Toxicology: Acute Toxicity Testing

Acute Toxicity Testing

• Acute toxicity testing determines short-term adverse effects of a drug.
• Testing involves administering a single dose or multiple doses within 24 hours to two mammals.
• One of the mammalian species should be non-rodent.

Toxicity Studies

• Types of toxicity studies include:
  • Acute toxicity (14 days)
  • Sub-acute toxicity (repeated doses) (28 days)
  • Sub-chronic toxicity (3 months)
  • Chronic toxicity (6 months to 2 years)
  • Special toxicity (carcinogenicity)

Acute Oral Toxicity Tests

• OECD Test Guidelines 420, 423, and 425 are used.
• Fixed Dose Procedure (OECD 420)
• Acute Toxic Class Method (OECD 423)
• Up and Down Procedure (OECD 425)

Toxicology

• Toxicology is the scientific study of adverse effects in living organisms due to chemicals.
• It involves observation, reporting of symptoms, mechanisms, detection and treatment of toxic substances, particularly in relation to humans.

OECD (Since 1961)

• The mission of OECD is to promote policies that improve economic and social well-being of people around the world.
• They work with governments to understand factors driving economic, social and environmental change.
• OECD sets international standards on a wide range of things, from agriculture to the safety of chemicals.

Acute Toxicity Symptoms

• Altered Respiration
• Weight loss
• Muscle spasm
• Salivation
• Convulsion
• Diarrhea
• Loss of righting reflex
• Tremor
• Lacrimation

Definition of Terms

• Acute oral toxicity: Adverse effects occurring after oral administration of a single or multiple doses within 24 hours.
• Delayed death: Animal does not die or appear moribund within 48 hours but dies later during the 14-day observation period.
• Dose: Amount of test substance administered. Expressed as weight of test substance per unit weight of test animal (e.g., mg/kg).
• GHS: Globally Harmonized System of Classification and Labelling of Chemicals (a joint activity of OECD, UN Committee of Experts on Transport of Dangerous Goods, and ILO).
• Impending death: Moribund state or death expected before the next planned observation.
• LD50 (median lethal oral dose): Statistically derived single dose expected to cause death in 50% of animals when administered orally, expressed in terms of weight of test substance per unit weight of test animal (mg/kg).
• Limit dose: Dose at an upper limit of testing (2000 or 5000 mg/kg).
• Moribund status: State of dying; unable to survive (even if treated), and is identified by particular clinical signs in rodents such as convulsions, lateral position, recumbence, and tremors.
• Predictable death: Presence of clinical signs indicative of death in the future. E.g. inability to reach water or food.

Guidelines

• Number of animals per test:
  • OECD 401: Up to 25
  • OECD 420: 5-7
  • OECD 423: Average 7
  • OECD 425: 6-9

OECD Guideline 423: Acute Oral Toxicity- Acute Toxic Class Method

• A stepwise procedure to assess acute oral toxicity of chemicals.
• Uses fewer animals than traditional methods.
• First adopted in 1996, revised in 2001.
• Supports hazard classification and regulatory compliance.

Principle of the Test

• Uses a minimum of 3 animals per step (typically females).
• Test substance is administered orally at fixed doses.
• Results determine toxicity class and LD50 cut-off values.
• Advantages include fewer animals used and 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 and Housing

• Preferred Species: Rats (females are slightly more sensitive).
• Age: 8-12 weeks, with consistent weight range (±20%).
• Housing Conditions:
• Temperature: 22°C ± 3°C.
• Relative humidity: 30-70%.
• Lighting: 12-hour light/dark cycle.
• Food: Standard laboratory diets.

Test Procedure

• Steps: Initial dose administered to 3 animals. Observe for mortality and signs of toxicity.
• Observation Period: At least 14 days; focus on first 24 hours.

Observation and Endpoints

• Key Observations:
  • Skin, fur, mucous membranes, and behavior.
  • Signs of distress (tremors, convulsions, lethargy).
  • Record body weight weekly.
• Humane Endpoints:
  • Moribund animals should be humanely euthanized.
  • Time of death recorded accurately.

Pathology and Data Recording

• Necropsy: All animals (moribund or dead) undergo gross necropsy; record pathological changes and perform 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 and Hazard Categorization

• Limit Test: Conducted at 2000 mg/kg (or 5000 mg/kg in specific cases)
• Determines if substance is likely non-toxic.
• Hazard Classification: Based on the Globally Harmonized System (GHS). Categories range from non-toxic to highly toxic.

Benefits and Limitations

• Benefits: Reduces animal use and enhances welfare, provides robust data for hazard classification.
• Limitations: Focused on acute toxicity, does not assess chronic effects. Requires experienced personnel for accurate observations.

