Exploring rabbit as a nonrodent species for general toxicology studies Free

Abstract

To avoid adverse events in humans, toxicity studies in nonclinical species have been the foundation of safety evaluation in the pharmaceutical industry. However, it is recognized that working with animals in research is a privilege, and conscientious use should always respect the 3Rs: replacement, reduction, and refinement. In the wake of the shortages in routine nonrodent species and considering that nonanimal methods are not yet sufficiently mature, the value of the rabbit as a nonrodent species is worth exploring. Historically used in vaccine, cosmetic, and medical device testing, the rabbit is seldom used today as a second species in pharmaceutical development, except for embryo-fetal development studies, ophthalmic therapeutics, some medical devices and implants, and vaccines. Although several factors affect the decision of species selection, including pharmacological relevance, pharmacokinetics, and ADME considerations, there are no perfect animal models. In this forum article, we bring together experts from veterinary medicine, industry, contract research organizations, and government to explore the pros and cons, residual concerns, and data gaps regarding the use of the rabbit for general toxicity testing.

Introduction

To support first-in-human use of new pharmaceuticals under an Investigational New Drug application (IND), sponsors are required to submit nonclinical pharmacology and toxicology data that form the basis on which the sponsor has concluded it is reasonably safe to conduct the proposed clinical investigations (21CFR312.23(a)(8) 2023). Different sources of data can be used (eg, in vivo, in vitro, in silico) depending on the context of the question being addressed. FDA’s ability to leverage advances in new approach methodologies (NAMs), such as those defined in the Food and Drug Omnibus Reform Act of 2022 (US, 2022) is limited by the science supporting an alternative method as appropriate for a given context of use. Although FDA has been able to adopt a number of animal-sparing approaches as appropriate for assessing certain risks (Avila et al., 2020, 2023; Brown and Wange, 2023; FDA, 2017; Wange et al., 2021), NAMs cannot, at this time, fully recapitulate the complexity of in vivo systems. Animals uniquely provide a fully integrated physiologic system, with intact homeostatic, metabolic, hormonal, and immune system responses; all of which can influence the toxicological effects of a pharmaceutical. Moreover, animal studies allow for a tissue-agnostic assessment of toxicity, unlike, for example, microphysiological systems or organotypic in vitro approaches. Although shortages in research primates and beagles are driving investment in advancing NAM approaches to pharmaceutical development, replacements for these scarce whole animal models necessarily need to focus on a shorter term transition via other nonrodent animal species.

There are ethical considerations to using nonhuman primates (NHPs) because of their similarity to humans; and dogs because they are companion species. Pigs and rabbits are also kept as pets; however, they have other purposes as food animals and research species. Although public opinion on species used in pharmaceutical research can be varied, the rabbit and pig are generally regarded as less-sensitive models in terms of public opinion compared with NHP and dog. Although minipig is covered in other publications (Jones et al., 2019) and is widely used in Europe for general toxicology studies, rabbits are not widely discussed in the published domain.

Domestic or European rabbits (Oryctolagus cuniculus) are lagomorphs in the family Leporidae. Although over 50 breeds of rabbits are recognized by rabbit hobbyists around the world, only a few are bred commercially and used in research settings, including the New Zealand white (NZW, a medium-sized breed) and the Dutch-belted (a smaller breed) rabbits.

Historically used as infectious disease and pharmacology models (Franco, 2013), to study neuroanatomy (Farias Serratos et al., 2017), and for cosmetic testing (Lee et al., 2017) the rabbit has never taken hold as a routine species in pharmaceutical development, except for embryo-fetal development studies (Fischer et al., 2012), ocular drug products (Ahn et al., 2016; del Amo and Urtti, 2015), vaccine development (Green and Hussain Al-Humadi, 2013), and medical device and implant testing (Schuh, 2008). NZW is a medium-sized white-coated rabbit originally bred for meat and fur production but are the most common breed used in laboratories because of their docile nature, medium size, consistent health, and genetic status when purchased from specific pathogen free sources, and similar reactions to human infectious agents, cosmetics, and pharmaceuticals (Esteves et al., 2018). Having a white coat enables assessment of skin and eye reactions and their large size allows for physical assessment and blood collections. Dutch-belted rabbits are generally used for intraocular drug delivery because they have pigmented eyes that can determine drug interactions with melanin similar to the human eye (Kiel and Kopczynski, 2015). Although there may be utility in the rabbit as a general toxicity model, there are also limitations. However, what is less clear are how these limitations compare with other species and if these limitations preclude the use of rabbits in general toxicology studies.

Although several factors affect the decision for species selection (ICH, 2011; Namdari et al., 2021; Prior et al., 2020, 2019) there are no perfect animal models. In addition to being pharmacologically relevant, criteria for an ideal nonrodent model in a general toxicology study include: (1) regulatory acceptance, (2) high survival rate in a repeat-dose toxicity study, (3) easily sourced, handled, and dosed by common routes of administration, (4) easily housed and assessed for toxicity with ready sampling mechanisms for blood, etc., and (5) robust historical control data with a high species concordance to humans. In this article, the practical value of the rabbit in general toxicology studies is highlighted while acknowledging the inherent limitations. Additionally, concerns with the rabbit for each of the above criteria are addressed in an effort to encourage companies to include rabbits in their species selection screens and use them when scientifically justified. Adding rabbits as a routinely utilized species and developing the necessary historical knowledge and control database could prevent future delays in pharmaceutical development by reducing the over-reliance on dogs and NHPs by diversifying the routine nonrodent species for general toxicology studies.

