LAORATORY ANIMALS IN TOXICOLOGY

Prof. Dr. Şahan SAYGI

NEU Faculty of PharmacyDepartment of Toxicology

CONTENTS

• Introduction• The Mouse • The Rat• The Hamster• The Guinea Pig• The Rabbit• The Ferret• The Dog• Primates• The Minipig• Alternative Species

Introduction

• Biomedical sciences’ use of animals as models to help understand and predict responses in humans, in toxicology and pharmacology.

• Scientists have used animal models for so long. Regulations governing the purchase, husbandry, and use of animals in research have continued to change over the course of the 21st century.

• In some countries such use has been banned and some sources made unavailable.

• The real and most apparent problems underlying the failure of animal models arise primarily from selecting the wrong model, not using an animal model correctly, or extrapolating results to humans poorly.

• The use of animals in experimental medicine, pharmacology, pharmaceutical development, safety assessment, and toxicological evaluation has become a well-established and essential practice.

The Mouse

• The domesticated mouse of North America and Europe (Mus musculus) is the most widely used animal in medical research.

• The use of the mouse in biomedical research has been shown for several hundred years.

Choice of the Mouse in Toxicological Research

• Ideally, if toxicity testing is intended to provide information on the safety of a test substance in or by humans, the species chosen for testing should be most similar to the human.

• Substantial differences in absorption, distribution, metabolism, or elimination (ADME) between test species and the target species (e.g., the human) will reduce the predictive value of the test results.

• Testing is usually conducted in at least two species. Generally, one of those species is usually a rodent and one a nonrodent.

• The two most commonly used rodent species are mice and rats.

• Mice have many advantages as test animals for toxicity testing. They are small; relatively economical to obtain, house, and care for; and generally easy to handle.

• Mice are generally more economical than rats in these respects. Other advantages of the species include a short gestation period and a short natural life span.

• There are some disadvantages to using mice, and most are related to the small size of the animal.

• Deviations in environmental conditions such as an air conditioning failure or failure in an automatic watering system typically have more severe effects on smaller species such as mice than they do on rats.

General and Reproductive Values of Mice

Life span Average 1–3 years

Adult weight Male 20–40 gFemale 18–40 g

Age, sexual maturity Male 50 days (20–35 g)Female 50–60 days, (20–30 g)

Estrus cycle 4–5 days

Gestation period Average 19 daysRange 17–21 days

Litter size Average 12

• Mice have a high metabolic rate compared to other species.

• This fact could result in increased or decreased toxicity of a test substance, depending on the specific mechanism of intoxication.

• In many cases, high metabolic rate may be associated with rapid ADME of a test substance.

• The small size of the mouse compared to other common laboratory species offers a significant advantage if the test substance is expensive or in short supply.

• Mice seem to be generally healthier at a temperature of about 21°C to 25°C.

• High relative humidity leads to increased production of ammonia in the urine and feces.

• Increased ammonia concentrations have been associated with the development of respiratory diseases in rodents.

• The recommended relative humidity for a room housing mice is 40% to 70%.

• The photoperiod for mouse rooms used for toxicity studies is typically with a cycle of about 12 hr light to 12 hr darkness.

• Mice can be housed singly or in groups for toxicity testing.

• The acute toxicity of a group of sympathomimetic amines was increased by 2- to 10 fold in mice that were group housed compared to mice housed singly.

• In addition, increased population in solid-bottom cages has been shown to lead to substantially higher levels of CO2 and ammonia within the cage.

• Increased levels of ammonia have been associated with hepatic microsomal enzyme induction, which can alter expected metabolism in a toxicity study.

The Rat

• The rat, considered to be the first animal to be domesticated for strictly scientific purposes, was first used experimentally in France in the study of adrenal gland function.

• Many of the rat strains commonly used in toxicology today, including the Wistar, Sprague-Dawley, and Long Evans. The Fischer 344, another commonly used strain, was developed for use in cancer research.

Choice of the Rat in Toxicology Research

• The rat has become a species of choice because of – metabolic similarities,– their small size, – relatively docile nature, – shortlife span, – short gestation period.

• Although the rat is a species of choice in toxicology research because of the many physiological similarities and anatomical characteristics, differences are also present.

