Methods of Toxicity Testing: Unit-Four PDF

Unit -Four 4. METHODS OF TOXICITY TEST AND ASSESSMENTS Content 4.1. Toxicity Testing in the Laboratory 4.2. Test Design and Selection of Test Species 4.3. Types of Exposure Systems 4.4. Exposure Assessment 4.5. Dose-Response Relationship in Laboratory Testing 4.6. Microcosm and Mesocosm in E...

Unit -Four 4. METHODS OF TOXICITY TEST AND ASSESSMENTS Content 4.1. Toxicity Testing in the Laboratory 4.2. Test Design and Selection of Test Species 4.3. Types of Exposure Systems 4.4. Exposure Assessment 4.5. Dose-Response Relationship in Laboratory Testing 4.6. Microcosm and Mesocosm in Ecotoxicological Studies 4.7. Field Evaluation/Studies in Ecotoxicology 4.1. Toxicity Testing in the Laboratory  Toxicity testing is a systematic evaluation of the toxic effects that a chemical can produce.  Toxicity tests are experiments or trials designed to assess or evaluate the dose/concentration of a toxicant and the duration of the exposure required to produce effect.  Toxicity tests compare the response of an organism exposed to a specific chemical at various dose to the response of the same organisms unexposed to the chemical.  Toxicity tests are mostly used to examine the specific adverse effect or specific end points such as cancer, cardiotoxicity, and skin/eye irritation.  Toxicity testing also helps to calculate the No observed adverse effect level (NOAEL) and is helpful for clinical trails.  Toxicity tests characterize the toxicity and the level of toxicity.  Toxicity tests help to determine the dose- response and the target organ  Toxicity tests are performed to assess the safety or hazards of several substances such as industrial chemicals, pharmaceuticals, and consumer care products.  Many of current toxicity tests use laboratory animals such as rats, rabbits, and rodents  Animals are useful models to predict the toxicity of chemicals in human because they have similar organelles, cells, and organs.  If the model is not close to human, uncertainty of the results will increase.  Many factors can affect the results of toxicity tests such as temperature, food, light, stressful environmental conditions. other factors related to the animal itself include age, sex, health, and hormonal status.  Toxicity test can be performed in several laboratory and the same results must be obtained in different lab.  Toxicity tests can range from cell cultures to ecosystem function. There are different types of toxicity tests  Toxicity test based on number and conditions of species: i) Single species test ii) Multispecies test, iii) Ecosystem test  Toxicity test types based on exposure of toxicants; i) Single dose test ii) Multiple dose test  Toxicity test types based on length of exposure: i) Acute toxicity test ii) Sub-acute toxicity test iii) Sub-Chronic toxicity test, and iv) Chronic toxicity test:  Toxicity test types based on specific condition: i) Teratogenic test, ii) carcinogenic test, iii) mutagenic test i) Acute toxicity test  Acute in toxicology relates to exposure that may be done once or may be done several times within or continuously throughout a 24-hour.  These studies utilize dose response curve to determine LD50 and/or LC50  The dose-response correlates exposures and the spectrum of induced effects.  Generally, the higher the dose, the more severe the response  Acute toxicity studies are based on the assumption that acute toxicity and lethality in animal models are relevant to humans. ii) Sub-acute toxicity test  This test predicts any cumulative effect of the drug or chemicals & play a role in the safety assessment of pharmaceuticals, pesticides, food additives, and other chemicals.  In this test, at least 3 species of animals are used: rats, mice and rabbits. Compound under test is given daily in 3 dose levels for 2 – 4 weeks.  Animals are observed for different parameters: physiological, clinical and chemical tests, behavior, CNS & autonomic profiles. iii) Sub-chronic Toxicity Test  The compound is given for 90 days by the intended route. Animals are observed all over the specified time.  For dead animals autopsy is performed by taking samples from different organs to be examined, chemically, microscopically & macroscopically  It provides information on target-organ toxicity and bioaccumulation potential and are designed to determine no-observed-adverse-effect levels (NOAELs), which are used to establish standards or guidelines for human exposure. iv) Chronic Toxicity Test  It is performed for compounds administered for a longer period. The compound is given for more than 90 days by the intended route.  A chronic toxicology study provides inferences about the long-term effect of a test substance in animals, and it may be extrapolated to the human safety of the test substance Methods of toxicity testing i) In vitro Tests  These tests are conducted using isolated cells, tissues, or biomolecules outside a living organism.  They would include studies using isolated enzymes, subcellular organelles, or cultured cells. Advantages of in Vitro Testing:  Controlled environment: Precise control over factors like temperature, pH, and nutrient levels allows for focused observation of specific cellular processes.  