Utility of rabbit in general toxicology, safety pharmacology, and juvenile animal studies

The ongoing shortages in research primates created urgency to reserve the use of primates for when they are the only appropriate model (Brown and Wange, 2023; ICH, 2011; NASEM, 2023). In the search for an alternative nonrodent model in addition to the dog and minipig, the rabbit has emerged as a potential candidate. Rabbits are a versatile toxicological and pharmacological model that are cost effective, well sized, docile, healthy, and readily available. Advantages of the rabbit in toxicology studies include practical considerations such as cost and timing. The size of the rabbit compared with other nonrodent species lends itself to be an efficient animal model (Table 1). The relatively small size compared with other nonrodent species allows for smaller quantities of test article to be administered per animal. Given the lower relative cost of rabbits and smaller amounts of test article required, use of rabbits can be an efficient way to increase the sample size to improve statistical power. Although the rabbit is a relatively small nonrodent species, they are large enough to sustain serial blood draws on study, negating the need for satellite animals for toxicokinetic analysis. Additionally, microsampling has been successfully employed in rodents (Esteves et al., 2018) and can be applied to rabbits to improve animal welfare when multiple samples are required.

Rabbits have been and continue to be used as a standard test system for vaccine safety testing (Barrow, 2012; Barrow and Allais, 2013; FDA, 2006; WHO, 2014) contraceptive safety testing (Castle et al., 1998), developmental and reproductive toxicity (ICH, 2020b), local tolerance studies, bone research (Moran et al., 2016; Wancket, 2015), and cholesterol pharmacology studies (Fan et al., 2015) as well as infectious disease models (viral, bacterial, and parasitic infectious diseases) (Peng et al., 2015). The rabbit is actively used as a pharmacology model for several noninfectious conditions, including intestinal immunity, lupus erythematosus, osteoarthritis, retinal cancer, and cognitive diseases. However, there are other opportunities to use the rabbit that are often overlooked or not routinely employed, such as general toxicology, safety pharmacology, and juvenile animal studies.

General toxicology studies

Although the use of rabbits in general toxicity studies is the exception, a handful of recently approved drugs were demonstrated to be safe using rabbits in general toxicity studies (BLA 761068, NDA 215014, and BLA 761235). As with any preclinical species, there are special considerations to optimize the use of the rabbit as a successful toxicology species. For example, rabbits are unique among the common nonrodent species as they are a prey species, and therefore, proper attention should be given to their needs as well as interpretation of their behaviors.

Most successful biologics and small molecule drug programs, regardless of nonrodent species chosen for the general toxicity studies, will use rabbit and rat in the embryo-fetal developmental toxicity study (EFD) prior to large scale clinical trials. Typically, a tolerability study in nonpregnant rabbits and a dose-range finding study in pregnant rabbits are conducted prior to EFD studies in rabbit. Tolerability study in nonpregnant rabbits (typically 1–2 weeks in duration) helps to identify any potential untoward effects following repeat dose administration of the test material, including vehicle control articles, gauge exposure, and guides decisions on dose selection for subsequent studies in pregnant rabbits. Such tolerability studies can also serve as a dose range finding study for subchronic general toxicology studies in rabbits, and with proper planning, some fertility assessments can be incorporated into the general toxicology study designs. Toxicity findings in maternal animals and their offspring in the EFD study can halt a drug development program if it is considered relevant to human safety. This can be problematic given the timing of EFD studies is in parallel with or after Phase 2 clinical trials. Identification of serious adverse toxicities after initiating clinical dosing results in potential safety concerns and the need for expedited safety reporting. Using rabbits as the nonrodent for general toxicity studies could help identify problematic toxicities early in drug development.

Age of the animals selected for general toxicology studies should be carefully considered. Using rabbits in general toxicology study designs prior to reaching sexual maturity can lead to problems, particularly during microscopic examination of the reproductive organs which will likely be immature (Frame et al., 1994). However, this issue also exists in NHP, minipig, and dog, where young animals are often used due to the shortages (Goedken et al., 2008; Haruyama et al., 2012; Taberner et al., 2016). Fortunately, rabbits mature quickly in comparison to NHP and dogs often reaching sexual maturity on a 3-month study.

For many biologics, the NHP is the only relevant species and, in some cases, there are no relevant species. Currently, transgenic mice are an acceptable alternative model for general toxicology testing when they are adequately characterized (ICH, 2011); however, their limitations include the inability to assess cardiovascular risk and their small size. Transgenic rabbit models of disease are available including transgenic models of lipid metabolism and atherosclerosis, cardiac failure, immunology, and oncogenesis (Doorbar, 2016; Fan et al., 2021; Matsuhisa et al., 2020; Peng, 2012). As biologics are most usefully tested in relevant species, the ability to engineer rabbits to express a functional human protein could make them invaluable to general toxicology testing. The cost-benefit of developing such a model should be carefully weighed. To generate a line of transgenic animals and characterize them for functional relevance is generally considered impractical for a single program. Efforts to reduce animal numbers using transgenic animals can be defeated when considering the low transgenesis efficiency and mosaic genotypes of born animals from pronuclear microinjection and lentiviral vector techniques. Although the only examples from the literature have leveraged transgenic rabbits to create disease models rather than toxicology models (Song et al., 2020), CRISPR/Cas9 and other gene editing technologies (Hay et al., 2022; Matsuhisa et al., 2020) provide more precise genomic manipulation with higher efficiency making the use of transgenic rabbits as a toxicology model a future possibility. As rabbits are regulated by USDA, genetically modified animals may need additional registration prior to shipping across state borders.

