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

Abstract

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.

INTRODUCTION

The Short Time Exposure (STE) test is used to assess the eye irritation potential on the basis of the cell viability calculated in the presence of formazan dye obtained by reduction of 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). This test is performed by a 5-min exposure of test chemicals (i.e., substances and mixtures) to the Statens Seruminstitute Rabbit Cornea (SIRC) cell line, arranged in a monolayer culture (Takahashi et al., 2008). The STE test has been adopted as Test Guideline (TG) 491 by the Organisation for Economic Co-operation and Development (OECD) to categorize chemicals. This guideline follows the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) and labels substances as Category 1 (Cat. 1) chemicals if they induce serious eye damage or as No Category (No Cat.) chemicals if they do not require classification for eye irritation or serious eye damage (OECD, 2020a). As the STE test cannot determine whether a specific chemical is not a Cat. 1 nor No Cat. chemical (i.e. GHS Category 2; Cat. 2, for chemicals that induce reversible eye irritation), additional testing is required. Hayashi et al. (2012a) combined the STE test and the Bovine Corneal Opacity and Permeability (BCOP) test as TG 437 (OECD, 2020b) to categorize Cat. 2. However, this combination of tests is the out of applicability domain for chemicals that did not dissolve in the test solvent. In response, Hayashi et al. (2012b) combined the STE and BCOP tests with the reconstructed human cornea-like epithelium (RhCE) test which has been adopted as TG 492 (OECD, 2019a). This approach can be used irrespective of a chemical’s solubility. Alépée et al. (2019) proposed a method that combined the STE test with a BCOP test that used measurements from a laser light-based opacitometer (LLBO) to classify three GHS category for liquid substances. The OECD (2022a) has adopted this combination of the STE test and BCOP LLBO test as TG 467.

The MTT assay used for the STE test was originally developed by Mosmann (1983). The MTT assay estimates cytotoxicity by calculating the proportion of viable cells using a formazan dye obtained from intracellular reductase. The RhCE test as TG 492 (OECD, 2019a) also uses the MTT assay to measure cell viability for in vitro eye irritation testing. Furthermore, MTT assays are also used with reconstructed human epidermis for in vitro skin corrosion and irritation testing as TG 431 (OECD, 2019b) and TG 439 (OECD, 2021), respectively, as well as for the in vitro testing of skin sensitization using the KeratinoSens and LuSens tests (TG 442D; OECD, 2022b). Thus, the MTT assay is a widely used in vitro test that is commonly used to evaluate cytotoxicity.

However, despite the wide usage of this assay, the presence of substances that directly reduce MTT (MTT reducers) may cause an underestimation of cytotoxicity for substances that possess chemical structures such as thiol group. This occurs because MTT reducers directly produce the formazan dye by reducing the test substance in the absence of intracellular reductase (Alépée et al., 2015). Strongly colored substances can also interfere with the absorption wavelength of MTT formazan dye and can therefore cause the underestimation of cytotoxicity (Alépée et al., 2015) by adsorbing to cells and/or wells. TG 492 as the RhCE test uses a method involving killed tissues for evaluating MTT reducers and strongly colored substances. The killed tissues are prepared by repeatedly freezing and thawing tissue and are used to evaluate the concerned substances. Another method to calculate cell viability describes measuring MTT formazan dye directly using high-performance liquid chromatography (HPLC) or ultra-performance liquid chromatography (OECD, 2019a). These methods are also described in TG 431 (OECD, 2019b) and TG 439 (OECD, 2021) for skin corrosion and irritation, respectively. A specific method has not yet been described to evaluate the degree of direct MTT reduction or strong color in TG 442D for skin sensitization. However, the PrestoBlue® assay, an alternative cytotoxicity test that uses a different operating mechanism from the MTT assay, has been used as a reference for the TG 442D Standard Operation Procedure (SOP) (OECD, 2022b).

To date, no reports have determined whether STE test results for MTT reducers and strongly colored substances reflect interference with absorbance. The aim of this study was to confirm whether the STE test can correctly assess the level of eye irritation potential without underestimation caused by the presence of MTT reducers and strongly colored substances.

MATERIALS AND METHODS

Materials

To determine whether the potential for eye irritation was correctly evaluated using the STE test and to confirm the applicability of this method for key substances of interest, we selected specific MTT reducers and strongly colored substances according to reports of the STE Summary Review Document (STE SRD; ICCVAM, 2013), Barroso et al. (2017), and Alépée et al. (2015, 2016). The MTT reducers were also selected based on whether they possessed chemical structures such as thiol groups that may directly reduce in the MTT assay. All MTT reducers and strongly colored substances selected for use in this study are shown in Table 1, and all were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Table 1. The list of substances evaluated in this study.

