Assessing skin and eye irritation is a crucial step in ensuring the safety of chemicals, cosmetics, biocides, detergents, and many industrial raw materials. OECD guidelines—international standards in regulatory toxicology—help determine whether a product can cause reversible inflammatory reactions on the skin or eyes, or whether it poses a risk of more serious damage requiring strict classification.
With evolving European regulations (REACH, CLP, Cosmetics Regulation) and the ban on animal testing in certain sectors, in vitro methods have become widely adopted, offering standardized, ethical, and scientifically robust approaches. Understanding the operation and implications of OECD 404, 405 , 431 , 439, and 492 is now essential to ensure regulatory compliance and user safety throughout a product's lifecycle.
Table of Contents
Understanding skin and eye irritation: toxicological definitions
What is skin irritation?
Skin irritation is a reversible inflammatory reaction that occurs after direct contact of a substance, mixture, or finished product with the skin. Unlike an allergic reaction, irritation does not involve an immunological mechanism; it is purely local and results from an alteration of the skin barrier or a disruption of cellular balance.
Commonly observed clinical signs include:
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erythema (redness),
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edema,
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dryness or peeling,
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burning or tingling sensations.
From a regulatory standpoint, skin irritation is clearly distinct from skin corrosion, which involves deep, irreversible damage, generally associated with the destruction of skin tissue. This difference is essential for CLP classification: an irritant substance will be classified as Skin Irrit. 2, while a corrosive substance will fall under categories Skin Corr. 1A, 1B, or 1C.
What is eye irritation?
Eye irritation refers to a reversible affecting the eye, including:
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the cornea,
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the conjunctiva,
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or the iris.
It generally manifests as redness, increased blood flow, tearing, transient corneal opacity, or iris irritation. Again, the aim is to differentiate these reversible from true corrosion or severe eye damage, which would cause irreversible tissue damage.
This distinction is the very basis of the regulatory classification into two main categories under CLP:
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Eye Irritant. 2 for eye irritants,
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Eye Dam. 1 for substances causing serious injury.
Because the eye is a particularly sensitive organ, small amounts of substances can trigger significant reactions, hence the importance of a precise and standardized toxicological assessment.
Why perform these tests?
Skin and eye irritation tests play a crucial role in product safety and regulatory compliance . They allow us to:
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to guarantee the protection of end users (workers, consumers),
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to prevent the risk of chemical accidents,
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determine the CLP classification and regulatory labelling,
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to meet the requirements of international regulations:
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REACH for chemical substances,
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Regulation 1223/2009 for cosmetics (exclusively in vitro methods),
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Biocidal Products Regulation (BPR),
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Detergents (EC 648/2004),
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industrial sectors (food processing, materials, polymers, chemicals).
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Beyond legal requirements, these tests have become a matter of industrial responsibility . For manufacturers, incorrect classification can lead to product recalls, penalties, or an increased risk of incidents in the field.
The standardization provided by the OECD guidelines therefore offers a reliable, reproducible and globally recognized framework for assessing these risks.
Overview of OECD guidelines for skin and eye irritation
The OECD guidelines are now the international standard for assessing skin and eye irritation. They define standardized, reproducible, and harmonized protocols across countries, enabling reliable results that are recognized by regulatory authorities and usable in REACH, CLP, BPR, or cosmetics dossiers.
All the relevant methods for these endpoints fall into two main categories:
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In vivo methods (historical, still used in specific cases)
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In vitro methods (currently preferred, particularly in Europe)
In vivo testing: historical methods still used in some cases
Although the European Union strongly encourages in vitro methods, particularly in the cosmetics sector where animal testing is prohibited, in vivo testing is sometimes still required in complex situations, when alternative methods do not allow for complete classification or when no existing data can shed light on the risk.
OECD 404 – Skin irritation/corrosion (in vivo)
The OECD 404 test is the historical method for assessing whether a substance causes irritation or corrosion in rabbits.
General principle:
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Application of the substance to the shaved skin.
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Observation at 24, 48 and 72 hours.
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Measurement of erythema, edema and any lesions.
Measured endpoints:
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Intensity and duration of inflammatory reactions.
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Possible irreversible damage (corrosion).
Current limitations and regulations:
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Increasingly replaced by in vitro methods (OECD 431 and 439).