OECD Guideline 420: Acute Oral Toxicity – Fixed Dose Procedure

• Introduction: Alternative to LD50; first adopted in 1992, revised in 2001.
• Key Objectives: Assess acute oral toxicity using fewer animals; reduce suffering by observing clear signs of toxicity instead of waiting for death.
• Approach: Fixed doses (5, 50, 300, and 2000 mg/kg). Observe for toxicity signs, pain, and distress.
• Outcome: Classify substances according to the Globally Harmonized System (GHS).
• Advantages: Fewer animals, reliable chemical classification, reduces animal suffering.
• Relevance: Supports regulatory compliance and human health protection.

Selection of Test Animals

• Species: Preferred rodents (rats, females often more sensitive) .
• Criteria: Age (8-12 weeks), health (healthy, nulliparous, and non-pregnant), weights (±20% of the group mean).

Sighting Study and Main Study

• Sighting Study: Administer a single dose to one animal; dosages are 5, 50, 300, or 2000 mg/kg. Observe for mortality.
• Main Study: Test groups of five animals sequentially. Observe for signs of toxicity or mortality.

Observation Period and Decision Making

• Observation Period: At least 14 days; frequent monitoring in the first 24 hours.
• Decision Criteria: Determines the next dose level; minimize errors during LD50 estimation.
• Stopping Criteria: Met when sufficient data is collected to estimate LD50; typical completion after 4 animals experience outcome reversal.

Dose Progression and LD50 Estimation

• Dose Progression Factor: Default value of 3.2 (equivalent to a half-log unit progression). Ensures methodical dose adjustments and reduces inaccuracies in initial estimations.
• LD50 Calculation: Calculated using the maximum likelihood method. Confidence intervals derived computationally.
• Outcome: LD50 estimate with associated precision levels.

OECD Guideline 407: Repeated Dose 28-Day Oral Toxicity Study

• Overview: Method for assessing toxic effects from repeated oral exposure over 28 days.
• Applications: Hazard identification and risk assessment, data provision regarding effects on nervous, immune, and endocrine systems.
• Key Objectives: Target organ identification, dose-response characterization, and establishment of NOAELs.
• Animal Selection and Housing: Rats (young, healthy adults); equal numbers of males and females; nulliparous females.

OECD Guideline 408: Repeated Dose 90-Day Oral Toxicity Study

• Purpose: Provides information on potential health hazards for repeated oral exposure to chemicals over 90 days; identifies toxic effects, potential target organs of accumulation; establishes the NOAEL.
• Key Updates (2018 Revision): Added endocrine sensitive endpoints (T3, T4, TSH and thyroid gland weight); assessment of LDL/HDL/cholesterol levels; emphasis on neurological, immunological and reproductive effects and careful clinical observations.
• Animal Selection and preparation: Rats, nulliparous non-pregnant females; similar housing and weight characteristics as 407.

OECD Guideline 452: Chronic Toxicity Studies

• Overview: Assess health hazards of prolonged chemical exposure; target organ identification; establishing dose-response relationships for chronic toxicity; determination of NOAEL and BMD.
• Key Goals: Assess chronic toxicity; identify target organs and their accumulation potential, characterize the dose–response relationship, and determine NOAEL and BMD; predict chronic toxicity at human exposure levels.
• Administration Routes: Oral or other relevant routes based on human exposure.
• Duration: Typically 12 months depending on regulatory needs; dose selection based on previous 28-day or 90-day studies.

Data Reporting

• Details and Analysis of Data: Collect individual animal data, detailed analysis of toxicity effects, organ weights, dose-response relationships, and group summaries.
• Statistical Analysis: Use pre-determined statistical methods for assessing dose-response relationships/survival data. Include adjustments as needed.

Strengths and Limitations

• Strengths: Long-term exposure insights, identification of cumulative and chronic toxicity effects.
• Limitations: Time-consuming and resource intensive; less relevant for quick acute effects; may not detect all endocrine disruptors.

Pathology and Histopathology

• Necropsy: Comprehensive examination of all animals, including gross lesions and weighing of organs (liver, kidneys, thyroid, heart).
• Histopathology: Comprehensive analysis of tissues, focusing on target organs; tissue preservation for histopathological analyses.

Haematology, Biochemistry, and Urinalysis

• Hematology: Erythrocyte count, hemoglobin, platelet count and clotting time.
• Biochemistry: Glucose, urea, liver enzymes, total proteins, etc.
• Urinalysis: Appearance, pH, specific gravity and protein levels.
• Frequency: Interim assessments at 3, 6, and 12 months for blood and urine tests.

Observations and Measurements

• Clinical observations: Daily monitoring of morbidity, mortality, toxic signs and detailed weekly behavioral and physical changes.
• Body weight and food consumption: Weekly measurements to observe health trends.
• Ophthalmology: Eye exams at the start and the end of the study.

OECD Guideline 425 (Up-and-Down Procedure)

• Introduction: Approach for acute oral toxicity testing. First Adopted 16 October 2008.
• Purpose: to determine the median lethal dose (LD50) but using fewer animals and less suffering.