review artcle a validation study of a new in vitro eye

Satoshi Nakahara et al., AATEX 24(1), 11-23, 2019

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A Validation Study of a New In Vitro Eye Irritation Test using the Reconstructed Human Corneal

Epithelial Tissue, LabCyte CORNEA-MODEL24

1Satoshi Nakahara, 2Hajime Kojima, 3Takashi Omori, 4Aimi Yamashita, 4Mai Endo, 4Maiko Satake, 5Hideki Nishiura,

6Shinsuke Shinoda, 6Saori Hagiwara, 7Toshihiko Kasahara, 7Haruna Tahara, 7Yusuke Yamamoto, 8Hidefumi Ikeda,

9Yuichiro Yoshitake, 10,11Jeong Ik Lee, 11Yuna Han, 11Soojung Lee, 12Katsura Sugawara, and 12Masakazu Katoh

1 Maruishilabo Corporation, Osaka, Japan

2Japanese Center for the Validation of Alternative Methods (JaCVAM), National Institute of Health Sciences, Tokyo, Japan

3Division of Biostatistics Department of Social/Community Medicine and Health Science, Kobe University School of Medicine, Kobe, Japan

4Faculty of Culture and Information Science, Doshisha University, Kyoto, Japan 5R&D, Nihon Kolmar Co., Ltd., Kashiwara, Osaka, Japan

6Examination Room 3, Drug Safety Testing Center Co., Ltd., Saitama, Japan 7Safety Evaluation Center, Fujifilm Corporation, Minamiashigara, Kanagawa, Japan

8Product Assurance, Mandom Corporation, Osaka, Japan 9Research & Development Division, Oppen Cosmetics Co., Ltd., Kusatsu, Shiga, Japan

10Department of Veterinary Obstetrics and Theriogenology, College of Veterinary Medicine, Konkuk University, Seoul, Korea

11Institute of Biomedical Science & Technology, Konkuk University, Seoul, Korea 12R&D Department, Japan Tissue Engineering Co., Ltd., Gamagori, Aichi, Japan

Abstract Eye damage is defined as the production of reversible changes in the eye, following the application ofa test chemical to the ocular surface. Chemicals causing such changes are classified as category 2 bythe United Nations Globally Harmonized System of Classification and Labeling of Chemicals (UNGHS). In 2015, an eye irritation test (EIT) method developed to identify category 2 chemicals wasscientifically validated and approved by the OECD as the test guideline 492 (OECD TG 492). A newEIT using LabCyte CORNEA-MODEL24, a commercially available recombinant human cornealepithelial (RhCE) tissue model developed by Japan Tissue Engineering Co., Ltd. (J-TEC), has beenestablished as a similar method to the OECD TG 492. Here we report the results of a validation studyconducted according to the performance standard for the OECD TG 492 to evaluate the accuracy andperformance reliability of LabCyte CORNEA-MODEL24 EIT at three independent laboratories.LabCyte CORNEA-MODEL24 EIT was highly predictive and reliable, showing sensitivity of 97%,specificity of 68.9%, and overall accuracy of 83.5%. These results satisfy the acceptance criteria ofthe performance standard for the OECD TG 492 (90% for sensitivity, 60% for specificity and 75% foraccuracy). The reproducibility of results within- and between the three participating laboratories wasalso within acceptable ranges. Altogether, the results from this validation study confirm that LabCyteCORNEA-MODEL24 EIT is a robust method to identify chemicals with the potential to trigger eyeirritation events. Key words: reconstructed human corneal epithelium, eye irritation, alternative methods, LabCyte

CORNEA-MODEL24, validation

REVIEW ARTCLE

Satoshi Nakahara et al., A Validation Study of Eye Irritation Test using LabCyte CORNEA-MODEL24, AATEX 24(1), 11-23, 2019

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Introduction The Draize eye test (Draize et al., 1944) has long been used to evaluate the ocular irritation potential of chemical substances. The test con-sists of a score determined based on macro-scopic changes observed in the rabbit cornea, conjunctiva and iris, and was officially adopted in the Organization for Economic Cooperation and Development test guideline (OECD TG) 405 (OECD, 2012a) as the means to classify eye-irritancy substances for regulatory purposes. However, the Draize eye test is often criticized for both ethical (animal welfare) and scientific reasons (subjective scoring, low inter-laboratory reproducibility, and sensitivity differences com-pared to humans) (Christian et al., 1996), result-ing in a strong need for an in vitro evaluation method that can be used to assess eye irritancy.