Confirmation of direct MTT reduction of undiluted substances

We first confirmed the direct MTT reduction of undiluted substances using the STE Test Protocol listed as an Appendix in the STE SRD (ICCVAM, 2013) as a reference. First, an MTT solution was prepared at a concentration of 0.5 mg/mL in medium. Next, 20 µL of an undiluted test substance was added to 200 µL of the MTT solution. After incubation in the dark at 37°C in 5% CO₂ for 2 hr, the color of the mixed solution was checked visually. If the reddish-orange unreduced MTT solution turned blue or purple, we concluded that the test substance directly reduced MTT; the absence of darkening indicated that the test substance did not directly reduce MTT. In addition, the degree of the color change was visually discriminated as marked or slight. The degree of color was assessed as per the standards of the Database Service on Alternative Methods to Animal Experimentation (DB-ALM) for SkinEthic™ HCE issued by the European Commission’s Directorate General Joint Research Centre (2017).

Confirmation of direct MTT reduction by MTT reducers at 5% and 0.05% under STE test conditions

Although direct MTT reduction was confirmed when the MTT reducers were present at undiluted concentrations, it was unclear whether these substances would directly reduce MTT at concentrations of 5% and 0.05% (5% and 0.05% MTT reducers). The direct MTT reduction of diluted substances at 5% and 0.05% was confirmed as same as confirmation at undiluted substances. Here, 5% and 0.05% indicate concentrations relative to 100%, representing the undiluted (i.e., commercial) form of the test substance. If the concentration was described as “10% in aqueous solution” in Table 1, for example, 5% and 0.05% mean active 0.5% and 0.005% in Table 3 and Table 4.

Estimation of interference with absorbance caused by strongly colored substances under STE test conditions

In this study, we defined strongly colored substances as follows: a substance that showed maximum absorption values at the MTT absorption wavelength of 570 nm, even after 5% and/or 0.05% dilution under STE test conditions. The selected strongly colored substances were prepared at 5% and 0.05% dilutions, and their absorbance was measured at wavelengths between 400 nm and 800 nm in visible light.

STE test

Cell culture

SIRC cell samples were purchased from the American Type Culture Collection and were cultured in Eagle’s minimum essential medium (Sigma-Aldrich) containing 10% (v/v) fetal bovine serum (MP Biomedicals LLC, Solon, OH, USA), 2 mM L-glutamine (Thermo Fisher Scientific Inc., Waltham, MA, USA), and 50 units/mL penicillin containing 50 µg/mL streptomycin (Thermo Fisher Scientific). After reaching confluence, cells were dispersed using trypsin-ethylenediaminetetraacetic acid solution (Sigma-Aldrich) and spread onto 96-well flat-bottomed plates (Corning Costar Co., Cambridge, MA, USA) at a density of 3.0 × 10³ cells/well (or at 6.0 × 10³ cells/well). After incubation at 37°C in 5% CO₂ for 5 days (or for 4 days at 6.0 × 10³ cells/well), the cells reached confluence.

Test protocol

The STE test was performed in accordance with the previously published STE Test Protocol (ICCVAM, 2013), referred to in OECD TG 491. We selected either saline (Otsuka Pharmaceutical Factory, Inc., Naruto, Tokushima, Japan) or mineral oil (CAS RN 8042-47-5; Sigma-Aldrich Co., LLC) as the test solvent based on confirmatory experiments performed prior to the test. For the STE test, dissolution indicated that the substance had dissolved or had become a uniform suspension within 5 min at 5% (w/w) and 0.05% (v/v) dilutions in the selected solvent.

For the exposure trial, test substance dilutions at relative concentrations of 5% and 0.05% were exposed to cell samples for 5 min. The cells were then washed twice with Dulbecco's phosphate-buffered saline (PBS) (−) (Sigma-Aldrich), and an MTT assay (Sigma-Aldrich) was performed. Next, MTT formazan dye was extracted from cells using 0.04 N HCl–isopropanol. The absorbance of the extract was measured at 570 nm using a plate reader (BioTek Instruments Inc., Winooski, VT, USA).