Rabbits have inherent limitations that may exclude that species for consideration during drug development. For instance, they are unable to vomit, which can be both a limitation and a benefit for orally administered drugs. Orally administered drugs in dogs have challenges in maintaining exposure as dogs readily vomit test article, preventing absorption. However, the lack of a gag reflex can eliminate one way of detecting toxicity in a species.

Rabbits are identified as a species that is sensitive to gastrointestinal disturbances (ICH, 2020a; Oglesbee and Lord, 2020), which, depending on the mechanism of action, may be translatable to humans. One inherent limitation of rabbits is their use for the development of antibiotics. As hindgut fermenters that rely on microbiota for digestion, high doses of antibiotics administered orally or parenterally to rabbits can cause profound adverse effects not translatable to humans. Irrelevant toxicities in rabbits with antibiotics could confound interpretation of toxicology studies. For example, lincomycin and clindamycin produce fatal enteritis in rabbits as a result of antibiotic-induced depression of lactobacilli and a rise in toxins produced by Clostridium perfringens and Clostridium spiroforme. Ampicillin causes fatal enteritis in rabbit, but penicillins and cephalosporins have a less clear toxicity in rabbit. Erythromycin, spectinomycin, and minocycline are toxic to rabbits but manifests as decreased growth rates. The rat appears to tolerate a wide range of antibiotics at super therapeutic doses, including lincomycin, not tolerated by guinea pigs, hamsters, or rabbits (Morris, 1995). However, mice have toxic responses to streptomycin at doses of 3–6 mg/kg (Morris, 1995) and alpha-chloralose administered intraperitoneally to rats causes a fatal ileus to develop at therapeutic doses (Fleischman et al., 1977). Despite the sited limitations of the rat and mouse, these species are the most used rodent species in general toxicology. This is just to illustrate that limitation of the rabbit for one class of medication does not preclude their broad use in drug development.

Safety pharmacology studies

Rabbits are not frequently used in safety pharmacology studies, but the rabbit Purkinje fiber assay (Aubert et al., 2006) has been a long-standing method for studying drug effects on cardiac conduction. The rabbit and human hearts have a comparable electrophysiological architecture (Ellermann et al., 2020) and some noteworthy similarities include, but are not limited to, similar cardiac ion currents, rectangular action potentials, repolarization reserve, and calcium fluxes. There are several reported applications for studying cardiac electrophysiology in rabbits, including single cell, tissue preparations, whole-heart arrythmia model, and in vivo models (Ellermann et al., 2020). In an analysis of 3290 approved drugs for which 1 637 499 adverse events were reported in both human and animals, the diagnostic power of various species was evaluated with rabbits having a high concordance of cardiac disorders (Clark and Steger-Hartmann, 2018). Wireless telemerized rabbits have been used to assess left ventricular pressure, cardiac contractility, and cardiac relaxation in Dutch-belted rabbits (Tate et al., 2011) or body temperature in Japanese White rabbits (Chen et al., 2023). Rabbit jackets with a pocket compatible with DSI and EMKA telemetry systems are commercially available. Telemetry techniques in rabbits have not been widely utilized for safety pharmacology evaluations in pharmaceutical development likely because of the inertia in other species given the large body of QTc in vivo data in dog, rather than an inability to obtain useful data from the rabbit.

Core respiratory function evaluations required by ICH S7 (ICH, 2000) include respiratory rate and other measures of respiratory function, such as hemoglobin oxygen saturation, which can be assessed in rabbits. There are well-established methods of evaluating more precise pulmonary endpoints, such as tidal volume, in rodents, dogs, and NHPs that have not been developed to the same extent in rabbits.

Central nervous system (CNS) function evaluation today is modified from the Irwin test first described in 1968 for mice. The modified Irwin test or functional observation battery has successfully been adapted to rats, but CNS safety pharmacology in primate, dog, and minipig rely mostly on qualitative clinical observations. Likewise, similar adaptations can be made to test CNS functions in the rabbit (Beck et al., 2012; Hurley et al., 1995).

Juvenile animal studies

There are pros and cons to working with any nonclinical test system, and the ICH S11 (ICH, 2020a) testing guidance highlights various advantages and disadvantages that should be taken into consideration when designing nonclinical studies in juvenile animals, including the rabbit. The basic core endpoints in any nonclinical juvenile animal study are akin to those of adult animals on a general toxicology study. These core endpoints include assessments of viability, clinical observations, growth, clinical pathology, anatomic pathology, and toxicokinetics. In juvenile animal studies, additional endpoints (eg, bone assessments, enhanced clinical or anatomic pathology, ophthalmologic examinations, or CNS, reproductive or immunologic assessments), are incorporated into the study design, based on weight of evidence, to address any compound-specific or safety concerns.

