Validation study of the Short Time Exposure (STE) test to assess the eye irritation potential of chemicals

doi:10.1016/j.tiv.2011.01.015

Toxicology in Vitro

Volume 25, Issue 4, June 2011, Pages 796-809

https://doi.org/10.1016/j.tiv.2011.01.015 Get rights and content

Abstract

Short Time Exposure (STE) test is a cytotoxicity test in SIRC cells (rabbit corneal cell line) that assesses eye irritation potential following a 5-min chemical exposure. This validation study assessed transferability, intra- and inter-laboratory reproducibility, and predictive capacity of STE test in five laboratories (supported by Japanese Society for Alternatives to Animal Experiments). Sodium lauryl sulfate, calcium thioglycolate, and Tween 80 were evaluated, in triplicate, using 5%, 0.5%, and 0.05% concentrations in physiological saline, to confirm transferability. Good transferability was noted when similar mean relative viabilities and rank classifications were obtained in all five laboratories and were comparable to data from test method developing laboratory. Good intra- and inter-laboratory reproducibility was obtained with four assay controls (three solvents and one positive control), and four assay controls and 25 chemicals, respectively. STE irritation category based on relative viability of a 5% solution of 25 blinded test chemicals showed good correlation with Globally Harmonized System (GHS) categories (NI; I: Cat. 1 and 2). The STE prediction model, using relative viability of the 5% and 0.05% solutions, provided an irritation rank (1, 2, or 3) that had a good correlation (above 80%), or predictive capacity, with GHS irritation ranks in all laboratories. Based on these findings, the STE test is a promising alternative eye irritation test that could be easily standardized.

Introduction

Eye irritation potential of chemicals has been evaluated mainly by the rabbit Draize test for many years. Recently, animal welfare considerations and enforcement of a new regulations like the banning of cosmetics in animal eye irritation tests in the EU (Directive 2003/15/EC, 2003) are drawing much attention in reducing or replacing animal experiments with alternative methods. In September 2009, the bovine corneal opacity and permeability (BCOP) assay and isolated chicken eye (ICE) assay were adopted by the Organisation for Economic Cooperation and Development (OECD) for identifying ocular corrosives and severe irritants, i.e., OECD Test Guideline 437 or 438, respectively (OECD, 2009a, OECD, 2009b). However, no in vitro test has been approved by OECD so far to evaluate minimal to moderate eye irritation potential. The STE test is a Short Time Exposure cytotoxicity test that uses SIRC cells (rabbit corneal cell line) to evaluate minimal, moderate or severe eye irritation potential (Takahashi et al., 2008). This test uses cell viability as an endpoint after 5 min of exposure and solves the problems associated with conventional cytotoxicity tests. For example, the STE test has the advantage of being able to evaluate the eye irritation potential of water insoluble chemicals using mineral oil as solvent. It is also very simple to use and provides rapid results. In collaborative research in three laboratories evaluating the STE test, similar test results were obtained for positive and negative controls, indicating that the STE test has excellent “transferability” (Takahashi et al., 2009). In addition, in an evaluation of 51 chemicals, high reproducibility and a good predictive capacity were found in each of the three laboratories (inter-laboratory accuracy was 98.0% or higher; Takahashi et al., 2009). However, each of the three laboratories was not blinded to the identity of the test chemicals. In the present validation study conducted by the Japanese Society for Alternatives to Animal Experiments (JSAAE), the number of laboratories performing evaluations was increased to five laboratories to confirm the transferability of the STE test using three standard chemicals. The usefulness of the alternative STE test for evaluating eye irritation was further determined by establishing the intra- and inter-laboratory reproducibility of four assay controls, and inter-laboratory reproducibility and predictive capacity of STE test (i.e., agreement with the irritation category and rank of the GHS) of the 25 blinded test chemicals.

