OECD acute toxicity tests are an essential step in assessing the safety of chemical substances. These standardized studies identify the potential toxic effects of a single exposure, whether oral, dermal, or inhaled. Internationally recognized, OECD guidelines ensure the reliability, comparability, and regulatory acceptance of the results.
This article provides a comprehensive overview of the main methods—OECD 402, 403, 420, 423, and 425—to help you understand their objectives, differences, industrial applications, and the criteria for selecting an assessment strategy tailored to your regulatory and technical needs.
Table of Contents
Introduction
Why is acute toxicity a critical stage?
Acute toxicity measures the adverse effects that can occur after a single, short-term exposure to a substance. In industrial projects, this information is used to ensure safe development, guide classification and labeling, and protect both end users and workers. It supports concrete operational decisions: selection of protective equipment, realistic exposure scenarios, usage restrictions, preparation of Safety Data Sheets (SDS), and compliance with regulatory requirements. Because it is performed early in a product's lifecycle, acute toxicity assessment helps reduce the risk of non-compliance, avoid costly iterations, and provide registration dossiers with robust and comparable data.
The role of OECD guidelines
The OECD guidelines offer harmonized and internationally recognized protocols for assessing acute toxicity via three major exposure routes: oral, dermal, and inhalation. They structure trial preparation, experimental conduct, clinical observations, statistical analysis, and interpretation of results. Their adoption facilitates the acceptance of studies by competent authorities, improves comparability between laboratories, and supports the 3Rs principles by reducing the number of animals used and refining the criteria for humane cessation. In a B2B context, these common references ensure secure communication between clients, laboratories, and authorities, accelerate decision-making, and limit technical or regulatory revisions.
Objectives and scope of the article
This article provides an operational overview of the OECD guidelines dedicated to acute toxicity most commonly used by industry:
– OECD 402 (dermal route)
– OECD 403 (inhalation)
– OECD 420 (predetermined dose method, oral route)
– OECD 423 (class method, oral route)
– OECD 425 (dose adjustment method, oral route)
For each test, we will describe its purpose, key implementation points, strengths and limitations, as well as its typical uses in regulatory submissions and risk management. The objective is to help quality, R&D, and regulatory managers choose the most relevant testing strategy based on the substance profile, the level of information required (GHS/CLP category versus LD50/LC50 estimation), ethical constraints, and regulatory acceptance requirements. Practical recommendations and comparative benchmarks will clarify the choice between tests 420, 423, and 425 for oral administration, and will position tests 402 and 403 when dermal or respiratory exposure is critical. Finally, we will outline how expert support compliant with GLP/GLP streamlines planning, improves the reliability of results, and accelerates data integration into industrial documentation systems.
General context
Definition of acute toxicity
Acute toxicity refers to adverse effects that occur following a single exposure or several closely spaced exposures over a short period (generally less than 24 hours). It aims to determine the dose or concentration capable of causing harmful effects, or even death, in a test animal.
The results allow the calculation of reference values such as the **LD50** (median lethal dose, expressed in mg/kg body weight) for oral and dermal exposures, or the LC50 (median lethal concentration) for inhalation exposures.
These parameters are then used to classify the substance according to the Globally Harmonized System (GHS) and, in Europe, to determine its labeling in accordance with the CLP Regulation (Classification, Labelling and Packaging – Regulation (EC) No 1272/2008).
Acute toxicity thus constitutes the first step in hazard characterization, before studies of subchronic, chronic, carcinogenic, or reproductive toxicity.
The ethical principles of the 3Rs
Acute toxicity studies traditionally involve animal experimentation. However, for several years now, the scientific and regulatory community has been committed to regulating and reducing this practice according to the 3Rs principle:
– Reduction: minimizing the number of animals used while maintaining the scientific relevance of the results.
– Refinement: improving housing conditions and experimental procedures to limit pain, distress, or suffering (humane stopping points, close observation, anesthesia during sampling).
– Replacement: using validated alternative methods when possible (in vitro models, in silico models, QSAR, cross-reading).