There are various routes of exposure that are feasible when administering test materials to preweaning juvenile rabbits (eg, oral gavage, intravenous, dermal, intraperitoneal, intramuscular, subcutaneous, and inhalation), but age at the initiation of administration is a key consideration as some of these routes might be technically challenging at a young age due to the continuation of their development ex utero. In the postweaning period, as well as in adult rabbits, additional nonstandard routes of administration (eg, penial, intravaginal, intra-auricular, intravitreal or ocular, intranasal instillation, and implanted osmotic mini pumps) have been validated in a research setting and incorporated into experimental designs and further justify the use of rabbits in nonclinical assessments. Overall, as with any toxicity studies, the intended clinical route of administration should be used when feasible and the chosen route should provide adequate systemic exposure.

As with any species, technical training is required to ensure proper dose administration regardless of the route. Even though rabbits have thin, delicate internal tissues compared with other species, oral gavage administration is accomplished either with a single technician and a properly restrained animal or with the use of a holder to restrain the animal during dose administration. If dose administration starts during the preweaning or early postweaning period, adjustments are necessary to account for the overall growth and development of the animal over time. Many laboratories across the industry have been administering test materials to pregnant rabbits orally via stomach tube without much consequence, and those same laboratories should be capable of dosing naïve male or female rabbits if used in a general toxicology setting. Rabbits can only tolerate limited volumes of oily formulations orally (usually about 1 mL/kg body weight) because of their sensitive gastrointestinal tract, and there are some other vehicle components that are not recommended for use in rabbits (Gad, 2006; Moxon et al., 2023).

Physical indicators of the onset of puberty are a core endpoint in nonclinical juvenile animal study designs. Examinations of the vaginal opening for patency, and of the penis for separation of the prepuce from the glans penis, are performed as markers of the rate of sexual development towards puberty. Disruption in the attainment of these endpoints could subsequently impact mating performance and fertility in adult animals. In male rabbits, attainment of balano-preputial separation is initiated on or around Postnatal Day (PND) 65 with most animals completing this process by PND 84. In female rabbits, attainment of vaginal opening is initiated on or around PND 28 with most animals completing this process by PND 38. Patency not only indicates imminent puberty but is also necessary for normal estrous cycles and for mating.

Rabbits reach skeletal maturity shortly after they are sexually mature. Although rabbits are used frequently for various types of bone research (ie, biofunctionality, biocompatibility/safety, vertebral fracture repair, oral surgical approaches), there are differences between rabbit and human bone as they relate to the secondary centers of ossification present at birth, macroscopic appearance, microscopic structure, osteonal remodeling, and cartilage thickness (Wancket, 2015). One possible advantage of the rabbit is that they are the smallest species to undergo Haversian cortical bone remodeling in the diaphysis similar to adult humans and large adult animals.

Unlike rodents, neurobehavior tests are limited in rabbits, but eyeblink classical conditioning has proven to be an appropriate learning and memory test that can be used in this species (Green and Woodruff-Pak, 2000; Hoberman and Barnett, 2012). Eye-blink conditioning involves the pairing of a conditioned stimulus (CS; such a tone) to an unconditioned stimulus (UC; air puff). The eye-blink response is known to be supported by well-defined neural circuits and the cerebellum is critically involved in the acquisition and retention of simple learned responses. With training, rabbits can learn to discriminate between a CS and US and eventually produce a conditioned response which would be the movement of the nictitating membrane.

Regulatory acceptance

After dogs and NHPs, the most commonly used nonrodent species for repeat dose general toxicity studies are the minipig and the rabbit (Brown and Wange, 2023). Even so, rabbits and minipigs together accounted for <10% of study reports submitted to module 4.2.3.2 (repeat dose toxicity) of INDs in CDER’s electronic document room between 2010 and 2022, inclusively. Although rabbits are more readily available and are commonly used in EFD studies, there are certain perceptions in the scientific community that tend to limit their use as a general toxicology model. For example, the perception that rabbits are too closely related to rodents. This perception is not necessarily mistaken; however, it fails to appreciate that, at least phylogenetically, humans (and other primates) are more closely related to the evolutionary lineage that gave rise to rabbits than we are to the lineage that gave rise to dogs and other carnivores (Nikolaev et al., 2007).

Clearly rabbits already can play an integral role in the safety assessment of pharmaceuticals, particularly for assessing embryo-fetal developmental toxicity, but also as a general toxicity species, at least for some classes of drug. There is no regulatory barrier to the broader use of the rabbit as the nonrodent species for toxicity testing, as long as its use is scientifically justified by the pharmaceutical developer.

Survival in repeat-dose toxicity studies

A common concern raised when considering rabbits in general toxicity studies is their survival during subchronic and chronic dosing. It is not clear when these concerns were first identified or if they are historical concerns that could be addressed with modern approaches in animal husbandry and restraint technology. The focus of this section will be on essential considerations of rabbit anatomy and their care, and several technical challenges, which should be understood to optimize success when working with rabbits in toxicologic research.

Rabbits have been used extensively for over 100 years in biomedical research (Fan et al., 2018) with just under 150 000 rabbits used annually for teaching, research, and testing in the United States and Canada in recent years (CCAC, 2021; USDA, 2021). Similar to some other species, the use of rabbits in research has declined significantly over the past 50 years, largely due to the development of replacement tests and methodology, for example, the Draize skin sensitivity and pyrogen tests, and polyclonal antibody production have now been replaced by in vitro approaches and biotechnology processes (Lee et al., 2017).