Section snippets

Organization for STE test validation

The Executive Committee formed a number of committees to ensure appropriate execution of the study. The STE Test Validation Executive Committee comprised of members of the JSAAE (Validation Committee members), biostatisticians, and representatives of the participating validation laboratories. The STE Test Validation Executive Committee undertook deliberations and made decisions regarding the study plan, test protocol, confirmed the data analysis results, and published the study results. The

Transferability data for three standard chemicals

Table 2 shows mean cell viability ± SD at three test concentrations and the eye irritation ranks calculated from the prediction model (i.e., results of the 5% and 0.05% test solutions) for the three standard chemicals obtained by the five laboratories. The background data for these chemicals obtained by the laboratory that developed the test method are also shown for comparison. The SLS had a cell viability of 7% or less; TW80 was 90% or higher at all three concentrations; and CT was 20% or less

Discussion

By now, a lot of eye irritation alternative methods are being developed around the world (Balls et al., 1999, Ohno et al., 1999, Eskes et al., 2005). However, it may not be likely to completely replace the Draize test by a single in vitro test. The tiered approach of several in vitro assays combined was proposed in order to estimate the irritation potential for a wide range of chemical classes (Hagino et al., 2008, McNamee et al., 2009, Scott et al., 2010). Many eye irritation alternative

Conclusion

On the basis of a unified test protocol, a validation study was performed in five laboratories. Three standard chemicals were first used to confirm transferability of method, and then four assay controls and 25 blinded test sample chemicals with GHS classification categories were used to confirm intra-/inter-laboratory reproducibility and predictive capacity. The results show that the STE test not only provided good transferability, but it also had good intra- and inter-laboratory

Acknowledgments

The Validation Executive Committee was organized under the JSAAE and performed the present study. The JSAAE, through its Validation Committee, provided both laboratory personnel and financial support for completion of this study. The authors are very grateful for this support. In addition, in performing the validation study, the authors received considerable support and advice from the Japanese Center for the Validation of Alternative Methods. The authors offer sincere thanks to them as well.

References (22)

E. Adriaens et al.
Successful prevalidation of the slug mucosal irritation test to assess the eye irritation potency of chemicals
Toxicol. in Vitro
(2008)
P.G. Brantom et al.
A summary report of the COLIPA international validation study on alternatives to the Draize rabbit eye irritation test
Toxicol. in Vitro
(1997)
P. Gautheron et al.
Bovine corneal opacity and permeability test: an in vitro assay of ocular irritancy
Fundam. Appl. Toxicol.
(1992)
J.W. Harbell et al.
IRAG working group 4: cell cytotoxicity assays
Food Chem. Toxicol.
(1997)
P. McNamee et al.
A tired approach to the use of alternative to animal testing for the safety assessment of cosmetics: eye irritation
Regul. Toxicol. Pharmacol.
(2009)
Y. Ohno et al.
Interlaboratory validation of the in vitro eye irritation test for cosmetic ingredients. (1) Overview of the validation study and Draize scores for the evaluation of the tests
Toxicol. in Vitro
(1999)
L. Scott et al.
A proposed eye irritation testing strategy to reduce and replace in vivo studies using Bottom-Up and Top-Down approaches
Toxicol. in Vitro
(2010)
Y. Takahashi et al.
Development of the short time exposure (STE) test: an in vitro eye irritation test using SIRC cells
Toxicol. in Vitro
(2008)
D. Bagley et al.
The SDA alternatives program phase III: comparison of in vitro data with animal eye irritation data on solvents, surfactants, oxidizing agents, and prototype cleaning products
J. Toxicol. Cut. Ocul. Toxicol.
(1994)
M. Balls et al.
Eye irritation testing: the way forward, The report and recommendations of ECVAM workshop 34
Altern. Lab. Anim.
(1999)

Directive 2003/15/EC of the European Parliament and of the Council of 27 February 2003. Official Journal of the...

Cited by (41)