• The placenta is considerably more porous in the rat. This difference could increase the chance of fetal exposure to an administered test material or increase the overall level of fetal exposure to an administered test material.

• The overall distribution of intestinal microflora is different in the rat, which could lead to differences in the metabolism of an orally administered test material.

General and Reproductive Values of Rats

Life span Average 2.5 –3.0 years

Weaning age/weight 21 days/40–50 g

Puberty (males and females) 50 ± 10 days

Estrus cycle 4–5 days

Gestation period 21–23 days 

Litter size 8-16 pups 

• Current specifications for temperature and humidity are 18°C to 26°C and 30% to 70% relative humidity.

• Toxicity might increase at temperature extremes, toxicity might increase linearly with temperature, or toxicity might remain constant with increasing temperature to a threshold, then begin to increase.

• Variations in light intensity should be taken into consideration when arranging animals on cage racks for toxicology studies.

• Most research facilities operate on a 12-hr light/12-hr dark cycle, but a 14-hr light/10-hr dark cycle is also acceptable.

• The rat is a social animal and whenever possible should be housed in pairs or groups of three.

• For the purpose of most toxicity studies, this might not be practical, but should be considered.

Study Design

• The length and design of toxicology studies used to predict human risk are governed by guidelines issued by regulatory bodies such as FDA, EPA, and their counterparts worldwide.

Maximum Tolerated Dose Study in Rats

• Subchronic and chronic toxicity studies are designed to assess the test compound effects following prolonged periods of exposure.

• The highest dosage level in each of these studies should produce a toxic effect such that a target organ can be identified.

• The lowest dosage level should provide a margin of safety that exceeds the human clinical dose and ideally allows for the definition of a no observable effect level (NOEL).

• In addition to the subchronic and chronic toxicity studies, reproductive safety studies might also be required. Reproductive toxicity studies are typically required for test compounds intended to be administered to women of childbearing age or that might affect male reproduction.

• These studies include an assessment of the potential effects of the test compound on:

– general fertility and reproductive performance (Segment I), – developmental toxicity (Segment II), – or perinatal and postnatal development (Segment III).

• Typically 18 months to 2 years in duration, study is designed to assess the potential of the test compound to induce neoplastic lesions.

• The highest dosage in a carcinogenicity study should cause minimal toxicity when administered via the intended route for clinical use.

• Rodents have several unique characteristics to be considered regarding the oral administration of test compounds.

• One of the most important characteristics is the lack of an emetic response.

• The lack of this response allows for a higher dose of a potential emetic compound to be administered and evaluated.

• Many compounds and excipients can cause emesis in dogs or other large animal species and could lead to a low level of exposure and erratic blood levels.

• Techniques for oral administration of test compounds include mixing in the diet, via gavage or stomach tube, via capsule, or in drinking water.

• The most widely used methods of oral administration are the dietary and gavage techniques.

• The gavage method can be used when the test compound is not stable in the diet or might not be palatable to the animals.

• The gavage method is also preferable when evaluating toxicokinetics or pharmacokinetics.

• Methods for solution or suspension might be easier to develop than those required for dietary mixtures.

• With the gavage method of dosing, a more precise amount of the test compound can be delivered, and this might reduce the amount of test compound required to complete the study.

• A disadvantage of the gavage method is that it involves handling of the rat for each dosing.

• Handling of the rat has been shown to increase corticosterone levels and could affect study results.

• Additionally, daily intubation might lead to death due to esophageal puncture or inhalation pneumonia.

Gavage dosing with infant feeding tube

• One of the most common methods of administration of test compound is via intravenous (IV) injection or infusion.

• The IV route is often the route of choice for compounds that have poor bioavailability via the oral route or have a short half-life.

Tail vein injection

• The tail veins are currently the most widely used for IV injections in the rat.

• The veins are easily visible, especially in young animals, and one person can perform injections without the use of anesthesia.

• Injection of 2 ml/100 g body weight can be performed without stress to the rat.

• Test compounds injected into the peritoneal cavity will be absorbed into the portal circulation and transported to the liver.

• Based on the level of blood flow and circulatory surface area in the peritoneal cavity, compounds injected intraperitoneally will be absorbed quickly.

Intraperitoneal injection technique

• Intramuscular injection of compounds will result in rapid absorption into general circulation due to the abundant supply of blood vessels.