Mean cell viability was expressed as the ratio of MTT formazan dye absorbance for each test substance relative to the test solvent control. All measurements were determined using three technical replicates. Moreover, for each exposure condition for each test substance, we calculated cell viability from the mean absorbance of three wells, and this calculated result was treated as a single technical replicate. The calculated overall mean cell viability was then used to predict the potential for eye irritation. Three additional independent runs were performed if the standard deviation of the measurements of mean cell viability was ≥15% for the original three technical replicates.

Prediction models

Table 2 shows two prediction models for the STE test. These two models treat the potential for eye irritation based on the overall mean cell viabilities obtained using 5% and 0.05% dilutions of the test substance. The first prediction model, based on TG 491, found that if the mean cell viability exceeds 70% under both the 5% and 0.05% dilutions, the correct GHS classification is ‘No Cat.’; if the mean viability is 70% or less under the 5% dilution but greater than 70% under the 0.05% dilution condition, the correct classification is “No stand-alone prediction can be made.” However, if the mean cell viability was 70% or less under both exposure conditions, the GHS classification is Cat. 1. The second prediction model classified a test substance as an irritant or a non-irritant based on the mean cell viability obtained for the 5% and 0.05% dilutions of the test substance. Substances with a mean cell viability greater than 70% at both the 5% and 0.05% dilutions were classified as non-irritants, and those with a mean cell viability of 70% or less at both the 5% and 0.05% dilutions or those that showed 70% or less at the 5% dilution and greater than 70% at the 0.05% dilution were classified as irritants (Takahashi et al., 2008).

Table 2. The prediction model by the STE test.

Table 3. The potency of the direct MTT reduction of the selected substances.

Table 4. The evaluation of 5% MTT reducers.

Absorbance interference of MTT reducers and strongly colored substances in the STE test at relative dilutions of 5% and 0.05%

Next, we examined the influence of interference with absorbance induced by direct MTT reduction at 5% and 0.05% dilutions. Here, we focused on whether identification as an irritant as shown Table 2 via the STE test was observed in the presence of MTT reducers. We therefore reasoned as follows: if a substance was classified as an irritant following the STE test (i.e., no false negatives), we considered this determination to be correctly evaluated without the interference of direct MTT reduction. Furthermore, cell viability measurements were carefully checked for whether they showed a dose-dependent relationship between concentration and viability because readings at 5% may have been higher than at those at 0.05% if the coloration of the test substance had interfered with absorbance. Next, if the substance was classified as a non-irritant following the STE test, the cell viability was carefully confirmed by focusing on whether cell viability was much higher than 100% based on color tones of the formazan dye in the 96-well microplates at the two dilution levels.

We also evaluated whether interference with absorbance by strongly colored substances occurred at 5% and 0.05% dilutions; this was determined based on whether specific test substances were identified as irritants by the STE test as shown Table 2. Those classified as irritants were considered to be correctly classified regardless of their coloration. Furthermore, we checked for dose dependence as with the evaluation of the MTT reducers. Next, for substances that were classified as non-irritants by the STE test, an additional test was performed in reference to the EpiOcular EIT SOP (MatTek Corporation, 2015). This test was performed in medium without MTT, unlike the normal STE test. After incubation for 2 hr in a no-MTT medium, cells were extracted from wells where the colored substances might have been adsorbed. Extraction was performed using 0.04 N HCl–isopropanol. Following the same calculation of cell viability used for the STE test, the bound color rates were determined for the 5% and 0.05% dilutions based on the absorbance of the solvent control. If the cell viability obtained by the no-MTT assay subtracted from the viability obtained from the normal STE test exceeded 70% (i.e., the STE test threshold) for both 5% and 0.05% dilutions, we determined that the test value was unaffected by absorbance interference.

Analytical evaluation by gas chromatography

We measured the volume of substance in wells washed with PBS after exposure for each dilution using a gas chromatography-flame ionization detector (GC-FID, Agilent Technologies Japan, Ltd., Tokyo, Japan). This measurement was performed if cell viability did not show a dose-dependent relationship, even if the substance was classified as an irritant. For GC-FID testing, 300 µL of methanol was added to 96-well plates after they had been washed out with PBS following 5 min of exposure in the STE test. The methanol solution was then sonicated for 1 min and further diluted ten times. After preparation, samples were analyzed using a GC-FID fitted with a HP-1 capillary column (30 m × 0.25 mm ID, 0.25 μm film). The oven temperature was held at 50°C for 1 min, then increased from 50°C to 260°C at a rate of 15°C/min. The temperature was then held at 260°C for 5 min. The inlet and detector temperatures were both 250°C. The injection volume was 1.0 µL, the flow rate of helium was 1.0 mL/min, and injection was performed in split mode (20:1).