Several ex vivo and in vitro ocular irritancy test methods have been developed to address the issues raised by the Draize test. The bovine cor-neal opacity and permeability (BCOP) test, and the isolated chicken eye (ICE) test were found to adequately predict severe irritancy and non-irritancy, but not irritancy, ex vivo, and were adopted as OECD TG 437 and 438, respectively (OECD, 2013a; OECD, 2013b). In addition, in vitro methods such as the fluorescein leakage (FL) test method, the short time exposure test using a monolayer culture system, and the eye irritation test method using reconstructed human corneal epithelial tissue were adopted as OECD TG 460, 491 and 492, respectively (OECD, 2012b; OECD, 2015c; OECD 2015a).

So far, four different EITs have been estab-lished using different RhCE models. Two of them, EpiOcular (MatTek, MA, USA), a model reconstructed using normal human epidermal keratinocytes (Stern et al., 1998; Kaluzhny et. al., 2011; Pfannenbecker et. al., 2013), and SkinEthic HCE (SkinEthic, France), a model reconstructed using an immortalized human corneal epithelial cell line (Van Goethem et. al., 2006, Cotovio et. al., 2010; Alépée et. al., 2013, Alépée et. al., 2016a; Alépée et. al., 2016b) have completed a formal validation study and were implemented as validated reference methods (VRM) into the OECD TG 492. Although not yet adopted by the OECD TG 492, LabCyte

CORNEA-MODEL24 (Japan Tissue Engineer-ing Co., Ltd, Japan), a model reconstructed us-ing normal human corneal epithelial cells (Katoh et. al., 2012; Katoh et. al., 2013); and MCTT-HCE (Modern Cell & Tissue Technolo-gies, Korea), a model reconstructed using nor-mal human corneal epithelial cells (Jung et. al., 2011; Jang et. al., 2015), are also EITs that measure cell viability as the endpoint of eye irritancy prediction.

The LabCyte CORNEA-MODEL24 has a representative corneal epithelium-like structure that contains the three major corneal epithelial layers: the superficial layer, the wing cell layer, and the basal layer. The reconstructed corneal tissue expresses the corneal epithelial marker Cytokeratin 3, mucins (mucin-1 and mucin-16), cell adhesion molecules (E-cadherin, Claudin-1 and Desmoglein-3), and the basement membrane component Laminin 332 in a pattern that resem-bles that of the human corneal epithelium (Katoh et al., 2013). Furthermore, electron microscope imaging reveals well developed microvilli in the superficial layer of the tissue, providing evidence that the histological structure of the LabCyte CORNEA-MODEL24 is highly similar to that of a native human corneal epithelium (Katoh et al., 2013).

As LabCyte CORNEA-MODEL24 pro-vides a promising alternative to corneal irritation assessment using animal testing, a protocol us-ing the tissue model for eye irritancy evaluation of chemicals was established and optimized for the application of liquids and solid substances. In short, liquid chemicals are applied to the tis-sue model for one minute, and incubated for 24 hours, whereas solid chemicals are applied for 24 hours without post-exposure incubation (Katoh et al., 2012).

The objective of this study is to confirm whether the LabCyte CORNEA-MODEL24 EIT adheres to the OECD TG 492. To this end, a validation study using 30 test chemicals listed in the performance standard for the OECD TG 492 (OECD, 2015b) was conducted at three inde-pendent facilities to assess the reproducibility of the results within and between laboratories (reliability), and the relevance (predictive capac-ity) of the new test method.


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Materials and Methods 1. Validation management team structure A validation management team (VMT) was or-ganized to coordinate the LabCyte CORNEA- MODEL24 EIT validation study. The VMT was responsible for the establishment of goal state-ments, project planning, including study proto-col writing and revision, quality control (QC) audits, data management procedures, monitoring of study progression, results interpretation, and publication of reports. In addition, the VMT was responsible for the selection of participating la-boratories. A subgroup of the VMT, the chemical selection group, was responsible for the selection, coding and distribution of chemicals, and liaising with suppliers. An independent biostatistics group was responsible for the col-lection, screening and analysis of the data. The following three laboratories participated in the validation study.

Laboratory A (Lab A): Drug Safety Testing Center Co., Ltd. Laboratory B (Lab B): Nihon Kolmar Co., Ltd. Laboratory C (Lab C): Fujifilm Corporation.

