Evaluation of MTT reducers and strongly colored substances in the Short Time Exposure test method for assessing eye irritation potential.

The Short Time Exposure (STE) test evaluates eye irritation potential using a 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT assays may underpredict results for some substances that directly reduce MTT (i.e., MTT reducers) or interfere with absorbance because of their strong color (i.e., strongly colored substances). Based on previous research, we selected 25 substances as MTT reducers. Of these, 13 were expected to be MTT reducers at 5% dilution (5% MTT reducers) of the STE test condition. These 13 substances were then tested to determine whether the results were interfered from direct MTT reduction. Those 5% MTT reducers that were classified as irritants based on in vivo data were identified as irritants by the STE test. In addition, the low cell viability results at 5% dilution suggested that direct MTT reduction had not occurred. Next, the remaining 5% MTT reducers that were classified as non-irritants based on in vivo data were identified as non-irritants by the STE test. We then examined two strongly colored substances. One was classified as an irritant based on in vivo data and was confirmed as an irritant by the STE test. The other was classified as a non-irritant by the STE test. This was further evaluated using a medium that did not contain MTT; the result indicated that it was a non-irritant correctly. In conclusion, the STE test is useful for evaluating eye irritation potential without the drawback of underprediction for MTT reducers and strongly colored substances.

Expansion of the applicability domain for highly volatile substances on the Short Time Exposure test method and the predictive performance in assessing eye irritation potential.

The Short Time Exposure (STE) test method is an in vitro method for assessing the eye irritation potential of chemicals and is used to classify the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) Category 1 and No Category (NC). The method has been adopted by the Organisation for Economic Co-operation and Development (OECD) as test guideline (TG) 491 since 2015. While this method can be used to classify GHS NC, it is not suitable for testing highly volatile substances and solids other than surfactants. Here we evaluated highly volatile substances to expand the applicability domain. According to TG 491, acetone, ethanol, iso-propanol, and methyl acetate as highly volatile substances resulted in false negatives. Saline was selected as a solvent of these false negatives. In this study, mineral oil was used as the solvent, because these false negatives were amphiphilic. Based on this change, four highly volatile substances were correctly evaluated. The predictive performance for classifying GHS NC was then verified using a substance dataset constructed in reference to the Draize eye test Reference Database and STE Summary Review Document. The accuracy and false-negative rate were 86.6% (194/224) and 3.8% (3/80), respectively. Collectively, the applicability domain was expanded by changing the solvent to mineral oil for highly volatile substances, and the predictive performance for the new applicability domain including highly volatile substances was excellent. The STE test method is suitable to classify GHS NC, indicating its applicability as a test method in a bottom-up approach.

Predictive performance and inter-laboratory reproducibility in assessing eye irritation potential of water- and oil-soluble mixtures using the Short Time Exposure test method.

The Short Time Exposure (STE) test method is an alternative method for assessing eye irritation potential using Statens Seruminstitut Rabbit Cornea cells and has been adopted as test guideline 491 by the Organisation for Economic Co-operation and Development. Its good predictive performance in identifying the Globally Harmonized System (GHS) No Category (NC) or Irritant Category has been demonstrated in evaluations of water-soluble substances, oil-soluble substances, and water-soluble mixtures. However, the predictive performance for oil-soluble mixtures was not evaluated. Twenty-four oil-soluble mixtures were evaluated using the STE test method. The GHS NC or Irritant Category of 22 oil-soluble mixtures were consistent with that of a Reconstructed human Cornea-like Epithelium (RhCE) test method. Inter-laboratory reproducibility was then confirmed using 20 water- and oil-soluble mixtures blind-coded. The concordance in GHS NC or Irritant Category among four laboratories was 90%-100%. In conclusion, the concordance in comparison with the results of RhCE test method using 24 oil-soluble mixtures and inter-laboratory reproducibility using 20 water- and oil-soluble mixtures blind-coded were good, indicating that the STE test method is a suitable alternative for predicting the eye irritation potential of both substances and mixtures.

Predictive performance of the Short Time Exposure test for identifying eye irritation potential of chemical mixtures.

The Short Time Exposure (STE) test is an in vitro eye irritation test based on the cytotoxicity in SIRC cells (rabbit corneal cell line) following a 5 min treatment of chemicals. This study evaluated the predictive performance of the STE test to identify the globally harmonized system (GHS) Not Classified category and other irritant categories (i.e., GHS Category 1 or 2) when used to test 40 chemical mixtures that included irritants. The STE test correctly identified 30 tested mixtures classified as GHS irritant categories and 5 out of 10 tested mixtures classified as GHS Not Classified. The sensitivity, specificity, positive predictivity, negative predictivity, and overall accuracy of the STE test were 100% (30/30), 50% (5/10), 86% (25/30), 100% (5/5), and 88% (35/40), respectively. These predictive performances were comparative to or greater than those in other in vitro eye irritation tests that have been accepted as test guideline by the Organisation for Economic Co-operation and Development. This suggests that the STE test has sufficient predictivity for identifying the eye irritation potential of chemical mixtures. Since no false negatives in this study were found, this indicates that the STE test is applicable as a part of the bottom-up approach.

Definition of the applicability domain of the Short Time Exposure (STE) test for predicting the eye irritation of chemicals.

