Acute oral toxicity – OECD 423 test (Method by acute toxicity class)

OECD Guideline 423 describes a step-by-step approach to assess acute oral toxicity and rapidly classify a substance according to GHS/CLP. Unlike historical LD50-centric methods, 423 assigns a toxicity class based on observed responses at standard dose levels, using a small number of animals and strict humane criteria. This method is widely accepted by regulatory authorities and integrates with GLP/GLP requirements for REACH, biocide, and phytosanitary dossiers, with a report usable in IUCLID.

Objectives and positioning of the method

OECD 423 aims to provide sufficient information for hazard classification after a single oral exposure, without estimating a precise LD50 value. It is suitable when the primary objective is effective GHS/CLP categorization of substances and mixtures, hazard communication, and regulatory compliance. The method is primarily used when data-based, QSAR-based, or read-across-based avoidance options do not allow for the avoidance of in vivo testing.

Methodological principle and sequential logic

The OECD 423 method administers the test substance orally at predefined dose levels, typically 5, 50, 300, and 2000 mg/kg bw. Administration is to one sex only, usually females, to limit variability and animal numbers. After each step, the next dose is decided based on the observed responses, primarily mortality and clinical signs. The results lead to classifying the substance in an acute toxicity class or to continuing the investigation at the higher or lower dose. The decision logic allows the study to be stopped as soon as the information necessary for classification is obtained, avoiding unnecessary exposure to doses likely to cause marked suffering.

Experimental procedure and test conditions

Animals are healthy young adult, nulliparous, non-pregnant females unless otherwise justified. At least five days of acclimatization are observed. Subjects are fasted before gavage, then the substance is administered as a single dose by gastric tube or suitable intubation cannula. The administered volumes are controlled, with water as the preferred vehicle; other vehicles may be used if justified and compatible. The study includes close observation in the first hours, then daily for at least 14 days, with systematic recording of clinical signs, mortality, onset times and reversibility of effects. Body weights are measured before the dose and then at least once a week; a macroscopic necropsy is performed on all animals, with targeted histology when necessary.

Choice of doses and management of limit points

The choice of the first tier is guided by the available data on the substance or its analogues. In the absence of information, an intermediate tier is often chosen to optimize progression. The sequence is adjusted according to observed mortality and overt toxicity, with humane criteria applied for euthanasia of moribund or severely distressed animals. Exposure to doses known to be corrosive or highly irritating should be avoided. A limit test may be considered at 2000 mg/kg if low toxicity is expected.

Data collected, interpretation and GHS classification

For each animal, clinical observations, time of effects, possible mortality, body weights, and necropsy findings are reported. Data are then summarized by dose to establish the acute toxicity class, in accordance with GHS/CLP criteria. The method is not intended to estimate the LD50; it provides a robust and reproducible categorization sufficient for labeling, risk management, and regulatory compliance. The study report includes substance and batch identification, purity, vehicle, formulation, housing conditions, dose regimen justification, results tables, discussion, and conclusions. In GLP/GLP, quality assurance, traceability, archiving, and a IUCLID-compatible format are ensured.

Key differences with OECD 420

OECD 423 and OECD 420 share the same classification objective, but differ in their operating logic. OECD 423 follows an acute toxicity class method based on observed mortality at predefined levels and strict sequential decision-making. OECD 420 relies on the predetermined dose method, searching for the lowest dose that induces overt toxicity, without necessarily observing mortality, and then deducing the category. In practice, OECD 423 is often preferred when mortality is the most relevant determining criterion for regulatory decision-making, while OECD 420 is useful for limiting deaths and reasoning on overt toxicity. The choice between the two is made according to regulatory expectations, animal ethics, and the anticipated toxicological profile.

Best practices and 3R principles

In accordance with the 3R principles, OECD 423 should be considered after a critical review of available information and alternatives. Animal reduction is achieved through sequential logic and early termination as soon as a class can be assigned. Refinement is expressed through the avoidance of doses likely to induce significant suffering, the use of humane criteria, and close clinical monitoring. When the weight of evidence allows for the trial to be abandoned or re-tested, these options are preferred.

YesWeLab support

YesWeLab manages your OECD 423 studies within a network of GLP/GLP and ISO 17025 accredited partner laboratories. We define with you the sequential strategy, the choice of the initial level, the vehicle and the observation plan, then ensure follow-up via our digital platform. You receive a complete, IUCLID-compatible report, immediately usable for REACH/CLP, biocides or phytosanitary products, with quality archiving and traceability.

Related analyses

• OECD 420 – Acute Oral Toxicity, Predetermined Dose Method
• OECD 425 – Acute Oral Toxicity, Dose Adjustment Method
• OECD 401 – Acute Oral Toxicity (Historical Method)
• OECD 402 – Acute Dermal Toxicity
• OECD 403 – Acute inhalation toxicity
• OECD 404/405 – Skin and Eye Irritation/Corrosion
• OECD 407 – Subchronic toxicity 28 days