Inhalation toxicity 28 days – OECD 412

The OECD 412 assesses the effects of a substance repeatedly inhaled over 28 days in rodents. It is intended for products whose human exposure occurs primarily through the respiratory tract: powders, aerosols, fumes, vapors, and gases.

This trial is not a transposition of an oral study to another route. Exposure is described by atmospheric concentration , not by an administered dose, and the fate of the substance depends closely on the size of the particles generated. This specificity makes the control of the exposure atmosphere the determining factor for the validity of the study.

Objective of the analysis

• Identify the target organs, distinguishing local effects on the respiratory system from systemic effects after passing into the circulation.
• Establish the concentration-response relationship and determine the NOAEC (no observed adverse effect concentration) as well as the LOAEC.
• Detect early lung inflammation, before the appearance of histologically visible lesions.
• To assess the reversibility of the effects using a satellite group maintained without exposure.
• Calibrate the concentrations for a possible 90-day subchronic study.

OECD Methodology 412

Atmospheric control: what makes the study acceptable or unacceptable

This is the first point on which an inhalation case is assessed, and the most frequent cause of rejection. The guideline imposes specific requirements, which the laboratory must document continuously.

Exposure method — exposure via the nose alone is the preferred method, particularly for liquid or solid aerosols and for vapors that may condense. Whole-body exposure remains possible but must be justified in the study report.

Particle size distribution —the median aerodynamic diameter by mass should be between 1 and 3 µm, with a geometric standard deviation between 1.5 and 3.0. This range ensures that the particles effectively reach the rodent's lower respiratory tract. The particle size distribution should be determined at least once a week for each concentration level.

Actual concentration — the nominal concentration does not characterize animal exposure; it is only used to assess the effectiveness of the generation system. The actual chamber concentration must be measured at least three times per day of exposure and for each level. Individual deviations from the mean must not exceed ±10% for gases and vapors and ±20% for liquid or solid aerosols.

Ambient conditions — temperature maintained at 22 ± 3 °C, relative humidity preferably between 30 and 70%, oxygen at a minimum of 19%, carbon dioxide capped at 1%. The airflow through the chamber is controlled, continuously monitored and recorded at least every hour during each exposure.

Parameters evaluated

Clinical monitoring — daily observations, body weight, food and fluid intake.

Biological analyses — hematology and clinical biochemistry.

Bronchoalveolar lavage — which can be performed as a complement, with total and differential white blood cell counts, total protein levels, and lactate dehydrogenase levels. These markers reveal a pulmonary inflammatory reaction at a stage where histology is still normal: this is often the most informative element of an inhalation study.

Necropsy, weighing of organs and histopathology — with particular attention paid to the entire respiratory tree, from the nasal passages to the alveoli.

Interpretation and regulatory consequences

NOAEC determines whether your substance will need to carry a STOT-RE under the CLP Regulation. Since CLP guideline values ​​are established for a 90-day study, they are multiplied by three for a 28-day study:

Under REACH, a repeated-dose toxicity study becomes mandatory for quantities exceeding 10 tonnes per year per registrant. The chosen method must reflect actual human exposure: when this is primarily respiratory, the 412 test is required, not a simpler oral study. Choosing the oral route for the sake of experimental convenience weakens the entire application.

Sectors and applications concerned

• Chemical industry — volatile substances, solvents, gases, powders handled in workshops.
• Biocides and plant protection products — formulations sprayed or applied as aerosol.
• Materials, polymers and nanomaterials — assessment of dust generated in production or use.
• Cleaning products and aerosols — sprays, cleaners, propellants.
• Animal health and animal feed — premix and additive dusts.

Why use YesWeLab?

Inhalation studies are the most technically demanding and equipment-intensive of all regulatory toxicology studies. They require exposure chambers, aerosol generation systems, and analytical instrumentation that few laboratories possess. The choice of service provider is not a variable to be adjusted: a poorly characterized atmosphere invalidates the entire study, regardless of the biological results obtained.

We connect you with partner laboratories equipped for inhalation exposure and operating according to Good Laboratory Practices, a condition for the study to be admissible in a regulatory file.

Our support focuses on the initial decision-making stages: the suitability of the respiratory route in light of your exposure scenario, the choice between 28 and 90 days, the exposure method, concentration plans, and parameters to be integrated from the design phase. Our platform then centralizes your requests, quotes, and reports, all with a single point of contact.

Frequently Asked Questions

What is the expected timeframe for an OECD 412 test?

The exposure phase lasts four weeks, but the development of the atmosphere generation system and its preliminary characterization often constitute the longest step. An overall timeframe of four to eight months is realistic, depending on the physicochemical properties of the substance.

Can an inhalation study be replaced by an oral study?

No, when human exposure is primarily respiratory. Local effects on the respiratory system do not appear in an oral study, and extrapolation from one route to another is not accepted by assessors for inhalable substances.

My product is a liquid: is it affected?

It is considered inappropriate if its use generates an aerosol or if its volatility leads to exposure through vapors. It is the actual usage scenario, and not the product's physical state at rest, that determines the appropriateness of the respiratory route.

Is bronchoalveolar lavage routine?

It can be performed as a complementary test and is highly recommended when the respiratory system is the primary site of deposition. It detects inflammation at an early stage and significantly enhances the interpretation of the study.

See also

• 90-day subchronic inhalation toxicity study – OECD 413
• 28-day subacute oral toxicity study – OECD 407
• Overview of subacute and subchronic toxicity trials
• Our complete catalogue of analyses