Each participating laboratory was responsi-

ble for complying with Good Laboratory Prac-tices (GLP) principles and QC specifications. The validation study was conducted in accor-dance with the principle and criteria documented in the OECD Guidance Document on the Valida-tion and International Acceptance of New or Updated Test Methods for Hazard Assessment (OECD, 2005), and according to the modular approach to validation described by Hartung et al. (2004).

2. Test Chemicals The 30 reference test substances (15 GHS no category and 15 GHS category 1 or 2) used in this study were selected in accordance with the performance standard for the OECD TG 492 (OECD, 2015a, Table.1). The test substances included both liquid and solid chemicals that were sourced, independently blind coded, and distributed the participant laboratories by the Japanese Center for the Validation of Alternative Methods (JaCVAM).

1) LabCyte CORNEA-MODEL EIT (1) LabCyte CORNEA-MODEL24

The LabCyte CORNEA-MODEL24 is a com-mercially available RhCE developed and manu-factured by Japan Tissue Engineering Co., Ltd. The tissue model is prepared with corneal epithelial cells acquired from the Rocky Moun-tain Lions Eye Bank (CO, USA), and cultured and expanded on a feeder layer of 3T3-J2 cells (Rheinwald and Green, 1975; Green, 1978). To reconstruct the corneal epithelial structure, pro-liferating corneal epithelial cells are cultured on a 0.3 cm2 inert filter substrate at the air-liquid interface for 13 days. The resulting tissue con-sists of a multilayered epithelial structured with features of the normal human corneal epithelial tissue (Katoh et al., 2013). Prior shipping, the LabCyte CORNEA-MODEL24 is embedded in agarose gel for transport. (2) LabCyte CORNEA-MODEL24 EIT test protocol Upon receipt, LabCyte CORNEA-MODEL24 tissues were aseptically removed from the aga-rose gel, and transferred into a 24-well plate containing 500 μL of assay medium. Tissues were incubated overnight at 37°C in a humidi-fied atmosphere of 5% CO2 in air. a) Procedure of the LabCyte CORNEA- MODEL24 EIT for liquid test chemicals LabCyte CORNEA-MODEL24 tissues were topically exposed to 50 μl of liquid chemicals for one minute, in triplicate. Dulbecco’s phos-phate buffered saline (D-PBS) and ethanol were applied under the same conditions as negative and positive controls, respectively. After expo-sure, the tissues were rinsed with D-PBS applied from a wash bottle for 10 times or more in order to remove any chemical residue from the tissue surface, and blotted on a paper towel before they were transferred to new wells containing 500 μL of fresh assay medium. Tissues were then incu-bated for 24 hours at 37°C in a humidified atmosphere of 5% CO2 in air. After incubation, tissues were transferred to new wells containing 300 μl of WST-8 solution freshly prepared by diluting Cell Counting Kit-8 (Dojindo Co., Japan) with Earle’s Balanced Salt Solution


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(Sigma-Aldrich, MO, USA) at 1:10 (Ishiyama et al., 1997; Tominaga et al., 1999). After tissues were incubated for four hours, 200 μL of culture supernatant were transferred to a 96-well plate, and the optical density was measured at 450 nm and 650 nm (reference), using WST-8 solution as blank. Cell viability was calculated as the percentage relative to the viability of the nega-

tive control, considered 100%. The mean of three replicates was used to classify the chemical irritation potential. b) Procedure of the LabCyte CORNEA- MODEL24 EIT for solid test chemicals LabCyte CORNEA-MODEL24 tissues were topically exposed to 10 mg of solid chemicals

Table 1 List of 30 reference chemicals required to determine accuracy and reliability for similar or modified RhCE eye

irritation test methods. 1) CAS No.: Chemical abstracts service registry number. 2) In vivo class was referred from the GHS classification. 3) TCI: Tokyo Chemical Industry Co., Ltd; WAKO: Wako Pure Chemical Industries, Ltd.