• However, the speed of absorption will not be as fast as with an intraperitoneal injection.

• A slow injection with a minimal volume will help to minimize pain. Approximately 1 ml/kg of solution can be injected per site. If larger volumes are required, multiple injection sites should be used.

• Absorption following subcutaneous injection is typically slower than following intramuscular injection.

• This could be advantageous if a relatively sustained period of absorption is desired.

• Another advantage of the subcutaneous route versus the intramuscular route is a much larger volume of test compound can be administered.

• The rat has not traditionally been used as a model in skin irritation or sensitization studies.

• The rectal route is not a routinely used method of administration in toxicology.

The Hamster

• The hamster is the third most frequently used laboratory animal following the rat and Mouse.

• It has many beneficial features as a laboratory animal because of its unique anatomical and physical features, reproductive ease, rapid physiological development, low incidence of spontaneous diseases, short life span, and a high susceptibility to induced pathological agents.

• The hamster is also a major model in diabetes research.

• 80 % of all hamsters used in research are Syrian, making them the most common laboratory hamster.

– The remaining 20% are primarily Chinese, followed distantly by– European, – Armenian,– Rumanian,– Turkish and South African hamsters.

• The Syrian hamster was first used in the laboratory in 1930 to study the Mediterranean disease kala-azar (Leishmania Enfeksiyonu: Şark Çıbanı ve Kala-Azar) .

• The Syrian hamster (chromosome number 44) has been involved in endocrinology, oncology, virology, physiology, parasitology, genetics, and pharmacology research.

• The cheek pouch of the Syrian hamster has provided the physiological technology for studying microcirculation and the growth of human tumors.

• The Chinese hamster is a native species to China. This hamster weighs 39–46 grams and is 9 cm long at adulthood. Its life span is approximately 2.5–3.0 years under standard laboratory conditions.

• The Chinese hamster has been used primarily in research for cytogenetics because of its low chromosome number (22).

• It is also used in diabetes mellitus because (a) some strains have very high incidences of the disease and (b) the course of the disease in this species is similar to that seen in humans.

• The Turkish hamster is native to Iran and Turkey and was originally trapped in 1962. At adulthood, its average body weight is 150 grams and its typical life span is a little less than 2 years.

• Some populations of the Turkish hamster have a diploid number of 42 chromosomes and others have 44.

• Turkish hamsters have been used in immunology, genetics, and reproductive behavior research.

• Hamsters should be housed individualy unless they have been housed together since weanlings.

• Hamsters are generally more adversely affected by higher temperatures than lower ones.

• Temperature ranges for the nonbreeding hamster are 20–24°C.. If temperatures drop below 4°C, the hamster will begin to hibernate.

• Hamsters are very suitable animals for carcinogenicity testing because of a low occurrence of spontaneous tumor development, but they are highly susceptible to experimentally induced carcinogenesis.

• The incidence of spontaneous tumors in Syrians is reported to be lower than the incidence seen in mice or rats.

• Although the hamster has a short lifespan, substance-related effects and neoplasms occur rapidly.

• Hamsters are recommended for long-term testing with aromatic amins, polycyclic hydrocarbons, and other agents suspected of being pulmonary carcinogens. Urinary bladder carcinomas induced by aromatic amines can take up to 7 years to induce in dogs, but can cause neoplasms in less than 1 year in hamsters.

• Hamsters are used widely in inhalation studies for toxicological research.

• The hamster is useful because it has a lower occurrence of spontaneous respiratory tumors and of respiratory diseases.

• Its respiratory epithelium is similar to that of the human than other laboratory rodents.

• The hamster provides a popular alternative species for teratology and reproductive toxicity studies due to its:

– short pregnancy period, – predictable estrus, – rapid embryonic development, and – a low incidence of spontaneous malformations.

• Toxicology Studies:

• The protocols used for rats in acute and long-term toxicity studies can be used for the hamster; however, blood collection should be kept to a minimum and the length of the test may need to be adjusted due to the shorter life span of the hamster.

The Guinea Pig

• Journalists often refer to human research subjects as human guinea pigs, and the public mind has long regarded the guinea pig as the classic laboratory animal for all biomedical research and safety assessment.