The most recent OECD guidelines (OECD 420, 423, and 425) fully incorporate these principles. They replace older approaches (such as the now-withdrawn OECD 401) that required large groups of animals and relied primarily on mortality. These modern methods significantly reduce the number of animals tested and emphasize detailed clinical observations to assess toxicity.
The international regulatory framework
Acute toxicity testing is an integral part of regulatory requirements in many international frameworks. It is a mandatory step in the evaluation of chemical substances, whether industrial, biocidal, plant protection, cosmetic, or transport chemicals.
Key frameworks concerned:
– REACH Regulation (EC No. 1907/2006): requires the provision of toxicological data for the registration of chemical substances produced or imported in quantities exceeding 1 tonne per year.
– CLP Regulation (EC No. 1272/2008): defines classification and labeling criteria according to acute toxicity (categories 1 to 5 for each route of exposure).
– Biocidal Products Regulation (EU No. 528/2012) and plant protection regulations: require acute toxicity studies to assess the safety of active substances and formulated products.
– ADR/UN transport regulations: the classification of hazardous materials is based, in particular, on acute toxicity.
– Occupational health and safety regulations: the results guide the preparation of Safety Data Sheets (SDS), occupational exposure limits, and precautions for use.
Tests conducted according to OECD guidelines, in compliance with Good Laboratory Practices (GLP), are recognized under the OECD Mutual Recognition Agreement (MRA). This means that a study conducted in one member country is accepted by all competent authorities in other signatory countries, preventing duplication of trials.
Towards an integrated approach to risk assessment
Before initiating an acute toxicity trial, it is recommended to evaluate the information already available:
– Existing data from the scientific literature or for similar substances.
– Read-across models that predict toxicity from structurally similar substances.
– Quantitative Structure-Activity Relationship (QSAR) and in silico models, which estimate toxicity from chemical structure.
– In vitro tests on cell lines or reconstructed tissues.
These approaches often make it possible to forgo in vivo testing or to adapt the experimental strategy by choosing the least invasive and most ethical method.
Risk assessment thus becomes an integrated process, combining existing data, predictions, and targeted testing, in accordance with the recommendations of the ECHA (European Chemicals Agency) and the requirements of the OECD guidelines.
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Overview of OECD guidelines on acute toxicity
The OECD guidelines provide an international methodological framework for assessing acute toxicity. They precisely define the experimental conditions, the animal species to be used, the exposure durations, and the observation parameters, thus ensuring the comparability of results between laboratories.
Each method is associated with a route of exposure (oral, dermal, or inhalation) and a distinct objective: to obtain a quantitative value (LD50/LC50) or a toxicity category for the GHS/CLP classification.
The main exposure pathways studied
Acute toxicity tests focus on three exposure pathways representative of the actual risks to humans and the environment:
– Oral route: accidental or intentional ingestion of a substance (e.g., food products, nutraceuticals, medications, additives).
– Cutaneous route: direct contact with the skin or mucous membranes, particularly for cosmetic, biocidal, or industrial products.
– Inhalation route: exposure via the respiratory tract to gases, vapors, aerosols, or dust.
For each route, specific methods are defined:
– OECD 402: acute toxicity via the dermal route
– OECD 403: acute toxicity via inhalation
– OECD 420, 423 and 425: acute toxicity via the oral route, according to three distinct approaches (predetermined dose, class method, dose adjustment method).
These protocols aim to characterize the systemic toxicity of a substance after a single exposure, while respecting the ethical principles of the 3Rs.
Acute oral toxicity tests: 401, 420, 423 and 425
The oral route is the most commonly studied in toxicology because it represents the most likely route of exposure for humans.
Historically, the OECD 401 test (now discontinued) aimed to determine an oral LD50 from groups of animals exposed to multiple doses. Although robust, this method used a large number of animals and resulted in significant mortality. It has been replaced by three modernized approaches:
• OECD 420 – Predetermined Dose Method
This method assesses the toxicity of a substance using fixed dose levels (5, 50, 300, 2000 mg/kg).
The objective is no longer to determine an exact LD50 value, but to classify the substance into a toxicity category.