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Still accepted in certain REACH cases or for complex substances, potentially corrosive substances or for which alternative data are insufficient.
OECD 405 – Eye irritation/corrosion (in vivo)
OECD 405 assesses eye irritation or corrosion in rabbits after instillation of a test substance.
Parameters evaluated:
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Corneal opacity
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Redness and swelling of the conjunctiva
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Iris reaction
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Level of reversibility of lesions
Scope of use:
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Still used only when in vitro methods (OECD 492) do not allow a conclusion.
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It is very controlled, and severely restricted in cosmetics where it is prohibited.
Although effective in some cases, OECD 405 is gradually being abandoned in favor of validated alternative tests.
In vitro testing: modern methods favored by regulators
In vitro methods have become the norm, thanks in particular to:
- their high reproducibility,
- their scientific relevance,
- the reduction of animal experimentation,
- their compliance with European regulatory requirements.
OECD 431 – In vitro skin corrosion
OECD 431 makes it possible to determine whether a substance is corrosive to the skin using reconstructed human epidermis (RhE) models.
Principle:
Application of the substance on an RhE model.
Measurement of cell viability (MTT or equivalent).
Classification as corrosive 1A, 1B, 1C or non-corrosive.
Approved models:
EPISKIN™
EpiDerm™
SkinEthic™ RHE
This test helps to identify corrosive substances before proceeding to an irritation test (OECD 439).
OECD 439 – In vitro skin irritation
Reference method to replace OECD 404 in the assessment of skin irritation.
Principle:
Application of the substance to a reconstructed human epidermis model.
Measurement of cell viability:
Viability < 50% → irritant substance
Viability ≥ 50% → non-irritating
OECD Strengths 439:
Compliant with cosmetic regulatory requirements.
A fast, reliable method, recognized by European and international agencies.
This avoids the need to use animals in the vast majority of cases.
OECD 492 – In vitro ocular irritation (RhCE)
OECD 492 assesses the irritant potential of a substance on the eye using a reconstructed human cornea model (RhCE).
Approved models:
EpiOcular™
SkinEthic™ HCE
Measured criteria:
Cell viability
Integrity of the corneal epithelium
Typical interpretation:
Viability < 60% → eye irritant
Viability ≥ 60% → non-irritating
Usage:
Preferred method in cosmetics (mandatory in animal substitutes).
Applicable to chemical ingredients, formulated mixtures, and industrial products.
OECD method comparison: skin irritation vs. eye irritation
OECD tests dedicated to skin and eye irritation address similar objectives—assessing a substance's ability to trigger a local inflammatory response—but rely on different biological models and evaluation criteria.
Understanding the specifics of each method is essential for selecting the most appropriate approach for a product, optimizing its regulatory strategy, and avoiding unnecessary or costly trials.
Choosing methods according to product type
The choice of the relevant OECD test depends on several parameters:
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nature of the matrix (liquid, powder, solid, paste)
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pH , potential corrosive properties
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regulatory status of the product (cosmetic, chemical, biocide, detergent…)
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existing data in technical files
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usage history or field feedback
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CLP classification requirements and sector restrictions
Here's how the methods are applied depending on the sector:
Cosmetics (Regulation 1223/2009)
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Tests must be in vitro (animal testing is prohibited).
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Typical combination:
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OECD 431 (skin corrosion)
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OECD 439 (skin irritation)
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OECD 492 (eye irritation)
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Chemical substances (REACH)
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Step-by-step approach (IATA).
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Possibility of combining in vitro + existing data + QSAR.
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In vivo tests (OECD 404/405) reserved for complex cases or cases with no alternative.
Biocides (BPR)
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Robust data is needed for the authorization file.
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In vitro alternatives are accepted, except for certain corrosive or difficult matrices.
Detergents and cleaners
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Very often assessed via OECD 439 and OECD 492.
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Highly alkaline/acidic mixtures may require a complementary approach.
Materials, polymers, and technical products industry
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Case-by-case analyses according to physico-chemical properties.
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Almost systematic passage through RhE and RhCE tests.
Advantages and limitations of different OECD methods
In vitro methods (OECD 431, 439, 492)
Benefits :
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Compliant with modern regulations
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Ethical (animal-free)
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Highly reproducible and standardized
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Speed of execution (48 to 72 hours)
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Costs generally lower than in vivo testing
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Excellent relevance for cosmetic, chemical and industrial products
Boundaries :
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Some matrices can interfere with RhE/RhCE models (dyes, particles, very aggressive surfactants).
