Animal Tests PDF

Basically, animal testing helps test the toxicity of a certain substance. - They are considered to be the only way to test toxicity (BEFORE) CE - PC EC- WC T- E MT- S...

Basically, animal testing helps test the toxicity of a certain substance. - They are considered to be the only way to test toxicity (BEFORE) CE - PC EC- WC T- E MT- S Animal Testing are now standardized- highly effective - makes use of humane procedures for animal welfare - only makes use of as much animals AS NEEDED only Not necessary 2 types of toxicity testing (SPECIFIC AND GENERAL) Animal Tests Animal tests for toxicity have been conducted prior to and in parallel with human clinical investigations as part of the non-clinical laboratory tests of pharmaceuticals. For pesticides and industrial chemicals, human testing is rarely conducted. Years ago, results from animal tests were often the only way to effectively predict toxicity in humans. before! Animal tests were developed and used because: Chemical exposure can be precisely controlled. Environmental conditions can be well-controlled. Virtually any type of toxic effect can be evaluated. The mechanism by which toxicity occurs can be studied. SAT- more effective, uses humane procedures Standardized Animal Toxicity Tests Animal methods to evaluate toxicity have been developed for a wide variety of toxic effects. Some procedures for routine safety testing have been standardized. Standardized animal toxicity tests have been highly effective in detecting toxicity that may occur in humans. As noted above, concern for animal welfare has resulted in tests that use humane procedures and only as many animals as are needed for statistical reliability. To be standardized, a test procedure must have scientific acceptance as the most meaningful assay for the toxic effect. Toxicity testing can be very specific for a particular effect, such as dermal irritation, or it may be general, such as testing for unknown chronic effects. Standardized tests have been developed for the following effects: Acute Toxicity 1. Evaluates the effects of a single or Subchronic Toxicity brief exposure within 24 hours. 2. Evaluates the effects of a single or Chronic Toxicity brief exposure over few weeks to several months. Carcinogenicity 3. Long term exposure typically animal's lifespan. Reproductive Toxicity 4. Cancer Developmental Toxicity 5. Impact of drugs to RS 6. Specific substance that may cause Dermal Toxicity birth defects or embryotoxicity 7. Potential skin irritation or inflammation Ocular Toxicity 8. Eye irritation or damage (Draize Test) 9. Effects on nervous system delayed Neurotoxicity neurotoxicity 10. Test for gene mutations Genetic Toxicity chromosomal changes Species Selection Species selection varies with the toxicity test to be performed. There is no single species of animal that can be used for all toxicity tests. Different species may be needed to assess different types of toxicity. The published literature (such as via PubMed) and online databases (such as TOXNET) 1 | Page Compared and Compiled by: Prof. MAG Antonio  should be searched for information from non-animal and animal studies, as well as for possible best approaches, most applicable species, and strains and gender of a species. Here are two examples: It would have been invaluable years ago for toxicologists and risk assessors to have known that carcinogenic effects in male rats are considered irrelevant for humans if the α(2u)- globulin protein is involved because humans lack that protein. Many physiological, pharmacological, and toxicological findings related to organic anion and cation transport and transporters in rodents and rabbits do not apply to humans. In some cases, it may not be possible to use the most desirable animal for testing because of animal welfare or cost considerations. For example, use of dogs and non-human primates is now restricted to special cases or banned by some organizations, even though they represent the species that may respond the closest to humans in terms of chemical and other exposures (however, note the examples above). Rodents and rabbits are the most commonly used laboratory species because they are readily available, inexpensive to breed and house, and they have a history of producing reliable results in experiments. The toxicologist attempts to design an experiment to duplicate the potential exposure of humans as closely as possible. For example: The route of exposure should simulate that of human exposure. Most standard tests use inhalation, oral, or dermal routes of exposure. The age of test animals should relate to that of humans. Testing is normally conducted with young adults, although in some cases, newborn or pregnant animals may be used. For most routine tests, both sexes are used. Sex differences in toxic response are usually minimal, except for toxic substances with hormonal properties. Dose levels are normally selected so as to determine the threshold as well as a dose-response relationship. Usually, a minimum of three dose levels are used. Acute Toxicity Historically, acute toxicity tests were the first tests conducted. They provide data on the relative toxicity likely to arise from a single or brief exposure, or sometimes multiple doses over a brief period of time. Standardized tests are available for oral, dermal, and inhalation exposures, and many regulatory agencies still require the use of all or some of these tests. Table 1 lists basic parameters historically used in acute toxicity testing. 