RESULTS

We first determined whether the selected substances were MTT reducers at each concentration. Table 3 shows the substances that were expected to be MTT reducers at 5%, at 0.05%, or when undiluted. Next, we checked whether the undiluted form of these 25 substances showed the potential to interfere with absorbance; here, all 25 substances were found to be MTT reducers (Table 3). Seventeen substances showed marked changes, while eight showed slight changes (Table 3). The 25 MTT reducers were then tested for whether they directly reduced MTT at dilutions of 5% and 0.05% using the STE test. In their respective diluted forms, 13 substances turned either blue or purple at 5% dilution (Table 3) and were labeled as 5% MTT reducers. In addition, four of the 5% MTT reducers showed marked changes. These included 2-ethylhexylthioglycolate, calcium thioglycolate [10% in aqueous solution], diethylethanolamine, and gamma-mercaptopropyl trimethoxysilane. In contrast, nine substances showed slight changes, including 3-(2-aminoethylamino)propyl]trimethoxysilane, bis-(3-aminopropyl)-tetramethyldisiloxane, diethylethanolamine [50% in aqueous solution], diethylethanolamine [25% in aqueous solution], di-isopropanolamine [10% in aqueous solution], gamma-aminopropyl triethoxy silane, n-butanal, n-octylamine, and monoethanolamine [10% in aqueous solution] (Table 3). Importantly, at the 0.05% dilution level, no substance turned either blue or purple, indicating that they did not reduce MTT in this highly diluted form (Table 3).

After checking whether the selected substances directly reduced MTT, we performed the STE test. Table 4 shows STE test results for the 13 5% MTT reducers. Seven of the thirteen substances that were classified as GHS Cat. 1 based on in vivo data were identified as irritants. Further confirmation of the cell viabilities of six of these substances at dilutions of 5% and 0.05% showed dose dependence. Their cell viabilities at 5% dilution were as follows: 0.2% for diethylethanolamine, 11.6% for [3-(2-aminoethylamino)propyltrimethoxysilane, 1% for diethylethanolamine (50% in aqueous solution), 0.8% for diethylethanolamine (25% in aqueous solution), 1.6% for gamma-aminopropyl triethoxy silane, and 2.9% for n-octylamine (Table 4). Furthermore, the cell viabilities of all of these substances were nearly 100% at 0.05% dilution; that is, we observed no false negatives. However, for bis-(3-aminopropyl)-tetramethyldisiloxane, the observed cell viability at a 5% dilution (i.e., 31%) was higher than that observed at 0.05% dilution (i.e., 18.7%). This result did not show dose dependence; therefore we hypothesized that MTT was directly reduced at 5% dilution because we did not observe any MTT reducers at 0.05% dilution (Tables 3 and 4). To confirm this hypothesis, we conducted additional tests. First, the optical densities (ODs) at 570 nm obtained by the substances exposed at 5% and 0.5% dilutions were confirmed (Table 5). Using the ODs after subtracting the reading of the blank, the cell viabilities were calculated: at 5% dilution we observed a cell viability of 33.8%, and at 0.5% dilution we observed a cell viability of 2.6% (Table 5). Here, the value as 2.6% at 0.5% dilution (without direct MTT reduction; data not shown) was used to understand easily the difference of the influence for direct reduction in comparison with the value as 18.7% at 0.05% dilution.

Table 5. The comfirmation for the interference of bis-(3-Aminopropyl)-tetramethyldisiloxane for each exposure condition.

Next, after washing twice with PBS following the STE test, the remaining volumes of bis-(3-aminopropyl)-tetramethyldisiloxane in wells exposed to 5% and 0.5% dilutions were measured using the GC-FID. We detected 0.524 mg of bis-(3-aminopropyl)-tetramethyldisiloxane administered to wells to which the 5% dilution was added, and the substance was not detected in wells to which the 0.5% dilution was added (Table 6). Moreover, the ODs of the wells exposed to the 5% and 0.5% dilutions were 0.330 and 0.080, respectively (Table 5). The latter value was similar to the OD of the blank (i.e., negative control) sample, which showed an OD reading of 0.059 (Table 5). These results indicate that the substance interfered with absorbance by direct MTT reduction at 5% dilution exposure but did not interfere at 0.5% dilution exposure.

