Two tiered approaches combining alternative test methods and minimizing the use of reconstructed human cornea-like epithelium tests for the evaluation of eye irritation potency of test chemicals

Journal Pre-proof Two tiered approaches combining alternative test methods and minimizing the use of reconstructed human cornea-like epithelium tests for the evaluation of eye irritation potency of test chemicals

Kyung Yuk Ko, Hye Lyun Jeon, Joohwan Kim, Tae Sung Kim, Yoon-hee Hong, Mi Kyung Jeong, Kyo-Hyun Park, Bae-Hwan Kim, Sera Park, Won-hee Jang, Sun-A Cho, Susun An, Ah. Rang Cho, Jung-Sun Yi, Ji-Young Kim, Hak Kim, Jong Kwon Lee, Ki Sook Park PII:

S0887-2333(18)30541-1

DOI:

https://doi.org/10.1016/j.tiv.2019.104675

Reference:

TIV 104675

To appear in:

Toxicology in Vitro

Received date:

18 September 2018

Revised date:

4 May 2019

Accepted date:

1 October 2019

Please cite this article as: K.Y. Ko, H.L. Jeon, J. Kim, et al., Two tiered approaches combining alternative test methods and minimizing the use of reconstructed human cornea-like epithelium tests for the evaluation of eye irritation potency of test chemicals, Toxicology in Vitro(2019), https://doi.org/10.1016/j.tiv.2019.104675

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 Published by Elsevier.

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Two Tiered Approaches Combining Alternative Test Methods and Minimizing the Use of Reconstructed Human Cornea-like Epithelium Tests for the Evaluation of Eye Irritation Potency of Test Chemicals Kyung Yuk Koa,1 , Hye Lyun Jeona,1 , Joohwan Kima, Tae Sung Kima, Yoon- hee Honga, Mi Kyung Jeongb, Kyo-Hyun Park b, Bae-Hwan Kimb, Sera Parkc, Won-hee Jangc, Sun-A Choc, Susun Anc, Ah Rang Choa, Jung-Sun Yia, Ji-Young Kima, Hak Kima, Jong Kwon Leed, Ki

Toxicological Screening & Testing Division, National Institute of Food and Drug Safety

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a

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Sook Parka,*

Evaluation, Ministry of Food and Drug Safety, Cheongju-si, Republic of Korea Major in Public Health, Faculty of Food and Health Sciences, Keimyung University, Daegu,

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b

Republic of Korea

AmorePacific R&D Center, Yongin-si, Republic of Korea

Toxicological Research Division, National Institute of Food and Drug Safety Evaluation,

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c

Ministry of Food and Drug Safety, Cheongju-si, Republic of Korea

These authors contributed equally to this work.

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* Correspondence to: [email protected]

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Abstract In order to overcome the limitations of single in vitro eye irritation tests, Integrated Approaches to Testing Assessment strategies have been suggested for evaluating eye irritation. This study developed two tiered approaches combining alternative test methods. They were designed in consideration of the solubility property of test chemicals and to use the RhCE tests at final steps. The tiered approach A is composed of the STE, BCOP, HET-CAM or RhCE tests, whereas the tiered approach B is designed to perform simultaneously two in vitro test methods at the first stage and the RhCE test at the final stage. The predictive capacity of

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the two tiered approaches was estimated using 47 chemicals. The accuracy, sensitivity, and

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specificity value of the tiered approach A were 95.7% (45/47), 100% (34/34), and 84.6% (11/13), respectively, whereas those of the tiered approach B were 95.7% (45/47), 97.1%

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(33/34), and 92.3% (12/13), respectively. The approach A and B were considered to be available methods for distinguishing test chemicals of Category 1 (all 73.3%) and No

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Category (84.6% and 92.3%), respectively. Especially, the approach B was considered as an

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efficient method as the Bottom-Up approach, because it predicted correctly test chemicals classified as No Category.

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Key words: Tiered approach, Predictive capacity, Eye irritation, Top-Down approach

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1. Introduction Animal welfare interests and issues have increased, and the ban on animal experiments with cosmetic products has emerged globally, beginning with the European Union (EU) (Directive 76/768/EEC, 2003). Based on such an international trend, selling and distributing cosmetic products or ingredients based on animal experiments has been prohibited since 2017, according to the Korean Cosmetic Act. Therefore, the need to develop an Integrated Approach on Testing and Assessment (IATA) in order to replace the rabbit Draize test for eye irritation has become an important issue in Korea.

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In order to replace the in vivo Draize test, Test Guidelines (TG) containing a variety of in

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vitro testing methods using organotypic models, cell lines, and re-constructed human cornealike epithelium (RhCE) have been developed and adopted by the Organization for Economic

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Co-operation and Development (OECD) as follows: Bovine Corneal Opacity and

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Permeability (BCOP) test (OECD TG 437, 2013a), Isolated Chicken Eye test method (OECD TG 438, 2013b), Fluorescein Leakage test (OECD TG 460, 2012a), Short Time Exposure

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(STE) test (OECD TG 491, 2015a), RhCE test using EpiOcularT M models (OECD TG 492, 2018), SkinEthicT M Human Corneal Epithelium (HCE) models (OECD TG 492, 2018), and LabCyte HCE models (OECD TG 492, 2018).

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The STE test, using Statens Seruminstitut Rabbit Cornea (SIRC) cells, was adopted as

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OECD TG 491 (2015a), and is a simple, rapid, low-cost testing method (Hayashi et al., 2012a), measuring cell viability after a short time of treatment with test chemicals using an

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MTT assay. However, the STE test method is known not to be appropriate for test chemicals that are insoluble or cannot be uniformly suspended for at least 5 minutes in physiological saline, 5% DMSO in saline, or mineral oil (OECD, 2015a). The BCOP test, using bovine corneas, was adopted as OECD TG 437 (2013a) and predicts test chemicals as Category (Cat) 1, No prediction can be made (No pre), or No Category (No Cat) based on an In Vitro Irritancy Score (IVIS) calculated by corneal opacity and permeability. The BCOP test is known to have high sensitivity (Hayashi et al., 2012b; ICCVAM, 2010a; Verstraelenet al., 2017). The Hen’s Egg Test-Chorioallantoic Membrane (HET-CAM) test uses chorioallantoic membranes (CAMs) of fertilized chicken eggs to predict eye irritation by scoring the severity of hemorrhage, lysis, coagulation, or response times after treatment with test chemicals to the CAM. The HET-CAM test, developed by Luepke (1985), has not been officially accepted as an OECD TG; however, it is generally recognized as a scientifically reasonable testing method for evaluating eye irritation (OECD

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GD 263, 2017a). The RhCE test is capable of distinguishing between irritants and nonirritants, and it can also be used in combination with other replacement methods to enable further classification of eye hazards in the framework of tiered testing approaches (Scott et al., 2010).The OECD adopted the EpiOcularT M eye irritation test as OECD TG 492 in 2015, which is known to be an RhCE test to assess eye irritation, and is a validated reference method (OECD, 2015b). Recently, the SkinEthicT M HCE model (OECD, 2017b) and the LabCyte cornea model (OECD, 2018) were included in OECD TG 492. Even though various in vitro eye irritation testing methods are reported, it is generally

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considered that one eye irritation method by itself cannot completely replace the in vivo

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Draize rabbit eye test (OECD TG 405, 2012b). Therefore, the OECD has suggested an IATA Guidance Document (GD) for serious eye damage and eye irritation hazard identification

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(OECD GD 263, 2017a). The OECD IATA analysis strategy of eye irritation integrates all data by combining i) existing testing and non-testing data, ii) weight of evidence on all

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collected information, iii) testing data obtained through direct performance.

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As a part of the IATA for serious eye damage and eye irritation, tiered approaches to evaluate eye irritation potency by combining in vitro eye irritation methods have been reported. Kolle et al. (2011) recommended combining the BCOP test and EpiOcularT M eye

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irritation test (EIT) in both Top-Down and Bottom-Up approaches. Hayashi et al. (2012a)

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suggested a Bottom–Up tiered approach combining the STE and BCOP tests. In a later study, Hayashi et al. (2012b) suggested a two-stage Bottom-Up tiered approach combining the STE,

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EpiOcularT M EIT and BCOP tests, which was designed by chemical solubility. Settivari et al. (2016) proposed a tiered approach of the neutral red release assay and EpiOcularT M EIT for evaluating eye irritation potentials of agrochemical formulations. In addition, Adriaens et al. (2018) developed two testing approaches to distinguish between Cat 1 and Cat 2 chemicals. One of the approaches is combined with the BCOP laser light-based opacitometer (LLBO) test and an RhCE test (SkinEthicT M EIT or EpiOcularT M EIT), and the second approach is comprised of the BCOP OP-KIT, Slug Mucosal Irritation assay, and an RhCE test method. Although several tiered approaches for eye irritation evaluations have been reported, this study aimed to develop more efficient strategies for predicting eye irritation. The tiered approaches were designed in consideration of the solubility property of test chemicals and the RhCE tests were employed at final steps of the tiered approach, instead of using them starting first step. The tiered approach A is composed of the STE, BCOP, HET-CAM, and RhCE tests, whereas the tiered approach B is designed to perform simultaneously two in vitro test

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methods at the first stage, and the RhCE test is used at the final stage if the two previous results are discordant. This study evaluated the predictive capacities of the two proposed approaches with 47 selected test chemicals in order to confirm whether they are efficient for predicting eye irritation potency.

