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.     

Comparative study of the ocular irritation potential of various alkyl polyglucoside surfactants

In the present work, we assessed the relationship between alkyl carbon chain length and ocular irritation potentials using the hen’s egg test–chorioallantoic membrane (HET-CAM) and bovine corneal opacity and permeability (BCOP) assays using 5 commercial alkyl polyglucoside surfactants with different compositions of alkyl chain lengths (C₆–C₁₆). With HET-CAM, there was a good correlation between the proportion of C₁₀ alkyl polyglucoside and the eye irritation potential Q score (r²?=?0.912, p?=?.011). There were no significant differences between the proportion of C₁₀ alkyl polyglucoside and corneal opacity in BCOP assays; however, there was a relatively high positive correlation between the proportion of C₁₀ alkyl carbon chain lengths and corneal permeability (r²?=?0.736, p?=?.063).     

BCOP法评价染发类化妆品眼刺激性的研究

目的 尝试采用牛角膜混浊和渗透性试验 (bovine corneal opacity and permeability,BCOP)对染发类化妆品进行眼刺激性的评价。方法 通过对染发剂与牛角膜的接触时间、染发剂各组成成分、染发剂配制后存放时间等检测条件的摸索,尝试建立一套标准的BCOP法评价染发类化妆品眼刺激性的方法,并用该法对市售样品进行评价性检测。结果 染发剂与牛角膜接触时间与刺激性大小成正比;染发剂各组成成分的刺激性不同,其中作为氧化剂的组分(通常为B剂或2剂)均为无刺激性,染发剂中的A剂或1剂的刺激性各品种存在较大的差异;染发剂配制后存放一定时间,刺激性会下降。以配制后即刻给样,接触牛眼时间为10 min,对市场销售的13种染发类化妆品进行评价性检测,结果体外刺激得分为3.5~78.2,分类为轻微刺激性的有10种,中度刺激性的有1种,严重刺激性的有2种。结论 用BCOP法可以对染发类化妆品的眼刺激性进行评价。     

Development of a human corneal epithelium model utilizing a collagen vitrigel membrane and the changes of its barrier function induced by exposing eye irritant chemicals

The brief TEER (trans-epithelial electrical resistance) assay after exposing chemicals to corneal epithelium in vivo is known as a suitable method for evaluating corneal irritancy and permeability quantitatively and continuously. A collagen vitrigel membrane we previously developed is a thin (about 20 μm thick) and transparent membrane composed of high density collagen fibrils equivalent to connective tissues in vivo, e.g. corneal Bowman’s membrane. To develop such a TEER assay system in vitro utilizing a human corneal epithelial model, HCE-T cells (a human corneal epithelial cell line) were cultured on the collagen vitrigel membrane substratum prepared in a Millicell chamber suitable for TEER measurement. Human corneal epithelium model possessing 5-6 cell layers sufficient for TEER assay was successfully reconstructed on the substratum in the Millicell chamber by culturing the cells in monolayer for 2 days and subsequently in air-liquid interface for 7 days. The exposure of chemicals to the model induced the time-dependent relative changes of TEER in response to the characteristic of each chemical within a few minutes. These results suggest that the TEER assay using the human corneal epithelial model is very useful for an ocular irritancy evaluation as an alternative to the Draize eye irritation test.     

Investigation of ingredient interactions in cosmetic formulations using isolated bovine corneas

The purpose of this paper is to report on use of a modified bovine cornea opacity and permeability assay (BCOP) to test the effects of several cosmetic formulations on eye-derived tissue in vitro. The results from these studies suggest that a BCOP protocol using prolonged exposure and repeated treatments may be useful for screening the eye effects of cosmetic formulations. Further work will be required, however, before the model is ready for formal validation. This series of experiments also provides an example of where the toxicity of one ingredient was significantly changed by its interaction with other ingredients in a mixture. As it was not possible to predict the highly reactive nature of the formulation in vitro based on an evaluation of ingredient toxicity data alone, this case illustrates the importance of obtaining adequate safety testing data on innovative mixtures of cosmetic ingredients before human exposure is allowed.     

The mucosal toxicity of different benzalkonium chloride analogues evaluated with an alternative test using slugs

Purpose. The objective of this study was to evaluate the mucosal toxicity of different benzalkonium chloride (BAC) analogues using slugs as the alternative test organism. Methods. The effect of different BAC analogues on the mucosal tissue of slugs was determined from the protein, lactate dehydrogenase, and alkaline phosphatase released from the foot mucosa after treatment. Additionally, mucus production and reduction in body weight of the slugs were measured. The eye irritation potency of the molecules was evaluated with the Bovine Corneal Opacity and Permeability (BCOP) assay. The antimicrobial activity of the different BAC analogues was also assessed. Results. All BAC analogues induced severe damage to the mucosal epithelium of the slugs, and the irritation increased with decreasing alkyl chain length: BAC-C16 < BAC-C14 < BAC-C12 BAC-mix. A similar ranking was obtained with the BCOP assay for eye irritation. The relative order of activities among the three BAC analogues was the same, i.e., BAC-C14 BAC-C16 > BAC-C12. The BAC-C14 exhibited higher activity than the BAC-mix. Conclusions. The toxicity and activity of BAC analogues depend on the alkyl chain length. The use of BAC-C14 as a conservative agent in pharmaceutical preparations instead of the BAC-mix should be considered.     

