Toxicology: Acute Toxicity Testing

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Questions and Answers

What does the limit dose refer to in toxicity testing?

The maximum dose for long-term studies.

A dose at an upper limitation on testing. (correct)

A dose that has no observable effects.

The lowest dose that can be administered safely.

Which animal species is preferred for OECD Guideline 423 testing?

Guinea pigs

Rats (correct)

Rabbits

Mice

What is the primary purpose of OECD Guideline 423?

To define the humane treatment of laboratory animals.

To assess the acute oral toxicity of chemicals. (correct)

To promote the use of alternative testing methods.

To establish long-term testing protocols.

How many animals are typically used per step in the OECD Guideline 423 testing?

3 animals (A) Signup and view all the answers

What does the term mymoribund status indicate in toxicity testing?

The animal is in a state of dying. (C) Signup and view all the answers

What preparation guideline is preferred for test substances?

Aqueous solutions (C) Signup and view all the answers

What is the effect of administering a fixed dose in toxicity testing?

It helps determine the LD50 cut-off values. (C) Signup and view all the answers

What is a common observable sign indicative of predictable death in toxicity testing?

Inability to reach water or food (D) Signup and view all the answers

What does acute oral toxicity refer to?

Toxic effects following a single dose of a substance. (A) Signup and view all the answers

Which term describes the state when death is expected prior to the next observation?

Impending death (D) Signup and view all the answers

What is the default dose progression factor used when there is no estimate of the slope for a substance?

3.2 (B) Signup and view all the answers

What does LD50 represent in toxicology?

The median lethal dose expected to kill 50% of test subjects. (D) Signup and view all the answers

What is the purpose of the dose progression factor?

To ensure proportional and methodical dose adjustments (D) Signup and view all the answers

In toxicity testing, what does the term 'dose' refer to?

The weight of the test substance per unit weight of the test animal. (A) Signup and view all the answers

What is the maximum number of animals that can experience an outcome reversal before testing concludes?

4 animals (D) Signup and view all the answers

Which method is used to calculate the LD50 estimate?

Maximum likelihood method (C) Signup and view all the answers

What does the Globally Harmonised Classification System (GHS) provide?

Standardized classifications for chemical substances and mixtures. (C) Signup and view all the answers

What could be an observable sign of impending death in rodents under toxicology testing?

Convulsions (C) Signup and view all the answers

For what primary purpose is the OECD Guideline 407 implemented?

Assess toxic effects from repeated oral exposure (A) Signup and view all the answers

Delayed death in toxicity testing means:

Death occurs more than 48 hours after administration. (B) Signup and view all the answers

What is the initial dose at which dosing should be initiated if no lethality estimate is available?

175 mg/kg (C) Signup and view all the answers

What specific outcome is aimed to be characterized in the study?

Dose-response relationships (B) Signup and view all the answers

What is the minimum observation period for the procedure mentioned?

14 days (C) Signup and view all the answers

What is the role of the Inter-Organization Programme for the Sound Management of Chemicals (IOMC)?

To coordinate efforts for the sound management of chemicals. (A) Signup and view all the answers

Which of the following is considered a humane endpoint for moribund animals?

Humanely euthanize the animal (B) Signup and view all the answers

Which species is preferred for the OECD Guideline 407 testing?

Rats (young, healthy adults) (A) Signup and view all the answers

What does the limit test conducted at 2000 mg/kg primarily determine?

Likely non-toxicity of the substance (B) Signup and view all the answers

What type of toxicity does the OECD 420 guideline focus on?

Acute oral toxicity assessment (D) Signup and view all the answers

Which feature is a focus of the OECD 420 guidelines?

Fewer animals required (D) Signup and view all the answers

What is the main benefit of adhering to the Globally Harmonized System (GHS) in hazard classification?

Reliable classification of chemicals (D) Signup and view all the answers

Which of the following does NOT represent a limitation of the tested acute toxicity procedures?

Assesses behavioral changes in humans (C) Signup and view all the answers

What type of examination is conducted on all animals post-mortem?

Gross necropsy (D) Signup and view all the answers

What is the preferred animal for toxicity testing according to the established criteria?

Rats (D) Signup and view all the answers

What is the age range for the animals selected for the toxicity test?

8–12 weeks (D) Signup and view all the answers

During the observation period of at least 14 days, which of the following is NOT a key observation?

Fur color (D) Signup and view all the answers

At which dose level may additional testing at 5000 mg/kg be conducted?

Only if significant regulatory needs arise (B) Signup and view all the answers

Which classification category indicates a highly toxic substance?

Category 1 (LD50 ≤ 5 mg/kg) (D) Signup and view all the answers

Which of the following is a limitation of the toxicity testing process described?

It cannot predict long-term effects (C) Signup and view all the answers

What is the primary purpose of conducting the limit test at dose levels of 2000 mg/kg?

For substances expected to be non-toxic (D) Signup and view all the answers

When the first animal in the step-dose testing dies or appears moribund, what is the next action?

Reduce the dose for the next animal (B) Signup and view all the answers

Which species is preferred for chronic toxicity studies under OECD Guideline 452?

Rats and mice. (C) Signup and view all the answers

How long do chronic toxicity studies typically last according to OECD Guideline 452?

12 months. (A) Signup and view all the answers

What measure is determined to assess chronic toxicity levels?

No-Observed-Adverse-Effect Level (NOAEL). (B) Signup and view all the answers

Which of the following routes of administration is NOT included in the OECD Guideline 452?

Intravenous. (C) Signup and view all the answers

What type of groups is established for monitoring reversibility in chronic toxicity studies?

Satellite groups. (C) Signup and view all the answers

Which observation is NOT typically monitored daily during chronic toxicity studies?

Weight gain. (A) Signup and view all the answers

What is the significance of adjusting doses based on body weight in chronic toxicity studies?

To account for metabolization differences. (D) Signup and view all the answers

Study Notes

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.

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This quiz covers the fundamentals of acute toxicity testing, including various types of toxicity studies and OECD guidelines. Explore the definitions, methodologies, and implications of acute toxicity in drug testing. Perfect for students and professionals in toxicology and pharmacology.