2. Materials and methods 2.1. Test Chemicals Prior to conducting the experiments, the information of the STE, BCOP, HET-CAM, and

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EpiOcularT M EIT test results were surveyed and collected through 16 literature sources to

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select test chemicals for eye irritation evaluations. Additionally, this study refers to a database (DB) called ChemEye DB (unpublished), which was created by the Center of Alternative

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Methods for Safety Evaluation of Cosmetics (CAMSEC), temporarily founded by the Korean

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Ministry of Food and Drug in 2013, in order to choose test chemicals for the present study. The ChemEye DB includes various test chemicals related to eye irritation listed in OECD

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TGs, OECD GDs, Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) reports, and international scientific papers over the last 10 years. It also provides information on in vivo categories based on the United Nations Globally Harmonized

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System of classification and labeling of chemicals (UN GHS), the European Union, and the

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United States Environmental Protection Agency (US EPA),results of various in vitro tests of

chemical listed.

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eye irritation, used vehicles, cut-off criteria, and information on human data for each

Based on the ChemEye DB, this study prioritized the selected chemicals containing results obtained from the EpiOcularT M EIT performed previously in our laboratory. Some of the test chemicals were randomly selected among reference chemicals suggested by the ChemEye DB and our literature survey. The 47 test chemicals selected in this study consist of 15 UN GHS Cat 1, 19 UN GHS Cat 2, and 13 UN GHS No Cat. The evaluations of eye irritation against some of the chemicals were conducted directly using each in vitro alternative test method in the present study. However, for a portion of the test chemicals, the eye irritation data were obtained from the ChemEye DB and literature survey. The list of the 47 test chemicals used in this study and existing information for several chemicals obtained from the literature are presented in Table 1. Among the test chemicals used in direct performance, di(2-ethylhexyl) sodium sulfosuccinate and diethyl toluamide were purchased from TCI (Tokyo, Japan), whereas the other chemicals were purchased from Sigma-Aldrich (St. Louis, MO,

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USA).

2.2. Short Time Exposure (STE) test The STE test was performed according to the procedure suggested by OECD TG 491 (2015a). Among the 47 chemicals, 35 test chemicals not having STE data or having discordant Category classification results in the in vivo UN GHS Category were selected for evaluation by direct performance. Additionally, the STE results of eye irritation against 12

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test chemicals were obtained by review of the literature.

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2.2.1. Cell culture and seeding

SIRC cells (American Type Culture Collection (ATCC), Manassas, VA, USA) were

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cultured in Eagle’s Minimum Essential Medium (ATCC) with 10% Fetal bovine serum (FBS)(Gibco-Invitrogen, Carlsbad, CA, USA), 100 IU/mL penicillin, and 100 µg/mL

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streptomycin (WelGENE, Daegu, Republic of Korea) at 37°C in a humidified atmosphere

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containing 5% CO 2 . The cells were sub-cultured 2 to 3 times per week. To perform the STE test, the cells were seeded at 6×103 cells per well in a volume of 200 µL in 96-well plates.

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The cells were maintained at 37°C in a humidified atmosphere containing 5% CO 2 for 4 days.

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2.2.2. Application of test chemicals and MTT assay Chemicals that have no STE data or showed inconsistent results with the in vivo UN GHS

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Category were evaluated by the STE test. However, insoluble chemicals in saline, 5% dimethyl sulfoxide (DMSO) in saline, or mineral oil (Sigma-Aldrich) were excluded. Test chemicals were prepared at a concentration of 5% and 0.05% (w/v) in the three solvents. Sodium lauryl sulfate (Sigma-Aldrich) at a concentration of 0.01% (w/v) in saline was used as a concurrent positive control. Saline, 5% DMSO in saline, or mineral oil were used as a concurrent vehicle control. The cells were exposed to 200 µL of each group (test chemicals at 5% and 0.05%, medium control, vehicle control, and positive control) for 5 min at room temperature. Three wells were used per group. The cells were washed twice with 200 µL of PBS and treated with 200 µL of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) solution (0.5 mg/mL in complete medium, Sigma-Aldrich) for 2 h at 37°C, 5% CO2 . After incubation, the MTT solution was removed and 200 µL of 0.04 N hydrochloric acid-isopropanol (Sigma-Aldrich) was added to each well for extraction of MTT formazan. The cells were then incubated for 1 h at room temperature in the dark, and the optical density

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(OD) was then measured at 490 nm using a spectrophotometer (Spectra MAX190, Molecular Devices, Sunnyvale, CA, USA). After subtracting the blank OD from the OD of the test chemical or solvent control, cell viability was calculated by dividing the test chemical OD by the solvent control OD.

2.2.3. Prediction and acceptance criteria Mean cell viability measurements obtained from three independent repetitions were used to evaluate eye irritation. If cell viabilities at 5% and 0.05% were greater than 70%, the

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chemical was determined as UN GHS No Cat, and if the cell viability at the two

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concentrations were less than 70%, the chemical was determined as Cat 1. However, if the cell viability at 5% was less than 70% and the cell viability at 0.05% was greater than 70%,

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the chemical was determined as No pre. The STE test was considered to be qualified for analysis if the following criteria were met: (1) OD of the medium control should be 0.3 or

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higher after subtraction of the blank OD; (2) Cell viability of the solvent control should be 80%

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or higher compared to that of the medium control; (3) The cell viability of the positive control should be within two standard deviations of the historical mean; and (4) The standard deviation of the final cell viability obtained from the three runs should be less than 15% for

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the two concentrations of the test chemical. If any criteria did not meet the acceptance criteria,

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another three runs were conducted.

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2.3. Bovine Cornea Opacity and Permeability (BCOP) test The BCOP test was conducted according to the procedure suggested by OECD TG 437 (2013a). Among the 47 test chemicals, the BCOP results of 18 test chemicals were obtained from the ChemEye DB and the literature survey. Four test chemicals among 18 test chemicals have discordant results from classification of the in vivo UN GHS Category. Thus, 33 test chemicals including the4 test chemicals were used for direct performance of eye irritation evaluation.

2.3.1. Preparation of corneas and treatment with test chemicals Bovine eyes were isolated from slaughtered cows (mean age of 31 months old) at an abattoir (Nonghyup Co. Ltd., Eumseong, Korea). The eyes were immersed in chilled Hank’s balanced salt solution with Ca2+ and Mg2+ (Gibco-Invitrogen) supplemented with 100 IU/mL penicillin and 100 µg/mL streptomycin (WelGENE). The eyes were transported to the

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laboratory while being kept chilled and then examined for damage. Corneas were excised, leaving a 2-3 mm rim of sclera, and each cornea was mounted in a corneal holder. The holder was filled with pre-warmed Minimum Essential Medium (MEM)(Gibco-Invitrogen) without phenol red containing 1% L-glutamine (Gibco-Invitrogen), 1% FBS, 100 IU/mL penicillin and 100 µg/mL streptomycin (WelGENE). The holders were incubated for 1 h at 32 ± 1°C. After incubation, the medium in the holder was removed and refilled with fresh complete MEM. Baseline opacity of the cornea in the holder was measured using a calibrated OP-KIT opacitometer (MC2, Clermont FerrandCedex, France). In case s where the opacity value of

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the cornea was more than seven, the cornea was excluded from the experiment.

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Distilled water (DW)(Gibco-Invitrogen), 0.9% sodium chloride solution (saline) or mineral oil were used as a vehicle control. Pure ethanol or 20% (w/v) imidazole in DW or

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saline were used as a positive control for liquid or solid chemicals, respectively. Prior to application of the test chemicals, liquids were prepared at neat form and solids were prepared

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at a concentration of 20% (w/v) in DW, saline, or mineral oil (Sigma-Aldrich). Three corneas

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were used per treatment group. The medium from an anterior chamber of the corneal holder was removed and 750 µL of the chemical was applied to the anterior chamber. However, an insoluble solid chemical in solvent was tested using the open-chamber method where the

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solid chemical was applied to the cornea surface directly. While the corneas treated with

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1°C.

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liquids were incubated for 10 min, those treated with solids were incubated for 4 h at 32 ±

2.3.2. Post-incubation of corneas and opacity and permeability measurements After removal of the test chemical, the holder was washed with complete MEM with phenol red three times and then with complete MEM without phenol red once or twice. The complete MEM without phenol red (5 mL) was injected into both chambers of the holder. The corneas treated with liquids were additionally post-incubated for 2 h at 32 ± 1°C. Corneal opacity was measured after post- incubation for liquids and complete medium injection for solids. After opacity measurements, the MEM in the anterior chamber of the holder was replaced with 1 mL of pre-warmed fluorescein solution (4 mg/mL for liquids; 5 mg/mL for solids) (Sigma-Aldrich). The holder was incubated for 90 min at 32 ± 1°C. The solution in the posterior chamber was transferred to 96-well plates and the OD was measured at 490 nm using a spectrophotometer (Spectra MAX190).