Table 6. The volume of bis-(3-Aminopropyl)-tetramethyldisiloxane in the well by each exposure condition using gas chromatography.

The two substances classified as GHS Cat. 2 using in vivo data were identified as irritants by the STE test (Table 4). The cell viabilities for two substances at 5% dilution were low, and we observed dose dependence for each result. We therefore concluded that the two Cat. 2 substances were correctly evaluated. In addition, all four No Cat. substances were evaluated as non-irritants (Table 4). Their obtained cell viabilities did not greatly exceed 100%. We then also considered that all four No Cat. substances were correctly evaluated by the STE test reported here.

Next, strongly colored substances were checked by measuring their absorbances for the range of wavelengths of visible light at 5% and 0.05% dilutions. Figs. 1 and 2 show the absorbance of acid red 92 and methylene blue solution at 5% and 0.05% dilutions. Each absorbance reading at 5% dilution exceeded the measurement limit at 570 nm. That is, the results indicate that the strongly colored substances may interfere with the absorbance in the STE test.

Fig. 1

The absorbance of Acid red 92 in visible light. The absorbance were measured at 5% and 0.05% dilutions between 400 nm and 800 nm. The substance would have the strongly potential to interfere O.D. at 5% dilution, because the absobance of mesurement limit was obtained at 570 nm as the absorption wavelength of MTT.

Fig. 2

The absorbance of Methylene blue solution in visible light. The absorbance were measured at 5% and 0.05% dilutions between 400 nm and 800 nm. The substance would have the strongly potential to interfere O.D. at 5% dilution, because the absobance of mesurement limit was obtained at 570 nm as the absorption wavelength of MTT.

The two strongly colored substances were evaluated using the STE test, and acid red 92 identified as Cat 1 by in vivo data was classified as an irritant. Furthermore, the observed cell viabilities at concentrations of 5% and 0.05% were both close to 0% (Table 7). Therefore, we considered that this substance was correctly evaluated. The STE test classified the methylene blue solution as a non-irritant (Table 7). Methylene blue solution was then confirmed for the influence of absorbance interference by the additional test as described in the Materials and Methods. The absorbance rates using the no-MTT assay were 6.9% and 7.4% at 5% and 0.05% dilutions, respectively (Table 8). From these results, the cell viabilities obtained from the no-MTT assay subtracted from the cell viabilities obtained from the normal STE test were 113.1% and 92.5% at 5% and 0.05% dilutions, respectively (Table 8). Both cell viabilities exceeded 70%, so we concluded that the irritancy of this substance was correctly evaluated.

Table 7. The evaluation of strongly colored substances.

Table 8. The comfirmation for the interference of Methylene blue solution by no MTT assay in the STE test.

DISCUSSION

In this study, we confirmed whether MTT reducers and strongly colored substances were correctly evaluated in the STE test.

First, we confirmed whether the test substances could directly reduce MTT. Here, we used a volume of 20 µL as a test substance solution, which was sufficient for a confirmatory under STE test conditions. That is, the test conditions assumed that the volume remained in the well was the 20 µL was enough conservative for the confirmation test. Other test protocols for the confirmation of direct MTT reduction—for example, the RhCE method by SkinEthic™ HCE —also use a similar condition in which 30 µL of test substance solution is mixed with 300 µL of MTT solution (OECD, 2019a). We therefore concluded that the excessive condition would be used to confirm direct MTT reduction in this study.

In the STE test conditions, we found that 13 of the 25 substances that directly reduced MTT at undiluted concentrations were 5% MTT reducers. Thus, almost half (i.e., 12 of 25) of the substances were identified as non-reducers. Furthermore, none of the substances were found to be MTT reducers at 0.05% dilution. When we examined the depth of the color induced during this test, we found that over half of the 5% MTT reducers elicited a “slight change.” These results mean that diluted substances can diminish the influence of absorbance interference by direct MTT reduction in the STE test conditions. In addition, it is also expected that the influence of interference would be lower in the test conditions than in other tests in which exposure to undiluted substances was performed (e.g., the RhCE test). Therefore, we concluded that the STE test was less susceptible to absorbance interference when evaluating MTT reducers.