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They do not always allow for a complete classification in borderline cases → possible recourse to additional data.
In vivo methods (OECD 404, 405)
Benefits :
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Historical models with significant scientific hindsight.
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Useful when in vitro data do not allow for a conclusion.
Boundaries :
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Animal use is strictly regulated or prohibited (cosmetics).
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Results are sometimes more variable than in vitro models.
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Regulatory acceptability is more restricted in Europe.
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Lengthy and costly procedures.
Are you looking for an analysis?
How is a laboratory study conducted?
Pre-analytical phase
A skin or eye irritation study according to OECD guidelines always begins with a rigorous pre-analytical phase. This step is essential, as it determines the relevance of the selected test and the reliability of the results. The laboratory first analyzes the nature of the product: raw material, formulated mixture, powder, liquid, gel, paste, or solid. Physicochemical properties are examined as a priority: pH, solubility, corrosive potential, presence of aggressive surfactants, volatility, and oxidizing character. Existing information may eliminate the need for a full test: for example, an extreme pH may be sufficient to conclude that the product is classified as corrosive, in accordance with CLP regulations. Already available data (SDS, results of previous analyses, QSAR, REACH history, literature) are also taken into account. The objective is to select the most suitable OECD method: OECD 431 for skin corrosion, OECD 439 for skin irritation, OECD 492 for eye irritation, or possibly an in vivo test when justified and authorized.
Performing the test
Once the test is defined, the study follows a strict protocol in accordance with OECD guidelines. For in vitro skin tests (OECD 431 or 439), the substance is applied to a reconstructed human epidermis (RhE) model. The tissue is then incubated for a time defined by the method, and cell viability is measured using a colorimetric assay (MTT or equivalent). The decrease in viability allows the substance to be classified as irritating or non-irritating, corrosive or non-corrosive. For OECD 492 (eye irritation), the principle is similar but applied to a reconstructed cornea (RhCE) model. The study includes positive and negative controls to validate the test's performance, ensuring that the tissues react as expected according to international standards. Each test adheres to strict acceptance criteria regarding tissue quality, replicate variability, control response, and measurement consistency.
Study report
Following the test, the laboratory prepares a comprehensive report, compliant with OECD standards. This report includes a description of the product, a justification for the chosen method, the experimental conditions, the raw results, viability tables, response curves, and qualitative observations where relevant. For certain models, photographs of the tissues before and after treatment may be added to visually document the observed effects. The conclusion provides the resulting toxicological classification (irritant, non-irritant, corrosive, etc.) and, where applicable, the associated regulatory interpretation, particularly for CLP classification and labeling implications. This report constitutes internationally recognized regulatory evidence and can be included in a REACH, biocide, or cosmetics dossier, or in any quality documentation intended for industrial clients.
Regulatory impact: how do OECD results influence CLP classification?
Skin irritation classification
The results obtained using OECD methods (404, 431, 439) determine whether a substance should be classified as a skin irritant under the CLP (Classification, Labelling and Packaging) Regulation. CLP clearly distinguishes between irritation and skin corrosion, which directly influences labelling, precautions for use, and safety obligations for manufacturers. A substance identified as a skin irritant is classified as Skin Irrit. 2 , accompanied by the hazard statement H315: Causes skin irritation . If the results show irreversible damage, the classification changes to skin corrosion (Skin Corr. 1A/1B/1C). In vitro tests allow for the precise determination of this distinction, avoiding the use of animals while ensuring a high level of confidence in the classification.
Classification: Eye irritation
For the eye, the CLP classification is based primarily on the results of OECD 405 (in vivo) and 492 (in vitro) tests. A substance is classified as Eye Irrit. 2 when it causes reversible ocular effects, such as redness, irritation, or mild to moderate opacity. The associated hazard statement is H319: Causes serious eye irritation . If tests reveal persistent damage or irreversible lesions to the cornea or iris, the classification becomes Eye Dam. 1 , with the statement H318: Causes serious eye damage . OECD 492 is now widely used to determine whether substances are irritants or non-irritators, particularly in the cosmetics sector where in vivo testing is prohibited. However, in cases where in vitro testing is inconclusive (highly alkaline substances, complex matrices), the regulatory authority may recommend additional testing or an integrated approach (IATA).