It is characterized by:
– A limited number of animals (often 5 per dose)
– An ascending or descending sequence depending on the observed response
– A 14-day observation period
– A significant reduction in experimental mortality
Discover the OECD 420 service offered by YesWeLab
• OECD 423 – Acute Toxicity Class Method
OECD 423 guidelines are based on a sequential approach that classifies substances into a GHS/CLP category (from 1 to 5) without determining a precise LD50.
They also rely on fixed dose increments (5, 50, 300, 2000 mg/kg) administered to a single sex (often females).
Observations of mortality and clinical signs guide the subsequent dose and the assigned category.
This method is rapid, cost-effective, and widely recognized by regulatory authorities.
Discover OECD service 423 – Acute Oral Toxicity
• Up-and-Down Procedure
OECD guideline 425 aims to estimate the LD50 using a sequential procedure.
Only one animal is exposed at a time; the subsequent dose is adjusted up or down based on observed survival or mortality.
This approach significantly reduces the number of animals used while providing a quantitative estimate with a statistical confidence interval.
It is particularly suitable when a numerical LD50 value is required for risk modeling or inter-product comparisons.
Non-oral toxicity tests: skin and inhalation
• OECD 402 – Acute dermal toxicity
. This test assesses systemic and local toxicity after a single application to the skin.
It involves applying the substance to a shaved area on the animal's back, generally covering 10% of the body surface area, for 24 hours, and then observing the effects for 14 days.
The resulting dermal LD50 is used to classify the substance according to GHS/CLP.
This test is essential for formulations likely to come into contact with the skin (cosmetics, biocides, adhesives, solvents, polymers, etc.).
Discover OECD service 402 – Acute Skin Toxicity
• OECD 403 – Acute Inhalation Toxicity
The OECD 403 test assesses the effects of a single respiratory exposure to a gas, vapor, aerosol, or dust.
Animals are exposed for 4 hours to a controlled test atmosphere (either "nose-only" or "whole chamber" mode).
Parameters monitored include the LC50 (median lethal concentration), respiratory, neurological, and systemic effects, as well as symptom reversibility.
This is a technically complex test, requiring a device for generating and characterizing the aerosol (particle size, homogeneity, concentration).
Criteria for choosing a method
The choice of guideline depends on several factors:
– Relevant route of exposure: ingestion, skin contact, inhalation.
– Desired level of information: LD50/LC50 value or simple GHS category.
– Expected degree of toxicity: low or high toxicity.
– Ethical constraints: minimize animal suffering and the number of tests.
– Regulatory requirements: some authorities require a numerical estimate.
– Existing data: QSAR, in vitro, cross-reading, previous studies.
– Laboratory technical capabilities: exposure chambers, dosing systems, GLP/GLP compliance.
In practice, OECD methods 420 and 423 are often preferred for substances of moderate to low toxicity, while 425 is recommended when a precise LD50 is required. Methods 402 and 403 apply when the dermal or inhalation route is deemed appropriate based on the product's intended use.
Interpretation and classification of results
The results obtained during an OECD acute toxicity test are translated according to the GHS/CLP classification criteria.
The categories range from 1 (extremely toxic) to 5 (slightly toxic), depending on the dose or concentration causing mortality in 50% of the test animals.
Examples of thresholds (oral route):
| GHS Category | LD50 (mg/kg body weight) | Hazard statement | Pictogram |
|---|---|---|---|
| Category 1 | ≤ 5 | Danger – Acutely Toxic 1 | Skull and crossbones on two crossed tibias |
| Category 2 | > 5 – 50 | Danger – Acute Toxic 2 | Skull and crossbones on two crossed tibias |
| Category 3 | > 50 – 300 | Danger – Acutely Toxic 3 | Skull and crossbones on two crossed tibias |
| Category 4 | > 300 – 2 000 | Warning – Harmful if swallowed | Exclamation mark |
| Category 5 | > 2 000 – 5 000 | May be harmful | Exclamation mark |
These classifications directly guide labeling, risk management, hazard communication, and regulatory compliance (REACH, CLP, GPR, ADR transport).
