Toxicity Testing 2.pdf

ACUTE TOXICITY TESTING Dr. Isaac Tabiri Henneh Department of Pharmacotherapeutics and Pharmacy practice School of Pharmacy and Pharmaceutical Sciences University of Cape Coast  Toxicology is the scientific study of adverse effectrs that occur in...

ACUTE TOXICITY TESTING Dr. Isaac Tabiri Henneh Department of Pharmacotherapeutics and Pharmacy practice School of Pharmacy and Pharmaceutical Sciences University of Cape Coast  Toxicology is the scientific study of adverse effectrs that occur in living organisms due to chemicals TOXICOLOGY It involves observing and reporting symptoms, mechanisms, detection and treatment of toxic substances, in particular relation to humans. Definition of terms Acute oral toxicity refers to Delayed death means that an those adverse effects animal does not die or appear occurring following oral moribund within 48 hours but dies administration of a single dose later during the 14-day of a substance, or multiple observation period. doses given within 24 hours. GHS: Globally Harmonised Classification System for Chemical Substances and Mixtures. A joint Dose is the amount of test activity of OECD (human health and the substance administered. Dose environment), UN Committee of Experts on Transport of Dangerous Goods (physical– is expressed as weight of test chemical properties) and ILO (hazard substance per unit weight of communication) and co-ordinated by the test animal (e.g. mg/kg). Interorganisation Programme for the Sound Management of Chemicals (IOMC). Definition of terms Impending death: when moribund LD50 (median lethal oral dose) is a state or death is expected prior to the statistically derived single dose of a next planned time of observation. substance that can be expected to Signs indicative of this state in cause death in 50 per cent of animals rodents could include convulsions, when administered by the oral route. lateral position, recumbence, and The LD50 value is expressed in terms tremor (See the Humane Endpoint of weight of test substance per unit Guidance Document (9) for more weight of test animal (mg/kg). details). Definition of terms Limit dose refers to a dose at an upper limitation on testing (2000 or 5000 mg/kg). Moribund status: being in a state of dying or inability to survive, even if treated (See the Humane Endpoint Guidance Document (9) for more details). Predictable death: presence of clinical signs indicative of death at a known time in the future before the planned end of the experiment for example: inability to reach water or food.  Purpose of OECD Guideline 423: OECD Guideline A stepwise procedure to assess the 423: Acute Oral acute oral toxicity of chemicals. Toxicity – Acute Uses fewer animals than traditional Toxic Class methods. Method Adoption: First adopted in 1996; revised in 2001. Application: Supports hazard classification and regulatory compliance.  Key Points: Uses a minimum of 3 animals per step (typically females). Test substance is administered orally at fixed doses. Principle of Results determine the toxicity class the Test and LD50 cut-off values. Advantages: Fewer animals used. Reproducible and reliable results.  Test Substance Preparation Preparation Guidelines: Test substance administered at a constant volume (e.g., 1 mL/100 g body weight). Preferred formulations: Aqueous solutions > oils > other solvents. Stability of the dosing preparation must be confirmed.  Animal Requirements: Preferred species: Rats (females are slightly more sensitive). Animal Age: 8–12 weeks, with consistent weight range (+/- 20%). Selection and Housing Housing Conditions: Temperature: 22°C ± 3°C. Relative humidity: 30–70%. Lighting: 12-hour light/dark cycle. Food: Standard laboratory diets.  Steps: Depending on outcomes: Initial dose administered Observe for mortality No further testing. Repeat with the same dose or to 3 animals. and signs of toxicity. a higher/lower dose. Test Procedure Observation Period: At least 14 days, with a focus on the first 24 hours.  Key Observations: Skin, fur, mucous membranes, and behavior. Observations Signs of distress: Tremors, convulsions, lethargy. and Record body weight weekly. Endpoints Humane Endpoints: Moribund animals should be humanely euthanized. Time of death recorded accurately.  