The cell viability of wells exposed to bis-(3-aminopropyl)-tetramethyldisiloxane was low, which agrees with the classification of this substance as an irritant by the STE test. Moreover, we observed absorbance interference by direct MTT reduction. As a result, we obtained cell viabilities of 31% at 5% dilution and 18.7% at 0.05% dilution. These cell viabilities did not show dose dependence, which was also not reported except for this substance in previous STE test data (Abo et al., 2018). When we performed an additional confirmation experiment using GC-FID, this substance was detected at 0.524 mg in wells exposed to 5% dilution but was not detected in wells exposed to 0.5% dilution (Table 6). Here, we hypothesized that the substance is not completely soluble in the solvent used. This hypothesis is consistent with visual observation in that 96-well plates showed deposition of this substance under the microscope after washing (data not shown). Moreover, insoluble residues present in the well may cause absorbance interference. Then, the oil droplets as residues showed migration to the top of the 5% dilution sample within 5 min of exposure time during the STE test. This phenomenon was not observed during the first test. Thus, we concluded that bis-(3-aminopropyl)-tetramethyldisiloxane is not an applicable substance in the STE test. Although solubility checks were described in the STE SOP, this result further emphasizes that solubility checks should be conducted carefully. Here, bis-(3-aminopropyl)-tetramethyldisiloxane was identified as an irritant. However, if the STE test used here is not suitable for assessing this substance, other in vitro tests should be conducted to evaluate its potential for eye irritation.

In Table 4, the six of seven Cat. 1 substances identified by in vivo data (i.e., all those except bis-(3-aminopropyl)-tetramethyldisiloxane) were identified as irritants in the STE test. The cell viability of these six substances ranged from 0.2% to 11.6% at 5% dilution (Table 4). These values suggested that exposure to these substances caused cell death and that this phenomenon was dose-dependent for all six substances. We conclude that these substances were correctly classified for their eye irritation potential. Moreover, for the degrees of color listed in Table 3, diethylethanolamine showed a “marked change” at 5% dilution, but its cell viability was close to 0%. In contrast, the remaining five substances elicited a “slight change” in color depth at 5% dilution (Table 3). This means that the influence of interference can have a negligible relationship with the results of the STE test. Therefore, these results indicate that all substances were correctly evaluated by the STE test, regardless of the degree of color intensity of the diluted test substance.

[3-(2-aminoethylamino)propyl]trimethoxysilane, gamma-aminopropyl triethoxy silane, and n-butanal were also evaluated by an RhCE test using EpiOcular^TM (Alépée et al., 2016). Here, the test was performed using live and killed tissues. Briefly, the cell viabilities of these three substances were calculated by subtracting the absorbance obtained in killed tissue from the absorbance obtained in normal tissue. These results showed almost the same values as the cell viabilities obtained using a normal RhCE test (Alépée et al., 2016). It was first expected that the cell viabilities would be different because of absorbance interference caused by direct MTT reduction. However, this result might also reflect the fact that these three substances were less adsorbed to corneal tissue and plate wells. Therefore, we consider that at least these three substances were further less adsorbed during the STE test.

We expected that the cell viability results for the No Cat. substances that were MTT reducers would be as high as 150% or 200%. However, the cell viability results did not record such high values for the four No Cat. substances examined in this study. As noted previously, the cell viability of diethylethanolamine, which showed a “marked change” in MTT solution at 5% dilution, was close to 0%. In the absence of large increases in cell viability, the influence of absorption interference was negligible for STE tests using diluted solutions. As a result, the four No Cat. substances mentioned were correctly evaluated. Furthermore, for calcium thioglycolate (10% in aqueous solution) and di-isopropanolamine (10% in aqueous solution), Takahashi et al. (2008) reported that the undiluted forms of these substances were classified as irritants using the STE test. Here, we found that their respective cell viabilities were close to 0% at 5% dilution and showed dose dependence at 5%, 0.5%, and 0.05% dilutions. Thus, the result for the undiluted substance, which classified these substances as irritants, was correct. Therefore, we conclude that both calcium thioglycolate (10% in aqueous solution) and di-isopropanolamine (10% in aqueous solution) were correctly evaluated as non-irritants when diluted, because of both being irritants at higher exposure concentrations.