2 | Page Compared and Compiled by: Prof. MAG Antonio Subchronic Toxicity Subchronic toxicity tests are employed to determine toxicity likely to arise from repeated exposures of several weeks to several months. Standardized tests are available for oral, dermal, and inhalation exposures. Detailed information is obtained during and after the study, ranging from body weight, food and water consumption measurements, effects on eyes and behavior, composition of blood, and microscopic examination of selected tissues and organs. Table 2 lists basic parameters previously used in subchronic toxicity testing. 3 | Page Compared and Compiled by: Prof. MAG Antonio Chronic Toxicity Chronic toxicity tests determine toxicity from exposure for a substantial portion of a subject's life. They are similar to the subchronic tests except that they extend over a longer period of time and involve larger groups of animals. Table 3 includes basic parameters previously used in chronic toxicity testing. Carcinogenicity Carcinogenicity tests are similar to chronic toxicity tests. However, they extend over a longer period of time and require larger groups of animals in order to assess the potential for cancer. Table 4 lists basic parameters used in the past in carcinogenicity testing. 4 | Page Compared and Compiled by: Prof. MAG Antonio Reproductive Toxicity Reproductive Toxicity Test is intended to determine the effects of substances on gonadal function, conception, birth, and the growth and development of offspring. The oral route of administration is preferable. Table 5 lists basic parameters historically used in reproductive toxicity testing. Developmental Toxicity Developmental toxicity testing detects the potential for substances to produce embryotoxicity and birth defects. Table 6 lists basic parameters previously used in developmental toxicity tests. 5 | Page Compared and Compiled by: Prof. MAG Antonio Dermal Toxicity Dermal toxicity tests determine the potential for an agent to cause irritation and inflammation of the skin. Those reactions may be a result of direct damage to the skin cells by a substance or an indirect response due to sensitization from prior exposure. In vitro approaches to dermal toxicity testing are being developed, in part because this type of testing has received so much publicity. Table 7 lists basic parameters historically used in dermal toxicity testing. Ocular Toxicity Ocular toxicity was at one time determined by applying a test substance for 1 second to the eyes of 6 test animals, usually rabbits. The eyes were then carefully examined for 72 hours, using a magnifying instrument to detect minor effects. An ocular reaction can occur on the cornea, conjunctiva, or iris. It may be simple irritation that is reversible and quickly disappears. 6 | Page Compared and Compiled by: Prof. MAG Antonio This eye irritation test was commonly known as the "Draize Test." This test has received much attention, such as the development of a "low volume" variation and in vitro approaches. Neurotoxicity A battery of standardized neurotoxicity tests were developed to supplement the delayed neurotoxicity test in domestic chickens (hens). The hen assay determines delayed neurotoxicity resulting from exposure to anticholinergic substances, such as certain pesticides. The hens are protected from immediate neurological effects of the test substance and observed for 21 days for delayed neurotoxicity. Table 8 lists measurements included in other neurotoxicity tests. Genetic Toxicity Genetic toxicity is determined using a wide range of test species including whole animals and plants (for example, rodents, insects, and corn), microorganisms, and mammalian cells. A large variety of tests have been developed to measure gene mutations, chromosome changes, and DNA activity. Table 9 lists parameters used for common gene mutation tests. 7 | Page Compared and Compiled by: Prof. MAG Antonio Chromosomal effects can be detected with a variety of tests, some of which utilize entire animals (in vivo) and some which use cell systems (in vitro). Several assays are available to test for chemically induced chromosome aberrations in whole animals. Table 10 lists common in vivo means of testing chromosomal effects. In Vitro Testing In vitro tests for chromosomal effects involve exposure of cell cultures and followed by microscopic examination of them for chromosome damage. The most commonly used cell lines are Chinese Hamster Ovary (CHO) cells and human lymphocyte cells. The CHO cells are easy to culture, grow rapidly, and have a low chromosome number (22), which makes for easier identification of chromosome damage. Human lymphocytes are more difficult to culture. They are obtained from healthy human donors with known medical histories. The results of these assays are potentially more relevant to determine effects of xenobiotics that induce mutations in humans. 