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2.3.3. Prediction Final opacity and permeability were calculated by subtracting the values of the vehicle control based on INVITTOX no 127 (EURL ECVAM, 2009) and ICCVAM-Recommended BCOP protocol (ICCVAM, 2012). The In Vitro Irritancy Score (IVIS) value for each group was calculated using the final opacity and permeability values as shown below: IVIS = mean opacity value + (15 × mean permeability OD490 value) If the IVIS value was less than 3, the chemical was evaluated as UN GHS No Cat, and if the IVIS valued was exceeded 55, the chemical was evaluated as Cat 1. However, if the IVIS

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value exceeded 3 or was less than 55, the chemical was evaluated as No pre. 2.4. Hen’s Egg Test-Chorioallantoic Membrane (HET-CAM) test

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The HET-CAM test was performed according to the procedure of Steiling et al. (1999). Among the 47 test chemicals used in the present study, 29 test chemicals were evaluated b y

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direct test performance, and the results of the HET-CAM test against 18 test chemicals were

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obtained from existing information in the literature and the ChemEye DB. The test chemicals were generally tested in an undiluted state. Solid chemicals were also tested in powder form. Fertilized eggs from white leghorn chickens were incubated at 37.5 ± 0.5°Cat 55% ± 7%

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humidity with automatic rotation until testing on day 9, and on day 10, the eggs (50-60 g)

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were used for the HET-CAM test. After the airspace of the egg was marked, the section of the shell was carefully removed and the shell membrane was moistened with saline at 37°C.

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After removal of saline, the shell membrane was carefully removed without injuring any underlying blood vessels using forceps. After exposure of the CAM, the blood vessels were observed and only intact CAMs were used in the experiment. Six eggs were used for each test chemical. Eye irritation potentials of test chemicals were evaluated by the reaction time method or endpoint assessment according to whether or not it was transparent. The reaction time method was used to evaluate transparent liquid test chemicals. After 300 µL of a test chemical was applied, the time for the first detection of hemorrhage, lysis, and coagulation for 5 min was measured. The evaluation of the reaction time method was performed by calculating a Q score comparing the possibility of stimulation of the test substance with that of the reference chemical (Texapon ASV®, Cognis, Dusseldorf, Germany) with specially designed software. The endpoint assessment was used when the CAM was not observed due to a non-transparent test chemical. The test chemical (300 µL) was applied to the CAM for 30 sec and the CAM

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was cautiously washed with saline for 20 sec. After waiting for 30 sec, the parameters (hemorrhage, lysis, and coagulation) were evaluated according to the DB-ALM Method Summary no 96 (2010). The results of the evaluation according to the endpoint assessments are shown by the sum of the S score of each parameter. Irritation classification by Q-Score was predicted as follows: (1) Slightly irritating, Q ≤ 0.8; (2) Moderately irritating, 0.8 < Q < 1.2; (3) Irritating, 1.2 ≤ Q < 2.0; (4) Severely irritating, Q ≥ 2.0. Irritation classification of the S-Score was predicted as follows: (1)

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Slightly irritating, S < 6; (2) Moderately irritating 6 ≤ S <12; (3) Irritating, 12 ≤ S < 16; (4)

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Severely irritating, S ≥ 16.

2.5. Reconstructed human Cornea-like Epithelium (RhCE) test

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This study utilized the EpiOcularT M EIT as the RhCE test, which was conducted ccording to the procedure described in the OECD TG 492 (2015b). In this study, 13 test chemicals

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were evaluated by direct performance using the EpiOcularT M EIT, whereas the results of 34 test chemicals were based on the information of eye irritation evaluations obtained from the

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ChemEye DB and literature surveys and from previous studies performed in our laboratory.

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2.5.1. Treatment of test chemicals

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EpiOcularT M EIT tissues were purchased from MatTek Co. (Ashland, OR, USA), which originated from human keratinocytes. On the day of receipt, the tissue models were equilibrated at room temperature for 15 min. After equilibration, the tissues were transferred to a 6-well plate containing 1 mL of fresh prewarmed assay medium and incubated at 37 ± 1°C in a humidified atmosphere of 5% ± 1% CO 2 in air (standard culture conditions, SCC) for 16-24 h. Before treatment, the tissues were pre-wetted with 20 μL of Dulbecco’s phosphate buffered saline (DPBS) without Ca 2+ and Mg2+ and incubated under SCC for 30 ± 2 min. Liquid chemicals (50 μL) were applied to the surface of each tissue and incubated under SCC for 30 ± 2 min. Solid chemicals (50 mg) which were crushed to a very fine powder were applied directly to the tissue and incubated under SCC for 6 ± 0.25 h. Each test chemical and concurrent control was treated onto two tissue models simultaneously. After treatment, the tissues were rinsed with 90 mL of DPBS without Ca 2+ and Mg2+ three times to

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remove any remaining test chemical from the tissue surface. After rinsing, the tissues were immediately immersed in pre-warmed assay medium in a 12-well plate at room temperature. The tissues treated with liquid or solid test chemicals were incubated for 12 ± 2 min or 25 ± 2 min, respectively. At the end of the post-soak step, the tissues were transferred to a 6-well plate containing 1 mL of fresh prewarmed assay medium and the tissues which were treated with liquid or solid test chemicals were incubated for 2 ± 0.25 h or 18 ± 0.25 h, respectively.

2.5.2. Viability measurement by the MTT assay

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After incubation, each tissue sample was transferred to a 24-well plate containing 300 μL

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of a 1 mg/mL MTT solution and then incubated under SCC for 180 ± 10 min in the dark. Following incubation, the tissues were blotted and transferred to a 24-well plate containing 2

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mL of isopropanol for liquid chemicals or a 6-well plate containing 2 mL of isopropanol for solid chemicals to extract the reduced MTT formazan from the tissues. The plates were sealed

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with parafilm to reduce evaporation and then placed on a plate shaker for 2 h at room

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temperature in the dark. The extracted solution was mixed, and two 200-μL aliquots were transferred into a 96-well plate for OD measurements using a plate reader at 570 nm. Isopropanol was used as the blank. The OD values obtained for each tissue were used to

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calculate the mean percent tissue viability normalized to that of the negative control, which

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was set at 100%. The results are expressed as the mean percent viability and the difference in

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viability between the two replicate tissues.

2.5.3. Prediction and acceptance criteria Based on the relative tissue viability results, the EpiOcular™ EIT can distinguish between chemicals that do not require classification and labeling according to the UN GHS (No Cat) from those requiring classification and labeling (Cat 1 and Cat 2). The test chemicals were identified as non-irritants (NI) if tissue viability was > 60%. The test chemical was identified as an irritant (Cat 1 and Cat 2) if tissue viability was ≤ 60%. Each run using EpiOcular™ tissue batches was considered to be qualified for analysis if the following criteria were met: (1) the negative control OD value was > 0.8 and < 2.5, (2) the mean relative viability of the positive control was < 50%, and (3) the difference in viability between the two replicate tissues for each chemical was < 20%. If a test did not meet the acceptance criteria in a run, the test chemical was re-tested.

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2.6. Predictive capacities of in vitro eye irritation test methods and the two tiered approaches To evaluate the predictive capacities of in vitro test methods and the two tiered approaches, this study calculated the sensitivity (the percentage of predicted irritants by test among irritants classified as UN GHS Cat 1 or Cat 2), specificity (the percentage of predicted nonirritants by test among non- irritants classified as UN GHS No Cat), and accuracy (the overall percentage of correct predictions according to in vivo UN GHS Category) against the 47 test

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chemicals. In addition, the pattern to predict eye irritation potency in each in vitro test method

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and the two tiered approaches was analyzed by comparison to in vivo UN GHS Category

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classifications.

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3. Results

3.1. Classification decision for eye irritation by direct test performance

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Among 47 test chemicals used in the present study, this study conducted directly experiment with 26 substances in STE test, 33 test chemicals in BCOP test, 29 test chemicals in HET-CAM test, and 9 test chemicals in RhCE test.

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In the STE test, 35 test chemicals were selected for evaluation by direct performance and

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conducted according to the procedure suggested in OECD TG 491. Prior to commencing the main test, solubility assays on the 35 test chemicals were performed with solvents such as

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saline, 5% DMSO in saline, or mineral oil. As a result, the 9 test chemicals, salicylic acid, chlorhexidine, tetraethylene glycol diacrylate, sodium oxalate, 4-formylbenzoic acid, 1,5Naphtahlenediol,

monostearin,

1-(4-chlorophenyl)-3-(3,4-dichlorophenyl)urea,

methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3,-tetramethylbutyl)-phenol),

were

2,2not

soluble in the three solvents, as shown in Table 1. Therefore, these chemicals were excluded from the STE test, and only 26 chemicals were finally evaluated. The results of predictive capacity against the 26 test chemicals evaluated by direct performance are shown in Table 1. According to these results, 16 out of the 26 chemicals were consistent with in vivo UN GHS classification. However, cyclohexanol, imidazole, promethazine hydrochloride, lactic acid, and 10% Di(2-ethylhexyl sodidumsulfosuccinate were under-predicted as No pre; and 2,5dimethyl-2,5- hexanediol,

ammonium

nitrate,

2,2-dimethyl-3-

methylenebicyclo[2.2.1]heptanes, sodium chloroacetate, and methyl acetate were underpredicted as No Cat.