When classifying substances as No Cat. in the STE test, solids other than surfactants cannot be reliably tested. Moreover, because most colored substances are solids, solids can be evaluated only when identifying Cat. 1 substances using the STE test. Although the colored substances tested here were selected in reference to the STE SRD and the study by Alépée et al. (2015), the two selected substances were the only ones that were applicable for the STE test and also available for purchase. Here, because acid red 92 was classified as an irritant by the STE test and showed dose dependence, this substance was correctly evaluated for its eye irritation potential. In contrast, methylene blue solution was classified as a non-irritant by the STE test, and an additional confirmation was performed using a medium that did not contain MTT. As a result, there was no absorbance interference that influenced the classification of the eye irritation potential of methylene blue solution. In addition, methylene blue solution was classified as No Cat. by Alépée et al. (2015). In that study, the cell viabilities calculated using optical density and HPLC analyses were close to 100%, which also suggests that there is no interference (Alépée et al., 2015). Taken together, these results show that two strongly colored substances did not adsorb to cells or to wells under the STE test. This result indicates that the STE test correctly evaluates the irritancy of strongly colored substances, although the number of substances evaluated was low.

In this study, we were able to determine whether there was no influence for judgment of the classification of eye irritation potential for concerned substances except for one MTT reducer as the out of applicability domain. In conclusion, we demonstrated in this study that the STE test is useful for evaluating the potential for eye irritation of various chemicals that contain MTT reducers and strongly colored substances.

Conflict of interest

The authors declare that there is no conflict of interest.

