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Acute Toxicity Studies - Study Design of Acute Inhalation Study in Rats (OECD 403)

The OECD Guideline 403 outlines the study design for assessing the acute inhalation toxicity of chemicals in rats. This information is crucial for ensuring the safety of chemicals before they enter the market. Here’s a breakdown of the key aspects:

Objectives:

Determine the concentration of a test substance inhaled by rats that causes evident toxic effects without severe toxicity or mortality.
Classify the substance according to its acute inhalation toxicity based on the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).
Provide initial information for further safety testing, if necessary.

Test System:

Healthy young adult rats of both sexes are used.
A minimum of 5 animals per sex per dose group are required.

Exposure Conditions:

Animals are exposed to the test substance as a gas, vapor, or aerosol in an inhalation chamber for a predetermined duration.

The study design features two protocols:

(a) Traditional LC50 protocol:

Objective: Determine the median lethal concentration (LC50) of the test article causing death in 50% of exposed animals.
Exposure: Groups of animals are exposed to the test article at four or more concentration levels for a predetermined duration, typically 4 hours.
Observation: Animals are observed for at least 14 days after exposure, monitoring mortality, signs of toxicity, and body weight changes.
Data analysis: LC50 and confidence intervals are calculated based on mortality data. The test substance is classified according to its acute inhalation toxicity based on GHS criteria.

(b) Concentration x Time (C x t) protocol:

Objective: Investigate the relationship between concentration, exposure time, and toxic effects.
Exposure: Groups of animals are exposed to the test article at varying concentrations and durations, creating a matrix of exposure conditions.
Observation: Similar to the LC50 protocol, animals are observed for 14 days, monitoring mortality, signs of toxicity, and body weight changes.
Data analysis: Dose-response relationships and “No Observed Adverse Effect Level” (NOAEL) are determined for various endpoints. This provides more comprehensive information on toxicity than just the LC50.
Particle size distribution and test substance preparation are carefully controlled.

Observation Period:

Animals are observed for at least 14 days after exposure.
Detailed observations are made at least hourly for the first 24 hours and at specific intervals thereafter.

Endpoints:

Mortality and time to death.
Signs of toxicity, including respiratory distress, labored breathing, cyanosis, weakness, tremors, and convulsions.
Body weight changes.
Clinical observations, including physical appearance, fur condition, eyes, and excreta.
Necropsy findings (at termination of the study).

Data Analysis:

The LC50 is calculated for the Traditional protocol using statistical methods.
The C x t protocol provides information on the relationship between exposure duration and toxicity.
Classification of the substance is based on the LC50 or other relevant data points and GHS criteria for acute inhalation toxicity categories.

Benefits:

Standardized protocol for international harmonization.
Provides a quick initial assessment of a substance’s inhalation toxicity potential.

Limitations:

Requires animal use, raising ethical concerns.
Only assesses acute effects and may miss delayed toxicity.
May not be suitable for all types of substances (e.g., highly volatile or reactive chemicals).