modelos de toxicología

1. LAORATORY ANIMALS IN
TOXICOLOGY
Prof. Dr. Şahan SAYGI
NEU Faculty of Pharmacy
Department of Toxicology

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

3. 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.

4. • 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.

5. 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.

6. 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.

7. • 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.

8. • 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.

9. General and Reproductive Values of Mice
Life span Average 1–3 years
Adult weight Male 20–40 g
Female 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 days
Range 17–21 days
Litter size Average 12

10. • 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.

11. • 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%.

12. • 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.

13. 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.

14. 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.

15. • 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.

16. 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

17. • 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.

18. • 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.

19. 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.

20. Maximum Tolerated Dose Study in Rats

21. • 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).

22. • 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).

23. • 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.

24. • 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.

25. • 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.

26. • 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.

27. • 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.

28. Gavage dosing with infant feeding tube

29. • 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

30. • 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.

31. • 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

32. • 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.

33. • 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.

34. The Hamster

35. • 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.

36. • 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.

37. • 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.

38. • 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.

39. • 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.

40. • 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.

41. • 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.

42. • 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.

43. • 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.

44. • 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.

45. 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.

46. • 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.

48. • 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.

49. • 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.

50. 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.

51. 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.

52. • 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.

53. • 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.

54. • 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.

55. 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.

56. • 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.

57. • 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.

58. 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.

59. • 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.

60. 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.

61. • 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.

62. 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).

63. • 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.

64. • 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.

65. • 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.

66. Nasogastric dose administration

67. The Minipig

68. • 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.

69. • 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.

70. Alternative Species
• EARTHWORMS
• Earthworms are invertebrate, cold-blooded animals.

71. • 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.

72. • 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

73. • 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.