Necropsy: Pathology and Data All animals (including moribund or Recordin dead) undergo gross necropsy. g Record pathological changes. Optional microscopic examination of affected organs. Data Reporting: Individual and summarized data for each test group. Includes dose levels, toxicity signs, and necropsy findings. Limit Test Limit Test: and Hazard Conducted at 2000 mg/kg (or 5000 Categorie mg/kg in specific cases). s Determines if substance is likely nontoxic. Hazard Classification: Based on the Globally Harmonized System (GHS). Categories range from non-toxic to highly toxic. Benefits Benefits: and Limitation Reduces animal use and enhances s welfare. Provides robust data for hazard classification. Limitations: Focused on acute toxicity; does not assess chronic effects. Requires experienced personnel for accurate observations.  Introduction OECD Guideline Background: 420: Acute Introduced as an alternative to the LD50 Oral test. First adopted in 1992, revised in 2001. Toxicity – Fixed Key Objectives: Dose Procedur Assess acute oral toxicity using fewer animals. e Reduce suffering by observing clear signs of toxicity rather than death. Principle of the Approach: Test Fixed doses: 5, 50, 300, 2000 mg/kg (optionally 5000 mg/kg). Observe for toxicity signs, pain, and distress. Outcome: Classify substances according to GHS (Globally Harmonized System). Key Features Advantages: of OECD Fewer animals required. 420 Reduces animal suffering. Reliable classification of chemicals. Relevance: Supports regulatory compliance and human health protection. Selection of Test Species: Preferred Animals rodents (rats); females often more sensitive. Criteria: Health: Healthy, Weight: Within ± Age: 8–12 weeks. nulliparous, and 20% of the non-pregnant. group mean.  Sighting Study: Administer a single dose to one animal. Fixed doses: 5, 50, 300, or 2000 mg/kg. Observations determine starting dose for the main study. Test Procedure Main Study: Groups of five animals tested sequentially. Observe for signs of toxicity or mortality.  Observation Period: At least 14 days. Frequent monitoring in the first Observations 24 hours. and Endpoints Key Observations: Skin, fur, mucous membranes, behavior, and weight. Signs: Tremors, lethargy, convulsions, coma, etc.  Data: Data Individual animal records. Collection Dose levels, toxicity signs, and mortality, and necropsy Reporting findings. Reporting Requirements: Test substance details, animal conditions, and results interpretation.  Purpose: For substances expected to be non-toxic. Conducted at the 2000 mg/kg dose level. Limit Test Criteria: Additional testing at 5000 mg/kg only under specific regulatory needs.  Using GHS: Substances categorized based on observed toxicity (Categories 1–5). Classification Example: Criteria Category 1: Highly toxic (LD50 ≤ 5 mg/kg). Category 5: Low toxicity (LD50 2000–5000 mg/kg).  Benefits: Ethical considerations (reduced suffering). Benefits and High reliability in chemical Limitations classification. Limitations: Does not assess chronic toxicity or long-term effects.   Main Test The first animal is dosed a step below the best preliminary estimate of the LD50. If the animal survives, the second animal receives a higher dose. If the first animal dies or appears moribund, the second animal receives a lower dose. The dose progression factor should be chosen to be the antilog of 1/(the estimated slope of the dose-response curve) and should remain constant throughout testing (a progression of 3.2 corresponds to a slope of 2). When there is no information on the slope of the substance to be tested, a dose progression factor of 3.2 is used. Using the default progression factor, doses would be selected from the sequence 1.75, 5.5, 17.5, 55, 175, 550, 2000 (or 1.75, 5.5, 17.5, 55, 175, 550, 1750, 5000 for specific regulatory needs) If no estimate of the substance’s lethality is available, dosing should be initiated at 175 mg/kg.  