Reconstructed Human Cornea-like Epithelium (RHCE) Test Method for Eye Hazard Identification (OECD 492B)

This test method is the first single non-animal eye irritation test method which can be used to distinguish between GHS Category 1, Category 2, and non-categorized substances and mixtures. Until recently, resolving GHS Category 2 Ocular Irritants had relied on a Weight-of-Evidence approach as the use of no single in vitro assay had been able to distinguish between Category 1, Category 2 and non-categorized test materials.With the approval of the OECD 492B test guideline, the determination of these classifications is now possible.

Test substances are classified for eye irritation hazards within the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS) according to the following categories:

  • GHS Category 1 – test substances or mixtures that induce serious eye damage, or decay of vision which is not fully reversible after a single exposure
  • GHS Category 2 – eye irritation and changes in the eye, which are fully reversible after a single exposure to a test substance
  • GHS No Category (No Cat) – test substances not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification.

SkinEthic™ HCE (Human Corneal Epithelium) tissues are treated with test substances following a time-to-toxicity protocol. Since corneal cell damage and loss of viability are highly related to adverse impacts on corneal function and visual acuity, viability of the HCE tissues is measured following test substance exposure using the MTT reduction viability endpoint.

Based on the Prediction Models presented below, a definitive categorization can be obtained. IIVS now offers this evaluation – please contact clientservices@iivs.org for further information.

Prediction Model for UN GHS Categorization

ProtocolNo CategoryCategory 2Category 1
TTL
Time-to-toxicity for Liquids
Mean tissue viability > 50% following all three exposuresAny other combination of viability outcomes 1Mean tissue viability ≤ 50% following all three exposures
TTS
Time-to-toxicity for Solids
Mean tissue viability > 40% after the 30 minute exposure
and
> 60% after the 120 minute exposure
Any other combination of viability outcomes 1Mean tissue viability ≤ 40% after the 30 minute exposure
and
≤ 60% after the 120 minute exposure
1 any combination of viability outcomes other than those defined for No Category and Category 1

OECD TG 492B Protocol for SkinEthic™ HCE Tissues

ProtocolExposure Times and ConditionsDose Volumes and TreatmentsTreatment TerminationPost Treatment Soaking
TTL
Time-to-toxicity for Liquids
5 min (neat)
16 min (20% w/v in diH2O)
120 min (20% w/v in diH2O)

37°C, 5% CO2, ≥ 95% RH
10 μL Ca2+/Mg2+-free DPBS
+ 80 μL test material

For viscous liquids, use a nylon mesh
25 mL of Ca2+/Mg2+-free DPBS
2 mL per rinse
10 min at RT in 4 mL culture medium
TTS
Time-to-toxicity for Solids
30 and 120 min (neat)

37°C, 5% CO2, ≥ 95% RH
80 mg test substance (ground to fine powder)
+ 80 μL diH2O

Use a nylon mesh
25 mL of Ca2+/Mg2+-free DPBS
2 mL per rinse
30 min at RT in 4 mL culture medium