8 | Page Compared and Compiled by: Prof. MAG Antonio Two widely used genotoxicity tests measure DNA damage and repair that is not mutagenicity. DNA damage is considered the first step in the process of mutagenesis. Common assays for detecting DNA damage include: 1. Unscheduled DNA synthesis (UDS) — involves exposure of mammalian cells in culture to a test substance. UDS is measured by the uptake of tritium-labeled thymidine into the DNA of the cells. Rat hepatocytes or human fibroblasts are the mammalian cell lines most commonly used. 2. Exposure of repair-deficient E. coli or B. subtilis — DNA damage cannot be repaired so the cells die or their growth may be inhibited. 3. In Silico Methods Also emerging are in silico methods, meaning "performed on computer or via computer simulation." This term was developed as an analogy to the Latin phrases in vivo and in vitro. Advanced computer models called "Virtual Tissue Models" are being developed by the U.S. EPA's National Center for Computational Toxicology (NCCT). The EPA's Virtual Tissue Models are described as using "new computational methods to construct advanced computer models capable of simulating how chemicals may affect human development. Virtual tissue models are some of the most advanced methods being developed today. The models will help reduce dependence on animal study data and provide much faster chemical risk assessments" (source). One example is the Virtual Embryo (v-Embryo™) research effort, aimed at developing prediction models to increase our understanding of how chemical exposure may affect unborn children. Researchers are integrating new types of in vitro, in vivo, and in silico models that simulate critical steps in fetal development. Virtual Embryo models simulate biological interactions observed during development and predict chemical disruption of key biological events in pathways that is believed to lead to adverse effects. "Chip" Models Also emerging are microphysiological systems (MPS) that are used in "tissue chip" and "organs-on- chips" models. Chip models include human cell cultures that are placed on a computer chip and studied there. The Wyss (pronounced "Veese") Institute for Biologically Inspired Engineering is a helpful resource for more information. For example, the "Lung-on-a-chip" is described as "combining microfabrication techniques with modern tissue engineering, lung-on-a-chip offers a new in vitro approach to drug screening by mimicking the complicated mechanical and biochemical behaviors of a human lung." Using a connected series of tissue chips as an integrated multi-organ system can allow for the creation of a "human-on-a-chip," to be used to model the metabolism and effects of drugs and other substances moving through a human. For example, a liver chip could provide fluids and metabolites 9 | Page Compared and Compiled by: Prof. MAG Antonio to a kidney chip, allowing for the assessment of the nephrotoxic (kidney damage) potential of a substance metabolized in the liver. Induced Pluripotent Stem Cells (iPSCs) Induced pluripotent stem cells (iPSCs) are an emerging approach using in vitro cultures of cells. The cells of mammals and plants can be reprogrammed via "cellular reprogramming" to generate iPSCs. Like human embryonic stem cells, iPSCs are pluripotent (capable of giving rise to several different cell types) and these cells can renew themselves. As examples, iPSC-derived hepatocytes, cardiomyocytes, and neural cells can serve as tools for the screening of drugs and other substances for potential toxicity, and also can be used to study disease mechanisms and pathways. Further, iPSCs have been studied in immunotherapy and regenerative cellular therapies. Figure 6. Promise of hiPSCs. Schematic representation of how somatic cells taken from a patient can be reprogrammed into induced pluripotent stem cells (iPSCs) using the ‘Yamanaka’ factors, OCT4, KLF4, c-MYC and SOX2. Subsequent differentiation of human iPSCs (hiPSCs) into neurons of define lineage allow for investigations into disease pathophysiology and identification of potential drug targets. In addition, hiPSC derived neurons may function as a cellular platform in which drug screens can be carried out using disease relevant neurons. (Image Source: Adapted under Creative Commons Attribution License (CC BY). doi: 10.1016/j.yhbeh.2015.06.014 Original image: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4579404/figure/f0005/) Combining "Chips" and iPSCs The emerging approaches of "chips" and iPSCs are being combined. One example is for the evaluation of drugs as potential countermeasures for biological and chemical threats that can be a substitute for human clinical trials. The "chips" and "humans on a chip" can be used as complex in vitro human models to simulate the biology and function of an organ. References: Toxtutor https://toxtutor.nlm.nih.gov/05-002.html 10 | Page Compared and Compiled by: Prof. MAG Antonio