The results must be accompanied by a comprehensive GLP/GLP report, including clinical observations, justification for dose selection, statistical analyses, and regulatory interpretation.
Critical comparison and recommendations
The OECD Acute Toxicity Guidelines (402, 403, 420, 423, and 425) share a common goal: to assess the hazard of a substance after a single exposure.
However, their experimental principles and analytical objectives differ depending on the exposure route and the level of information required. Understanding how these complement each other allows for the adoption of the most appropriate testing strategy, taking into account the nature of the substance, regulatory constraints, and the company's ethical commitments.
Comparison of the main OECD methods
Oral toxicity tests (420, 423, 425) are the most commonly used and employ distinct approaches:
| Method | Main objective | Type of result | Number of animals | Benefits | Boundaries |
|---|---|---|---|---|---|
| OECD 420 | Identify the category of acute toxicity | GHS/CLP Category | Reduced | Simple, inexpensive method, reducing the number of animals | No precise LD50 value |
| OECD 423 | Classify the substance according to toxicity levels | GHS/CLP Category | Reduced to moderate | A fast, widely recognized, ethical method | Less precise in borderline cases |
| OECD 425 | Estimate the DL50 using a sequential procedure | Quantitative value (LD50) | Moderate | Provides a useful numerical estimate for calculating the safety margin | A more statistically complex method |
| OECD 402 | Evaluate toxicity after cutaneous application | cutaneous LD50 | Moderate | Covers the dermal route, essential for contact products | Less relevant for substances poorly absorbed by the skin |
| OECD 403 | Assess inhalation toxicity | CL50 (gases, vapors, aerosols) | Moderate to high | Required for volatile or aerosolized products | Expensive and technically complex |
Thus, methods 420 and 423 are often preferred for rapid classification and ethical assessment , while 425 is chosen when a numerical estimate is required. Tests 402 and 403 complement the strategy when the dermal or respiratory route is relevant for the product's use.
Choose the appropriate method
The choice of guideline depends on several criteria:
Substance profile : volatile liquids, dusts or aerosols often require an inhalation test (OECD 403), while formulations intended for skin contact warrant an OECD 402 study.
Regulatory objective : some frameworks (transport, biocides, CLP) require a numerical value, others are satisfied with a toxicity category.
Availability of existing data : if reliable results are already available on similar substances, a cross-reading approach may avoid a new trial.
Ethical and economic constraints : OECD methods 420 and 423 are designed to minimize the number of animals, while reducing the cost and duration of trials.
Before any planning, it is recommended to carry out a preliminary toxicological strategy analysis : compilation of available data, evaluation of the most relevant mode of exposure and scientific justification of the methodological choice.
Complementary and alternative methods
Advances in toxicological knowledge and digital tools are enabling the complementation or replacement of certain in vivo tests. The following approaches are increasingly being integrated:
QSAR (Quantitative Structure-Activity Relationship) : predictive modeling of toxicity from chemical structure.
Read-across : extrapolation from similar substances already tested.
In vitro tests : use of cell models or reconstructed tissues.
In silico methods and toxicological AI : prediction algorithms combining molecular, physicochemical and pharmacokinetic data.
Waiving : scientific justification for the absence of testing when existing data are sufficient to conclude on non-toxicity or the absence of relevant exposure.
These approaches help to reduce the use of animal testing , while maintaining the scientific robustness required by the authorities (ECHA, EFSA, OECD).
Good experimental and regulatory practices
The quality of an acute toxicity test depends on the rigor of the protocol and the laboratory's regulatory compliance. The essential elements to verify before implementation are:
GLP/GLP compliance : required for any study submitted to the authorities under the REACH or GLP regulations.
Complete traceability of raw data and experimental conditions.
Validation of equipment (exposure chambers, dosing systems, concentration control).
Internal quality assurance and independent supervision of the analytical process.
Drafting a comprehensive report that complies with OECD standards and is directly usable in IUCLID.
Rigorous planning helps to optimize the budget, avoid duplication of trials and ensure international recognition of results.