Anatomically, lagomorphs have open-rooted (ie, constantly growing) incisors and cheek teeth, a large diastema (gap) between the cheek teeth and the incisors, a second set of incisors behind the upper ones (peg teeth), a large fleshy tongue, and a narrow mouth and jaw. Because of their constantly growing teeth, it is essential that rabbits be provided with appropriate feed, hay, and other resources to gnaw and chew to ensure even wear of their teeth over time to prevent malocclusion (ie, dental problems arising from overgrowth or uneven wear).

Rabbits are hindgut fermenters with a simple stomach and a large cecum and they require a high fiber diet for optimal health. Approximately 50% of their lymphoid tissue can be found within the gastrointestinal tract. Rabbits produce 2 types of feces, the first being a soft, mucoid material (cecotrophs) that they re-ingest directly from the anus to derive further nutrition (a process known as cecotrophy), leading to the second type, a hard, dry pellet.

Additionally, rabbits are known to be susceptible to mucoid enteritis which can result in adverse clinical observations and eventually death if not managed well. The doe nurses her kits for brief periods once daily until the kits are big enough to start leaving the nest and nibbling pelleted feed and the doe’s fecal pellets at around 14–16 days. At this point, when they transition from a milk-based diet to a plant-based diet, and until they have acquired adult gastrointestinal bacterial flora, kits are susceptible to developing enteritis and mucoid enteropathy, the latter being a condition characterized by diarrhea and subsequently, jelly-like feces and often acute death. Fatal gut infections likely progressed to damage the integrity of the intestinal barrier resulting in permeability of pathogens leading to systemic inflammation, a condition known as “leaky gut” or intestinal wall leakage syndrome (Di Vincenzo et al., 2023). Ensuring an appropriate environment and minimizing stress during this time both help to reduce the incidence of mucoid enteropathy. However, life-threatening gastrointestinal diseases are not unique among rabbits: the most common spontaneous findings in NHP used in toxicology studies is diarrhea and diseases of the gastrointestinal tract, which can lead to mortality on study (Johnson et al., 2022).

Rabbits have a high muscle to bone ratio for their size, and therefore, they are susceptible to lumbar vertebral luxations or fractures if they are poorly restrained and their back legs kick out. This generally results in hindlimb paresis requiring euthanasia. This is unique among the other more common nonrodent toxicity species and a reason for animal loss on study; however, these injuries are not considered test article related. The risk for subluxation and struggling during restraint can be mitigated through habituating animals to restraint and employing low stress handling methods when working with rabbits.

For large molecule therapeutics designed for humans, nonclinical species often mount an immunogenic response against the human test article resulting in antidrug antibodies (ADA). ADAs complicate general toxicity studies as they can cause neutralization of the test article blunting on-target effects, exposure loss through increased clearance of the test article, or acute phase responses that are adverse to the animal. Rabbits, commonly used to make antibodies for the life sciences, are perceived to be more likely to build immunogenic responses than other species. However, it is important to remember that all species could mount an immunogenic response against human-derived biologics. In 3 recently approved large and small molecules using rabbits and cynomolgus macaque as their test species, it was noted that the rabbit did not consistently have higher ADA formation than cynomolgus macaque (BLA 761068, NDA 215014, and BLA 0761235). It should be noted that this conclusion is drawn from a very small sample size of approved drugs and may represent a biased sample of successful programs using rabbits.

Overall, understanding the rabbits’ needs and providing proper housing and care can help mitigate some of the cliche concerns of using rabbits.

Sourcing, handling, and dosing rabbits

Availability of docile, parasite free, colonies make the rabbit easily sourced for general toxicology studies. There are long-standing colonies of NZW rabbits that consistently test negative for Bordetaella bronchiseptica and Pasterurella multocida for over 2 decades (Yanabe et al., 1999). Rabbits socialized by technicians from birth in these colonies allow for receipt of more docile animals that can be handled on general toxicology studies with less risk of injury to the animal and technician. Due to the current demand for rabbits, the capacity of the breeding facilities is not sufficient at this time to replace dogs and NHP used in general toxicology studies for pharmaceutical development. However, because of the short intergeneration interval and large litter sizes, rapid scale up of rabbit colonies appears more feasible than research NHP colonies.

Prior to using rabbits in research, it is important to understand the model needs proper housing and care. Conventionally raised rabbits are commonly infected with a number of respiratory and enteric bacteria and parasites whereas rabbits acquired from commercial sources breeding for research are free of these subclinical infections, but they are very susceptible to acquiring bacterial and viral infections from human caregivers. Thus, a good biosecurity program is needed and personnel handling rabbits should wear appropriate personal protective equipment and avoid caring for rabbits when they are ill or have active cold sore lesions.

Occupational hazards to staff when working with animals should also be considered. Rabbit fur allergies are moderately common amongst laboratory animal personnel, such that a risk assessment may be needed. In comparison, NHP harbor infectious zoonotic pathogens that can be hazardous to technicians including, Herpesvirus simiae (NRC, 2003). To avoid injury and infection, advanced training techniques are necessary for handling NHPs (McMillan et al., 2014; Salian-Mehta et al., 2023).

