Acute Inhalation Toxicity Test

OECD TG 403, OECD TG 433 and OECD TG 436

Acute Inhalation Toxicity: Scientific and Regulatory Assessment of Respiratory Hazards

Inhalation is an important route of chemical exposure because gases, vapours, aerosols, dusts and mists can interact directly with the respiratory tract. Depending on the physicochemical properties of the test article, deposition pattern, solubility, reactivity and clearance, inhalation exposure may cause local respiratory effects, systemic toxicity, or both. Acute inhalation toxicity studies are therefore used when inhalation is a relevant exposure route and the applicable regulatory data requirement cannot be adequately addressed by existing information, alternative methods, read-across or an accepted waiver.

Selection of the appropriate acute inhalation test method should be scientifically justified and should reflect the regulatory objective, available toxicity information, expected human exposure, physical form of the test article, technical feasibility of generating a suitable atmosphere and the principles of replacement, reduction and refinement (3Rs). The current OECD TG 403 specifically emphasizes consideration of existing information and more refined acute inhalation approaches before conducting a traditional TG 403 study. Where classification and labelling are the principal regulatory objective, OECD TG 436 is generally recommended by TG 403, subject to the needs of the receiving authority.

Scientific Basis of Acute Inhalation Toxicity Testing

Acute inhalation toxicity testing evaluates adverse effects arising after a short-duration inhalation exposure. The relevant endpoint depends on the OECD Test Guideline selected. A precise LC50 or other quantitative concentration-response estimate is principally associated with OECD TG 403. OECD TG 433 instead uses evident toxicity as the key decision point, whereas OECD TG 436 uses a stepwise Acute Toxic Class procedure in which mortality or moribundity determines progression between predefined concentration levels.

Inhaled material does not necessarily pass directly and rapidly from the alveoli into systemic circulation. Respiratory tract deposition and absorption depend on factors such as aerodynamic particle size, vapour pressure, water and lipid solubility, reactivity, regional deposition, mucociliary clearance and alveolar clearance. Consequently, an acute inhalation study should be interpreted for both local respiratory toxicity and systemic toxicity rather than as a purely systemic toxicity test.

OECD Acute Inhalation Test Guidelines: Correct Regulatory Distinction

OECD TG 403 – Acute Inhalation Toxicity

OECD TG 403 contains two distinct study designs: the Traditional LC50 protocol and the Concentration × Time (C × t) protocol. The Traditional protocol generally uses a fixed exposure period, normally 4 hours, and at least three concentration levels in the main study. A main study is typically conducted with five males and five females per concentration, or five animals of the more susceptible sex when that susceptibility is known. The design is intended to generate a robust quantitative concentration-response relationship and may provide LC50 and other lethality estimates with confidence limits.

The C × t protocol is also part of OECD TG 403. It exposes animals at several concentration levels and multiple exposure durations, normally using a nose-only system. This approach may be selected when a regulatory or scientific need exists to characterize toxicity across different exposure durations, for example in emergency-response or land-use planning. It is not an OECD TG 436 procedure.

OECD TG 433 – Acute Inhalation Toxicity: Fixed Concentration Procedure

OECD TG 433 is the Fixed Concentration Procedure (FCP). Its central refinement is the use of evident toxicity rather than death or moribundity as the intended endpoint, while avoiding concentrations expected to cause lethality or marked pain and distress. The procedure is stepwise and uses predefined fixed concentrations appropriate to gases, vapours or aerosols. A sighting study may be used to identify the most sensitive sex and an appropriate starting concentration. The main study normally uses five animals of one sex at each concentration level investigated, in addition to animals used in the sighting study.

A 4-hour exposure is recommended, although other durations may be needed for specific regulatory requirements. The method is intended primarily to support acute toxicity hazard assessment and classification rather than to generate a precise point estimate of LC50.

OECD TG 436 – Acute Inhalation Toxicity: Acute Toxic Class Method

OECD TG 436 is the Acute Toxic Class (ATC) method. It is a stepwise classification procedure in which each step uses three animals of each sex, with the rat as the preferred species. Animals are exposed for 4 hours to a predefined concentration selected from fixed levels corresponding to GHS acute inhalation toxicity categories. The presence or absence of test-article-related mortality or moribundity at a step determines the next step.