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for 24 hours, in triplicate. Non-treated tissues and tissues exposed to 10 mg of Lauric Acid were used as negative and positive controls, re-spectively. After exposure, the tissues were rinsed with D-PBS applied from a wash bottle for 10 times or more in order to remove any chemical residue from the tissue surface, and blotted on a paper towel before they were trans-ferred to new wells containing 300 μL of WST-8 solution, as described above. c) Eye irritation prediction model A chemical is identified as irritant (i.e. Category 1 or Category 2 according to the GHS classifica-tion), if the mean tissue viability of three indi-vidual tissues exposed to the chemical is below 40%, relative to the mean viability of the respec-tive negative control. d) Detection of chemical interference with WST-8 endpoints and correction procedures Chemicals that may stain epithelial tissues or directly reduce WST-8 may affect the eye irrita-tion protocol endpoint. To identify such sub-stances, 50 mL (liquids) or 10 mg (solid) of the test chemical were added to 300 mL of WST-8 solution in a 24-well plate and incubated for 4 hours at 37°C in a humidified atmosphere of 5% CO2 in air. Untreated WST-8 was used as control. Upon naked eye observation, if the test chemical stained the WST-8 solution, an additional test was conducted to correct the measured OD. First, tissues were freeze-killed at -80°C or lower, for 30 minutes twice, and used to test liquid and solid chemicals as described in sessions 2.3.2.1 and 2.3.2.2, respectively. The corrected OD was then calculated using the following formula: Corrected OD = Each tissue OD (viable tissue) test chemical - [Mean OD (freeze-killed tissue) test chemical - Mean OD (freeze-killed tissue) negative control]. A tissue OD below zero was regarded as zero. (3) Acceptance criteria The absolute OD of the negative control for liq-uid test chemicals or for solid test chemicals (D-PBS-treated tissues and untreated tissues, respectively) was used as an indicator to assess tissue viability after shipping and storing proce-dures and under specific conditions of use at the

participating laboratories. Moreover, a test was considered acceptable if the following three cri-teria were met: (1) the mean OD of the negative control must be ≥ 0.5 and ≤ 1.3; (2) the viability of the positive control must be < 40% and; (3) the standard deviation (SD) of tissue viability of three identically treated replicates for negative control, positive control and test chemicals must be ≤ 18%. 3) Defined reliability and accuracy value The VMT established that the target value for within-laboratory reproducibility, between labo-ratory reproducibility, and predictive capacity (sensitivity, specificity, and overall accuracy) should be equal to, or better than, a value derived from the EpiOcular EIT, a VRM of the OECD TG 492.

Since the results for the EpiOcular EIT were 93%, 93% and 97% at three participating laboratories, the within-laboratory reproducibil-ity target for this validation study was set to 90% or higher. Likewise, the VMT used the EpiOcu-lar result (90%) as a reference and set the between-laboratory reproducibility target for this validation study to 85% or higher. The target value for sensitivity was set to be equal to, or higher than, 90% (EpiOcular EIT: 93%), and the target value for specificity was set to be equal to, or higher than, 60% (EpiOcular EIT: 63%). No restrictions to the sensitivity or specificity values were applied, and non-concordance of results with the in vivo classification was acceptable just as long as the final sensitivity and specific-ity of the test method achieved the target values and the overall accuracy was equal to, or higher than, 75% (EpiOcular EIT: 78%). One restric-tion was added, however, that none of the UN GHS Category 1 reference chemicals were to be under-predicted as “no category” based solely on valid test results from all participating laborato-ries. Results 1) LabCyte CORNEA-MODEL24 QC The histological observation performed as a QC test demonstrated that all LabCyte CORNEA- MODEL24 used in this validation study were composed of a multilayered corneal epithe-


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lium-like tissue constituted of viable cells under a non-keratinized surface. The cell viability, measured by both MTT and WST-8 assays, as well as the barrier function, determined as the IC50 for SLS, showed that little variation was observed between lots provided to each partici-pating laboratory. All batches used in this vali-dation study passed the manufacturer’s release criteria (data not shown).

2) Positive and negative controls All negative control OD values met the acceptance criteria established for this study (0.5 ≤ Mean OD ≤ 1.3, and SD ≤ 18%). Based on negative controls alone, the frequency of non-qualified tests was 0% (Table 2). Likewise, positive control data also met the criteria established (cell viability ≤ 40%, and SD ≤ 18%), and the frequency of non-qualified tests was 0% (Table 3).

Table 2 Mean OD value (n=3) and SD of cell viability of negative controls for the different tissue batches used in

the validation study. Upper rows show mean OD values, lower rows show SD of cell viability in brackets.

Table 3 Mean cell viability (n=3) and SD of positive controls for the different tissue batches used in the vali-

dation study. Upper rows show mean cell viability, lower rows show SD of cell viability in brackets.


