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

OECD Guideline 423 describes a sequential approach to assessing acute oral toxicity and rapidly classifying a substance according to the GHS/CLP. Unlike historical methods centered on the LD50, 423 assigns a toxicity class based on observed responses to standard dose levels, using a small number of animals and strict humane criteria. This method is widely accepted by regulatory authorities and is integrated into GLP/GLP requirements for REACH, biocides, and plant protection product dossiers, with a report usable in IUCLID.

Objectives and positioning of the method

OECD 423 aims to provide sufficient information for hazard classification following a single oral exposure, without estimating a precise LD50 value. It is suitable when the primary objective is the effective GHS/CLP categorization of substances and mixtures, hazard communication, and regulatory compliance. The method is used as a priority when foregone conclusions based on existing data, QSAR, or cross-reading do not allow for avoiding in vivo testing.

Methodological principle and sequential logic

OECD Method 423 administers the test substance orally at predefined doses, typically 5, 50, 300, and 2000 mg/kg bw. Administration is limited to one sex only, usually females, to minimize variability and the number of animals required. After each dose step, the next dose is determined based on observed responses, primarily mortality and clinical signs. The results lead to classifying the substance in an acute toxicity class or continuing the investigation at the next higher or lower dose. This decision-making logic allows the study to be stopped as soon as the information needed for classification is obtained, thus avoiding unnecessary exposure to doses that could cause significant suffering.

Experimental procedure and test conditions

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

Dose selection and management of endpoints

The choice of the first dose level is guided by the available data on the substance or its analogues. In the absence of information, an intermediate dose level is often chosen to optimize the progression. The sequence is adjusted according to observed mortality and manifest toxicity, with the application of humane criteria for the euthanasia of moribund or severely distressed animals. Exposure to doses known to be corrosive or highly irritating should be avoided. A limit test at 2,000 mg/kg may be considered if low toxicity is expected.

Data collected, interpretation and GHS classification

For each animal, clinical observations, timing of effects, mortality, body weights, and necropsy results are recorded. The 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 lot identification, purity, vehicle, formulation, housing conditions, rationale for the dosing schedule, results tables, discussion, and conclusions. In GLP/GLP, quality assurance, traceability, archiving, and an IUCLID-compliant format are ensured.

Key differences with OECD 420

OECD 423 and 420 share the same classification objective but differ in their operational logic. OECD 423 follows an acute toxicity class method based on mortality observed at predefined thresholds and a strict sequential decision-making process. OECD 420 relies on the predetermined dose method, identifying 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 factor for regulatory decisions, while OECD 420 is useful for limiting deaths and focusing on overt toxicity. The choice between the two depends on regulatory requirements, animal ethics, and the anticipated toxicological profile.

Best practices and 3R principles

In accordance with the 3Rs principles, OECD 423 should be considered after a critical review of available information and alternatives. Reducing the number of animals is achieved through a sequential approach and early termination as soon as a class can be assigned. Refinement is achieved by avoiding doses likely to cause significant suffering, using humane criteria, and close clinical monitoring. When the weight of evidence allows for abandoning the trial or repeating it, these options are preferred.

YesWeLab support

YesWeLab manages your OECD 423 studies within a network of partner laboratories accredited to GLP/GMP and ISO 17025 standards. We work with you to define the sequential strategy, the initial assessment level, the vehicle, and the observation plan, then monitor the results via our digital platform. You receive a comprehensive, IUCLID-compliant report, immediately usable for REACH/CLP, biocides, or plant protection 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