Acute oral toxicity – OECD 420 test (Predetermined Dose Method)

OECD Guideline 420 describes an alternative method for assessing acute oral toxicity, designed to reduce the number of animals used and avoid relying solely on mortality as the endpoint. Based on administering predetermined doses to a single sex (usually females), it allows for rapid classification of a substance according to GHS/CLP standards without requiring an exact LD50. This approach is recognized by regulatory authorities (REACH/CLP) and is easily integrated into GLP/GLP requirements, with a report directly usable in IUCLID.

Objectives and scope of the trial

OECD 420 provides the information necessary for hazard assessment following a single oral exposure and for GHS/CLP classification in acute toxicity. It aims to determine the relevant toxicity range by limiting exposure to moderately toxic doses. The study is indicated for REACH registration, biocidal or phytosanitary dossiers, transport/UN compliance, and more broadly for hazard communication and product risk assessment.

Methodological principle

The method is based on fixed dose levels of 5, 50, 300, and 2,000 mg/kg bw (with, exceptionally and with due justification, 5,000 mg/kg). The initial dose is chosen based on a preliminary study and available information (physicochemical properties, in vitro/in vivo data, QSAR, analogs). The objective is to elicit clear signs of toxicity without causing serious adverse effects or mortality. Depending on the observed response (absence of toxicity, overt toxicity, mortality), the sequence continues at the next higher or lower dose until a GHS category can be assigned. The method prioritizes reducing animal suffering by avoiding doses known to be severely corrosive/irritating and by applying humane limit points.

Experimental procedure

The animals are healthy young adults (preferably rats), non-pregnant, nulliparous females, acclimated for at least five days. Subjects are fasted before gavage, and the substance is then administered in a single dose (which can be divided over 24 hours if necessary) via a gastric tube. Volumes are controlled (typically ≤ 1 mL/100 g, up to 2 mL/100 g for aqueous solutions), and water is the preferred vehicle whenever possible. After administration, the animals are monitored for at least 14 days, with close observation in the first few hours, then daily. Parameters monitored include clinical signs (respiratory, neurological, behavioral), the occurrence of local/systemic toxicity, mortality, body weights (before dose and then at least weekly), and, at the end of the study, a systematic macroscopic examination (and targeted histology if necessary).

Initial dose assessment and selection

The pilot study uses one animal per dose level (5, 50, 300, 2000 mg/kg), with an interval of at least 24 hours between subjects, until the most informative initial dose for the main study is determined. In the absence of information, 300 mg/kg is often recommended. If mortality occurs at 5 mg/kg, a confirmation procedure can be initiated, sequentially administering the same dose to a limited number of animals to confirm the most severe category, while stopping immediately if a second death occurs.

Main study, levels and group size

The primary study typically includes five animals at the dose level under consideration (including the pilot animal already treated at that same level). After the first dose, three routes are possible depending on the observed response: direct classification, administration of the lower dose, or administration of the higher dose. A dose level that resulted in mortality during the pilot phase is not repeated for welfare reasons. The interval between groups (often 3–4 days) can be adjusted to detect any delayed effects. A limit trial at 2,000 mg/kg (exceptionally 5,000 mg/kg if required by regulation and if there is a clear health benefit) may be conducted when low toxicity is expected.

Observations, humanitarian criteria and pathology

Observations are performed at least once within the first 30 minutes, regularly during the first 24 hours, and then daily until day 14 (or longer if toxicity is delayed). Moriform or severely distressed animals are euthanized and taken into account in the interpretation. Weights are monitored individually (before dose, then weekly), and changes are documented. All animals (deceased or surviving) are necropsied; targeted histopathological examinations may be conducted in cases of obvious lesions.

Data, classification and deliverables

The results are presented individually and summarized by dose: number of animals, signs of toxicity (nature, intensity, timing, reversibility), mortality and time to onset, weight, and macro/histological lesions. The GHS/CLP classification is assigned based on the responses observed at predetermined thresholds, without requiring an LD50 estimate. The study report documents the substance (identity, purity, lot), vehicle, formulation, housing conditions, rationale for the initial dose, administration methods, results tables, discussion, and conclusions. In GLP/GLP, quality assurance, traceability, archiving, and an IUCLID-compliant format are provided.

Strengths of the OECD 420 method

The procedure offers robust classification while reducing the number of animals and their suffering compared to traditional LD50 approaches. It is particularly well-suited when the regulatory objective is rapid GHS/CLP categorization and advocates a cautious escalation strategy aligned with the 3R principles. It integrates easily with strategies of foregoing or cross-referencing with existing data (in vitro, QSAR, analogues), limiting in vivo testing to justified cases.

YesWeLab support

YesWeLab coordinates your OECD 420 trials with accredited partner laboratories (GLP, ISO 17025). We help you define the dose strategy (target, initial plateau), select the vehicle, and document the scientific rationale. You benefit from digital monitoring, rigorous quality control, and a comprehensive IUCLID-ready report, usable in REACH/CLP, biocides, or plant protection product dossiers.

Related analyses

• OECD 423 – Acute oral toxicity, method by acute toxicity class
• 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
• Non-clinical evidence: in vitro data, QSAR, cross-readings for decision-making