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In the BCOP test, the evaluation results of eye irritation by direct performance with 33 test chemicals are shown in Table 1. As a result, 28 out of the 33 chemicals were consistent with in vivo UN GHS classification; however, sodium chloroacetate was predicted as a false negative. Also, the 4 test chemicals classified as UN GHS No Cat, polyethylene glycol (PEG40) hydrogenated

castor oil,

potassium tetrafluoroborate,

propylene

glycol,

and

triethanolamine, were over-predicted as Cat 1 or No pre (Table 1). In the HET-CAM test, the evaluation results of eye irritation by direct performance with 29 test chemicals are shown in Table 1. As a result, 13 out of the 29 chemicals were

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consistent with in vivo UN GHS classification. However, zinc gluconate, salicylic acid,

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chlorhexidine, methylthioglycolate, tetraethylene glycol diacrylate, and 1,5- naphthalenediol were under-predicted as moderately irritating, irritating, or slightly irritating. Benzyl alcohol,

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citric acid, 1-octanol, cyclopentanol, sodium chloroacetate, di(propylene glycol) propyl ether, 1-ethyl-3-methylimidazolium ethylsulphate, polyethylene glycol (PEG-40) hydrogenated

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castor oil, and propylene glycol were over-predicted as severely irritating or moderately

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irritating (Table 1).

In the EpiOcularT M EIT, 9test chemicals were evaluated by direct performance in the present study, and 23 test chemicals were evaluated through previous stud ies performed in

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our laboratory. As a result, 30 out of the 32 test chemicals conducted were consistent with in

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vivo UN GHS classification. However, two chemicals, methanol and triethanolamine,

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classified as UN GHS No Cat, were shown to be false positives (Table 1).

3.2. Predictive capacities of each in vitro eye irritation test method against 47 test chemicals

This study collected and summarized the eye irritation results obtained by direct experiment, the literature survey, and the ChemEye DB against 47 test chemicals as shown on Table 1.This study performed directly experiment about some chemicals, which have different eye irritation potency in literature survey or that we could not search any results of eye irritation. The final decision of eye irritation potency for such chemicals was made according to the results of direct performance in priority. Also, the final decision against a part of chemicals, which were not performed directly in the present study, was made by the results of literature survey. The final decision (FD) for each test chemicals were described as shown Table 1. That is, the results obtained by direct experiment performance do not have reference numbers, whereas those obtained by literature survey have reference numbers in

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Experiment & Literature section of Table 1. The accuracy, sensitivity, and specificity of the STE test were 86.8% (33/38), 82.1% (23/28), and 100% (10/10), respectively; and those of the BCOP test were 89.4% (42/47), 97.1% (33/34), and 69.2% (9/13), respectively. In the HET-CAM test, the accuracy, sensitivity, and specificity values were 83.0% (39/47), 91.2% (31/34), and 61.5% (8/13), respectively. The accuracy, sensitivity, and specificity values of the EpiOcularT M EIT were 95.7% (45/47), 100% (34/34) and 84.6% (11/13), respectively (Table 2A). Based on the results of the 5 in vitro eye irritation test methods, the sensitivities, in decreasing order, were

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EpiOcular (100%) > BCOP (97.1%) > HET-CAM (91.2%) > STE (82.17%), whereas the

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specificity were STE (100%) > EpiOcular (84.6%) > BCOP (69.2%) > HET-CAM (61.5%) (Table 2A).

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This study calculated the predictive capacities of eye irritation of 38 test chemicals in order to compare precisely the predictive capacities of each in vitro test method under the sample

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with the same size. As shown on Table 2B, the STE had an accuracy of 86.8% (33/38), a

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sensitivity of 82.1% (23/28) and a specificity of 100% (10/10). The BCOP showed an accuracy of 84.2% (32/38), a sensitivity of 96.4% (27/28), and a specificity of 50.0% (5/10). In the HET-CAM test, the accuracy, sensitivity, and specificity values were 86.8% (33/38),

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100% (28/28), and 50.0% (5/10), respectively, whereas the EpiOcularT M EIT were an

(Table 2B).

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accuracy of 95.7% (36/38), a sensitivity of 100% (28/28), and a specificity of 80.0% (8/10)

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Also, the present study analyzed the characteristics of each in vitro eye irritation test method based on the results obtained from direct test performance and non-testing data by literature review. The STE test, accepted officially as OECD TG 491 (OECD, 2015a), is known to have high specificity, low false positive rates, and low Cat 2 over-prediction rates (Takahashi et al., 2009; Takahashi et al., 2011; Sakaguchi et al., 2011; Kojima et al., 2013).The OECD IATA (OECD, 2017a) also recommends that eye irritation test methods with low false positive prediction rates be preferentially used in Top-Down approaches. According to the results obtained from the 47 test chemicals, the STE test indicated that only 5 test chemicals were false negatives, without false positives. The chemicals determined to be false negatives were 2,5-dimethyl-2,5-hexanediol, ammonium nitrate, 2,2-dimethyl-3methylenebicyclo[2.2.1] heptanes, sodium chloroacetate, and methyl acetate. In the STE results, both 2,5-dimethyl-2,5- hexanediol, classified as UN GHS Cat 1, and sodium chloroacetate, classified as UN GHS Cat 2, were predicted as No Cat. As shown in our results

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and in other previously reported studies, the STE test shows under-prediction for 2,5dimethyl-2,5- hexanediol, ammonium nitrate, and sodium chloroacetate (Takahashi et al., 2009; Kojima et al., 2013). According to the results of the STE test, the rate of correctly predicting a chemical as Cat 1 was shown to be only 36.4%. However, the STE test has the highest value in predictive capacity in distinguishing test chemicals of No Cat among three single in vitro eye irritation test methods (Table 3A and 3B). Therefore, this study employed it at the first stage of the proposed approaches for soluble test chemicals. The BCOP test, accepted officially as OECD TG 437, is known to have high sensitivity

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(OECD, 2013a; Hayashi et al., 2012b; ICCVAM, 2010a; Verstraelen et al., 2017). Similarly,

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this study confirmed that the BCOP test had the highest sensitivity compared to the other in vitro testing methods, having a value of 97.1% (33/34). In this study, the BCOP results had 1

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false negative and 4 false positives among the 47 test chemicals. The false negative test chemical was sodium chloroacetate, and the over-predicted test chemicals were polyethylene

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glycol (PEG-40) hydrogenated castor oil, potassium tetrafluoroborate, propylene glycol, and

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methanol. For sodium chloroacetate, classified as Cat 2 in the UN GHS, the BCOP test predicted the test chemical as No Cat, which was concordant with the STE test results.

classification.

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However, the HET-CAM and RhCE tests showed results identical to the in vivo UN GHS

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The HET-CAM test evaluating the eye irritation potency of test chemicals, based on vascular changes in the CAM, indicated high sensitivity at 91.2% (31/34); however, it

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showed low specificity at 61.5% (8/13). In the HET-CAM results, the 3 false negative chemicalsare tetraethylene glycol diacrylate, 4- formylbenzoic acid, and 1,5- naphthalenediol, whereas the 5 false positive chemicals are 1-ethyl-3- methylimidazolium ethylsulphate, polyethylene glycol (PEG-40) hydrogenated castor oil, propylene glycol, methanol, and triethanolamine. The EpiOcularT M EIT is officially validated and accepted as OECD TG 492 (OECD, 2015b) and is known to have high specificity or low false negative rates (Kolle et al., 2011; OECD, 2017a). In our study, this test method had high sensitivity, with a value of 100% (34/34) and a specificity value of 84.6% (11/13). Compared to other RhCE test results known to effectively distinguish between No Cat test chemicals (Kolle et al., 2011), the sensitivity value obtained from this study seems to be comparatively high. The high sensitivity results are assumed to be ascribed to the selection of the test chemicals, which were mainly chosen from RhCE results acquired through previous studies in our laboratory. The EpiOcularT M EIT

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resulted in 2 false positive results, methanol and triethanolamine. These results demonstrated that the BCOP and HET-CAM test predict positive test chemicals more correctly, whereas STE test predict negative test chemicals correctly. Also, RhCE test was proved to distinguish positive test chemicals from negative test chemicals. These results of Table 2A and 2B showed that the number and property of test chemicals could influence the predictive capacities. Therefore, it seems to be important to acquire various range and sufficient number of test chemicals in prediction evaluation of in vitro test

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method.

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3.3. Predictive capacities for eye irritation potency in each in vitro test method As shown in Table 3, the test methods correctly predicting Cat 1 chemicals were HET-

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CAM, BCOP (60%, 9/15) > STE (36.4%, 4/11), in decreasing order, for the 47 test chemicals used in the present study. The test methods correctly predicting Cat 2 were STE (76.5%,

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13/17) > BCOP (57.9%, 11/19) > HET-CAM (15.8%, 3/19), in decreasing order; and the

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methods correctly predicting No Cat were STE (100%, 10/10) > BCOP (69.2%, 9/13) > HETCAM (61.5%, 8/13), in decreasing order (Table 3A). Based on these results, we found that the STE test predicted correctly test chemicals of No Cat (100%, 10/10). The STE had higher

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prediction rate against Cat 2 test chemicals (76.5%, 12/17) than BCOP and HET-CAM,

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whereas it had the lowest prediction rate against Cat 1 chemicals. Also, the HET-CAM tests tended to over-predict Cat 2 chemicals as Cat 1 and had highest prediction rate against Cat 1

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chemicals (Table 3B). This study confirmed that there was no great single in vitro eye irritation test method, which can predict Cat 1 test chemicals correctly as Cat 1.