Advancing ocular safety research: A comprehensive examination of benzocaine acute exposure without animal testing
2024, Toxicology Letters
Benzocaine is a widely employed local anaesthetic; however, there is a notable dearth of preclinical and clinical evidence regarding its safety in ophthalmological products. To address this, a comprehensive strategy incorporating in silico and in vitro methodologies was proposed for assessing benzocaine's ocular toxicity without animal testing. To collect the in silico evidence, the QSAR Toolbox (v4.5) was used. A single exposure to two benzocaine concentrations (2% and 20%) was evaluated by in vitro methods. Hen’s Egg Chorioallantoic Membrane Test (HET-CAM) was performed to evaluate the effects on the conjunctiva. To study corneal integrity, Short Time Exposure test (STE) and Bovine Corneal Opacity and Permeability (BCOP) assay, followed by histopathological analysis, were carried out. Results from both in silico and in vitro methodologies categorize benzocaine as non-irritating. The histopathological analysis further affirms the safety of using benzocaine in eye drops, as no alterations were observed in evaluated corneal strata. This research proposes a useful combined strategy to provide evidence on the safety of local anaesthetics and particularly show that 2% and 20% benzocaine solutions do not induce eye irritation or corneal damage, supporting the potential use of benzocaine in the development of ophthalmic anesthetic products.
Corneal epithelium models for safety assessment in drug development: Present and future directions
2023, Experimental Eye Research
The human corneal epithelial barrier plays a crucial role in drug testing studies, including drug absorption, distribution, metabolism, and excretion (ADME), as well as toxicity testing during the preclinical stages of drug development. However, despite the valuable insights gained from animal and current in vitro models, there remains a significant discrepancy between preclinical drug predictions and actual clinical outcomes. Additionally, there is a growing emphasis on adhering to the 3R principles (refine, reduce, replace) to minimize the use of animals in testing. To tackle these challenges, there is a rising demand for alternative in vitro models that closely mimic the human corneal epithelium. Recently, remarkable advancements have been made in two key areas: microphysiological systems (MPS) or organs-on-chips (OoCs), and stem cell-derived organoids. These cutting-edge platforms integrate four major disciplines: stem cells, microfluidics, bioprinting, and biosensing technologies. This integration holds great promise in developing powerful and biomimetic models of the human cornea.
Development of a Defined Approach for Eye hazard identification of chemicals having surfactant properties according to the three UN GHS categories
2023, Toxicology in Vitro
The purpose of this study was to develop a defined approach (DA) for eye hazard identification according to the three UN GHS categories for surfactants (DASF). The DASF is based on a combination of Reconstructed human Cornea-like Epithelium test methods (OECD TG 492; EpiOcular™ EIT and SkinEthic™ HCE EIT) and the modified Short Time Exposure (STE) test method (0.5% concentration of the test substance after a 5-min exposure).
DASF performance was assessed by comparing the prediction results with the historical in vivo data classification and against the criteria established by the OECD expert group on eye/skin.
The DASF yielded a balanced accuracy of 80.5% and 90.9% of Cat. 1 (N = 22), 75.0% of Cat. 2 (N = 8), and 75.5% of No Cat. (N = 17) surfactants were correctly predicted. The percentage of mispredictions was below the established maximum values except for in vivo No Cat. surfactants that were over-predicted as Cat. 1 (5.6%, N = 17), with a maximum value set at 5%. The percentage of correct predictions did meet the minimum performance values of 75% Cat. 1, 50% Cat. 2, and 70% No Cat. established by the OECD experts. The DASF has shown to be successful for eye hazard identification of surfactants.
Development of an oral mucosal irritation test using a three-dimensional human buccal oral mucosal model
2023, Toxicology in Vitro
Citation Excerpt :
We also evaluated the transferability of our test at the three laboratories using SLS and these materials as standards.In addition, we selected and analysed 32 chemicals with known UN-GHS eye irritation data (Table 2) as substitutes for in vivo oral mucosal irritation data. These chemicals were classified according to the UN-GHS categories based on the results of the Draize eye irritation test and labeled as Category 1 (Cat 1: irreversible effects on eyes), Category 2A (Cat 2A: irritating to eyes), Category 2B (Cat 2B: mildly irritating to eyes), No Category (No Cat: non-irritant) (Barroso et al., 2017; Sakaguchi et al., 2011). These were purchased from Sigma-Aldrich Corp., or FUJIFILM Wako Pure Chemical Corporation or Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).