Practical aspects for manufacturers
Selecting a testing provider
Choosing a partner laboratory is a crucial step in ensuring the quality and regulatory validity of the results. Before placing an order, it is recommended to verify several key points:
GLP/GMP accreditation (Good Laboratory Practices) : essential for the study to be recognized by European authorities (ECHA, EFSA, ANSES).
Experience in the relevant field : some substances (gases, complex formulations, polymers) require specific skills.
Suitable equipment : exposure chambers for inhalation tests, automated dosing systems, atmospheric concentration control.
Regulatory competence : the laboratory must be able to provide usable reports in IUCLID , compatible with REACH or BPR requirements.
Transparency and communication : regular monitoring of the steps (preparation, testing, validation, QA review) is essential to anticipate any difficulties.
YesWeLab rigorously selects its partner laboratories according to these criteria. This approach guarantees its clients a network of accredited experts, capable of meeting a variety of needs (chemicals, biocides, materials, cosmetics, etc.) while respecting international standards.
Planning, budget and deadlines
Implementing an acute toxicity test requires methodical preparation:
Framing phase : analysis of the substance, identification of the relevant exposure route, choice of the most appropriate OECD protocol (420, 423, 425, etc.).
Experimental phase : preparation of sample batches, exposure period and post-exposure observation (often 14 days).
Analysis and validation : review by internal quality assurance (QA) and drafting of the final report.
Depending on the complexity of the test, the total timeframe typically ranges from 6 to 12 weeks .
Costs may vary depending on:
the route of exposure (inhalation tests being more expensive than oral tests),
the nature of the substance (powder, liquid, gas),
and the need for further analyses (histopathology, biochemistry).
YesWeLab supports its clients in planning these tests, evaluating technical feasibility and optimizing the budget through its network of partner laboratories in France and Europe.
Analysis and integration of results
Once the final report is validated, the acute toxicity data is used at several levels:
CLP classification and labelling : assignment of toxicity categories, hazard statements and pictograms.
Preparation or updating of Safety Data Sheets (SDS).
Regulatory submission REACH , BPR , phytosanitary or cosmetic dossiers .
Calculation of safety margins (SOM) to assess the risks of human exposure.
Transport compliance (ADR/UN) for substances classified as dangerous.
A rigorous interpretation is essential: the result is not limited to an LD50/LC50 value, but must be placed within a context of use and exposure. YesWeLab's regulatory expertise allows this scientific data to be transformed into directly actionable information for risk management and product compliance.
Examples of strategies according to product profiles
Slightly toxic substance or already studied : use of an OECD 420 or 423 test to confirm low toxicity and avoid duplication of tests.
Formulation containing solvents or polymers : perform a skin test (OECD 402) to assess the risk of occupational contact.
Sprayed or gaseous product : implementation of an inhalation test (OECD 403) to characterize respiratory toxicity.
New molecule with no existing data : combination of an oral test (OECD 425) and QSAR modeling to obtain an accurate estimate while respecting the 3Rs.
These examples show that the choice of a test depends not only on regulations, but also on the context of exposure , the expected toxicological profile and the level of evidence required .
Conclusion
OECD acute toxicity tests are a key pillar of chemical safety assessment . Through harmonized protocols, they make it possible to characterize the hazard associated with a single exposure, determine the relative toxicity of a compound, and ensure compliance with international regulations such as REACH, CLP, BPR, and ADR for the transport of dangerous goods.
These guidelines — OECD 402 (dermal), 403 (inhalation), 420, 423 and 425 (oral) — provide manufacturers with a reliable scientific basis for classifying their substances, developing safety data sheets and anticipating regulatory requirements. Each guideline has specific features that address distinct needs:
the oral tests (420, 423, 425) are mainly aimed at GHS classification and estimation of systemic toxicity;
Skin and inhalation tests ( 402, 403) complete the evaluation according to the routes of exposure relevant to the use of the product.
The evolution of methods over time also reflects a strong commitment to scientific ethics. Modern guidelines are based on the 3Rs (Reduce, Refine, Replace) and encourage the use of integrated approaches, combining existing data, QSAR modeling, cross-reading, and in vitro testing. This international dynamic aims to limit animal experimentation while maintaining scientific rigor and regulatory reliability.
