Animal Models in Toxicologic Research: Dog

2.1

Genetics of Canines and Background for Their Use in Toxicity Testing

The domestic dog, Canis familiaris , is a direct descendant of the gray wolf, Canis lupus . DNA analysis suggests that the modern-day dog is derived from multiple regional wolf populations ( ). The genome sequence of a 35,000 years old ancient Siberian Taimyr wolf fossil represents the most recent common ancestor of modern wolves and dogs.

There are small changes in the genome that distinguishes the various breeds. Based on genome data, the majority of dog breed diversity has taken place within the last 200 years. Results of the Dog Genome Project indicate that dog breeds are generally closed populations, and a given dog is more closely related to others within that breed than to dogs of other breeds. Variations among breeds accounts for more than 27% of the total genetic diversity observed in dogs. The dog breed variation is much higher than that reported within human populations (5%–10%). Across domesticated animal species, the dog has the greatest physical diversity, based upon differences in the ranges of appearance, size, and weight. Some differences in size have been traced to a single gene encoding insulin-like growth factor 1 which is common to all small breeds yet is practically absent from giant breeds ( ).

94% of the dog genome is in the same order within individual chromosomes as is present in the human, mouse, and rat. There are many similarities when comparing the genome of domesticated dogs and humans, as suggested by over 360 genetic diseases that are observed in both humans and the domesticated dog ( ). Although first identified in humans, many of these genetic defects involve a mutation of the same, or in an analogous gene, in the dog ( ).

2.2

The Evolution of the Laboratory Beagle

Dogs are used as a model in animal research primarily due to their size, friendly disposition, and ease of handling. Randomly sourced canines have an unknown genetic background which could potentially increase the variability of data collected from the study subjects. The purpose-bred beagle has become the dog breed of choice for in vivo research, although other breeds or breed mixes are also purpose bred for investigative work ( ).

The first US laboratory beagle colony was established in Utah in 1952. Most likely, these dogs were derived from champion stock based on their hunting performance. In the Utah colony, 18 dogs accounted for 98% of the colony's genetic pool. The prevalence of congenital defects would be amplified with such a restricted gene pool as evidenced by spontaneous seizures being reported in several laboratory beagle colonies in the late 1960s ( ). As a result, breeding practices across the industry were refined to reduce these occurrences. Commercial breeders of purpose-bred dogs manage their facilities based on governmental regulations and industry guidelines. This includes maintaining a breeding program to track the health and behavior of colony progeny, providing housing units allowing normal species-specific behavior and socialization, access to environmental enrichment, regularly scheduled husbandry, access to a clean home environment, appropriate nutrition, and adequate veterinary care.

2.3

Basic Biological Characteristics

Beagle dogs are medium-sized members of the domestic dog family, C. familiaris , with a lifespan of between 11 and 15 years with an average age of 12 years when kept under “controlled” conditions ( ). Their chromosome number is 39 pairs, 38 diploid pairs and 2 sex chromosomes. Male adult beagles have a body weight range of 10–12 kg at 1 year, while females are smaller, ranging between 9 and 10 kg at the same age. There appears to be two basic sizes of beagles in the literature ( https://www.akc.org/dog-breeds/beagle/ ), the smaller size reaching 13 inches at the shoulder with the larger breed being between 13 and 15 inches. These sources state that pups of both sizes can be born within the same litter.

The rectal temperature varies between 38 and 39°C (100–102.5° F) in adults, with pups having a lower temperature at birth and during the first week (37°C; 94–97°F), increasing to 36–38°C (97–100°F) during the second and third weeks. The respiration rate for puppies varies between 15 and 40 breaths/min, while adults have a lower rate, with a normal range of 10–30 breaths/min ( ).