The oral mucosa can become irritated by oral care products and lip cosmetics. Therefore, it is important to determine the irritation potential of their ingredients and products during safety evaluations. We developed a method for oral mucosal irritation test using EpiOral, which is a three-dimensional cultured model. Exposure of sodium lauryl sulphate (SLS) to EpiOral showed a dose-dependent decrease in cell viability. Under 120 min exposure conditions, SLS irritation was detected when 60% cell viability was set as a criterion. Evaluation of the irritancy of SLS and four other raw materials used in oral products at three laboratories under the above conditions confirmed good transferability of the test. Focused on the similarity of the oral and eye mucous, 32 chemicals categorised by the UN-GHS eye-irritation classification were evaluated to ensure the reliability of our criteria at these laboratories. The concordance rate between the UN-GHS classification and our test results was 100% for irritants and 60% for non-irritants. The good intra-laboratory reproducibility of our test was confirmed from the evaluation results of negative and positive controls, and the good inter-laboratory reproducibility was confirmed from the results of 32 chemicals. These findings showed that oral mucosal irritation can be evaluated using EpiOral.
Evaluation of in vitro cornea models for quantifying destructive effects of chemicals
2022, Toxicology in Vitro
Citation Excerpt :
Another alternative in vitro model is the short time exposure (STE) test (OECD, 2020; Takahashi et al., 2011). Using a rabbit corneal cell line (SIRC from rabbit cornea), physiological saline or mineral oil as the test solvent, and 5-min chemical exposure, the STE cytotoxicity test was suggested to be effective in evaluating damages to the eye due to chemical exposure situations (Sakaguchi et al., 2011; Takahashi et al., 2008; Verstraelen et al., 2013). These testing methods are simple, promising, and of value as standardized alternative eye irritation tests.
In vitro models are available as alternatives for the Draize eye irritation test. However, most of the alternative models are not quantitative nor designed to evaluate the effects of chemicals on the corneal barrier such as those encountered in pharmaceutical and cosmetic products. The objective of the present study was to investigate tissue electrical resistance to provide sensitive in vitro testing of tissue alteration caused by chemicals in pharmaceutical and cosmetic formulations as a potential eye irritation testing approach. The experimental protocols for effective tissue resistance measurements were examined using two in vitro eye models: porcine cornea and EpiCorneal. In these models, a test chemical was applied to the cornea or EpiCorneal tissue for 1 min, and tissue resistances/conductances were measured at 1–60 min after the application. The changes in conductance of the tissues after exposure to the chemicals were shown to provide quantitative evaluations to the influence of the chemicals. A correlation was found between the two in vitro models. The results suggest that these models can provide quantitative in vitro assessments of eye-irritating chemicals.
An alternative predictor of eye irritation that utilizes potential parameters of the human corneal epithelium model calculated based on Hansen solubility parameters
2021, Toxicology Letters
Eye irritation predictions are very important in the development of cosmetics and pharmaceuticals. For animal protection, alternative tests are being developed to replace the Draize test, which involves the use of rabbits to test eye irritation. The Vitrigel-eye irritancy test (Vitrigel-EIT), is one such alternative. As a preliminary study, we evaluated if Hansen solubility parameter (HSP) values can be used to predict Vitrigel-EIT results. An Hansen sphere was created based on the HSP values and Vitrigel-EIT results from 61 substances. Substances inside and outside of the sphere were designated as dangerous and safe substances, respectively. The safety of each test substance was predicted by comparing the center point (R_o) of the sphere with the relative energy difference, i.e., the ratio of each test substance (R_a). The accuracy, false negativity, and false positivity of the “irritant” and “nonirritant” designations, as determined by the Vitrigel-EIT results and Hansen sphere, were 91.8% (56/61), 2.3% (1/43), and 22.2% (4/18), respectively. These results indicated that HSP values can be used to predict Vitrigel-EIT results with high reproducibility, and thus are useful for evaluating the safety of substances.

View all citing articles on Scopus

View full text

Validation study of the Short Time Exposure (STE) test to assess the eye irritation potential of chemicals

Abstract

Introduction

Section snippets

Organization for STE test validation

Transferability data for three standard chemicals

Discussion

Conclusion

Acknowledgments

Toxicol. in Vitro

Toxicol. in Vitro

Fundam. Appl. Toxicol.

Food Chem. Toxicol.

Regul. Toxicol. Pharmacol.

Toxicol. in Vitro

Toxicol. in Vitro

Toxicol. in Vitro

The SDA alternatives program phase III: comparison of in vitro data with animal eye irritation data on solvents, surfactants, oxidizing agents, and prototype cleaning products

J. Toxicol. Cut. Ocul. Toxicol.

Eye irritation testing: the way forward, The report and recommendations of ECVAM workshop 34

Altern. Lab. Anim.