipment. There have been instances of hereditary cancer predispositions within source colonies leading to multiple rare tumors arising in chronic bioassays ( ).
Toxicologic pathologists that perform nonhuman primate pathology evaluation must pay particular attention to the source of their animals. In these species, there is far less genetic and microbial standardization within colonies and there have been many noted differences in study endpoints depending on the source of animals. For many years, the pharmaceutical industry has utilized cynomolgus monkeys from the Indian Ocean island of Mauritius due to the fact that these animals are free of a number of adventitious viral agents such as Herpes simiae . This relatively healthy stock of outbred animals is only 400-500 years old and is derived from original animals of Southeast Asian origin deposited on the island by Dutch traders. Because the cynomolgus monkey is not native to Mauritius and all animals from this source are derived from an original group, these animals demonstrate genetic homogeneity compared to cynomolgus monkeys of other origins (e.g., China, Indonesia, Philippines) due to a narrow genetic base. There are known immunologic variations and other differences in responses noted that can be important in certain types of studies depending on source of macaque ( ; ) (see also Animal Models in Toxicologic Research: Nonhuman Primate , Vol 1 , Chap 21 ).
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