Obligations according to industrial sectors
The results of skin and eye irritation tests directly influence a product's regulatory compliance on the European market. Under REACH , they are essential for the technical dossier of substances manufactured or imported in quantities exceeding one tonne per year. The cosmetics sector, governed by Regulation 1223/2009 , requires validated in vitro methods (OECD 431, 439, 492) to demonstrate the safety of ingredients and finished products. In the field of biocides (BPR), irritation data are crucial for establishing the complete toxicological profile of active substances and formulations. Detergents and cleaning products, governed by Regulation 648/2004, must also include a robust irritation assessment, particularly for surfactants. Finally, the chemical, food, pharmaceutical, and agricultural industries, as well as manufacturers of polymers and materials, must rely on OECD results to ensure compliant labeling, user safety, and facilitate market access.
How to choose the right OECD method for your product
Strategic approach by sector
The choice of the most relevant OECD method depends on a combination of factors: the nature of the product, its intended use, its regulatory status, and the data already available. In the cosmetics sector, governed by Regulation 1223/2009, in vitro tests are mandatory: the assessment of irritation therefore relies almost exclusively on OECD 431 (skin corrosion), OECD 439 (skin irritation), and OECD 492 (eye irritation). For chemical substances subject to REACH, the authorities recommend a phased approach, based on IATA (Integrated Approaches to Testing and Assessment), integrating existing data, QSARs (Quality Safety Assessment Reports), physicochemical information, and alternative methods. In vivo tests are considered only in cases where no reliable conclusion can be drawn from in vitro methods or prior data. The highly regulated biocides sector requires robust and validated data and accepts in vitro approaches when they are scientifically relevant. For detergents and cleaning products, irritation is a key criterion given the frequent presence of surfactants, and OECD 439 and 492 are the reference methods. Finally, in the materials, polymer, technical products, or agrochemical industries, the test selection often depends on the matrix, solubility, corrosion risks, or specific interactions of the product with RhE and RhCE models.
Importance of Integrated Assessment (IATA)
IATA guidelines play a central role in modern toxicological assessment strategies. They allow for the combination of multiple information sources to determine a product's classification without multiplying experimental tests. This approach includes the analysis of physicochemical data, such as pH or the presence of corrosive agents, the use of historical results or data available in the literature, the application of QSAR models, and the interpretation of in vitro tests. For example, a strongly acidic or basic product can be classified as corrosive without a full test, in accordance with CLP regulations. The integration of several OECD guidelines is also common: a product may first require a skin corrosion assessment (OECD 431), then a skin irritation test (OECD 439), or even an eye test (OECD 492), depending on its properties and intended use. A well-structured IATA strategy reduces costs, avoids unnecessary testing, accelerates market access, and ensures reliable classification that complies with international regulatory requirements.
Have these tests done with YesWeLab: expertise, partner laboratories and support
A network of expert and certified laboratories
YesWeLab collaborates with a broad network of partner laboratories specializing in toxicology, in vitro testing, chemical safety, cosmetics, biocides, and European regulations. All are equipped with materials compliant with Good Laboratory Practices (GLP), and the majority are ISO 17025 , guaranteeing the reliability, reproducibility, and conformity of analyses. OECD methods dedicated to skin and eye irritation—OECD 431, 439, 492, and, when necessary, 404 and 405—are performed there according to official protocols, using validated reconstructed epidermis or cornea models (RhE, RhCE). This expertise ensures manufacturers receive scientific quality aligned with European and international standards.
A digital platform to centralize and expedite your requests
YesWeLab offers an online platform that allows manufacturers to centralize all their analytical needs: quote requests, sample tracking, results viewing, and test management. Our network of laboratories is integrated, facilitating coordination and the rapid execution of OECD tests. Thanks to our database and automation tools, requests for skin and eye irritation tests are processed quickly and efficiently. Companies thus benefit from significant time savings and direct access to the best analytical expertise on the market.
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Whether you're developing a chemical raw material, a cosmetic product, a detergent, a biocide, or a mixture for industrial use, YesWeLab supports you in conducting your OECD tests under optimal conditions.
Entrust us with your OECD 404, 405, 431, 439, or 492 analyses and ensure the regulatory compliance of your products.
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