DEVELOPMENT OF THE EPIOCULAR™ EYE IRRITATION TEST FOR HAZARD IDENTIFICATION AND LABELLING OF EYE IRRITATING CHEMICALS IN RESPONSE TO THE REQUIREMENTS OF THE EU COSMETICS DIRECTIVE AND REACH LEGISLATION.

The recently implemented 7th Amendment to the EU Cosmetics Directive and the EU REACH legislation have heightened the need for in vitro ocular test methods. To address this need, the EpiOcular™ eye irritation test (EpiOcular-EIT), which utilises the normal (non-transformed) human cell-based EpiOcular tissue model, has been developed. The EpiOcular-EIT prediction model is based on an initial training set of 39 liquid and 21 solid test substances and uses a single exposure period and a single cut-off in tissue viability, as determined by the MTT assay. A chemical is classified as an irritant (GHS Category 1 or 2), if the tissue viability is ≤ 60%, and as a non-irritant (GHS unclassified), if the viability is > 60%. EpiOcular-EIT results for the training set, along with results for an additional 52 substances, which included a range of alcohols, hydrocarbons, amines, esters, and ketones, discriminated between ocular irritants and non-irritants with 98.1% sensitivity, 72.9% specificity, and 84.8% accuracy. To ensure the long-term commercial viability of the assay, EpiOcular tissues produced by using three alternative cell culture inserts were evaluated in the EpiOcular-EIT with 94 chemicals. The assay results obtained with the initial insert and the three alternative inserts were very similar, as judged by correlation coefficients (r2) that ranged from 0.82 to 0.96. The EpiOcular-EIT was pre-validated in 2007/2008, and is currently involved in a formal, multi-laboratory validation study sponsored by the European Cosmetics Association (COLIPA) under the auspices of the European Centre for the Validation of Alternative Methods (ECVAM). The EpiOcular-EIT, together with EpiOcular’s long history of reproducibility and proven utility for ultra-mildness testing, make EpiOcular a useful model for addressing current legislation related to animal use in the testing of potential ocular irritants.

ECVAM, EpiOcular-EIT. EpiOcular™ Eye irritation test, Eye irritation test, Hazard identification, In vitro reconstructed ocular tissue model, Intra-laboratory reproducibility, Liquids, Prediction model, QC acceptance criteria, REACH, Testing set, Training set, Validation

1,3-Diiminobenz-IIJ-isoindoline, 1,3-Diisopropylbenzene, 1,6-Hexadiene, 1,9-Decadiene, 1-Phenyl-3-pyrazolidinone, 1-Octanol, 2,2-Dimethyl-3-pentanol, 2,3-Dimethyl-2,3-dinitrobutane, 2,4-Dichloro-5-sulfamoylbenzoic acid, 2,4-Difluoronitrobenzene, 2,4-Pentanediol, 2,6-Dichloro-5-fluoro-beta-oxo-3-pyridinepropanoate, 2,6-dichlorobenzoyl chloride, 2-Aminophenol, 2-Ethyl-1-hexanol, 2-Ethylhexyl p-dimethyl-amino benzoate,2-Mercaptopyrimidine, 2-Methyl-1-pentanol, 2-Methylbutanoic acid, 2-methylpentan 3,3-Dimethylpentane, 3,3-Dithiodipropionic acid, 3-Chloropropanenitrile, 3-Methoxy-1,2-propanediol, 3-methylhexane, 3-Methyl-pentynol, 4-Fluoroaniline, 4-Formylbenzoic acid, 4-Formylbenzoic acid (100%), 4-tert-Butylphenol, Acetone, Aluminum Hydroxide, Ammonium nitrate, Anthracene, Benzalkonium chloride (10%), Benzalkonium chloride, 0.01% wlv, Benzalkonium chloride, 0.1% w/v, Betaine monohydrate, beta-Resorcylic acid, Butanol, Camphen, Cetylpyridinium bromide (6%), Cetylpyridinium bromide, 0.1% w/v, Cetylpyridinium bromide, 1% w/v, Cetyltrimethyl ammonium bromide, 10% w/v, Chlorendic anhydride, Chlorhexidine, cis-Cyclooctene, Cyclohexanol, Cyclopentanol, Di(propylene glycol) propyl ether, Dibenzoyl-L-tartaric acid, Dibenzyl phosphate, Diethylethanolamine, Diisobutyl Ketone, Dodecane, Ethanol, Ethanol, 10% vlv, Ethyl acetate, Ethyl trimethyl acetate, Ethyl-2-methylacetoacetate, Ethylenediaminetetraacetic acid dipotassium salt dihydrate, Gluconolactone, Glycerol, Granuform, Imidazole, 20% wlv, Iminoch"benzyl, Iso-octyl acrylate, Isopropanol, Isopropyl bromide, Lactic Acid (100%), Magnesium carbonate hydroxide pentahydrate, Maneb, m-Dinitrobenzene, Methoxyethyl acrylate, Methyl acetate, Methyl amyl ketone, Methyl cyanoacetate, Methyl cyclopentane, Methyl ethyl ketone (MEK), Methyl isobutyl ketone, Methyl trimethyl acetate, n,n-Dimethylguanidine sulfate, n-Butyl acetate, n-Hexanol, n-Octyl bromide, Nonylphenyl-polyethylene glycol, Phenothiazine, Phenylbutazone, p-Nitrobenzoic acid, Poly(ethylene glycol) monolaurate, Polyethylene glycol 400, Potassium tetrafluoroborate, Promethazine Hydrochloride, Propyl4-hydroxybenzoate, Propylene glycol, Quinacrine, Sodium deoxycholate, 10% w/v, Sodium hydroxide (1%), Sodium lauryl sulphate, 1% w/v, Sodium monochloroacetate, Sodium oxalate, Styrene, Tetraaminopyrimidine sulphate, Tetrahydrofuran, Tetraoctylammonium bromide, Toluene, Triton X-100 (5%), Triton X-100, 1% vlv, Tween 80, 10% v/v, Tween 80®, Xylene