Rabbits are obligate nose breathers and it is important not to inadvertently occlude the nostrils during handling and restraint for procedures. An anatomically narrow mouth and jaw can make visualization of the oropharynx difficult for oral gavage or intubation when compared with other nonrodent species, such as dogs or primates and the esophagus can easily be ruptured, as is the trachea, compared with rodents and dogs, and therefore more care is required during oral gavage. When dosing, stressed rabbits may hold their breath, making it difficult to detect whether the gavage apparatus is in the esophagus or the trachea. It is essential that animals are relaxed during dosing, but that applies to all species. Having specially trained technicians can mitigate challenges with oral dosing and it is important that technical staff are trained to identify signs of pain or distress in rabbits, either by visual observation or by monitoring clinical signs or food intake.

Rabbit skin is thin compared with other animals and it can tear easily if clipper blades become too hot or dull when shaving hair on a rabbit. Tearing skin when preparing for subcutaneous or intramuscular dosing will disqualify an animal from use on study. They can also bruise easily such that habituation and gentle handling are important for welfare. This emphasizes that rabbits are a unique species and not just big rats but can still be easily integrated into research programs once aware of the differences.

One of the basic principles of toxicology is that agents produce the greatest effect and the most rapid response when given directly into the systemic circulation (Eaton and Gilbert, 2012). The percentage of drug absorption varies based on the route of administration. In conscious, restrained adult rabbits, intravenous administration of drug products directly into the systemic circulation is feasible via bolus injection, intermittent or long-term continuous infusion, with or without surgical implants or indwelling catheters. The marginal ear vein is the vessel of choice, but care must be exercised to minimize air embolisms, hematomas, infection, local irritation, or extravascular dosing. Alternative vessels include the cephalic or saphenous vein in an anesthetized or sedated rabbit.

Assessing toxicity in rabbits

Rabbits have a reputation of being a difficult or highly sensitive species to manage and work with in research settings. However, because of the lack of publications documenting failed rabbit studies and the authors success using rabbits, it is inaccurate to broadly state that rabbits are more fragile than any other toxicology species. Instead, rabbits are a species with their own unique physiology, anatomy, and behavioral characteristics that require consideration on toxicology studies. Successful management requires an understanding of rabbit behavioral and nutritional needs in captive situations to prevent nontest article related stress. Toxicology testing facilities using rabbits should have a comprehensive program of care that focuses on animal-based indicators of success (Turner and Bayne, 2023).

It is unclear where the perception of rabbits as a fragile species started and why it continues. The use of rabbits as a food animal in some parts of the world as well as their traditional use in infectious diseases are a testament to their robust health and heartiness. As a prey species, rabbits are stoic often masking underlying disease or pain. The stoicism of the rabbit can prevent recognition of health issues resulting in delayed medical care or even sudden death syndrome. In clinical practice, sudden death during veterinary exams occurs sporadically in rabbits. This usually occurs in extremely sick animals which is compounded by stress from a long ride to the clinic followed by long periods in loud waiting rooms with barking or whining dogs. The clinical setting is less relevant from a research perspective because toxicology studies generally start dosing in healthy animals and are closely monitored for clinical signs, food intake, fecal output, and body weight by trained technicians. However, the clinical experience with rabbits needs to be acknowledged and distinguished from experiences in toxicology studies.

Other classical uses of rabbits in toxicology studies may perpetuate unfair perceptions of rabbits as a fragile species may include the use of rabbits in EFD studies. Rabbits and rats are the classical species used in EFD studies and traditionally have not been well habituated before dosing in such toxicology studies. Rabbits require an acclimation period after transport (ideally 7–10 days) to readjust to their new environment. This is not possible for most EFD studies where time-pregnant animals need to start dosing within 24–48 h after arrival. Without adequate habituation, rabbits can become stressed. When rabbits are afforded adequate acclimation and habituation, such as at many university facilities, they have been used successfully for decades for difficult and invasive studies related to infectious disease, ocular instrumentation and implantation, and bone and dental implants. The authors have not personally experienced additional challenges with rabbits compared with other species either through site visits or conducting their own infectious disease studies. Understanding the species used in toxicology studies and providing proper desensitization and habituation are crucial to a successful study.

As previously mentioned, rabbits can suffer fatal enteritis after administration of therapeutic doses of antibiotics. Although rats and mice appear to be the least susceptible to antibiotic toxicity, the rabbit along with the guinea pig and hamster is particularly sensitive. It is curious that the 3 species susceptible to antibiotic induced toxicity (ie, guinea pig, hamster, and rabbit) are not commonly used in oral or parenteral pharmaceutical development and may have propagated the perception that rabbits are a fragile species. The rabbits may have limited value as a toxicology model for antibiotic drug development; however, the rabbit may be a suitable toxicology model for other drug classes.

In toxicology studies, knowing the needs of the test species will prevent undue stress in the laboratory environment. Likewise, understanding how a species presents under stress or distress can improve animal care, monitoring, and mitigation. Both preventing and recognizing stress in the test species improve the overall interpretation and data quality of toxicity findings. Refining techniques and animal care environments to reduce stress and discomfort prior to and after dosing are more than good stewardship and compliance with the 3Rs, it also improves data quality (Cait et al., 2022). Properly addressing or preventing sources of stress in rabbits requires an understanding of their natural physical and psychological needs. Therefore, here we discuss the unique nature of the rabbit.

