Eye irritation – OECD 492b

OECD Guideline 492B describes an in vitro test method using reconstituted human corneal epithelium (RhCE) for identifying ocular hazards . Adopted in 2022 and updated in June 2024, it allows a substance or mixture to be classified into one of the three GHS ocular hazard categories — category 1 (serious eye damage), category 2 (eye irritation) or no classification — from a single test, without animal testing.

This is what radically distinguishes it from OECD492, its predecessor, which uses the same type of tissue model but only allows the classification to be ruled out, without being able to discriminate between irritation and serious injury.

The "time-to-toxicity" principle: what changes everything

Whereas the 492 measures cell viability after a single exposure time, the 492B relies on a so-called "time-to-toxicity" (TTT) : the product is applied to the corneal tissue for several increasing durations, and viability is measured after each.

The logic is simple and biologically sound: a product causing severe eye damage destroys tissue rapidly, while a simple irritant only reduces viability after prolonged exposure. It is the kinetics of the damage, and not just its final stage, that provides the classification information. By cross-referencing the results obtained at different time points, the predictive model distinguishes between the three categories.

Prediction model: how to read the result

The validated model for this guideline is the SkinEthic™ HCE TTT (reconstituted human corneal epithelium, time-to-toxicity protocol), with two distinct protocols depending on the product form.

Liquids Protocol

The three exposures in the liquid protocol are carried out at increasing durations and concentrations in severity: 5 minutes on pure product, then 16 minutes and 120 minutes on a 20% (w/v) water dilution.

Solid Protocol

Solid products are tested in their pure form, as a finely ground powder, at 30 and 120 minutes of exposure.

Each trial includes a negative control and a positive control, the viability values ​​of which must meet the guideline acceptance criteria for the series to be validated.

OECD 492 or OECD 492B: a comparison

To learn more about this choice, see our article OECD 492 or 492B: which method to choose to assess eye irritation?

Scope of application

The OECD 492B test applies to both substances and mixtures, and covers a wide range of physical forms: liquids, solids, semi-solids, and waxes. This versatility makes it a particularly suitable method for formulated finished products, which are often difficult to test using other approaches.

It is relevant for:

• the cosmetics industry — ingredients and finished products, within the framework of Regulation (EC) No 1223/2009 which prohibits animal testing;
• chemical substances subject to REACH, for registration and CLP classification;
• detergents, cleaners and maintenance products, whose eye profile directly influences the labeling;
• biocides, in BPR authorization files;
• plant protection products and industrial raw materials.

Why choose the 492B over a sequence of tests?

Historically, classifying a product as category 1 or 2 required a series of methods: an exclusion test, followed by an ex vivo such as OECD 437 (BCOP) or OECD 438 (ICE). Each step adds time, cost, and uncertainty.

The 492B renders this sequence unnecessary in many cases:

• one single trial, one single classification — no more waiting between two stages;
• only one sample to provide, only one experimental setup;
• no animal-derived tissue, unlike BCOP (bovine corneas) and ICE (chicken eyes) — an argument that counts more and more in cosmetic dossiers and CSR policies;
• a model of human origin, therefore more physiologically relevant to predict the response in humans.

Why conduct this trial with YesWeLab?

• Properly equipped laboratories — 492B is a recent method: not all toxicology laboratories offer it. We work with those who use it routinely;
• The right protocol for your matrix — liquid, solid, semi-solid or wax: the choice of protocol and sample preparation determine the validity of the result. We define this point before launch;
• A consistent testing strategy — if your case combines eye irritation, skin irritation and corrosion, we build the most economical sequence rather than piling up studies;
• A report that can be directly used in your REACH, CLP, cosmetics or biocide files;
• A single point of contact and a centralized quote request, regardless of the number of laboratories involved.

Frequently Asked Questions

What is the difference between OECD 492 and OECD 492B?

OECD 492 is an exclusion method: it only allows the identification of products that do not require classification. OECD 492B, through its multi-time exposure approach, discriminates between category 1 (serious eye damage), category 2 (irritation), and no classification in a single test.

Can OECD 492B replace OECD 405 in vivo?

In the cosmetics sector, animal testing is prohibited: in vitro testing is the only permitted method. For other regulations, 492B is a recognized standalone method, to be integrated into an Integrated Testing and Evaluation Approach (IATA). Its acceptance depends on the application and the relevant authority; we can help you define this aspect from the outset.

My product is a formulated mixture. Is it testable?

Yes. The guideline explicitly covers mixtures, including those in semi-solid or waxy form. This is even one of its main advantages over older methods.

How long does the trial last?

The experimental phase takes several days. The overall timeframe depends on the availability of the reconstituted tissues and the chosen test plan; it will be clearly communicated to you with the quote.

How much product should be supplied?

A few grams or milliliters are usually sufficient. The exact quantity depends on the number of replicates, the applicable protocol, and any preliminary interference tests.

What happens if my product interferes with the MTT test?

Colored products or products capable of spontaneously reducing MTT can distort the reading. This is a known interference, addressed by additional controls outlined in the guideline—provided it is anticipated. Please inform us of any significant coloring when requesting a quote.

Is 492B accredited?

The accreditation applies to the laboratory, not the methodology. We will inform you of the scope of accreditation of the laboratory selected for your study.

Request your quote

Describe your product, its physical form, and your regulatory objective. We will identify the appropriate laboratory and provide you with a detailed quote and a firm deadline.