The Short Time Exposure (STE) test is a simple and easy-to-perform in vitro eye irritation test, that uses the viability of SIRC cells (a rabbit corneal cell line) treated for five minutes as the endpoint. In this study, our goal was to define the applicability domain of the STE test, based on the results obtained with a set of 113 substances. To achieve this goal, chemicals were selected to represent both different chemical classes and different chemical properties, as well as to cover, in a balanced manner, the categories of eye irritation potential according to the Globally Harmonised System (GHS). Accuracy analysis indicated that the rates of false negatives for organic/inorganic salts (75.0%), hydrocarbons (33.3%) and alcohols (23.5%) were high. Many of the false negative results were for solid substances. It is noteworthy that no surfactant resulted in a false negative result in the STE test. Further examination of the physical property data and performance showed a significant improvement in the predictive accuracy, when substances with vapour pressures over 6kPa were excluded from the analyses. Our results indicate that several substances - i.e. certain solids such as salts, alcohols, hydrocarbons, and volatile substances with a vapour pressure over 6kPa - do not fall within the applicability domain of the STE test. Overall, we are encouraged by the performance and improved accuracy of the STE test.

Two-stage bottom-up tiered approach combining several alternatives for identification of eye irritation potential of chemicals including insoluble or volatile substances.

For the assessment of eye irritation, one alternative test may not completely replace the rabbit Draize test. In the present study, we examined the predictive potential of a tiered approach analyzing the results from several alternatives (i.e., the Short Time Exposure (STE) test, the EpiOcular assay, the Hen's Egg Test-Chorioallantoic Membrane (HET-CAM) assay and the Bovine Corneal Opacity and Permeability (BCOP) assay) for assessing Globally Harmonized System (GHS) eye irritation categories. Fifty-six chemicals including alcohols, surfactants, and esters were selected with a balanced GHS category and a wide range of chemical classes. From a standpoint of both assessable sample numbers and predictive accuracy, the more favorable tiered approach was considered to be the two-stage bottom-up tiered approach combining the STE test, the EpiOcular assay followed by the BCOP assay (accuracy 69.6%, under prediction rate 8.9%). Moreover, a more favorable predictive capacity (accuracy 71.4%, under prediction rate 3.6%) was obtained when high volatile alcohols/esters with vapor pressures >6 kilopascal (kPa) at 25°C were evaluated with EpiOcular assay instead of the STE test. From these results, the two-stage bottom-up tiered approach combining the STE test, the EpiOcular assay followed by the BCOP assay might be a promising method for the classification of GHS eye irritation category (Not classified (NC), Category 2 (Cat. 2), and Category 1 (Cat. 1)) for a wide range of test chemicals regardless of solubility.

A tiered approach combining the short time exposure (STE) test and the bovine corneal opacity and permeability (BCOP) assay for predicting eye irritation potential of chemicals.

For the evaluation of eye irritation, one in vitro alternative test may not completely replace the Draize test. Therefore, a tiered approach combining several in vitro assays, including cytotoxicity assays, is proposed in order to estimate the eye irritation potential of a wide range of chemical classes. The Short Time Exposure (STE) test, a relatively newer alternative eye irritation test, involves exposing Statens seruminstitut rabbit cornea (SIRC) cells for 5 min to two concentrations (5% and 0.05%) of test material. In the present study, we examined the predictive capacity of a tiered approach analyzing the results from the STE test and then the results of the bovine corneal opacity and permeability (BCOP) assay for assessing globally harmonized system (GHS) eye irritation rankings of various chemicals. The accuracy of predicting the GHS rankings was slightly improved when the tiered approach combination of STE test and BCOP assay was used compared to when the STE test irritation rank classification was used alone. Moreover, the under prediction rate was substantially improved when this tiered approach was used. From these results, the tiered approach of combining the data analysis of the STE test and BCOP assay might be a promising alternative eye irritation test strategy.

The Short Time Exposure (STE) test for predicting eye irritation potential: intra-laboratory reproducibility and correspondence to globally harmonized system (GHS) and EU eye irritation classification for 109 chemicals.

Short Time Exposure (STE) test is an easy in vitro eye irritation test that assesses cytotoxicity in SIRC cells (rabbit corneal cell line) following a 5 min dose treatment. To assess intra-laboratory reproducibility, medium control, three vehicles (saline, saline containing 5% (w/w) dimethyl sulfoxide, and mineral oil) and three standard chemicals (sodium lauryl sulfate, calcium thioglycolate, and Tween 80) were evaluated. Assessments were repeated 30 times for vehicles and 18 times for standard chemicals; resulting in almost the same cell viability and a low coefficient of variation value. In addition, the STE eye irritation rankings of three standard chemicals, as calculated on the cell viabilities in 5% and 0.05% solutions were in agreement in all tests. Based on these results, high intra-laboratory reproducibility was confirmed. In addition, the irritation category (irritant and non-irritant) was evaluated for 109 chemicals with STE test, globally harmonized system (GHS) classification, and European Union (EU) classification. The results of the evaluation found the STE classification to have an accuracy with GHS classification of 87% and with EU classification of 83%, which confirmed the excellent correspondence. The correspondence of STE rankings (1, 2, and 3) based on the prediction model by STE test with the eye irritation rankings by GHS (non-irritant, categories 2 and 1) and EU (non-irritant, R36, and R41) was 76% and 71%, respectively. Based on the above results, STE test was considered to be a promising alternative method for assessing eye irritation that has high intra-laboratory reproducibility as well as an excellent predictability of eye irritation.