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3) Eye irritation prediction of reference chemicals The mean cell viability for each of the chemicals tested at the three participating laboratories is shown in Table 4. One data point at Lab A, 2,2'-[[3-Methyl-4-[(4-nitrophenyl)azo]-phenyl]imino]bis-ethanol (No. 26), generated SD higher than 18%, thus failing to meet the acceptance criteria. One the other hand, all data points from Lab B met the acceptance criteria and the fre-quency of non-qualified tests was 0% (0/90). At

Lab C, however, one non-qualified test was produced for Methylthioglycolate (No. 2), 2-Ethoxyethyl methacrylate (No.17), 3- Phenoxybenzyl alcohol (No. 18), and Ethyl thioglycolate (No. 21), and three were produced for 2,2'-[[3-Methyl-4-[(4-nitrophenyl) azo]-

phenyl] imino]bis-ethanol (No. 26). Complete data matrices for chemicals No. 2, No. 18, and No. 21 were submitted upon being re-tested twice. Although chemical No. 26 was tested for five times at Lab C, only two qualified results

Table 4 Within-laboratory reproducibility in the LabCyte CORNEA-MODEL24 EIT validation study. Mean cell viability(± SD) for 30 chemicals and concordance of prediction within each laboratory. Cells with a grey background correspond to classified prediction (mean cell viability ≤40%). 1) WST-I: WST-8 interfering chemicals. 2) WLR: Within-laboratory reproducibility; C: Concordance; NC: Non-concordance.


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were obtained. Furthermore, chemical No. 26 at Lab A and chemical No. 17 at Lab C produced only two qualified results, as these chemicals were not re-tested. Overall, the frequency of non-qualified results was 1% (1/90) for Lab A, and 7% (7/95) for Lab C.

(Ethylenediaminepropyl)-trimethoxysilane (No. 1), Methylthioglycolate (No. 2), 1,5- Naph-thalenediol (No. 10), Ethyl thioglycolate (No. 21), 2,2'-[[3-Methyl-4- [(4-nitrophenyl) azo]-phenyl] imino]bis-ethanol (No. 25) and Trisodium mono- (5-(1,2 -dihydroxyethyl)-4- oxido-2-oxo- 2,5- di-

Table 5 Between-laboratory reproducibility in the LabCyte CORNEA-MODEL24 EIT validation study.Mean cell viability of 3 independent runs for 30 chemicals and concordance of prediction betweenparticipating laboratories. 1) CLASS: classification according LabCyte CORNEA-MODEL EIT. 2) I: Irritant; NI: Non irritant. 3) BLR: Between-laboratory reproducibility; C: Concordance; NC: Non-concordance.


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hydro-furan-3-yl) phosphate (No. 30) were de-tected as chemicals that interfere in the WST-8 endpoint at all participating laboratories (Table 4). The ODs of these chemicals were corrected using freeze-killed tissues.

4) Reliability (1) Within-laboratory reproducibility Following the performance standard require-ments, within-laboratory reproducibility was determined as the concordance of classification (UN GHS Category 1 or 2, and No Category) of three independent experiments for the subset of 30 reference chemicals tested in this study. As shown in Table 4, Lab A consistently classified the 30 reference chemicals in three independent experiments and achieved a reproducibility rate of 100%. Lab B produced two non-concordant classifications (chemicals No. 13 and No.14), resulting in within-laboratory reproducibility of 93.3% (28/30). Finally, Lab C produced one non-concordant classification (chemical No. 16), resulting in within-laboratory reproducibility of 96.7% (29/30). Altogether, the results from all laboratories were acceptable, having a repro-ducibility rate equal to or higher than 90%.

(2) Between-laboratory reproducibility Between-laboratory reproducibility was deter-mined as the concordance of predictions (UN GHS Category 1 or 2, and No Category) obtained from replicate tests of 30 reference chemicals tested in this study, at all three par-ticipating laboratories. As shown in Table 5, with four non-concordant prediction in total (chemi-cals No. 13, No. 14, No. 16, and No. 17), between-laboratory reproducibility was 86.7% (26/30), thus meeting the target established for this study (≥ 85%). 5) Predictive capacity Lab A and Lab C correctly predicted all 15 UN GHS Category 1 or 2 chemicals. Lab B, on the other hand, correctly predicted 13, generating two false negatives in two valid test runs of Diethyl toluamide (No. 13), and two valid test runs of 1,4-Dibutoxy benzene (No. 14) (Table 4). Furthermore, all three participating laboratories accurately predicted the seven UN GHS Cate-gory 1 test chemicals. Sensitivity was 100% at Lab A, 93.3% at Lab B, and 100% at Lab C, and a two-by-two cumulative table resulted in 97.8% sensitivity (Table 6).