High throughput: Large numbers of samples can be analyzed quickly and efficiently, making it cost-effective for initial screening and basic toxicity assessments.  Ethical considerations: No live animals are involved, aligning with animal welfare concerns. Limitations of in Vitro Testing:  Lack of complexity: Isolated cells lack the intricate interaction of organs and systems present in a living organism, potentially missing important interactions and feedback loops.  Species-specific differences: Human cell lines may not accurately reflect responses in the whole human body. ii) In vivo Tests  These tests are conducted on living organisms, ranging from simple invertebrates like worms to complex mammals like mice and primates.  This approach allows for studying the effects of a substance on the entire organism, encompassing its intricate physiological and biochemical pathways.  The results of these tests are then used, by a variety of extrapolation techniques, to estimate hazard to humans Advantages of in Vivo Testing  Physiological relevance: Provides a more accurate picture of how a substance interacts with the complex interaction of organs and systems within a living organism.  Species-specific data: Valuable for understanding the effects of a substance in the target species, such as humans.  Identification of unforeseen effects: Can reveal unexpected interactions and side effects that might be missed in vitro. Limitations of in Vivo Testing  Ethical concerns: Animal welfare considerations are paramount, requiring careful planning and adherence to ethical guidelines.  Cost and time: Experiments can be expensive and time-consuming, often involving complex protocols and specialized facilities.  Species differences: Results obtained in one species may not directly translate to another, including humans.  Importance of toxicity testing  To have an idea of toxic doses of xenobiotic for certain organisms.  Evaluation of safe doses of the toxicants for certain organisms.  Recommendation of maximum permissible limits of those substances in the ambient air and drinking water.  The data on long term toxicity tests may be reliable for the evaluation of safe level of toxicants.  Evaluation and recommendation of maximum acceptable daily intake.  Developing good air and water quality.  Toxicity tests usually focus on:  cytotoxicity (damages cells),  mutagenicity (alters genetic materials),  carcinogenicity (causes cancer)  teratogenicity (causes birth defects). 4.2. Test Design and Selection of Test Species 1. Test Design for toxicity studies  The followings should be considered for design of toxicity test i) Selection of animal species  Choice of test species for predictive toxicity testing is often affected by economic consideration, limitation of laboratory facilities or by problems associated with real target species.  Two rodent species are most often used for the study, the laboratory rat and the laboratory mouse. ii) Route of administration  The toxicant should be administered through the route by which the animals are exposed. iii) Dose selection  The selection of the appropriate doses for the test animals is another step in design toxicity test.  The doses should be chosen to provide information about the full range of possible effects of the test substance, from no effect to severe toxicity. iv) Endpoints  The endpoints of a toxicity study are the measures that will be used to assess the effects of the test substance. These may include clinical signs, body weight, organ weights, blood chemistry, and histopathology.  The choice of endpoints will depend on the type of study and the specific concerns about the toxicity of the test substance. 2) The selection of species for toxicity test  The selection of the appropriate test species is an important consideration in designing a toxicity study.  The species should be chosen to be as relevant as possible to humans, but ethical and practical considerations must also be taken into account i) Relevance to Humans  The test species should be similar to humans in terms of anatomy, physiology, and metabolism..  This will help to ensure that the results of the study are relevant to human health ii) Ethical and practical considerations  The availability, cost, and animal welfare of the test species must also be considered.  Some species are more readily available and less expensive than others.  It is also important to choose a species that can be housed and cared for in a humane way. iii) Other factors  The sensitivity of the test species to the test substance, the existing knowledge base on the species and the test substance, and any regulatory requirements must also be considered. 4.3. Types of Exposure Systems  Exposure is the process by which an organisms come in contact with the toxicants for a specified period of time.  Exposure to chemicals may come from many sources: environmental, occupational, therapeutic, dietary, and accidental  Exposure media: air, water, soil, and food. Exposure or concentration is often expressed in units of μg/m3 (air), μg/L (water), or μg/cm2 (skin).  