3.4. Prediction models of the proposed two tiered approaches The two proposed tiered approaches in the present study are shown in Fig. 1 and Table 4. The proposed tiered approaches were designed to use the RhCE test at the final stage in order to minimize the use of the RhCE test. Also two approaches was made in consideration of the solubility of test chemicals, because the STE test employed at the first stage could not be employed for test chemicals, which are not dissolved or suspended in saline, 5% DMSO, or mineral oil. In application of each tiered approach, a solubility test for each chemical should be conducted prior to test performance in order to determine a proper in vitro eye irritation test method employed at the first stage. That is, in proposed tiered approach A, the STE test is employed for soluble test chemicals

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at the first stage. If the result of STE had “No pre” or “No Cat”, the BCOP should be performed at the second stage. If the results of the BCOP tests showed “No pre” or “No Cat”, the RhCE test should be conducted. At the final step, if the results of the RhCE test was predicted as “irritant” or “Non- irritant”, the final decision of eye irritation potency was classified as “No pre” or “No Cat” respectively. In proposed tiered approach A, the BCOP test is used for insoluble test chemicals at the first stage. If the BCOP results indicated “Cat 1”, further test performance was not required and classified as “Cat 1”. If the results of BCOP test had “No pre” or “No Cat”, the HET-

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CAM test should be conducted. At the next step, if the result of the HET-CAM test indicated

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“Severe”, the eye irritation potency of the chemical was classified as “Cat 1”, whereas if they showed “Slight” or “Mild” results, the RhCE test was required and then the final decision

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was determined by the results of RhCE test as shown on Fig 1. .

In tiered approach B, the STE and BCOP tests are employed simultaneously for soluble

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test chemicals at the first stage. If the results of two tests are either positive (Cat 1or No pre)

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or negative, the RhCE test was not required and the eye irritation potency for test chemicals was finally determined by the results obtained from the STE and BCOP tests. If the test chemical was predicted as “Cat 1” in the STE test and “No pre” in the BCOP test or “No pre”

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in the STE test and “Cat 1” in the BCOP test, the eye irritation potency of the test chemicals

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were predicted as “Cat 1”. However, if both the BCOP and STE tests had discordant hazard identifications, the RhCE test should be employed at the final step. The final decision of

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hazard identification is determined by the result of RhCE test and the eye irritation potency is classified as “No Cat” when the result of RhCE test is “Non- irritant. Also if the results of the STE, BCOP and RhCE tests indicated i) “Cat 1”, “No Cat”, and “irritant” or ii) “No pre”, “No Cat”, and “irritant” or iii) “No Cat”, “Cat 1”, and “irritant” or iv) “No Cat”, “No pre”, and “irritant”, the eye irritation potency was determined as Cat 1, Cat 2, Cat 1, or Cat 2, respectively (Table 4). If the test chemicals were insoluble in tiered approach B, both the HET-CAM and BCOP tests should be used simultaneously at the first stage. When the results of two tests are either positive (Cat 1 or No pre) or negative, the RhCE test is not required. The hazard identification is determined to be identical to that of two results; however, the decision for eye irritation potency is based on the results of the BCOP test. However, if the two results had different hazard identifications in the BCOP and HET-CAM tests, the RhCE test should be employed in the final step. The final decision of hazard identification is classified based on

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the result of RhCE test, whereas the eye irritation potency is determined as “No Cat” when the result of RhCE test is “Non- irritant. Also if the results of the HET-CAME, BCOP and RhCE tests indicated i) “Severe”, “No Cat”, and “irritant” or ii) “Mild”, “No Cat”, and “irritant” or iii) “Non- irritant”, “Cat 1”, and “irritant” or iv) “Non- irritant”, “No pre”, and “irritant”, the eye irritation potency was classified as Cat 1, Cat 2, Cat 1, or Cat 2 respectively according to the prediction model of tiered approach B(Table 4).

3.5. Final decision of eye irritation potency against 47 test chemicals using proposed

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tiered approach A or B

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The final decision of eye irritation potency against 47 test chemicals using the prediction models of the tiered approach A and B was summarized as shown on Table 5. In the tiered

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approach A, as the results of the STE test conducted at the first stage were predicted as Cat 1, the final decision of eye irritation potency was made as Cat 1. However, as the STE test

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showed No pre or No Cat and then the BCOP test was performed, its result was predicted as

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Cat 1, the final decision of eye irritation potency was classified as Cat 1. At second step, the BCOP results had No pre or No Cat and the RhCE test was conducted. At final stage, the final decision for the chemicals that had the results of Non- irritant in the RhCE test were

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classified as No Cat, whereas that of some chemicals that have the results of irritant in RhCE

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test were classified as Cat 2 (Table 5). The final decision for insoluble chemicals was made by the prediction model described at Fig. 1.

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In tiered approach B, the STE and BCOP tests were conducted simultaneously with soluble chemicals, whereas the BCOP and HET-CAM tests were conducted simultaneously with insoluble chemicals at the first step. As the results of two tests were either positive (Cat 1or No pre) or negative, the RhCE test was not conducted and the eye irritation potency was finally calssified as Cat 1, Cat 2, or No Cat by the results of two tests by the prediction model suggested at Table 4. As the eye irritation potency for the test chemical in two tests conducted at the first stage were discordant positive results (Cat 1 and No pre), the final decision of eye irritation potency was classified as Cat 1. Also when the results of two tests performed in the first stage had different hazard identifications, the RhCE test was performed at the second step and then the final decision was made by the results of the RhCE test.

3.6. Predictive capacities of two tiered approaches against 47 test chemicals As shown in Table 6, the A tiered approach for the prediction of eye irritation shows a 95.7%

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(45/47) accuracy, 100% (34/34) sensitivity, and 84.6% (11/13) specificity, whereas the B tiered approach shows a 95.7% (45/47) accuracy, 97.1% (33/34) sensitivity, and 92.6% (12/13) specificity. Based on the results of eye irritation potency, the approaches A and B demonstrate a 73.3% prediction rate for test chemicals of Cat 1, whereas they have prediction rates for test chemicals of No Cat as 84.6% and 92.3%, respectively (Table 6). This study confirmed the increase of the prediction rate (73.3%) against Cat 1 test chemicals in the two tiered approaches, compared with the results of single BCOP or HET-CAM test, with approximately 60%. Also, both approaches A and B seem to be effective methods that can

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predict test chemicals of Cat 1 or No Cat.

4. Discussion

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In the case of sodium chloroacetate, the BCOP and STE tests did not correctly identify the

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classification; however, the HET-CAM and RhCE tests were shown to have consistent results with in vivo UN GHS classification. In the case of methanol, classified as UN GHS No Cat,

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the BCOP test correctly identified this chemical as Cat 1 by direct test performance, which is classified as a severe irritant in the literature. Like the BCOP test results, the HET-CAM and RhCE tests classified methanol as an irritant. However, only the STE test showed consistent

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results with classifications of the UN GHS.

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For 1,5-naphthalenediol, classified as UN GHS Cat 2 and identified as an insoluble test chemical, the BCOP test and the EpiOcularT M EIT predicted the chemical as No pre and

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irritant, respectively; however, the HET-CAM test predicted the chemical as a false negative. According to a reported study related to this chemical, Alépéeet al. (2016) asserted that 1,5naphthalenediol was under-predicted in 4 out of 9 runs in the SkinEthicT M HCE EIT. In the case of methanol, classified as UN GHS No Cat, only the STE test indicated consistent results with in vivo UN GHS classification; however, the BCOP, HET-CAM, and RhCE tests gave false positive results. According to the report of the US EPA (ICCVAM, 2010a), they classify methanol as Cat 2. In addition, Hayashi et al. (2012b) report that triethanolamine was predicted as a false positive, similar to the result of the EpiOcularT M EIT. For potassium tetrafluoroborate classified as UN GHS No Cat, only the BCOP test was discordant with classification by the in vivo UN GHS, whereas the other test methods correctly identified the test chemical as No Cat. Propylene glycol and polyethylene glycol, classified as UN GHS No Cat, were predicted consistently with the UN GHS classification in the STE and RhCE tests; however, the BCOP and HET-CAM tests over-predicted two

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chemicals by indicating “moderately irritating” and “severely irritating”, respectively. In general, as alcohols are known to have frequent false positive responses (OECD TG 437, 2013a; ICCVAM, 2006), propylene glycol, which is classified as an alcohol group, was overpredicted in this study. Tetraethylene glycol diacrylate, which is classified as UN GHS Cat 1 and an insoluble chemical, was predicted as No pre in the BCOP test and an irritant in the RhCE test; however, only the HET-CAM test indicated slightly irritating. Additionally, 1ethyl-3-methylimidazole, classified as UN GHS No Cat, was correctly predicted in the STE, BCOP, and RhCE tests, whereas only the HET-CAM test over-predicted as severely irritating

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(Table 1).

