This is the second in our run through the regulatory safety testing suite, and it picks up where the last one left off.
If a skin irritation test comes back showing tissue viability below the threshold, you know the substance is hazardous. What you don’t yet know is whether it’s an irritant or something considerably worse. Corrosion means irreversible damage, visible necrosis, and it carries a different classification and a different label.
Test Guideline 431 is the test that answers that question, and depending on what you already know about a substance, it can also be the place to start. It uses reconstructed human epidermis, and the OECD’s own description notes that the guideline does not require the use of live animals or animal tissue.
Why corrosion was solved before irritation
TG 431 arrived six years ahead of TG 439, the equivalent guideline for skin irritation, despite both using essentially the same tissue system. That gap isn’t an accident.
Corrosion is the cruder endpoint. It’s defined as irreversible tissue damage, visible necrosis, and the underlying mechanism is relatively straightforward: a corrosive chemical penetrates the stratum corneum by diffusion or erosion and kills the cells beneath. A reconstructed epidermis with a functional barrier will show that clearly.
Irritation is harder. It’s reversible by definition, which means the method has to distinguish damage that will heal from damage that won’t, and do it from a fixed timepoint reading. Getting the RhE models to that level of discrimination took another six years of work.
The two guidelines are designed to work together, and the OECD’s Guidance Document 203 sets out how. It describes an integrated approach built in three parts: review of existing data, weight-of-evidence analysis, and additional testing where it’s needed. Testing isn’t always necessary.
Where it is, the sequence depends on what you already expect. If there’s reason to suspect a substance is corrosive, the top-down approach starts with TG 431 and moves to TG 439 only if the corrosion result is negative. If a substance is expected to be non-irritant, the bottom-up approach starts with TG 439 instead and escalates to TG 431 only if it comes back positive.
Either way, a substance confirmed as corrosive doesn’t need an irritation test, and one confirmed as non-irritant doesn’t need a corrosion test. The entry point is chosen to reach the answer in as few studies as possible.
The MTT problem
The original TG 431 gave a binary answer: corrosive or non-corrosive. Sub-categorisation into GHS Category 1A and 1B/1C came later, and the reason why is a good illustration of a guideline improving.
The method measures cell viability through MTT conversion. Viable cells reduce the dye to a blue formazan salt, and the amount of formazan indicates how much living tissue remains. The problem is that some chemicals reduce MTT directly, by their own chemistry, with no cells involved. That produces a colour change indistinguishable from cellular viability, and the tissue looks healthier than it is.
In the original ECVAM validation studies, controls for direct MTT reduction weren’t performed. Category 1A and 1B/1C chemicals that were direct MTT reducers came out underpredicted as a result, and the same issue affected all the validated RhE corrosion methods.
Once that was identified, the OECD recommended retesting 82 chemicals across the validated methods with adapted controls in place, using killed tissues to isolate the non-cellular reduction. The retesting confirmed the hypothesis, and TG 431 was revised in 2015 to permit sub-categorisation.
The guideline has been updated several times since, in 2016 and 2019, as further RhE models were added.
What the test does
Reconstructed human epidermis is pre-warmed, then test items are applied directly to the tissue surface. Triplicate models are dosed at two exposure durations, three minutes and 60 minutes, with ultrapure water as the negative control and potassium hydroxide as the positive.
Viability is measured by MTT conversion, with the formazan extracted and quantified by absorbance spectrophotometry at 570nm, and readings compared against negative controls.
The classification logic uses both timepoints. Viability below 50% at three minutes indicates a corrosive. If viability holds above 50% at three minutes but falls below 15% at 60 minutes, the item is still classified as corrosive. Only if it clears both thresholds is it non-corrosive. Sub-categorisation into 1A or 1B/1C is available where required.
Where it sits now
This is largely a chemicals endpoint rather than a cosmetics one, and the relevant framework is REACH and the CLP Regulation 1272/2008.
The position there has shifted decisively. Following the May 2016 update to the REACH annexes, in vivo testing for skin irritation and corrosion is no longer a standard information requirement. It sits as an Annex VIII column 2 adaptation, meaning it is required only where the in vitro methods are not applicable or the results are not adequate for classification. ECHA’s endpoint-specific guidance is clear that in vitro methods should be used wherever possible.
In practical terms, the in vitro test is now the default and the animal test is the exception you have to justify.
We run TG 431 to GLP under our MHRA accreditation, in fully animal-product-free conditions, which means no animal-derived serum or components anywhere in the culture system. Data carries recognition across all territories participating in the OECD’s Mutual Acceptance of Data arrangement.
If you need corrosion data for a REACH registration or a CLP classification, get in touch and we’ll talk through what your substance needs.
Email info@x-cellr8.com or call +44 (0)1925 607 134.
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