Beagles mature between 6 and 12 months, with the majority reaching maturity by 10 months of age ( ; ; ). Females show a wide range of maturation times, with puberty beginning around 6 months of age and the first “cycle” occurring between 8 and 14 months ( ). Body weight, strain, geographical location, and housing, as well as many other factors, can affect the age of maturity in both sexes. The Covance laboratories (Covance Laboratories, Huntingdon, UK) have noted that Harlan sourced beagles of both sexes mature sooner than Marshall beagles, at least partially related to their higher body weights at equivalent ages. At a laboratory in France (Charles River Laboratories—formerly MDS Pharma Services, Saint-Germain-sur-L'Arbresle, France), noted that males sourced from Harlan breeders in France grew faster, and reached a higher body weight sooner, than males from Marshall Farms, USA. They also noted that 90% of Harlan males aged between 31 and 40 weeks were sexually mature, whereas only 10% of Marshall males had reached sexual maturity at this age. , however, noted that 11 dogs from Marshall Farms, USA, aged between 8 and 11 months, were all sexually mature, reflecting the variability of this parameter and the need for caution especially where reproductive parameters may be important in the study.

Beagles are cyclical reproducers, with one cycle occurring roughly every 7–8 months. Despite this, they cannot be classified as either seasonal or continuous breeders given the highly variable timings found across geographical locations and interindividual variability seen in the species, at least in a laboratory setting. Each individual cycle comprises proestrus and estrous phases, followed by a long diestrus period. Whereas proestrus and estrus each lasts between 1 and 2 weeks, diestrus lasts between 2 and 3 months although this duration can be highly variable. This cycle is then followed by a variable period of anestrus, which can last between 3 and 5 months.

The reproductive system of the male and female dog is routinely examined as part of the screen incorporated into routine toxicity studies for the evaluation of the safety of pharmaceutical products. For practical purposes, most safety assessment studies are conducted with prepubescent dogs and because of this there can be wide variability in maturity status at the termination of a 1 or 3 month study. Evaluation is also complicated by the variable breeding cycle, with females often being at different stages of the estrus cycle in longer-term studies. Combined with the small group sizes used in dog toxicity studies, this can make a thorough evaluation of effects on the estrus cycle problematic but not impossible. The gestation period is between 60 and 65 days, with an average litter size of five to six puppies. Puppies weigh between 140 and 280 g at birth and are toothless. Eyes which are closed at birth open at approximately 2 weeks, although vision is not completely developed until week 8 postpartum. They are also deaf at birth due to closed ear canals, with hearing beginning between week 2 and 3 postpartum as the ear canal opens. Deciduous teeth begin to erupt at approximately 3 weeks and eruption is complete by the end of the fifth week following birth ( ). The dental formula for deciduous teeth is 3/3 incisors, 1/1 canines, and 3/3 premolars. The dental formula of the adult dog is 3/3 incisors, 1/1 canines, 4/4 premolars, and 2/3 molars, and permanent teeth are generally complete and functional by 7 months of age. Incisors and canines have single roots, premolars have one to three roots (only the carnassial teeth have three roots), and molars have three roots ( Table 19.1 ).

Adult beagles consume between 25 and 40 gms of food per kg body weight each day. Unlike most other animal species, the dog's saliva lacks α-amylase ( ) even though, as in most other species, beagles possess mandibular, parotid, and sublingual salivary glands, lying in close proximity to each other. In addition, they possess a pair of smaller salivary glands known as the zygomatic glands. These are present more anteriorly than the other salivary glands and are located below the orbit representing a combination of the dorsal buccal glands present in other animals ( ). These zygomatic glands are composed of long, branching, mucous cell-lined tubules with occasional small, poorly developed serous demilunes ( ). As in other animal species, the primary function of saliva in dogs is lubrication of food and protection of the oral mucosa, but as opposed to the case in other species, digestion within the oral cavity plays a very minor role overall because dog saliva does not contain amylase.