Table 6 Participating laboratories’ and overall predictive capacity for the set of 30 chemicals based on individual

laboratory predictions from the LabCyte CORNEA-MODEL24 EIT validation studies.

Sensitivity (%) = Number of chemicals predicted as category 1/2 by In vitro method / Number of chemicals classified as category 1/2 in UN GHS. Specificity (%) = Number of chemicals predicted as no category by In vitro method / Number of chemicals classified as no category in UN GHS. Accuracy (%) = Number of chemicals predicted in same category as UN GHS by In vitromethod / Number of reference chemicals.


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Lab A and Lab C correctly predicted 10 of 15 UN GHS No Category chemicals, whereas Lab B correctly predicted 11. Four substances gener-ated false positives in all participant laborato-ries: Ethyl thioglycolate (No. 21), 2,2'-[[3- Me-thyl-4-[(4-nitrophenyl)azo]-phenyl] imino] bis-ethanol (No. 25), Cellulose, 2-(2-hydroxy-

3-(trimethylammonium)propoxy) ethyl ether chloride (91%) (No. 28), and Trisodium mono-(5-(1,2-dihydroxyethyl)-4-oxido-2-oxo-2,5-dihydro-furan-3-yl) phosphate (No. 30). Addi-tionally, 2-Ethoxyethyl methacrylate (No. 17) generated a false positive at Lab A, and 1-Ethyl-3-methylimidazolium ethylsulphate (No. 16) generated false positives in two valid test runs at Lab C. Therefore, specificity was 65.9% at Lab A, 73.3% at Lab B, and 67.4% at Lab C, and a two-by-two cumulative table resulted in 68.9% specificity (Table 6).

Overall accuracy was 83.1% at Lab A, 83.3% at Lab B, and 84.1% at Lab C, and a two-by-two cumulative table for all three par-ticipating laboratories resulted in 83.5% overall accuracy of (Table 6).

Discussion

As all test runs for negative and positive con-trols met the acceptance criteria, the VMT con-cluded that these results were highly reproduci-ble over the time this validation study was con-ducted (Table 2 and Table 3).

One test run each for Methylthioglycolate (No.2), 2-Ethoxyethyl methacrylate (No.17), 3-Phenoxybenzyl alcohol (No.18), and Ethyl thioglycolate (No. 21) at Lab C, as well as one test run at Lab A and three test runs at Lab C for 2,2'- [[3-Methyl-4- [(4-nitrophenyl) azo]-phenyl] imino]bis-ethanol (No.26) generated SD > 18%, therefore failing to meet the acceptance criteria and resulting in invalid runs. This can be ex-plained by the fact that Methylthioglycolate (No.2) and Ethyl thioglycolate (No.21) are both strong WST-8 reducers, which activity might have influenced the measured endpoint. As for 3-Phenoxybenzyl alcohol (No.18) and 2,2'-[[3- Methyl-4- [(4-nitrophenyl) azo] -phenyl] imino] bis- ethanol (No.26), residual test chemicals in the cell culture inserts due to adhesion to the plastic surface might have caused some variabil-

ity in cell viability. This suggests that the wash-ing process was not enough to eliminate all traces of chemicals from the tissue.

Based on the rationale above, the run sequences for three chemicals (No.1, No.18 and No.21) were augmented by retesting with an improved washing and standardized washing procedure proposed by the VMT. Additionally to the new approach to decrease variability in cell viability, the VMT recommended that a suitable video presentation of the washing procedure should be prepared as an effective means to train naïve laboratories planning to perform the Lab-Cyte CORNEA-MODEL24 EIT. The VMT fur-ther requested the protocol to be updated to include precautions in the description of the washing process.

On the other hand, although three incom-plete test run sequences (chemical No. 17 at Lab C, and chemical No. 26 at Lab A and Lab C) were produced, the data quality of this validation study met the criteria established in the OECD TG 492 performance standard (OECD, 2015b). The robustness and reliability of LabCyte CORNEA-MODEL24 EIT was also demon-strated by the within-laboratory reproducibility (Lab A: 100%, Lab B: 93.3%, Lab C: 96.7%), and between-laboratory reproducibility (86.7%) achieved, as these rates also met the criteria established in the OECD TG 492 performance standard (OECD, 2015b).