TG 436 is therefore not a concentration × time method and does not use evident toxicity as its primary decision endpoint. Its principal purpose is hazard classification using fewer animals than a classical quantitative LC50 design.

Comparison of the OECD Acute Inhalation Approaches

Generation and Characterization of the Test Atmosphere

The generation and characterization of the inhalation atmosphere are central to study validity. The physical form tested should reflect the physicochemical properties of the test article and, where relevant, the form likely to be encountered during handling or use. Gases and vapours may be generated by controlled dilution or vaporization, whereas dusts and liquid aerosols require suitable generation systems capable of producing a stable and respirable atmosphere.

Actual test-article concentration should be determined in the animals’ breathing zone using an appropriate validated or scientifically justified analytical method. For OECD TG 403, the exposure atmosphere should be held as constant as practicable. When intermittent sampling is used in a 4-hour study, at least two chamber samples are generally expected, with more sampling if substantial variability is observed. Individual concentration samples should generally not deviate from the mean chamber concentration by more than ±10% for gases and vapours or ±20% for liquid or solid aerosols.

For OECD TG 433, actual concentration is likewise measured in the breathing zone. When intermittent sampling is used, the guideline specifies at least five samples at approximately hourly intervals where feasible. The same ±10% criterion for gases/vapours and ±20% criterion for liquid/solid aerosols applies to individual chamber concentration samples relative to the mean.

Aerosol Particle-Size Characterization

Particle-size characterization should be expressed using aerodynamic parameters, not simply as the percentage of particles in a nominal size band. Under OECD TG 403, aerosols with a mass median aerodynamic diameter (MMAD) of 1-4 µm and a geometric standard deviation (GSD) of 1.5-3.0 are recommended to permit exposure of relevant regions of the respiratory tract. Particle-size distribution should be determined at least twice during each 4-hour exposure.

Under OECD TG 433, an aerosol MMAD of ≤4 µm with a GSD of 1.0-3.0 is recommended, and particle-size distribution should also be determined at least twice during each 4-hour exposure. Any scientifically justified deviation from the recommended particle-size range should be explained in the study documentation.

Exposure Systems: Nose/Head-Only and Whole-Body

Both nose/head-only and whole-body exposure systems may be appropriate depending on the test article, study design and regulatory objective. Nose/head-only systems can reduce non-inhalation exposure and generally provide tighter control over the inhaled atmosphere, but restraint can introduce stress and requires careful acclimatization and monitoring. Whole-body systems avoid restraint but may increase test-article consumption and can permit additional dermal exposure or oral exposure through grooming after deposition on the fur.

Accordingly, nose-only exposure should not be described as completely preventing dermal or ocular exposure. A more accurate statement is that it minimizes exposure by non-inhalation routes.

GHS and EU CLP Classification of Acute Inhalation Toxicity

Acute inhalation toxicity classification depends on the physical form of the test material and the 4-hour acute toxicity estimate (ATE) or equivalent LC50 information. For EU CLP, Categories 1-4 are used. The UN GHS also includes an optional Category 5, but Category 5 is not an acute toxicity category under EU CLP.

Note: The numerical criteria shown are the EU CLP acute inhalation toxicity categories for 4-hour exposure. For other jurisdictions, the applicable adopted GHS edition and national/regional implementation should be checked.

Conduct of a Regulatory Acute Inhalation Study

A scientifically defensible acute inhalation program normally begins with review of all available information, including test-item identity and composition, purity, physicochemical properties, volatility, particle-size characteristics where relevant, flammability/explosibility, existing toxicology data, anticipated use and exposure, and the analytical method for atmosphere measurement.

Technical feasibility should be demonstrated before animal exposure. This includes generation of the intended atmosphere, confirmation of stability and homogeneity, appropriate sampling at the animals’ breathing zone, and verification that environmental conditions such as airflow, temperature, relative humidity, oxygen and carbon dioxide remain within acceptable ranges.