Observation Period: Animals monitored for up to 48 hours post-dose. Decision based on cumulative survival Observatio patterns. Decision Criteria: n and Determines the next dose level. Adjustments made to minimize errors in Decision- LD50 estimation. Making Stopping Criteria:Met when sufficient data is collected to estimate LD50 confidently. Typically, testing concludes after 4 animals experience an outcome reversal. Dose progression and LD50 estimation Dose Progression Factor Default Value: 3.2 (equivalent to a half-log unit progression). Purpose: Ensures dose adjustments are proportional and methodical. Reduces the impact of poor initial dose estimates. LD50 Calculation: Calculated using the maximum likelihood method. Confidence intervals derived computationally. Outcome: LD50 estimate with associated precision levels.  Introduction OECD Guideline Overview: 407: Repeated First adopted in 1981; updated in 1995 and Dose 28-Day Oral 2008. Toxicity Study in Provides a method for assessing toxic effects from repeated oral exposure over 28 Rodents days. Applications: Hazard identification and risk assessment. Provides data on potential effects on the nervous, immune, and endocrine systems. Key Objectives of the Study Primary Identify target organs for toxicity. Characterize dose-response relationships. Goals: Determine the No-Observed-Adverse-Effect Level (NOAEL). Additional Identify chemicals with endocrine-disrupting potential. Focus: Provide preliminary data for long-term studies. Animal Selection and Housing Preferred Species: Rats (young, healthy adults). Criteria: Equal numbers of males and females. Nulliparous and non-pregnant females. Housing: Temperature: 22°C ± 3°C. Humidity: 30–70%. Light cycle: 12 hours light, 12 hours dark. Test Procedure Dosing: Control Groups: Dose Levels: Oral Include vehicle- Minimum of administration only and three dose levels via gavage, diet, untreated plus control. or drinking control groups. Designed to water. induce toxic Daily for 28 days. effects without causing death.  Daily Observations: Mortality, morbidity, behavioral changes, and physical appearance. Observations Weekly Assessments: and Body weight, food, and water intake. Endpoints Functional observations in Week 4 (e.g., sensory reactivity, grip strength). Endpoints: Signs of neurotoxicity, immunotoxicity, and endocrine disruption.  Pathology and Necropsy Optional Necropsy: Histopathology: Measurements: Comprehensive Examination of Hormonal assays examination of all preserved tissues (e.g., T3, T4, TSH). animals. to identify cellular Collection and changes. weighing of organs (e.g., liver, kidneys, brain, gonads).  Key Data: Body weight and changes. Food and water consumption. Toxicity signs (nature, severity, duration). Data Organ weights and histopathological Reporting findings. Statistical Analysis: Summarize results using appropriate statistical methods. Strengths and Limitations Strengths: Identifies target organs for toxicity. Provides NOAEL for regulatory purposes. Offers preliminary data for long-term studies. Limitations: Cannot detect all endocrine disruptors. May not capture long-term toxic effects. OECD Test Guideline 408: Repeated Dose 90-Day Oral Toxicity Study in Rodents Purpose of Guideline 408: Provides information on potential health hazards from repeated oral exposure to chemicals for 90 days. Identifies toxic effects, target organs, and potential chemical accumulation. Establishes the No Observed Adverse Effect Level (NOAEL). History: Adopted in 1981; revised in 1998 and updated in 2018. Key Updates in 2018 Revision Addition of endocrine Emphasis on: -sensitive endpoints: Neurological, Measurement of immunological, T3, T4, TSH, and and thyroid gland reproductive weight. effects. Assessment of Careful clinical cholesterol, HDL, observations. and LDL levels.  Animal selection and preparation Preferred Species: Rats (other rodents like mice may be used with justification). Selection Criteria: Young, healthy adults, nulliparous, non- pregnant females. Weight variation ≤ ±20% of the mean. Housing Conditions: 22 ± 3°C, relative humidity 30–70%. 12-hour light/dark cycle.  