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This study aimed to develop an efficient tiered approach to minimize the use of RhCE tests by combining several alternative test methods in order to predict eye irritation potency

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of test chemicals. They were designed in consideration of the solubility property of test chemicals and the RhCE tests were used at the final steps of the tiered approach. In the

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proposed approach A, the STE, BCOP and RhCE tests are employed for soluble chemicals,

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whereas for insoluble chemicals the BCOP, HET-CAM and RhCE tests are used. However, the proposed approach B uses simultaneously two in vitro eye irritation test methods, which consist of the STE and BCOP for soluble chemicals or the BCOP and HET-CAM tests for

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insoluble chemicals at the first step. When two results obtained at the first step were

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discordant, the final decision was made based on by considering the results of RhCE test. The proposed approach A had high accuracy and sensitivity values, with 95.7% (45/47) and

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100% (34/34), respectively, whereas approach B indicated an accuracy of 95.7% (45/47), a sensitivity of 97.1% (33/34), and a specificity of 92.3%. Hayashi et al. (2012a) proposed a tiered approach combining the STE and BCOP tests. The accuracy, sensitivity, and specificity of their proposed approach were 8.4% (41/51), 76.9% (25/26), and 84.0% (21/25), respectively. Hayashi et al. (2012b) tried to design 4 tiered approaches and finally recommended a two-stage Bottom- Up tiered approach combining the STE test (for soluble chemicals), the EpiOcularT M EIT (for insoluble chemicals), and the BCOP test. The accuracy, sensitivity, and specificity of this proposed approach was reported to be 80.3% (45/56), 86.2% (25/29), and 74.1% (20/27), respectively. In tiered approach A, the prediction rates to correctly distinguish between Cat 1, Cat 2, and No Cat test chemicals were 73.3% (11/15), 63.29% (12/19), and 84.6% (11/13), respectively, while the prediction rates of approach B were 73.3% (11/15), 57.9% (11/19), and 92.3% (12/13), respectively. Adriaens et al. (2018) developed several approaches for the

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evaluation of eye irritation potency by using 7 different tests. The two-tiered and three-tiered Top-Down/Bottom-Up approaches used one of the RhCE test methods, such as the EpiOcularT M EIT and SkinEthicT M EIT, in combination with the BCOP LLBO in the twotiered approach or the BCOP OP-KIT and the Slug Mucosal Irritation assay in a three-tiered approach. They report that the prediction rates for Cat 1, Cat 2, or No Cat chemicals were 71.1-82.9%, 64.2-68.5%, or ≥80%, respectively In the prediction of Cat 1 test chemicals, this study confirmed that approaches A and B had increased prediction rates, 73.3%, compared to only the BCOP, STE, or HET-CAM tests,

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having 36.4%, 60%, or 60%, respectively. Comparing the several tiered approaches proposed

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in previous studies, the proposed approaches A and B are considered to be an appropriate method for distinguishing Cat 1 or No Cat test chemicals. Especially, the proposed approach

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B seems to be useful for distinguishing negative chemicals for the Bottom-Up approach.

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However, further study is required to confirm the specificity of the proposed tiered approaches using No Cat test chemicals including various drivers of classification (corneal

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opacity (CO) > 0** , CO > O, CO = 0** , and CO = 0) (Barroso et al., 2017) with a large number. Furthermore, as both approaches A and B give a low prediction rate to correctly distinguish Cat 2 test chemicals, further studies to develop standards or additional test

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methods that can accurately predict between Cat 1 and Cat 2 are required. In conclusion, the

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proposed approaches A and B seem to be effective strategies to replace the in vivo Draize

test chemicals.

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rabbit eye test and single in vitro test method for the evaluation of eye irritation potency of

Conflicts of interest None.

Acknowledgements This research was supported by a grant (17181MFDS403) from the Ministry of Food and Drug Safety in 2017.

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for Eye Irritation or Serious Eye Damage. OECD Guidelines for the Testing of chemicals, Section 4, OECD Publishing, Paris. OECD, 2015b. 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. OECD Guidelines for the Testing of chemicals, Section 4, OECD Publishing, Paris. OECD, 2017a. No. 263 Guidance Document on an Integrated Approach on Testing and Assessment (IATA) for Serious Eye Damage and Eye irritation. OECD Publishing, Paris. OECD, 2017b. 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. OECD Guidelines for the Testing of chemicals, Section 4, OECD Publishing, Paris. OECD, 2018. 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. OECD Guidelines for the Testing of chemicals, Section 4, OECD Publishing, Paris. Pfannenbecker, U., Bessou-Touya, S., Faller, C., Harbell, J., Jacob, T., Raabe, H., Tailhardat, M., Alépée, N., De Smedt, A., De Wever, B., Jones, P., Kaluzhny, Y., Le Varlet, B., McNamee, P., Marrec-Fairley, M., Van Goethem, F., 2013. Cosmetics Europe multilaboratory pre- validation of the EpiOcularT M reconstituted human tissue test method for the prediction of eye irritation. Toxicol.in Vitro 27, 619-626. Sakaguchi, H., Ota, N., Omori, T., Kuwahara, H., Sozu, T., Takagi, Y., Takahashi, Y., Tanigawa, K., Nakanishi, M., Nakamura, T., Morimoto, T., Wakuri, S., Okamoto, Y., Sakaguchi, M., Hayashi, T., Hanji, T., Watanabe, S., 2011.Validation study of the short time exposure (STE) test to assess the eye irritation potential of chemicals. Toxicol.in Vitro 25, 796-809. Schrage, A., Kolle, S.N., Rey Moreno, M.C., Norman, K., Raabe, H., Curren, R., Ravenzwaay, B.V., Landsiedel, R., 2011. The bovine corneal opacity and permeability test in routine ocular irritation testing and its improvement within the limits of OECD test guideline 437. ATLA.39, 37-53. Scott, L., Eskes, C., Hoffmann, S., Adriaens, E., Alepée, N., Bufo, M., Clothier, R., Facchini, D., Faller, C., Guest, R., Harbell, J., Hartung, T., Kamp, H., Le Varlet, B., Meloni, M., McNamee, P., Osborne, R., Pape, W., Pfannenbecker, U., Prinsen, M., Seaman, C., Spielmann, H., Strokes, W., Trouba, K., Den Berghe, C. V., Van Goethem, F., Vassallo, M., Vinardell, P., Zuang, V.,2010. A proposed eye irritation testing strategy to reduce and replace in vivo studies using Bottom–Up and Top–Down approaches.Toxicol.in Vitro 24, 1-9. Settivari, R.S., Amado, R.A., Corvaro, M., Visconti, N.R., Kan, L., Carney, E.W., Boverhof, D.R., Gehen, S.C., 2016. Tiered application of the neutral red release and EpiOcular™ assays for evaluating the eye irritation potential of agrochemical formulations.Regul.Toxicol. Pharm. 81, 407-420. Steiling, W., Bracher, M., Courtellemont, P., de Silva, O., 1999.The HET-CAM, a Useful in vitro Assay for Assessing the Eye Irritation Properties of Cosmetic Formulations and

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Ingredients.Toxicol.in Vitro. 13, 375-384. Takahashi, Y., Hayashi, K., Abo, T., Koike, M., Sakaguchi, H., Nishiyama, N., 2011. 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. Toxicol.in Vitro 25, 1425-1434. Takahashi, Y., Hayashi, T., Watanabe, S., Hayashi, K., Koike, M., Aisawa, N., Ebata, S., Sakaguchi, H., Nakamura, T., Hirofumi, K., Nishiyama, N., 2009.Inter- laboratory study of short time exposure (STE) test for predicting eye irritation potential of chemicals and correspondence to globally harmonized system (GSH) classification. J. Toxicol. Sci. 34, 611-626. Verstraelen, S., Jacobs, A., De Wever, B., Vanparys, P., 2013.Improvement of the bovine corneal opacity and permeability (BCOP) assay as an in vitro alternative to the draize rabbit eye irritation test.Toxicol.in Vitro 27, 1298-1311. Verstraelen, S., Maglennon, G., Hollanders, K., Boonen, F., Adriaens, E., Alépée, N.,

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Drzewiecka, A., Gruszka, K., Kandarova, H., Willoughby, J.A.Sr., Guest, R., Schofield, J., Van Rompay, A.R., CON4EI: Bovine Corneal Opacity and Permeability (BCOP) test for

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hazard identification and labeling of eye irritating chemicals. Toxicol.in Vitro 44, 122-13

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Table 1. Prediction capacities of eye irritation potency obtained by direct test performance (represented in supplementary data) and literature survey against 47 test chemicals No.

Test chemicals

Physical state

UN GHS Category

4468-02-04

Solid

CAS no.

Predictions ST E Exp & Liter.

FD

BCOP Exp & Liter.