• Actually, their use is now proportionately constant at 2% of the annual total of laboratory animals.

• This makes them only the third or fourth most popular species in toxicology and safety assessment.

• Guinea pigs have long been used as experimental animals in biomedical research because they are small, tame, and easy to handle.

• The popularity of the guinea pig as a pet and research animal owes much to their docile nature. They seldom bite or scratch and will respond to attention with frequent and gentle handling.

• In the broad range of biomedical research, the guinea pig has been

employed as the test animal in a wide range of investigations: nutrition, pharmacology, allergy, radiology, and immunology.

• Life span 2–6 years, Gestation 59–70 days;, Litter size 3–4 average.

• Developmental Toxicity

• Guinea pigs have characteristics that make them unlike any of the other species commonly used for developmental toxicity studies (rabbits, rats, and mice).

• Their endocrine control of reproduction is similar to that of the human, even to its trimester characteristics.

• The guinea pig is used in a wide variety of studies in toxicology. The most common are the various sensitization and photosensitization studies.

• The closed patch procedure is performed when a test substance either is highly irritating to the skin by the intradermal injection route of exposure or it cannot be dissolved or suspended in a form allowing injection.

The Rabbit

• Domestic rabbits are similar to rodents in many respects.

• The principal anatomical difference is that rabbits have two pairs of upper incisor teeth, whereas rodents have only one pair.

Choice of the Rabbit in Toxicological Research

• A number of size, shape, and color variations derived from centuries of selective breeding constitute the more than 50 well-established breeds recognized by the rabbit breeders’ associations.

• The New Zealand White albino is the rabbit most commonly used for research purposes.

• Compared to the high cost of cats, dogs, and monkeys and the problems associated with their proper care and maintenance, rabbits are relatively inexpensive, hardy, small, clean, and more easily housed and handled.

• Thus, they are readily used for a wide variety of experimental procedures and testing situations, including immunology, teratological, dermal, ocular, and implant studies.

• Although rabbits are frequently used to study dermal toxicity, they might not be the best species.

• Because human skin has a thicker stratum corneum, it is more resistant to the dermal absorption of foreign substances and is penetrated much less easily by xenobiotics than the skin of the most widely used animal models, including the rabbit and the rat.

• It appears that because the permeability of the skin of miniature swine is close to that of human skin, for studies in which dermal toxicity data are to be used to predict toxicity in humans, in some respects the miniature swine appears to be a more suitable test animal than the rabbit.

• In general, for a single dermal dose, total exposure of the outer surface of the skin to the applied compound will be of shorter duration in the rabbit than in humans.

• However, because of the higher penetration rate in the rabbit, temporarily higher concentrations of compound might occur within the rabbit skin as compared to human skin.

Study Designs

• The rabbit is used in numerous study designs to evaluate toxicological responses to pesticides, drugs, and industrial chemicals.

• The rabbit is also the nonrodent species most frequently used to evaluate developmental toxicity.

• In the 1960s, the drug thalidomide was tested and shown to be safe in rats, but caused severe birth defects in humans.

• When given to pregnant rabbits, this drug caused fetal malformations, and now it is required that chemicals must be tested for developmental toxicity in both rodents and nonrodent species.

• Ocular and dermal irritation studies and dermal toxicity studies are types of acute studies that are routinely done using rabbits.

• Eye, or ocular, irritation studies generally last 72 hr, but can be continued for up to 21 days if irritation persists.

• If the pH of the test material evaluated is less than 3.0 or greater than 11.5, consideration should be given to not performing the test, as the

• material can be considered corrosive.

• Acute dermal irritation and toxicity studies last 14 days.

• The length of exposure is 24 hr and the numbers of animals and dose levels depend on the specific testing guidelines.

• As with the ocular study, if the pH of the material is less than 3.0 or greater than 11.5, consideration should be given to not performing the test, because the test material can be considered corrosive.

• Rabbits are frequently used as the nonrodent species for teratogenicity studies. In this study design, the pregnant rabbit is treated during fetal organogenesis, days 7 to 19 of gestation.

The Ferret

• Interest in the ferret as a laboratory animal has grown in direct public opposition to the use of “domestic” animals in scientific research.

