Acute inhalation toxicity – OECD 403 test

The OECD 403 test assesses the effects of acute inhalation exposure to a substance or mixture to determine its immediate hazard to human health after a single exposure. Conducted according to OECD Guidelines and in compliance with GLP/GMP, the study provides the data necessary for GHS/CLP classification and labeling, regulatory registration (REACH, biocides, pesticides), and occupational risk assessment. It allows for the estimation of acute inhalation toxicity and guides risk prevention and management measures.

When to conduct an OECD 403 and for what purpose

The test applies to gases, vapors, aerosols, and dusts when acute inhalation data are required for marketing or industrial handling. It determines a median lethal concentration (LC50) or acute hazard category, identifies target organs and the severity of effects, and contributes to the GHS/CLP classification. Where data can be waived or cross-checked (e.g., through alternative methods or sufficient data on similar substances), these options are considered to avoid unnecessary in vivo testing.

Principle of the method and scope

The OECD 403 test relies on a single exposure, typically lasting four hours, to different concentrations of the test substance generated in a controlled system. Depending on the nature of the product, the exposure is conducted in nose-only mode (preferred for controlling the inhaled dose) or in a whole-body chamber. The generation and characterization of the test atmosphere are crucial: a stable target concentration, controlled temperature and humidity, and, for aerosols/dusts, a particle size distribution appropriate for the respirable fraction (MMAD typically 1–4 µm, documented GSD). The study must prevent any condensation or adsorption that would distort the inhaled concentration and ensure spatial homogeneity.

Experimental procedure

Animals (often adult rats, females preferred unless otherwise justified) are randomized into groups and acclimated. After a single four-hour exposure to each selected concentration, the animals are monitored for at least 14 days. Observations include respiratory and neurological clinical signs, behavior, body weight, and mortality. Humane stopping points are applied to limit suffering; any moribund animal is euthanized with precise documentation of the time and signs. At the end of the study, a routine macroscopic necropsy is performed; targeted histopathological examinations of the respiratory tract or organs with lesions may be added depending on the observations.

Choice of concentrations and testing strategy

A preliminary study can help determine target concentrations. The selected levels must cover a relevant range to establish the concentration-effect relationship without inducing excessive mortality. For aerosols, the generated mass, temporal stability, and alveolar fraction are documented. For vapors and gases, vapor pressure, formulation, and volatility guide the generation device. The conformity of the measured concentrations to the targets is verified analytically during exposure.

Evaluation criteria and classification

Individual and group results (clinical signs, time of onset, reversibility, weight, mortality) are summarized. Depending on the strategy used, an LC50 is estimated or an acute GHS/CLP category is directly assigned based on observed responses. The study report documents the substance, lot, purity, vehicle, generation method, atmospheric characterization (concentrations, MMAD/GSD for aerosols), exposure device, environmental conditions, rationale for concentrations, raw data, statistical analysis, and interpretation. In GLP, quality assurance, traceability, archiving, and IUCLID compliance are ensured.

Animal welfare, renunciation, and complementary approaches

In accordance with the 3Rs principles, OECD 403 aims for the responsible use of animals and the refinement of procedures (nose-only testing, close monitoring, stop points). Before any in vivo test, a critical review of the weight of evidence is conducted: existing data, QSAR, cross-reading, oral/dermal data, physicochemical information, and intended uses. Alternative or complementary guidelines may be considered depending on the regulatory context (toxicity class methodologies or fixed-concentration procedures) and the waiver options provided for in the assessment guidelines.

Sectors and use cases

OECD 403 testing is required for substances and mixtures in the chemical industry, biocides and plant protection products, intermediate and specialty products, as well as for materials likely to emit vapors, aerosols, or dust during their manufacture, transport, or use. The results inform GHS/CLP classification, hazard communication, and the definition of workplace exposure control measures.

What YesWeLab supports

YesWeLab selects a suitable partner laboratory (GLP/GLP compliant if required), defines the experimental strategy with you (exposure method, atmospheric characterization, concentration levels), coordinates the planning, monitors progress via the platform, and delivers a comprehensive report compliant with regulations. Our partners have dedicated inhalation facilities, analytical resources for real-time concentration monitoring, and recognized expertise in respiratory safety.

Related analyses

• OECD 401 – Acute oral toxicity
• OECD 402 – Acute dermal toxicity
• OECD 404 – Skin Irritation/Corrosion
• OECD 405 – Eye irritation
• OECD 407 – Subchronic toxicity 28 days (oral)
• Repeated inhalation studies 28/90 days