Exposure paths: inhalation, ingestion, and dermal contact  Based on duration, exposure can be classified as  Acute: Short-term, high-dose exposure, resulting in rapid onset of symptoms. Occurs from accidental spills, poisoning events, or high- intensity occupational exposure.  Subacute: Exposure lasting days or weeks, with potential for cumulative effects.  Chronic: Long-term, repeated exposure over months or years, leading to gradual accumulation of toxins and potentially irreversible damage.  Based on frequency, exposure can be classified as:  Continuous: Constant exposure with no breaks, posing the highest risk of toxicity.  Intermittent: Exposure with periods of non-exposure, allowing for partial recovery and potentially reducing overall risk.  Occasional: Rare or infrequent exposure events, typically posing a lower risk, but still requiring consideration. 4.4. Exposure Assessment  Exposure assessment is the process of estimating or measuring the magnitude, frequency and duration of exposure to an agent, along with the number and characteristics of the population exposed.  It describes the sources, pathways, routes, and the uncertainties in the assessment.  An exposure assessment is performed to identify the affected population and, if possible, calculate the amount, frequency, length of time, and route of exposure.  Exposure to chemicals can occur anywhere, including the home (cleaning products, paints, pesticides, etc.). Outside the home, exposure to chemical pollutants in the air occurs through inhalation.  Exposure assessment is a part of risk assessment process  Exposure can be measured directly but more commonly is estimated indirectly through:  Measuring the concentrations in the environment  modeling chemical transport and fate in the environment  Estimating human intake over time.  Exposure assessment considers three facts: i. Exposure pathway (the course an agent takes from its source to the person(s) being contacted) ii. Exposure route (means of entry of the agent into the body) iii. It is described as intake (taken in through a body opening) or uptake (absorption through tissues). 4.5. Dose-Response Relationship in Laboratory Testing  The dose-response relationship is a fundamental tool used to determine the relationship between the amount of a substance administered and its effect on the body.  The dose – response relationship provides an estimation of the relationship between the dose of a chemical agent and incidence of effects in a population.  The knowledge of the dose-response relationship is important as it helps determine the threshold for both effective dose and toxic dose  Dose-response relationships observed from animal studies must be extrapolated from that animal species to humans.  The slope of the curve indicates the percent of the population responding per unit change in dose, and the shape of the curve is important additional information for predicting the toxicity of a substance.  The dose-response relationship is used to establish causality, the threshold effect, and the slope for the dose response  The relationship is used to determine safe levels and dosages for drugs, pollutants, foods, and other substances to which humans or other organisms are exposed.  Threshold dose is a dose below which there are no adverse effects from exposure to the chemical.  Thresholds based on acute responses, such as death, are more easily determined, while thresholds for chemicals that cause cancer or other chronic responses are harder to determine.  LD (50) is standard measure of the toxicity of a material that will kill half of the sample population of a specific test animal in a specified period.  LD50 is measured mg of chemicals /kg of the test animal’s body weight; lower the amount, more toxic the material.  LD50 is used in comparison of toxicities, LD50 values cannot be directly extrapolated from one species to the other or to humans. Figure: dose- response function with a no-effect region  The concentration of the chemical in air that kills 50% of the test animals during the observation period is the LC50 value.  The threshold value can never be derived with absolute certainty and therefore the lowest observed adverse effect level (LOAEL) or the NOAEL have normally been used instead of the threshold value in deriving regulatory standards.  Now, there is a move to replace the value of LOAEL or NOAEL value by Benchmark dose (BMD).  Both NOAEL and BMD can be used as point of departure (POD) to derive human health guidance value such as reference dose (RfD) or acceptable daily intake (ADI).  A benchmark dose (BMD) is a dose or concentration that produces a predetermined change in the response rate of an adverse effect (called benchmark response, BMR) compared to background.  Normally, the default BMR is 5% or 10% change in the response rate of an adverse effect compared to background, defined as 0%.  The BMD is a range, rather than a fixed number. For example, the benchmark dose (lower confidence limit) (BMDL) can be regarded as a dose where the observable physical effect is less than the predetermined benchmark response (BMR).  The main goal of calculating BMDL is to use it to estimate a daily oral or dermal exposure level to the human population (including sensitive subgroups) that is likely to be without an appreciable risk of deleterious effects during a lifetime. RFD or ADI = ( ) Note: UF ranges from 100 to 10,00.  