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 using mortality as the sole endpoint. Based on the administration of predetermined doses to a single sex (usually females), it allows for rapid classification of a substance according to GHS/CLP without the need for an exact LD50. This approach is recognized by the authorities (REACH/CLP) and easily integrates with GLP/GLP requirements, with a report that can be directly used in IUCLID.

Objectives and scope of the trial

OECD 420 provides the information needed for hazard assessment after single oral exposure and for GHS/CLP classification of acute toxicity. It aims to determine the relevant toxicity range by limiting exposure to moderately toxic doses. The study is indicated for REACH registration, biocide 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 2000 mg/kg bw (with, exceptionally and duly justified, 5000 mg/kg). The initial dose is chosen based on a scoping study and available information (physicochemical properties, in vitro/in vivo data, QSAR, analogues). The objective is to induce clear signs of toxicity without causing serious effects or mortality. Depending on the observed response (absence of toxicity, overt toxicity, mortality), the sequence continues at the higher or lower dose, until a GHS category can be assigned. The method prioritizes the reduction of animal suffering by avoiding doses known to be severe corrosives/irritants and by applying humane endpoints.

Experimental process

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

Orientation study and choice of initial dose

The scoping study uses one animal per dose level (5, 50, 300, 2000 mg/kg), with an interval of at least 24 h 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 main study generally includes five animals at the dose level considered (including the orientation animal already treated at this level). After the first dose, three routes are possible depending on the observed response: classify directly, administer the lower dose, or administer the higher dose. A level resulting in mortality during orientation is not repeated for welfare reasons. The time between groups (often 3–4 days) can be adjusted to detect possible delayed effects. A limit test at 2000 mg/kg (exceptionally 5000 mg/kg if a regulatory requirement dictates it and if it has a clear health value) can be performed when low toxicity is expected.

Observations, humanitarian criteria and pathology

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

Data, classification and deliverables

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

Advantages of the OECD 420 method

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

YesWeLab support

YesWeLab coordinates your OECD 420 testing with accredited partner laboratories (GLP/GLP, ISO 17025). We help you define the dose strategy (orientation, initial level), select the vehicle, and document the scientific justification. You benefit from digital monitoring, rigorous quality control, and a complete IUCLID-ready report that can be used in REACH/CLP, biocide, or phytosanitary dossiers.

Related analyses

• OECD 423 – Acute Oral Toxicity, Acute Toxicity Class 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
• Non-clinical support: in vitro data, QSAR, read-across for waiver