The esophagus is lined by squamous epithelium which, like that of humans, is nonkeratinized. The capacity of the adult beagle's stomach is approximately 1 L, with lipase and pepsin being the main digestive enzymes in the gastric juices. The resting pH can vary between one and eight depending upon the dog breed ( ; ), although the average gastric pH of laboratory beagle dogs is reported to range from pH 1.5 to 2.1 within a couple of hours of consuming a meal ( ). A further complication is that the dog stomach has a very high capacity for acid secretion, with the fed stomach pH reported to be equal to or even lower than that of the resting stomach depending upon the nature of the meal. The small intestines measure between 3 and 4 m in length and have a pH close to neutral. The total gastrointestinal transit time is usually reported as 24 h, while the individual transition times in the stomach, small intestines, and large intestines are reported as being 3–5 h, 1 h, and greater than 10 h, respectively.

The beagle's heart weight is approximately 0.84%–0.85% of the body weight in young adults ( ), the age used in most safety assessment studies. The heart rate for a young adult beagle is between 70 and 90 beats per minute. In females, the heart rate increases during early pregnancy, reaching a peak shortly before parturition, and remaining above normal throughout lactation ( ). Spontaneous cardiac arrhythmias in laboratory beagles are reported to be rare, with <2% showing irregularities of the electrocardiogram (ECG) ( ).

The beagle's liver weight is approximately 7% of the body weight in young puppies reducing to 4% in adults. The liver consists of six lobes, left lateral and left medial, right lateral and right medial, and quadrate and caudate, of which the left lateral is the largest ( ). The lung and kidney weight of 6-month-old male and female beagle dogs is reported as being approximately 1% and 0.5% of body weight, respectively, with females generally having slightly lower organ weights than male dogs ( ). There are seven lobes in the lung of the dog which are divided into left and right sides. The left lung is further divided into a cranial lobe and a caudal lobe, while the right lung is divided into cranial, middle, caudal, and accessory lobes. Other organs are essentially the same as those present in other laboratory animals.

The reader is referred to the excellent publication by for a complete listing of organ weights and a description of the gross anatomy of the dog.

2.4

Housing and Care of the Beagle Dog

In terms of housing of dogs used for laboratory animal toxicity studies, minimal requirements have changed significantly over the last 10 or so years and are fairly precisely defined in various guidelines (for example, ; ). Institutions are mandated to house all research animals in clean, well-maintained enclosures that allow enough space for the animals to exhibit normal behavior and activities. Access to species-appropriate food and clean water is a primary component of daily animal husbandry. Dogs require room to exercise and to have the opportunities to interact socially with both other dogs and humans. Exercise and enrichment programs are expected components of each institution's husbandry program along with timely access to veterinary care. Proper housing conditions include the appropriate environmental temperature, humidity, ventilation and air quality, and room illumination. The ultimate objective is to provide an environment that supports animal welfare, which can have a direct effect on study conduct. Findings in laboratory animal studies from dogs with compromised welfare have been shown to be misleading as a result of reduced sensitivity, reduced reliability, and inability to repeat data due to the complex physiological effects induced by the stress responses ( ). The imperative to enforce the scientific method has led to improved conditions of care for laboratory animals as a whole, and dogs in particular, and has led to substantial improvements in the value of the data obtained ( , see Issues in Laboratory Animal Science that Impact Toxicologic Pathology , Vol 1, Chap 29 ).

3.1

Drug Metabolism, Disposition, and Excretion of Drugs/Chemicals in the Dog—Comparison with Other Species

Potential species-specific differences in drug metabolism need to be taken into consideration when selecting any animal model for toxicology testing since significant differences from the human situation can seriously affect the extrapolation of any data derived from experimental studies. The rate of metabolism by cytochrome P450 (CYP)–dependent mixed-function oxidase has been shown to be inversely proportional to the subject's body weight with rodents clearing compounds at a considerably faster rate than larger animals including human beings. In contrast, for compounds that are not metabolized by oxidation, the results would be expected to be very different. The function of other organs affecting exposure to administered drugs also differs between different animal species, sometimes in a protective way and occasionally increasing the subsequent toxicity of a given drug/chemical. For example, renal function in the dog is considerably less affected by high concentrations of sulfonamide drugs in comparison to humans ( ). In contrast, dogs are extremely susceptible to nabilone and tolbutamide metabolites, suggesting that other animal models would make more appropriate test subjects to predict safe application of these agents for humans.