Dose selection and administration Dose Levels: At least three dose levels plus a control group. High dose should induce toxicity but not death. Methods of Administration: Oral gavage, diet, or drinking water. Limit test: Single dose of 1000 mg/kg/day if no adverse effects are expected. Observations and Measurements Clinical Observations: Daily checks for morbidity, mortality, and toxic signs. Weekly detailed observations, including behavior and physical changes. Body Weight and Food Consumption: Measured weekly to monitor health trends. Ophthalmology: Eye exams at study start and termination. Biochemical and Hematological Testing Hematology: Tests include hematocrit, hemoglobin, leukocyte count, and clotting time. Clinical Biochemistry: Key metrics: liver/kidney function, cholesterol, triglycerides, and enzymes. Thyroid-related hormones: T3, T4, TSH. Pathology and Histopathology Necropsy: Full examination of external and internal organs. Organs weighed: liver, kidneys, thyroid, heart, etc. Histopathology: Detailed examination of organs for microscopic changes. Focus on endocrine-sensitive tissues and target organs.  Data Analysis and Reporting Data Collection: Individual animal data for toxicity, clinical signs, and organ weights. Statistical methods for dose-response relationships. Test Report Includes: Test chemical details, methods, and results. NOAEL determination and toxicological conclusions. OECD Guideline 452: Chronic Toxicity Studies Introduction Overview: Purpose: First adopted in 1981; Identify target organs, revised in 2018. establish dose-response Aims to assess health relationships, and hazards from prolonged determine the No- chemical exposure. Observed-Adverse- Effect Level (NOAEL).  Key Goals: Assess chronic toxicity. Objectives of Identify target organs and accumulation potential. Chronic Characterize dose-response Toxicity relationships. Determine NOAEL and Benchmark Studies Dose (BMD). Predict chronic toxicity at human exposure levels.  Administration Routes: Oral (common), dermal, or inhalation based on human exposure routes. Test Chemical Duration: and Typically 12 months but may vary Administration depending on regulatory needs. Considerations: Dose selection based on previous studies (e.g., 28-day or 90-day studies).  Preferred Species: Rodents, primarily rats, and mice; non- rodents as necessary. Animal Criteria: Selection Young, healthy adults; nulliparous and non- pregnant females. and Housing Weight variation within ±20%. Housing Conditions: Temperature: 22°C ± 3°C. Humidity: 30–70%. Light cycle: 12-hour light/dark.  Groups: At least three dose levels plus control group. High dose chosen to identify toxic effects without causing mortality. Dose Groups Dose Administration: and Daily for 12 months; adjustments for body Treatment weight considered. Special Groups: Satellite groups to monitor reversibility. Interim kills for toxicological progression insights. Observations and Endpoints Clinical Observations: Daily monitoring for morbidity, mortality, and signs of toxicity. Specialized Assessments: Sensory reactivity, grip strength, and motor activity (if needed). Neurotoxic and immunotoxic effects. Endpoints: Target organ identification, cumulative toxicity, and NOAEL determination. Pathology and Necropsy Necropsy: Full examination of all animals, including gross lesions and organ weights. Tissue preservation for histopathology. Histopathology: Comprehensive analysis of tissues, focusing on target organs. Haematology, Biochemistry, and Urinalysis Parameters: Haematology: Erythrocyte count, haemoglobin, platelet count, etc. Biochemistry: Glucose, urea, liver enzymes, total proteins, etc. Urinalysis: Appearance, pH, specific gravity, protein levels. Frequency: Interim assessments at 3, 6, and 12 months. Data Reporting and Interpretation Data Requirements: Statistical Analysis: Individual animal data Pre-determined and group summaries. methods, including Detailed analysis of survival adjustments toxic effects, lesions, if required. and dose-response relationships. Strengths and Limitations Strengths: Long-term exposure insights. Identification of cumulative and chronic toxicity effects. Limitations: Requires significant time and resources. Limited relevance for acute effects.