During and after exposure, animals are assessed for mortality/moribundity and clinical signs of toxicity according to the selected guideline. Body weights are recorded at specified intervals. Post-exposure observation is generally continued for approximately 14 days, with the exact duration adjusted when justified by the onset, persistence or recovery of toxic effects. All animals are subjected to gross necropsy as required by the applicable guideline; histopathological examination may be appropriate when indicated by lesions, study objectives or regulatory requirements.

For regulatory submissions, the study should be performed under the applicable Good Laboratory Practice (GLP) framework when required by the receiving authority. The final report should clearly distinguish target, nominal and actual concentrations, provide the atmosphere-generation and analytical methods, document particle-size data for aerosols, report individual-animal observations and body weights, describe necropsy findings, and provide the statistical analysis appropriate to the selected study design.

Frequently Asked Questions

Do all gases, vapours, aerosols, dusts or volatile liquids automatically require an acute inhalation study?

No. The need for testing depends on the applicable regulatory framework, the expected route of human exposure, available acute toxicity information, physicochemical properties, and whether accepted adaptations, read-across, alternative methods or waivers can address the endpoint. Testing should not be presented as universally mandatory for every inhalable material.

Which OECD guideline should be selected when only GHS classification and labelling are required?

The current OECD TG 403 notes that TG 436 is generally recommended when information for classification and labelling is the principal need. TG 433 may also be appropriate where the Fixed Concentration Procedure and its evident-toxicity endpoint meet the regulatory objective. The final choice should be justified against the requirements of the receiving authority.

When is OECD TG 403 appropriate?

TG 403 is appropriate when a quantitative concentration-response relationship, a point estimate such as LC50/LCx, or concentration-time information over multiple exposure durations is scientifically or regulatorily required. TG 403 should not be used automatically when a more refined approach can satisfy the data requirement.

Can a precise LC50 be calculated from TG 433 or TG 436?

These methods are principally classification procedures and are not designed to generate the same precise point estimate of LC50 as a conventional TG 403 quantitative design. They provide information sufficient to assign or bracket acute inhalation hazard categories according to their decision schemes.

How long does an acute inhalation study take?

The animal phase normally includes the exposure followed by an approximately 14-day observation period. Total project duration depends on test-item characterization, analytical method readiness, atmosphere-generation feasibility, study scheduling, pathology requirements, data review, QA review and final reporting. A fixed universal turnaround time should therefore not be stated without study-specific confirmation.

Regulatory Conclusion

Acute inhalation toxicity assessment should be designed around the actual regulatory question rather than a single default method. OECD TG 403 provides quantitative Traditional LC50 and C × t options; OECD TG 433 provides a Fixed Concentration Procedure centred on evident toxicity; and OECD TG 436 provides an Acute Toxic Class procedure based on predefined concentrations and mortality/moribundity. Correct method selection, technically controlled atmosphere generation, guideline-compliant aerosol characterization and accurate interpretation of GHS/CLP criteria are essential for a scientifically valid and regulatorily acceptable study.

Authoritative References

1. OECD (2024). Test No. 403: Acute Inhalation Toxicity. OECD Guidelines for the Testing of Chemicals, Section 4. Corrected 25 June 2024. DOI: 10.1787/9789264070608-en. Link
2. OECD (2018). Test No. 433: Acute Inhalation Toxicity: Fixed Concentration Procedure. OECD Guidelines for the Testing of Chemicals, Section 4. DOI: 10.1787/9789264284166-en. Link
3. OECD (2009). Test No. 436: Acute Inhalation Toxicity – Acute Toxic Class Method. OECD Guidelines for the Testing of Chemicals, Section 4. DOI: 10.1787/9789264076037-en. Link
4. OECD (2018). Guidance Document on Inhalation Toxicity Studies – Second Edition. OECD Series on Testing and Assessment No. 39. DOI: 10.1787/fba59116-en. Link
5. European Parliament and Council. Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (CLP), Annex I, Section 3.1 – Acute Toxicity. Link
6. United Nations Economic Commission for Europe (UNECE). Globally Harmonized System of Classification and Labelling of Chemicals (GHS), 11th revised edition. Link