Acute inhalation toxicity – OECD 403 test

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

When to carry out 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 allows for the determination of a median lethal concentration (LC50) or an acute hazard category, the identification of target organs and severity of effects, and the input to the GHS/CLP classification. Where waiver or cross-checking of data is possible (e.g., through alternative methods or sufficient data on analogous substances), these options are considered to avoid unnecessary in vivo testing.

Principle of the method and scope

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

Experimental process

Animals (often adult rats, females preferred unless otherwise justified) are randomized to groups and acclimated. After a single four-hour exposure to each selected concentration, animals are monitored for at least 14 days. Observations include respiratory and neurological clinical signs, behavior, body weight, and mortality. Humane endpoints apply to limit suffering; any moribund animal is euthanized with precise recording of time and signs. At the end of the study, a systematic gross 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 test strategy

A preliminary study can help position concentrations. The levels selected 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. Compliance of measured concentrations with targets is analytically verified during exposure.

Evaluation criteria and classification

Individual and group results (clinical signs, time of onset, reversibility, weight, mortality) are synthesized. Depending on the strategy chosen, an LC50 is estimated or an acute GHS/CLP category is directly assigned from the observed responses. The study report documents the substance, batch, purity, vehicle, generation method, atmospheric characterization (concentrations, MMAD/GSD for aerosols), exposure device, environmental conditions, justification of 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 3R principles, OECD 403 aims for a rational use of animals and a refinement of procedures (nose-only, close monitoring, breakpoints). Before any in vivo test, a critical review of the weight of evidence is conducted: existing data, QSAR, read-across, oral/dermal data, physicochemical information and uses. Alternative or complementary guidelines may be considered depending on the regulatory context (methodologies by toxicity classes or fixed concentration procedures) and the possibilities of waiver provided for by the assessment guides.

Sectors and use cases

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

What YesWeLab supports

YesWeLab selects a suitable partner laboratory (GLP/GLP if required), defines the experimental strategy with you (mode of exposure, atmospheric characterization, concentration levels), coordinates the planning, ensures monitoring via the platform and delivers a complete report that can be used for regulatory purposes. Our partners have dedicated inhalation facilities, analytical means for real-time monitoring of concentrations 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