Although the LabCyte CORNEA- MODEL24 EIT was able to correctly identify eye irritation (UN GHS Category 1 or 2) chemi-cals, two UN GHS Category 2B chemicals (one test of Diethyl toluamide, No. 13, and two tests of 1,4-Dibutoxy benzene, No. 14) were pre-dicted as false-negatives in Lab B. On the other hand, the LabCyte CORNEA-MODEL24 EIT misclassified four UN GHS no classified chem-ical as false-positives; Ethyl thioglycolate (No. 21), 2,2'-[[3-Methyl-4-[(4-nitrophenyl) azo]-phenyl]imino]bis-ethanol (No. 25), Cellu-lose, 2-(2-hydroxy-3-(trimethylammonium) pro-poxy) ethyl ether chloride (91%) (No. 28), and Trisodium mono- (5- (1, 2 – dihydroxyethyl ) - 4- oxido-2-oxo-2,5-dihydro-furan-3-yl) phosphate (No. 30). EpiOcular EIT, the VRM for the OECD TG 492 ha also reported a false negative


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(chemical No. 14) and three false positives (No. 21, No. 25, and No. 30) (OECD, 2015b). From the results of this study, in which 26 out of 30 chemicals were correctly predicted, the tendency of the eye irritation prediction between LabCyte CORNEA-MODEL24 EIT and EpiOcular EIT can be considered similar.

The sensitivity, specificity, and accuracy of LabCyte CORNEA-MODEL24 EIT were 97.8%, 68.9%, and 83.5%, respectively. Some devia-tions from the OECD performance standard (sensitivity of 90%, specificity of 60%, and ac-curacy of 75%) were due to specific adaptations for the LabCyte CORNEA-MODEL24. Finally, predictive capacity at all laboratories was within the acceptance criteria established in the OECD performance standard.

The VMT determined that LabCyte CORNEA-MODEL24 EIT is a method func-tionally similar to the EpiOcular EIT. At the classification of 30 reference chemicals, the LabCyte CORNEA-MODEL24 EIT misclassi-fied four GHS No Category chemicals as false-positives, and the EpiOcular EIT misclas-sified one GHS Category 2B chemicals as false-negative, and five GHS No Category chemicals as false-positive. Both assays mis-classified four GHS No Category chemicals as false-positives.

Only the LabCyte CORNEA-MODEL24 EIT, but not EpiOcular EIT, over-predicted one reference chemical, Cellulose, 2-(2-hydroxy- 3-(trimethylammonium) propoxy) ethyl ether chloride (91%) (No. 28). On the other hand, only the EpiOcular EIT, but not the LabCyte CORNEA-MODEL24 EIT, under-predicted one reference chemical, 1,4-Dibutoxy benzene (No. 14), and also over-predicted two chemicals, 2-Ethoxyethyl methacrylate (No. 17) and 3-Phenoxybenzyl alcohol (No. 18). Predictions for the remaining 26 chemicals were concordant between LabCyte CORNEA-MODEL24 EIT and EpiOcular EIT. From the results above, it is suggested that LabCyte CORNEA-MODEL24 EIT has a similar predictive performance com-pared to the EpiOcular EIT.

The LabCyte CORNEA-MODEL24 EIT underwent a validation study focused on the ful-fillment of the performance standards defined by

the OECD TG 492 for similar or modified in vitro RhCE EIT methods, based on the EpiOcu-lar EIT. Finally, the results in this validation study suggested that the LabCyte CORNEA- MODEL24 EIT is a robust and reliable method to address the initiating event of eye irritation. Most importantly, these data provide relevant information to propose the LabCyte CORNEA- MODEL24 EIT method as a me-too method for inclusion in the OECD TG 492. References Alépée, N., Bessou-Touya, S., Cotovio, J., de Smedt,

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(Received: May 2, 2019/ Accepted: October 25,2019)

Corresponding author: Masakazu Katoh, D.D.S., Ph.D Japan Tissue Engineering Co., Ltd. 6-209-1 Miyakitadori, Gamagori, Aichi443-0022, Japan Tel: +81-533-66-2128 E-mail: [email protected]

review artcle a validation study of a new in vitro eye

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