FD

Cat 1

Cat 1

O

No pre

∆

Moder

Exp

1

Zinc gluconate

2

Cetyltrimethylammonium bromide

57-09-0

Solid

Cat 1

Cat 1

O

Severe irritant 2,3 , moderate irritant 4

O

Seve

3

Salicylic acid

69-72-7

Solid

Cat 1

– Moderate irritant5,6,7, I3 No pre Moderate irritant7,10, I2 No pre

–

Cat 1

O

Ir

Cyclohexanol

108-93-0

Liquid

Cat 1

5

Imidazole

288-32-4

Solid

Cat 1

∆

Severe irritant , Moderate irritant 4

O

Seve

∆

Severe irritant 2,3,4 , Cat 1 11 , very severe irritant12

O

Seve

∆

Severe irritant 4

O

Seve

O

Very severe irritant 4

O

Severe

O

Moder

Mo irritat

f

4

Promethazine hydrochloride

58-33-3

Solid

Cat 1

7

10% Benzalkonium chloride (w/v)

8001-54-5 (63449-41-2)

Liquid

Cat 1

Cat 1

Methylthioglycolate

2365-48-2

Liquid

10

Tetraethylene glycol diacrylate

17831-71-9

Liquid

11

2,5-Dimethyl-2,5-hexanediol

12

Sodium oxalate

13

2-Methoxyethyl acrylate

62-76-0

Lactic acid

15 16 17

10% Di(2-ethylhexyl) sodium sulfosuccinate (w/v) Benzyl alcohol Citric acid

18

Acetone

Ethanol

Cat 1

–

–

Cat 1

Cat 1

O

Cat 1

O

Cat 1

–

–

No pre

∆

No

Solid

Cat 1

Minimal irritant 6,10 No Cat

X

Mild irritant 4

∆

Ir

Liquid

4

Cat 1

3121-61-7

Liquid

50-21-5

Liquid

Cat 1

Liquid

Cat 1

Jo u

14

19

110-03-2

Pr

9

pr

Solid

Very severe irritant 4,12 , Cat 1 11

e-

55-56-1

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Chlorhexidine

rn

8

oo

6

Severe irritant 10 , Moderate irritant5,10, I3 No pre

577-11-7

Cat 1

100-51-6 77-92-9

Liquid Solid

Cat 2 Cat 2

67-64-1

Liquid

Cat 2

64-17-5

Liquid

Cat 2

20

2-Ethyl-1-hexanol

104-76-7

Liquid

Cat 2

21

Methyl ethyl ketone

78-93-3

Liquid

Cat 2

22

4-Formylbenzoic acid

619-66-9

Solid

3,4

Cat 2

–

Moderate irritant6,10 Cat 1 Moderate irritant 5,6 No pre No pre 5,6

Moderate irritant Moderate irritant6,10 Moderate irritant 5,6 , minimal irritant 7 , I 3 Minimal irritant NI 3

5,6,7

–

Mild irritant , Cat 1/2 11 , minimal irritant 12 No pre

∆

Seve

∆

No pre

∆

Severe

∆

Cat 1

O

Seve

∆

No pre

∆

Seve

∆ ∆

No pre Cat 1 Very severe irritant 4,12 , Cat 1 11 Severe irritant 3,4 , moderate irritant4,12 , not Cat 1 11 Cat 1 Moderate irritant 4 No pre

∆ O

Severe Severe

O

Seve

O

Seve

∆

Seve

∆

Severe irritant 4

O

Seve

–

Severe irritant 3,4,12 , moderate irritant4,12 , Cat 1 11

O

∆

, ∆

No pre Moderate irritant5,6,7, I3 Moderate irritant 6,7 , I 3 , minimal irritant 7 –

∆

10

23

Ammonium nitrate

6484-52-2

Solid

Cat 2

Minimal iritant , NI 3 No Cat

X

No pre

∆

Seve

24

2,4,11,13Tetraazatetradecanediimidam ide,N,N''-bis(4chlorophenyl)-3,12-diimino,di-Dgluconate(20%, aqueous)

18472-51-0

Liquid

Cat 2

No pre

∆

Cat 1

O

Seve

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Solid

1-Octanol

111-87-5

Liquid

Cat 2

134-62-3

Liquid

Cat 2

79-92-5

Solid

Cat 2

29

Diethyl toluamide 2,2-Dimethyl-3methylenebicyclo[2.2.1] heptane Cyclopentanol

96-41-3

Liquid

Cat 2

30

Sodium chloroacetate

3926-62-3

Solid

Cat 2

31

29911-27-1

Liquid

Cat 2

32

Di(propylene glycol) propyl ether 3-Chloropropionitrile

542-76-7

Liquid

Cat 2

33

Methyl acetate

79-20-9

Liquid

Cat 2

34 35

78-83-1 123-94-4

Liquid Solid

Cat 2 No Cat

541-02-6

Liquid

No Cat

44 45

Isobutanol Monostearin Decamethylcyclopentasiloxa ne Isopropyl myristate 1-Ethyl-3methylimidazolium ethylsulphate Dipropyl disulphide Piperonyl butoxide Polyethylene glycol (PEG40) hydrogenated castor oil 1-(4-Chlorophenyl)-3-(3,4dichlorophenyl)urea 2,2'-Methylene-bis-(6-(2Hbenzotriazol-2-yl)-4(1,1,3,3-tetramethylbutyl)phenol) Potassium tetrafluoroborate Propylene glycol

46

Methanol

39 40 41 42

43

47

T riethanolamine

–

∆

Slight

∆

Moderate irritant

No pre Minimal irritant 6,10

∆

No pre

∆

Severe

∆

Ir

X

No pre

∆

Moder

∆

Cat 1

O

Severe

X

No Cat

X

Severe

Moderate irritant6,10

∆

No pre

∆

Severe

No pre Minimal irritant 6,10 , NI 3 No Cat Moderate irritant6,10 –

∆

No pre

∆

Ir

X

Moderate irritant4,12

∆

Seve

∆ –

Moderate irritant 4 No Cat

∆ X

Severe Slight

X

No pre

∆

Slight

X

No Cat

X

Slight

No Cat

X

No Cat

X

Severe

No Cat No Cat

X X

No Cat No Cat

X X

Slight Slight

5,6,10,12

No Cat No pre Moderate irritant 10 , minimal irritant 6 No Cat

Minimal irritant 6

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– Moderate irritant

5,6,10

No pre 4

110-27-0

Liquid

No Cat

342573-75-5

Liquid

No Cat

629-19-6 51-03-6

Liquid Liquid

61788-85-0

Viscous

No Cat

Minimal irritant 5,6

X

No pre

∆

Moder

101-20-2

Solid

No Cat

–

–

No Cat

X

Slight

Soild

No Cat

–

–

No Cat

X

Slight

Soild Liquid

No Cat No Cat

No Cat Minimal irritant 6,7

X X

∆ ∆

Slight Severe

Liquid

No Cat

No Cat

X

No pre No pre Severe irritant 4 Cat 1

103597-45-1 14075-53-7 57-55-6 67-56-1

102-71-6

Liquid

Minimal irritant

pr

37

No Cat No Cat

e-

36

Cat 2

Pr

28

al

27

1,5-Naphthalenediol

f

83-56-7

26

rn

25

No Cat

Minimal irritant NI 3

5,6

, X

NI 4

No Cat

Exp & Liter : Experimental data & Literature data; FD : Final decision O: Severe irritant, severely irritating, Cat 1; △ : M oderate irritant, mild irritant, no prediction can be made, irritating, moderately irritating; X: No Category, minimal irritant, slightly irritating -: Insoluble chemicals in saline, 5% DM SO in saline, or mineral oil; I: Irritant; NI: Non-irritant; No pre: No prediction can be made; 1 Ko et al., 2018; 2 Verstraelen et al., 2013; 3 Hayashi et al., 2012b; 4 ICCVAM , 2010a; 5 Takahashi et al., 2009; 6 Takahashi et al., 2011; 7 Sakaguchi et al., 2011; 8 Kaluzhny et al., 2011; 9 Pfannenbecker et al., 2013; 10 Kojima et al., 2013; 11 Kolle et al., 2011; 12 Schrage et al., 2011; 13 Kaluzhny et al., 2015; 14 OECD TG 492, 2015; 15 ICCVAM , 2010b; 16 ICCVAM , 2006; 17 Gilleron et al., 1996

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Table 2A. The predictive capacity of each eye irritation test method calculated by the test chemicals employed with different sample size. Predictive capacity* Accuracy Sensitivity Specificity n % n % n % ** STE 33/38 86.8 23/28 82.1 10/10 100 BCOP 42/47 89.4 33/34 97.1 9/13 69.2 HET-CAM 39/47 83.0 31/34 91.2 8/13 61.5 TM EpiOcular 45/47 95.7 34/34 100 11/13 84.6 *

These values of predictive capacities were calculated based on Cat 1/Cat 2 vs No Cat. As nine test chemicals were not dissolved in solvents recommended from the OECD TG 491, they were excluded from the STE test.

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**

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Table 2B. The predictive capacity of each eye irritation test method calculated with the same number of 38 test chemicals. Predictive capacity* Accuracy Sensitivity Specificity n % n % n % STE 33/38 86.8 23/28 82.1 10/10 100 BCOP 32/38 84.2 27/28 96.4 5/10 50.0 HET-CAM 33/38 86.8 28/28 100 5/10 50.0 TM EpiOcular 36/38 94.7 28/28 100 8/10 80.0 *

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These values of predictive capacities were calculated with 38 substances in order to compare their predictive cap acities more precisely based on Cat 1/Cat 2 vs No Cat.