• The use of ferrets in biomedical research areas as cardiology, ophthalmology, virology, bacteriology, toxicology, and developmental toxicology has become frequent and important.

• The most obvious advantages to the use of the ferret as an animal model in toxicology include their relatively low cost, small body size, ease of handling and maintenance, and ability to adapt to existing facilities and laboratory equipment.

• Less obvious advantages: the current lack of opposition to their use, and their apparent physiological and biochemical similarities with humans.

• Disadvantages of the ferret: large relative heterogeneity, current lack of extensive databases, and the small number of vendors selling the animal.

The Dog

• Advantages of using the dog in the laboratory were recognized by researchers as early as the 17th century.

• The dog’s internal system, organs, and muscles are similiar to those of man, a fact that has stimulated the development of canine models in numerous areas such as circulation and cardiovascular research.

• Because of the large amount of background data available on the dog, it is a commonly used nonrodent species in acute, limited, repeated-dose (2- or 4-week studies), subchronic (up to 13 weeks of duration), or chronic (26 weeks or longer) toxicology studies.

• For teratology studies, the dog does not appear to be as sensitive an indicator of teratogens as other nonrodent animal models such as primates and ferrets.

• The dog is an appropriate nonrodent animal model for use in pediatric studies to assess the effects of various treatments on postnatal development.

Primates

• Only a few of almost 200 primate species are utilized in toxicology studies.

• The most commonly used species are Old World monkeys (cynomolgus monkey, rhesus monkey, and baboon), New World monkeys (squirrel monkey and marmosets), and the great apes (chimpanzees).

• Because of the genotypic and phenotypic resemblance to humans, nonhuman primates have been used in the study of induced or naturally occurring human diseases such as acquired immunodeficiency syndrome (AIDS), hepatitis, diabetes mellitus, and atherosclerosis.

• Our understanding of the human brain, vision, aging, reproductive function, and behavior has been enhanced by studies in primates.

• Safety evaluations of drugs, vaccines, and biotechnology products are conducted in nonhuman primates prior to approval for general use by the public.

• In toxicological studies in which many compounds are novel to primates, animal availability and industry trends have had a significant impact on the choice of species used for these studies.

• From the early research into polio vaccine development (during the 1940s) and continuing until approximately 20 years ago, the rhesus macaque was the monkey of choice for most research programs.

• Today the cynomolgus macaque is the most commonly used monkey in toxicological studies, although the rhesus monkey still remains a suitable alternate from a scientific standpoint.

Nasogastric dose administration

The Minipig

• The use of pigs (Sus scrufa) in biomedical research is well established.

• In toxicology, although the use of pigs in the United States was historically limited to dermal studies, since the mid-1990s they have become very popular for pharmaceutical studies in place of dogs and primates.

• They have been extensively used for surgical and physiological (primarily cardiovascular, renal, and digestive) research for years.

• They are already more frequently used in nutritional toxicology studies.

• Among the more common experimental animals, pigs are the only one whose use is on the increase.

• In short, there are scientific, economic, and sociological reasons that make minipigs good toxicological models.

• Use in general toxicity testing and reproduction, teratological and behavioral toxicity.

Alternative Species

• EARTHWORMS

• Earthworms are invertebrate, cold-blooded animals.

• Earthworms have been one of the more common species used to test chemicals for potential hazardous impact on the environment.

• FDA, for example, includes protocols for the study of earthworms in its Environmental Assessment Handbook.

• Earthworms could also be used for lethality assessment in place of rodents.

• Earthworms are highly specialized for life in the soil.

• FISH

• Fish, like earthworms have historically been commonly used to assess potential environmental impact. Most fish used in toxicity studies are rainbow trout, zebrafish, and Japanese medaka.

• Rainbow trout in particular have been extensively used in carcinogenicity and mechanistic cancer research.

Rainbow trout Japanese medaka

zebrafish

• There are three main advantages to using fish in carcinogenicity testing.

• First, they have an extremely low background tumor rate, which enhances the sensitivity of the assays.

• Second, fish are less expensive to purchase and maintain than rodents.

• Third, a positive carcinogen will generally show up in fish within 1 year’s time. Rodent studies generally last for 18 months (mice) to 30 months (rats).

• On the basis of current data, fish would be best used in confirming potential hepatic carcinogens.