REFERENCES

•  Abo,  T.,  Yuki,  T.,  Xu,  R.,  Araki,  D.,  Takahashi,  Y.,  Sakaguchi,  H. and  Itagaki,  H. (2018): 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. J. Toxicol. Sci., 43, 407-422.
•  Alépée,  N.,  Barroso,  J.,  De Smedt,  A.,  De Wever,  B.,  Hibatallah,  J.,  Klaric,  M.,  Mewes,  K.R.,  Millet,  M.,  Pfannenbecker,  U.,  Tailhardat,  M.,  Templier,  M. and  McNamee,  P. (2015): Use of HPLC/UPLC-spectrophotometry for detection of formazan in in vitro Reconstructed human Tissue (RhT)-based test methods employing the MTT-reduction assay to expand their applicability to strongly coloured test chemicals. Toxicol. In Vitro, 29, 741-761.
•  Alépée,  N.,  Leblanc,  V.,  Adriaens,  E.,  Grandidier,  M.H.,  Lelièvre,  D.,  Meloni,  M.,  Nardelli,  L.,  Roper,  C.S.,  Santirocco,  E.,  Toner,  F.,  Van Rompay,  A.,  Vinall,  J. and  Cotovio,  J. (2016): Multi-laboratory validation of SkinEthic HCE test method for testing serious eye damage/eye irritation using liquid chemicals. Toxicol. In Vitro, 31, 43-53.
•  Alépée,  N.,  Adriaens,  E.,  Abo,  T.,  Bagley,  D.,  Desprez,  B.,  Hibatallah,  J.,  Mewes,  K.,  Pfannenbecker,  U.,  Sala,  À.,  Van Rompay,  A.R.,  Verstraelen,  S. and  McNamee,  P. (2019): Development of a defined approach for eye irritation or serious eye damage for liquids, neat and in dilution, based on cosmetics Europe analysis of in vitro STE and BCOP test methods. Toxicol. In Vitro, 57, 154-163.
•  Barroso,  J.,  Pfannenbecker,  U.,  Adriaens,  E.,  Alépée,  N.,  Cluzel,  M.,  De Smedt,  A.,  Hibatallah,  J.,  Klaric,  M.,  Mewes,  K.R.,  Millet,  M.,  Templier,  M. and  McNamee,  P. (2017): Cosmetics Europe compilation of historical serious eye damage/eye irritation in vivo data analysed by drivers of classification to support the selection of chemicals for development and evaluation of alternative methods/strategies: the Draize eye test Reference Database (DRD). Arch. Toxicol., 91, 521-547.
• European Commission’s Directorate General Joint Research Centre. (2017): DB-ALM Protocol n° 190: SkinEthic™ HCE Eye Irritation Test Liquid (EITL) <http://cidportal.jrc.ec.europa.eu/ftp/jrc-opendata/EURL-ECVAM/datasets/DBALM/LATEST/online/DBALM_docs/190_P_SkinEthicHCE%20Eye%20Irritation%20Test%20Liquid.pdf> (accessed 26. Jan. 2023).
•  Hayashi,  K.,  Mori,  T.,  Abo,  T.,  Koike,  M.,  Takahashi,  Y.,  Sakaguchi,  H. and  Nishiyama,  N. (2012a): 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. J. Toxicol. Sci., 37, 269-280.
•  Hayashi,  K.,  Mori,  T.,  Abo,  T.,  Ooshima,  K.,  Hayashi,  T.,  Komano,  T.,  Takahashi,  Y.,  Sakaguchi,  H.,  Takatsu,  A. and  Nishiyama,  N. (2012b): Two-stage bottom-up tiered approach combining several alternatives for identification of eye irritation potential of chemicals including insoluble or volatile substances. Toxicol. In Vitro, 26, 1199-1208.
• ICCVAM. (2013): Short Time Exposure (STE) Test Method Summary Review Document. adopted 2013, <https://ntp.niehs.nih.gov/pubhealth/evalatm/test-method-evaluations/ocular/ste/index.html#srd> (accessed 22. Dec. 2022).
• MatTek Corporation. (2015): EpiOcular™ Eye Irritation Test (OCL-200-EIT), <https://www.mattek.com/wp-content/uploads/OCL-200-EIT-Eye-Irritation-Test-Protocol-MK-24-007-0055_02_02_2021.pdf> (accessed 22. Dec. 2022).
•  Mosmann,  T. (1983): Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods, 65, 55-63.
• OECD. (2019a): Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage. Test Guideline 492, Organisation for Economic Co-operation and Development. adopted 18 June 2019, <https://www.oecd-ilibrary.org/environment/test-no-492-reconstructed-human-cornea-like-epithelium-rhce-test-method-for-identifying-chemicals-not-requiring-classification-and-labelling-for-eye-irritation-or-serious-eye-damage_9789264242548-en> (accessed 22. Dec. 2022).
• OECD. (2019b): In vitro skin corrosion: reconstructed human epidermis (RHE) test method. Test Guideline 431, Organisation for Economic Co-operation and Development. adopted 18 June 2019, <https://www.oecd-ilibrary.org/environment/test-no-431-in-vitro-skin-corrosion-reconstructed-human-epidermis-rhe-test-method_9789264264618-en> (accessed 22. Dec. 2022).
• OECD. (2020a): Short Time Exposure In Vitro Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage. Test Guideline 491, Organisation for Economic Co-operation and Development. adopted 26 June 2020, <https://www.oecd-ilibrary.org/environment/test-no-491-short-time-exposure-in-vitro-test-method-for-identifying-i-chemicals-inducing-serious-eye-damage-and-ii-chemicals-not-requiring-classification-for-eye-irritation-or-serious-eye-damage_9789264242432-en> (accessed 22. Dec. 2022).
• OECD. (2020b): Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage. Test Guideline 437, Organisation for Economic Co-operation and Development. adopted 26 June 2020, <https://www.oecd-ilibrary.org/environment/test-no-437-bovine-corneal-opacity-and-permeability-test-method-for-identifying-i-chemicals-inducing-serious-eye-damage-and-ii-chemicals-not-requiring-classification-for-eye-irritation-or-serious-eye-damage_9789264203846-en> (accessed 22. Dec. 2022).
• OECD. (2021): In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method. Test Guideline 439, Organisation for Economic Co-operation and Development. adopted 17 June 2021, <https://www.oecd-ilibrary.org/environment/test-no-439-in-vitro-skin-irritation-reconstructed-human-epidermis-test-method_9789264242845-en> (accessed 22. Dec. 2022).
• OECD. (2022a): Defined Approaches for Serious Eye Damage and Eye Irritation. Test Guideline 467, Organisation for Economic Co-operation and Development. adopted 30 June 2022, <https://www.oecd-ilibrary.org/environment/test-no-467-defined-approaches-for-serious-eye-damage-and-eye-irritation_28fe2841-en> (accessed 22. Dec. 2022)
• OECD. (2022b): In vitro skin sensitisation assays addressing the Adverse Outcome Pathway Key Event on Keratinocyte activation. Test Guideline 442D, Organisation for Economic Co-operation and Development. adopted 30 June 2022, <https://www.oecd-ilibrary.org/environment/test-no-442d-in-vitro-skin-sensitisation_9789264229822-en> (accessed 22. Dec. 2022)
•  Takahashi,  Y.,  Koike,  M.,  Honda,  H.,  Ito,  Y.,  Sakaguchi,  H.,  Suzuki,  H. and  Nishiyama,  N. (2008): Development of the short time exposure (STE) test: an in vitro eye irritation test using SIRC cells. Toxicol. In Vitro, 22, 760-770.