BMDL is more reliable than NOAEL since it is less dependent on dose selection and sample size.  The value of calculated BMDL can be bigger or smaller than NOAEL. When test sample (i.e, no. of tested animals) is very big, BMDL is bigger than NOAEL. When the size of sample is small, BMDL may be smaller than NOAEL. 4.7. Microcosm and Mesocosm in Ecotoxicological Studies  Microcosms and mesocosms are two important tools used in ecotoxicological studies to assess the effects of pollutants on ecosystems.  They offer a more realistic and complex environment compared to traditional single-species laboratory tests, providing valuable insights into how contaminants might impact real-world ecosystems.  Microcosms (“micro “small”, and cosm “world”), small world contained systems, often housed in glass containers or aquariums.  They typically contain a simplified representation of an ecosystem, with a few species of organisms interacting with each other and their non-living environment.  Microcosms are relatively quick and easy to set up and maintain, making them ideal for screening potential pollutants or studying specific ecological processes.  A mesocosm (meso- or medium and cosm ‘world’) is any outdoor experimental system that examines the natural environment under controlled conditions. in this way mesocosm studies provide a link between field surveys and highly controlled laboratory experiments.  Mesocosms are larger and more complex than microcosms, often encompassing outdoor ponds, artificial streams, or even large enclosures covering several hectares.  Mesocosms have been used to evaluate how organisms or communities might react to the environmental changes through deliberate manipulation of environmental variables such as increased temperature, carbon dioxide.  The two key concepts addressed when studying a mesocosm include:  A mesocosm as a model of a larger ecosystem  A closed ecosystem, one in which energy enters and leaves but matter does not.  They aim to capture a wider range of species and ecological interactions, providing a more comprehensive picture of how a pollutant might affect an entire ecosystem.  However, mesocosms are also more expensive and time-consuming to establish and maintain compared to microcosms. Feature Microcosm Mesocosm Large (e.g., cubic meters or Size Small (e.g., liters) hectares) Simplified ecosystem with few More complex ecosystem Complexity species with diverse species Cost and setup Relatively low cost and quick Higher cost and longer time setup setup time Screening pollutants, studying Assessing ecosystem-level Suitability specific processes effects, long-term studies  Both microcosms and mesocosms have their own strengths and weaknesses, and the choice of which tool to use depends on the specific research question and available resources.  Microcosms and mesocosms are not perfect imitations of natural ecosystems. They are simplified systems with inherent limitations, and their results may not always translate directly to the real world.  Careful design and control are essential to ensure that the results of microcosm and mesocosm studies are reliable and meaningful.  Microcosms and mesocosms can be used in conjunction with other ecotoxicological tools, such as field studies and modeling, to provide a more comprehensive understanding of the effects of pollutants on ecosystems. 4.8. Field Evaluation/Studies in Ecotoxicology Field evaluation in ecotoxicology involves conducting studies in natural environments to assess the impact of contaminants on ecosystems and the organisms within them. Field studies are designed to evaluate survival and reproductive success of nontarget wildlife species under actual conditions of use of the chemicals like pesticides.  Potential outcomes of pesticides in field tests can include  direct poisoning by ingestion, dermal, or inhalation exposure  sublethal effects indirectly causing death by reducing resistance to natural environmental stresses such as disease, weather, or predators  altered behavior such as abandonment of nests or young and change in parental care  habitat alteration that results in reduced food resources or greater vulnerability to predators; and  reduced productivity.  Field studies consist of two types:  screening field studies to evaluate whether impacts are occurring and  definitive field studies to estimate the magnitude of the impact.  These studies often require extensive monitoring of reproductive success and survival of young.  In most instances screening studies monitor for overt signs of toxicity such as mortality or aberrant behavior and changes in biochemical and histological indicators of toxicity.  In field studies the presence of interacting environmental factors, such as ambient temperature and weather conditions, complicate establishing a cause-and-effect relationship.  Field studies should also be considered to characterize bioaccumulation potentials of chemicals in situ.  An inherent limitation of ecotoxicological field studies stems from the difficulty in isolating the biological effects of one contaminant exposure from the effects of other contaminants. …………………….The end……………………