Improvement of the Bovine Corneal Opacity and Permeability (BCOP) assay as an in vitro alternative to the Draize rabbit eye irritation test

Measurement of ocular irritancy is a necessary step in the safety evaluation of both industrial and consumer products. Assessment of the acute eye irritation potential is therefore part of the international regulatory requirements for testing of chemicals.The Bovine Corneal Opacity and Permeability (BCOP) assay is generally accepted as a valid in vitro alternative method to the Draize eye irritation test to detect corrosive and severe eye irritants (category 1), but has not proven sensitive enough to discriminate accurately moderate (category 2A/2B) to mild and non-irritating compounds. In the currently accepted BCOP assay, opacity is determined by the amount of light transmission through the cornea, and permeability is determined by the amount of sodium fluorescein dye that passes through all corneal cell layers. Both measurements are used to assign an In Vitro Irritancy Score (IVIS) for prediction of the in vivo ocular irritation potential of a test substance. Nowadays, opacity is measured by an OP-KIT opacitometer providing a center-weighted reading of light transmission by measuring changes in voltage when the transmission of white light passes through the cornea alters. As a consequence, this may underestimate opacity that develops as spots or heterogeneous opaque areas on the periphery of an isolated cornea.A prototype of a laser light-based opacitometer (PLLBO) allowing better measurement of opacities was developed by Van Goethem et al. (2010). This new device showed improved sensitivity to detect subtle changes in corneal transparency. Furthermore, the new opacitometer allowed the analysis of the complete corneal surface and was able to detect more efficiently opaque spots located along the sides of the excised corneas.A further improved prototype of the PLLBO was constructed in combination with a camera and a speckle noise reducer. Treatment conditions of the corneas in the cornea holders were optimized in order to mimic more the real in vivo situation. A set of test compounds with irritancy potencies especially in the mild and moderate range was tested. The improved LLBO showed some promising features which potentially could improve the usefulness of the BCOP test. Adaptation of cornea holders showed to be of limited value and only restricted to concentrations up to 15% which mimics more test conditions in industry.This 3-year research project was sponsored by the Stavros Niarchos Foundation (Greece).     

Eye irritation potential: palm-based methyl ester sulphonates

Aim: To evaluate eye irritation potential of palm-based methyl ester sulphonates (MES) of different chain lengths; C12, C14, C16, C16:18.

Methods: The Bovine Corneal Opacity and Permeability test method (BCOP), OECD Test Guideline 437, was used as an initial step to study the inducing effect of palm-based MES on irreversible eye damage. The second assessment involved the use of reconstructed human corneal-like epithelium test method, OECD Test Guideline 492 using SkinEthic? Human Corneal Epithelium to study the potential effect of palm-based MES on eye irritancy. The palm-based MES were prepared in 10% solution (w/v) in deionized water and tested as a liquid and surfactant test substances whereby both test conducted according to the liquid/surfactant treatment protocol.

Results: The preliminary BCOP results showed that palm-based MES; C12, C14, C16, C16:18 were not classified as severe eye irritants test substances with in vitro irritancy score between 3 and the threshold level of 55. The second evaluation using SkinEthic? HCE model showed that palm-based MES; C12, C14, C16, C16:18 and three commercial samples were potentially irritants to the eyes with mean tissue viability ≤ 60% and classified as Category 2 according to United Nations Globally Harmonized System of Classification and Labelling of Chemicals. However, there are some limitations of the proposed ocular irritation classification of palm-based MES due to insolubility of long chain MES in 10% solution (w/v) in deionized water.

Conclusion: Therefore, future studies to clarify the eye irritation potential of the palm-based MES will be needed, and could include; methods to improve the test substance solubility, use of test protocol for solids, and/or inclusion of a benchmark anionic surfactant, such as sodium dodecyl sulphate within the study design.     

牛角膜混浊和渗透性试验方法与家兔法评价染发类化妆品眼刺激性的比较研究

目的 探讨在染发类化妆品备案审查中,用牛角膜混浊和渗透性试验(BCOP)代替家兔法(Draize 试验)进行眼刺激性评价的可能性。方法 分别用BCOP与Draize 试验对14种市售染发剂进行眼刺激性评价,并对评价结果进行比较。结果 对14种染发剂眼刺激性检测的结果表明,Draize 试验所得的刺激反应等级为微刺激性―轻刺激性,按现行评审标准,通过率为100%(14/14);BCOP所得的刺激反应等级为轻微刺激性―严重刺激性,与Draize法分类一致性达到78.6%(11/14),以体外刺激评分值(IVIS)≤25为限值,通过率为78.6%(11/14);以IVIS≤55为限值,通过率为85.7%(12/14)。结论 用BCOP代替Draize 试验对染发类化妆品进行眼刺激性评价可行。