3.2

Genetic Variability within Beagles and Impact on Testing

Numerous dog breeds, including the beagle, are reported to have a genetic predisposition for idiopathic epilepsy ( ). Spontaneous seizure activity was first reported in laboratory beagle colonies in 1969 with subsequent research identifying the ADAM23 gene as a risk factor in the development of epilepsy in dogs ( ). While the risk remains within the gene pool of the beagle and other dog breeds, recognition of the underlying cause has highlighted the need to monitor breeding to identify and remove susceptible individuals from the gene pool, thus keeping the problem under control for laboratory-bred animals. However, the complex interaction of the gene and the relatively low penetrance of the seizure predisposing variant in ADAM23 have made it difficult to eliminate entirely ( ).

Immunogenicity distinctions have been identified between pet beagles and purpose-bred laboratory beagles with the latter showing a different haplotype frequency of the dog leukocyte antigen when compared to the more diverse population of pet beagles. For those laboratory-bred beagles involved in establishing safety and efficacy during vaccine trials, the results obtained from a relatively closed breeding colony involved in providing dogs for laboratory animal studies may not accurately reflect the responses that might occur with clinical use in the genetically diverse dog breeds in the general population.

Beagles are generally considered a good animal model for testing drug toxicity although certain strains may present a higher risk than others when evaluating contraceptive steroids ( ). Gastric motility in beagles appears to be more affected by anticholinergic and prokinetic drugs than other breeds such as Labradors. Within strains of purebred beagles, there are subpopulations of fast and slow metabolizers of celecoxib, a nonsteroidal antiinflammatory drug ( ). This genetic polymorphism is associated with isoenzymes CYP2D15 and CYP3A12, resulting in strain-related differences in drug responses.

3.3

Use of the Dog in Drug Discovery and Development

In 2015, the US FDA finalized their guidance entitled “Product Development Under the Animal Rule.” The premise of this document was to provide a path forward for drug development when human efficacy studies were not ethical or feasible. This guidance provided information on the use of animal models of disease from an efficacy perspective, but there were no guidelines describing the use of animal models in general toxicology studies. As described by , the use of animal models of disease in routine toxicology studies may be a beneficial approach but is not without its drawbacks. The use of disease models would permit evaluation of efficacy and toxicity in the same animals. A drawback to this approach is that efficacy is usually assessed at dose levels that are relevant multiples of the anticipated human dose, whereas safety is normally evaluated at significantly higher doses. The combination of efficacy and safety in single studies could also allow for the detection of rare or unexpected adverse safety events in human disease/patient populations, thus increasing the value of such models. In addition, the effects of compounds in development could be compared directly in normal and diseased animals, thus facilitating the investigation of mechanisms of toxicity. However, the use of animal models of disease in safety studies could also result in unexpected findings that could be difficult to interpret, particularly as robust historic control databases for such models generally do not exist. Several considerations should be taken into account when considering the utility of disease models in safety assessment: (1) a clearly defined scientific rationale should be established, (2) a determination should be made as to whether the model will replace or accompany traditional toxicity studies, (3) the impact of safety findings on compound development should be understood, (4) the endpoints should be clearly defined, and associated tests should be qualified and/or validated for that species/model, and (5) it should be determined whether the safety endpoint(s) of interest can be evaluated appropriately in an efficacy study ( ).