Rabbits are prone to gastrointestinal disturbances (Oglesbee and Lord, 2020) (eg, gastrointestinal stasis syndrome, dysbiosis, or leaky gut) which are often evident by signs of inappetence, reduced water intake, and/or changes in fecal output (ie, frequency and consistency). Gastrointestinal disturbances can be triggered by stress (ie, alone or in response to pain), inappropriate diet (eg, low fiber, high carbohydrates, high-fat enrichment, diets without hay supplementation) which disrupts the balance of the cecal microflora, or underlying disease. Clinically, rabbits with gastrointestinal disturbances may appear normal (ie, alert, active, and/or responsive). Therefore, it is important to frequently monitor the quantity of food consumed in combination with behavioral and clinical assessments (ie, reluctance to move around in the home cage, lack of social interaction, depression, grinding of teeth, change in posture) and the presence and appearance of fecal output. Indigestible fiber is also an essential component of the rabbit diet to ensure proper peristalsis. In addition, the cecal microflora which are responsible for converting indigestible fiber to digestible nutrients are critical to the health of this animal model. For this reason, oral antibiotics that target microflora could cause adverse findings in a rabbit that may not be translatable to humans. Most species will go off their feed when stressed, but they can recover once pain and other factors inducing sickness behavior are addressed. Social buffering (ie, social housing) is implemented with rabbits with enormous success (Kikusui et al., 2006; Turner and Bayne, 2023). It is not possible to socially house intact males, but castrated males can be pair house similar to minipigs.

Stress can be induced in rabbits in a variety of ways (eg, pain, trauma, sudden changes in environment, exposure to predators, social isolation), and the responses are variable depending on the circumstance (eg, transitory immobility response, fear response, inappetence or overeating, behavioral changes, changes in body posture). However, the effects of heat stress are often overlooked or not frequently discussed in the context of nonclinical research. Higher ambient temperatures (above 36°C) have been shown to increase mortality, decrease food intake, and alter immune function and reproduction, and milk production in rabbits (Sirotkin et al., 2021). Rabbits have few functional sweat glands, which limits their ability to properly thermoregulate (Oladimeji et al., 2022) and the thickness of the fur coat can complicate dissipation of body heat. Instead, rabbits eliminate excessive body heat through their breath (ie, panting), and their ears help them to regulate their body temperature. The negative impacts of heat stress include but are not limited to hormonal changes in males and females, fragmented ovarian follicles, decreased semen quality, lower pregnancy rates in females and seasonal infertility in males, increased mortality (adult and offspring), and lower growth rate (Marai et al., 2002). Adequate climate control minimizes stress and heat stress related sequalae in rabbits. Similar to other species, disturbances to immune function from stress may cause rabbits to be more susceptible to pathogens or result in metabolic disorders. Adequate prestudy habituation and desensitization with regularly scheduled short handling sessions and introduction to procedures planned for the study can minimize stress from study procedures.

Being a prey species, rabbits do not make extensive vocalizations and much of their expression is seen through their ear position and body posture. For example, teeth grinding (bruxism) is a sign of pain, while thumping with their hind feet indicates fear or displeasure. Loud squealing is only heard when rabbits experience severe fear or pain. Relaxed rabbits will display horizontal lying if comfortable quarters are provided.

Rabbits require sufficient space to perform their full repertoire of behaviors, including jumping, hopping, running, rearing, lying, and standing with their ears fully erect (periscoping). Current U.S. housing requirements for laboratory rabbits are insufficient for performing many of these behaviors (9CFR3(a) 2023; NRC, 2011; UK, 2006). If the rabbit enclosure is not of sufficient size, they should be provided with periodic opportunities for exercise, for example, through use of a floor or mobile pen. Periodic provision of exercise helps to relieve boredom and frustration, but also helps to ensure adequate bone density, reducing fracture incidence. Rabbits have a significant need to chew and forage, which can be met by providing timothy hay, wood blocks or balls, and other manipulanda. Hay can be autoclaved or irradiated to address biosecurity concerns.

Rabbits are curious and social animals and they will seek positive interactions with conspecifics as well as their human handlers if handled gently using nonaversive techniques and permitted to habituate to researchers and procedures. Rabbits can quickly learn to perform certain desired behaviors, for example, sitting quietly and without restraint on a weighing scale through simple behavioral management techniques, such as positive reinforcement. Incorporation of simple training procedures to better prepare animals for study work can improve the accuracy of observations as animals will be less fearful when being handled.

In terms of social housing, intact bucks cannot be housed with other rabbits because of significant territoriality, fighting, and resultant injuries; however, castrated bucks and intact females can readily be co-housed with rabbits of the same sex on study, provided animals have sufficient space and the space is structured to enable choice to hide from, avoid or otherwise escape each other (Thurston et al., 2018). Remixing of established social pairs or groups should be avoided, ideally with randomization of animals occurring on intake. Pregnant does can also be pair housed up to parturition, but preferably not throughout as reduced litter sizes (due to cannibalization) can be seen periodically if they are socially housed during this process.

Rabbits have altered calcium homeostasis compared with other mammals, in that calcium uptake from the gastrointestinal tract is largely unregulated and is proportional to the level of calcium in their diet. For this reason, plasma calcium levels may be higher and urinary secretion is proportional to dietary intake, such that the urine may be thick and opaque. Porphyrins from the diet are also secreted into the urine, which may be orange to red colored as a result.

