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.

An interlaboratory study of the short time exposure (STE) test using SIRC cells for predicting eye irritation potential.

We have developed the short time exposure (STE) test using a rabbit corneal cell line (SIRC cells) as an alternative eye irritation test. The STE test uses relative viability as the endpoint after cells are exposed to the test material at 5%, 0.5%, and 0.05% concentrations for 5 minutes. In this interlaboratory study, 2 laboratories conducted the test using 70 raw materials in order to evaluate transferability, between-laboratory reproducibility, and predictive capacity of the STE test as an alternative eye irritation test. Transferability was assessed using saline as a negative control and 0.01% sodium lauryl sulfate as a positive control. The relative viabilities obtained for the 2 laboratories were almost the same. Therefore, transferability was considered to be excellent. The 2 laboratories showed similar relative viabilities for all 70 raw materials at each test concentration. The correspondence rates of the eye irritation categories (irritants and nonirritants) were over 97% for each concentration tested, exhibiting high between-laboratory reproducibility. The correspondence rates for predicting eye irritation potential of undiluted raw materials and a diluted solution (10%) were over 85% at each laboratory for the 5% and 0.05% test concentrations in the STE. Lastly, the correspondence rate for the rank classification by the STE test prediction model at each laboratory was over 72%, and the correspondence rate became almost 90% when acids, amines, and alcohols were excluded from the analysis. From the above data, excellent transferability, high between-laboratory reproducibility, and high predictive capacity of the STE test were observed in the interlaboratory study by 2 laboratories.

Inter-laboratory study of short time exposure (STE) test for predicting eye irritation potential of chemicals and correspondence to globally harmonized system (GHS) classification.

Short time exposure (STE) test using rabbit corneal cell line (SIRC) cells was developed as an alternative eye irritation test. STE test uses relative viability as the endpoint after cells are exposed to the test material at constant concentrations for 5 min. In this inter-laboratory study with 3 laboratories, 44 chemicals with a wide range of classes were evaluated for the transferability, between-lab reproducibility and predictive capacity of the STE test as an alternative eye irritation test. Globally harmonized system (GHS) classification based on Draize eye irritation test data was used as the comparative in vivo data. Transferability was assessed using standard chemicals (sodium lauryl sulfate, calcium thioglycolate, and Tween 80) and the coefficient variations (CVs) of relative viabilities between 3 labs were less than 0.13. The irritation category (Irritant or Non irritant) at each test concentration (5% and 0.05%) in STE test was the same in 3 laboratories for all 44 tested chemicals. The predictive capacity irritation category classification between STE test and GHS were compared, and a good correlation was confirmed (accuracy was 90.9% at all laboratories). In addition, the STE rankings of 1, 2, and 3 classified by the prediction model (PM) based on the relative viability at two concentrations (5% and 0.05%) were highly correlated with the GHS ranks of non-irritant, category 1, and category 2, respectively (accuracy was 75.0% at all laboratories). These results suggest that the STE test possessed easy transferability, reproducibility, good predictive performance.

Development of the short time exposure (STE) test: an in vitro eye irritation test using SIRC cells.

Using SIRC (rabbit corneal cell line) cells, we developed an alternative eye irritation test: the short time exposure (STE) test. This STE test is a cytotoxicity test using physiological saline or mineral oil as the test solvent. Evaluation exposure time is short (5 min), which is similar to actual exposure situations, and uses the cell viability (CV) at a constant concentration as the endpoint for irritation potential. First, in order to confirm the usefulness of this STE test in assessing eye irritation potential of chemicals, 51 raw materials were tested and the correlation between CV in the STE test and the eye irritation score in the Draize test was examined. For the undiluted raw materials tested in the Draize test, the 5% test concentration in the STE test gave irritation classes that correlated well with the irritation classes from the Draize test (accuracy: 89.6%). For those materials tested as a 10% solution in the Draize test, STE irritation classes with 0.05% test concentration corresponded well with the Draize irritation classes (accuracy: 80.0%). Next, using the cell viabilities at these two concentrations, the STE prediction model (PM) was developed. A score of 1 or 2 was given for the results from each tested concentration in the STE test and Draize test. The scores from each test were then summed to yield a 3-level (Rank 1: minimally irritant, Rank 2: moderate irritant, Rank 3: severe irritant) eye irritation potential classification. Rank classification in the STE test showed a good correlation mostly to that in the Draize test (irritation class correspondence rate: 70.2%, but after exclusion of data of alcoholic materials, the rate was 91.7%). In most cytotoxicity test, the cytotoxicity of acids and amines is generally underestimated due the use of medium as the solvent. This is the result of the buffering capacity of the media. On the other hand, the STE test could predict the eye irritation potential by evaluating the chemical with a 5% test concentration. Eleven water insoluble materials such as toluene, octanol, and hexanol could be evaluated by using mineral oil as test solvent in the STE test. The STE test demonstrated itself to be simple, promising, have great potential, be of value, and to be an easily standardized alternative eye irritation test.