3.4

Duration of Studies/Age at Start of Studies in Dogs

The duration of any toxicity study is determined by the scientific objective. Dogs are commonly used for both single and multiple administration for periods up to 12 months ( ) by any of the major exposure routes: oral (both gavage and feeding), injection (intravenous, intramuscular, or subcutaneous), inhalation, or dermal although the latter route is rarely used. Where studies require intravenous administration of test items, indwelling catheters can readily be placed and maintained in superficial veins for administration of test compounds and for subsequent blood collection ( ). The guidelines describe the use of sexually mature animals for standard toxicity studies, but the precise definition of “sexually mature” has been a contentious one with regards to dogs in particular but is also pertinent to the use of nonhuman primates. Until recently, dosing was initiated in dogs aged 4–6 months, when the vast majority of the male, laboratory-bred, beagle dogs were still immature ( Fig. 19.1 ). This led to questions regarding the ability of such studies to evaluate potential chemical-induced effects on fertility and prompted a Society of Toxicologic Pathology survey that resulted in a recommendation that the degree of sexual maturity be routinely documented when examining the dependent organ systems such as the reproductive and associated organs ( ). As a consequence of this concern, current recommendations and most common practices are to use both male and female dogs of approximately 12 months of age ( ). Since dogs can mature at different relative rates, there still exists the possibility, albeit less than in younger dogs, that some of the individuals may be immature at the end of a 1-month toxicity study, but concern is less for longer duration toxicity studies.

In terms of the numbers of dogs per sex per group, there are different recommendations dependent upon which guidelines are referenced. While most regulatory guidance, including that of the OECD guideline, for the repeat dose 90-day oral toxicity study in nonrodents ( ) recommends that a minimum of four dogs/sex/group should be used with four dose groups (including the control group), a more typical protocol utilizes a minimum of three dogs/sex/dose group ( ). Clearly, the use of even four dogs/sex/group places severe limitations on the value of statistical analysis on data such as body weight, organ weight, clinical chemistry, and hematology. While analysis of mean changes among these parameters is still recommended on a per group and per sex basis, analysis of individual animal data, utilizing scatter plots, is invaluable in identifying changing trends in parameters with increasing doses which a simple analysis of the mean changes by statistical analysis might conceal ( ).

3.5

Use of the Dog for Assessing Clinical Pathology and Pharmacokinetic Changes

The dog has considerable advantages for taking serial samples of blood for serum chemistry, hematology, and drug kinetics assessments. Large peripheral veins and a large blood volume permit multiple sampling timepoints for the conduct of longitudinal studies on the toxicity and fate of drugs and industrial chemicals following administration to laboratory animals ( ; ; ). Inclusion of a predosing blood sample in the dog also provides an intraanimal reference value against which to compare subsequent drug/chemical-induced changes in the respective parameters, a protocol that is not available to studies in rodents. For pharmacokinetic studies in particular, the dog is probably the most widely used animal species in drug development ( ). There has been criticism of the use of the dog for ADME-type studies (absorption, disposition, metabolism, and excretion), because of significant qualitative and quantitative differences in the presence of hepatic CYP450 between those present in dog liver and those present in human liver ( ; ). Since this enzyme group constitutes the main metabolic system for drugs and chemicals, these differences have the potential to mislead attempted extrapolations from dog studies to subsequent human trials ( ; ). A clear understanding of these differences does exist and it is therefore incumbent upon the scientist to determine which CYPs are responsible for metabolizing the particular chemical, and then to use appropriate judgment in determining whether or not the dog can satisfy these requirements. Clearly, for some chemicals, the dog will not be the appropriate species to use.

One major concern in utilizing any laboratory animal species is the existence of good background data that can be used to understand the normal variations in hematological and serum chemistry data with age, gender, and strain. There is extensive published information on a vast range of reference data for the dog ( ; ; ), whereas this is not always the case for the alternative nonrodent species. As with all experimental data obtained from animal studies, the experiments need an expert interpretation, with a knowledge of the vagaries and species differences present in the model. Despite these reservations, the results of a recent survey from confirmed the value of the dog in predicting specific human safety concerns in subsequent clinical trials for drugs. Exceptions to the simple direct extrapolation of dog data to humans clearly exist, but the knowledge of which toxicities are predictive, and which are not, has been gained over many decades of dog use for safety evaluation. It has been clearly demonstrated that canines are a reliable model provided the necessary caveats for interpretation are understood and rigorously applied ( ).