Rabbits are a unique nonrodent toxicology species as it is truly a prey species compared with NHP, dog, and minipig. Rabbits have specialized gastrointestinal systems that make them susceptible to antibiotics and, as a prey species, can present with subtle outward clinical signs. Understanding how to work with the rabbit can minimize stress on study and provided higher quality data. The key to any successful rabbit study is to minimize stress with adequate prestudy habituation and desensitization, understanding their housing and dietary needs, as well as recognizing normal and abnormal rabbit behaviors. Rabbits have been used in pharmacokinetic studies and novel infectious disease studies with significant morbidity without animal loss due to stress or inappetence. The perception that rabbits are a fragile species is not an opinion held by the authors.

Historical control data for rabbits

Another perception is that historical control data are not available for rabbits, outside of their use in embryo-fetal development studies. Although repeat dose general toxicity studies in the rabbit are in the minority (compared with dogs and NHPs), it would not be accurate to state that there is no experience with this model in such studies. The perception that there is no historical control data in rabbits contributes to the hesitancy to incorporate the rabbit in IND-enabling programs. A search of CDER’s electronic document room found a number of repeat dose toxicity studies that used the rabbit as a nonrodent species for the general toxicity assessment. Although the majority of studies involved administration of the pharmaceutical to the eye (topically, intravitreally, subconjunctivally), subcutaneous, intravenous, dermal, and oral routes of administration were also common.

There are decades worth of historical control data for rabbit clinical signs, ophthalmology, hematology, clinical chemistry, urinalysis, and histopathology at contract research organizations in addition to published resources (Barthold et al., 2016; Bradley, 2012; Bradley et al., 2021; Pritchett-Corning et al., 2010; Suckow et al., 2012). Big data analyses are ongoing to determine whether and how it can be leveraged. Currently, there is ample historical background and reference information available to support general toxicity data interpretation.

Although historical controls are valuable to understand the low incidence lesions and variability of a population, in-study controls are considered most relevant. Additionally, the cost effectiveness of using rabbit on study could facilitate increasing the number of controls on study to overcome the perceived lack of historical control data on rabbits. Alternative designs may include a double-sized control group or 2 equal-sized control groups (negative control and vehicle control) to ensure a sufficient background data are available for comparative purposes.

However, a better strategy could be to publish toxicity findings in rabbits and share any efforts to de-risk the findings as nontranslatable to humans. Industry surveys could help understand the common and uncommon toxicities associated with administration of certain drug classes in rabbits.

Conclusion

All nonclinical animal models have their advantages and disadvantages when it comes to study outcomes, available assays, and translatability of nonclinical findings to the clinical situation. Overall, the choice of an appropriate species is complex and must be well planned and scientifically justified. The most common applications for the rabbits in a toxicology setting have been embryo-fetal developmental toxicity studies, systemic evaluation of vaccines, dermal and ocular irritation, dermal toxicity, contraceptive agents, medical devices, and ophthalmology products. However, there might be additional opportunities to use the rabbits beyond these standard applications.

Increased awareness of the rabbit as a feasible nonrodent species for general toxicity testing of pharmaceuticals can stimulate efforts to include them in species selection screens, supplement the historical control database for this species, and alleviate reliance on dog and NHP which have recently shown vulnerability to shortages, are more expensive, and generally considered less favorably from an ethics point-of-view.

Laboratory strains of rabbits include NZW and Dutch Belted, with colonies of the docile NZW demonstrating consistent healthy rabbits free of parasites and viruses. Advantages to this species include the small size that is comparable to the cynomolgus macaque while allowing for sample collection, spontaneous polyestrous cycling and large litters that make colony expansion feasible, availability of domesticated strains from healthy colonies with historical control data, and capable of supporting various routes of administration. Rabbits also appear to have comparable concordance rates as other test species for predicting human toxicities (Clark and Steger-Hartmann, 2018; Leenaars et al., 2019). Bioanalysis of rabbit tissues and blood to support pharmacokinetic and biodistribution of test articles to support general toxicology studies are also feasible (Gao et al., 2013; Pandey et al., 2010).

It is also recognized that there are disadvantages to this species including sensitivity to changes in gastrointestinal microbiome and stress, limited experience for behavioral and use in safety pharmacology studies. It is also important to recognize the inherent biological limitations of the species (eg, inability to provide relevant safety data for antibiotics) and avoid their use when they are not relevant to humans through species selection screens. However, many limitations are technical or commercial that can be addressed with specialized training and management, additional use to build a historical control database, and commercial efforts to expand breeding colonies. In Table 2, specific areas of research are outlined that would facilitate and reinforce the use of rabbits as a general toxicology test species.

Considering the current climate for more responsible use of animal models, the continued need to reduce and refine study designs, and the limited sources for larger animal models such as cynomolgus macaques and dogs, there is an increasing urgency to think outside of the box. Evaluating the utility of rabbits as a viable animal model and openly discussing current and future challenges should be part of a global strategy, which includes in vitro and in silico approaches, to ensure the sustainability of our drug development armamentarium and ensure patient safety and timely progression to the clinic and beyond.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Disclaimer: This article reflects the views of the authors and should not be construed to represent FDA’s views or policies.

References