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eye irritation potency in each alternative test method calculated by STE(38* ) n % 4/11 36.4 6/11 54.5 1/11 9.1 0/17 0 13/17 76.5 4/17 23.5 0/10 0 0/10 0 10/10 100

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BCOP(47) n % 9/15 60 6/15 40 0/15 0 7/19 36.8 11/19 57.9 1/19 5.3 1/13 7.7 3/13 23.1 9/13 69.2

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Table 3A. Predictive capacities of different test chemical numbers. UN GHS Prediction Category Cat 1 Cat 1 No pre** No Cat*** Cat 1 Cat 2 No pre No Cat Cat 1 No Cat No pre No Cat *

HET-CAM(47) n % 9/15 60.0 5/15 33.3 1/15 6.7 14/19 73.7 3/19 15.8 2/19 10.5 3/12 25.0 1/12 8.3 8/13 61.5

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As nine test chemicals were not dissolved in solvents recommended from the OECD TG 491, they were excluded from the STE test so that the number of test chemicals conducted in STE test was 38. The BCOP and HET-CAM tests used 47 test chemicals for evaluating eye irritation hazard and potency identification. ** No pre: No prediction can be made; *** No Cat: No Category

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Table 3B. The predictive capacity of eye irritation potency in each alternative test method calculated by the same number (38) of test chemicals.

Cat 1

Cat 2

No Cat

Prediction Cat 1 No pre No Cat Cat 1 No pre No Cat Cat 1 No pre No Cat

STE (38* ) n 4/11 6/11 1/11 0/17 13/17 4/17 0/10 0/10 10/10

% 36.4 54.5 9.1 0 76.5 23.5 0 0 100

BCOP (38) n % 7/11 63.6 4/11 36.4 0/11 0 6/17 35.3 10/17 58.8 1/17 5.9 1/10 10.0 4/10 40.0 5/10 50.0

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UN GHS Category

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*

HET-CAM (38) n % 8/11 72.7 3/11 27.3 0/11 0 14/17 82.4 3/17 17.6 0/17 0 3/9 33.3 1/9 11.1 5/10 50.0

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These values of predictive capacities were calculated with 38 substances in order to compare their predictive capacities more correctly.

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Table 4. The tiered approach B combining 3 in vitro eye irritations test methods Hazard identification 1 st stage STE

Potency identification

2 nd stage Decision

Decision

-

I

Cat 1

-

I

Cat 1

I

I

Cat 1

NI

NI

No Cat

Cat 1

-

I

Cat 1

No pre

-

I

Cat 2

I

I

BCOP

RhCE

Cat 1 No pre Cat1

Soluble chemicals

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No pre No Cat I NI No pre

1 st stage

-

Cat 1

NI

No Cat

I

Cat 2

NI

No Cat

NI

No Cat

Decision

Decision

BCOP

RhCE

Cat 1

-

I

Cat 1

-

I

Cat 2

I

I

Cat 1

NI

NI

No Cat

Cat 1

-

I

Cat 1

No pre

-

I

Cat 2

I

I

Cat 2

NI

NI

No Cat

I

I

Cat 1

NI

NI

No Cat

I

I

Cat 2

NI

NI

No Cat

-

NI

No Cat

rn No pre

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Severe

I

2 nd stage

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HET-CAM

Pr

NI

No Cat

No Cat

e-

I

No Cat

Cat 2

NI

pr

NI Cat 1

f

No Cat

No Cat

M ild Insoluble chemicals

No Cat

Cat 1 Slightly irritant, no irritant

No pre

No Cat -: test is not needed;

I: irritant;

NI: Non irritant;

No pre: no prediction can be made;

No Cat: No Category

Journal Pre-proof Table 5. Final decision of the eye irritation potency against 47 test chemicals in applying the tiered approach A or B. No.

Test chemicals

ST E

4468-02-04

Cat 1

57-09-0

Cat 1

69-72-7

Cat 1

1

Zinc gluconate

2 3

Cetyltrimethylammonium bromide Salicylic acid

4

Cyclohexanol

108-93-0

Cat 1

5

Imidazole

288-32-4

6

Promethazine hydrochloride

58-33-3

8 9

10% Benzalkonium chloride (w/v) Chlorhexidine Methylthioglycolate

8001-54-5 (63449-41-2) 55-56-1 2365-48-2

10

Tetraethylene glycol diacrylate

11

7

A approach

UN GHS Category

CAS No.

BCOP

HETCAM

B approach RhC E

FD

ST E

BCOP

O

O

O

∆

O

O

O

O

O

O

∆

O

∆

O

O

O

O

O

O

O O

O O

∆

O

∆

X

∆

O

O

Cat 1

∆

O

Cat 1

∆

O

Cat 1

O

Cat 1 Cat 1

O ∆

17831-71-9

Cat 1

2,5-Dimethyl-2,5-hexanediol

110-03-2

Cat 1

12

Sodium oxalate

62-76-0

Cat 1

13

2-Methoxyethyl acrylate

3121-61-7

Cat 1

∆

14

50-21-5

Cat 1

∆

16 17 18

Lactic acid 10% Di(2-ethylhexyl) sodium sulfosuccinate (w/v) Benzyl alcohol Citric acid Acetone

19

al

X

n r u

∆ ∆ ∆

r P X

O

∆

o r p

I

∆

I

∆

O

X

I

O

O

O

O

O

∆ ∆

O

O

∆

FD

O

O

e

RhCE

f o

O

O

HET-CAM

O O I

∆

I

∆

O

O

∆

∆

∆

∆

O

∆

O

O

577-11-7

Cat 1

∆

∆

I

∆

∆

∆

∆

100-51-6 77-92-9 67-64-1

Cat 2 Cat 2 Cat 2

∆ ∆ ∆

∆ O O

I

∆ O O

∆ ∆ ∆

∆ O O

∆ O O

Ethanol

64-17-5

Cat 2

∆

O

O

∆

O

O

20

2-Ethyl-1-hexanol

104-76-7

Cat 2

∆

∆

21 22

Methyl ethyl ketone 4-Formylbenzoic acid

78-93-3 619-66-9

Cat 2 Cat 2

∆

O O

23

Ammonium nitrate

6484-52-2

Cat 2

X

∆

24

2,4,11,13Tetraazatetradecanediimidamide, N,N''-bis(4-chlorophenyl)-3,12diimino-,di-Dgluconate(20%, aqueous)

18472-51-0

Cat 2

∆

O

15

o J

I

I

∆

∆

∆

∆

O O

∆

O O

I

O O

∆

X

∆

I

∆

O

∆

O

X

O

Journal Pre-proof 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

1,5-Naphthalenediol 1-Octanol Diethyl toluamide 2,2-Dimethyl-3methylenebicyclo[2.2.1] heptane

83-56-7 111-87-5 134-62-3

Cat 2 Cat 2 Cat 2

∆ ∆

∆ ∆ ∆

79-92-5

Cat 2

X

Cyclopentanol Sodium chloroacetate Di(propylene glycol) propyl ether 3-Chloropropionitrile Methyl acetate Isobutanol Monostearin Decamethylcyclopentasiloxane Isopropyl myristate 1-Ethyl-3-methylimidazolium ethylsulphate Dipropyl disulphide Piperonyl butoxide Polyethylene glycol (PEG-40) hydrogenated castor oil 1-(4-Chlorophenyl)-3-(3,4dichlorophenyl)urea 2,2'-Methylene-bis-(6-(2Hbenzotriazol-2-yl)-4-(1,1,3,3tetramethylbutyl)-phenol)

96-41-3 3926-62-3 29911-27-1 542-76-7 79-20-9 78-83-1 123-94-4 541-02-6 110-27-0

Cat 2 Cat 2 Cat 2 Cat 2 Cat 2 Cat 2 No Cat No Cat No Cat

∆ X ∆ ∆ X ∆

342573-75-5

44 45 46

Potassium tetrafluoroborate Propylene glycol Methanol

47

T riethanolamine

I I I

∆ ∆ ∆

∆ ∆

∆ ∆ ∆

∆

I

∆

X

∆

I I I I I NI NI NI

O ∆ ∆ ∆ ∆ ∆ X X X

∆ X ∆ ∆ X ∆

X X

O X ∆ ∆ ∆ ∆ X ∆ X

O X ∆ ∆ ∆ ∆ X ∆ X

No Cat

X

X

629-19-6 51-03-6

No Cat No Cat

X X

X X

61788-85-0

No Cat

X

∆

101-20-2

No Cat

103597-45-1

No Cat

14075-53-7 57-55-6 67-56-1

No Cat No Cat No Cat

102-71-6

No Cat

o J

ur

l a n X

X

X

NI

-p

I

∆

I

O X ∆ ∆ ∆ ∆ X X X

X NI

X

X X

X X

X X

X X

NI

X

X

∆

X

NI

X

X

X

X

X

NI

X

X

X

X

X X X

∆ ∆ O

NI NI

X X O

X X X

∆ ∆ O

X

X

I

∆

X

X

FD : Final decision O: Severe irritant, severely irritating, Cat 1; △ : M oderate irritant, mild irritant, no prediction can be made, irritating, moderately irritating; X: No Category, minimal irritant, slightly irritating; I: irritant; NI: Non irritant;

∆ ∆ ∆

X

NI NI

X

X X

I

X

e r P

X

ro

f o

X

NI

NI NI I

X

X X O X

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Table 6. Comparison of the predictive capacities obtained from the tiered approach A and B A approach B approach Predictive capacity* n % n % Hazard Accuracy 45/47 95.7 45/47 95.7 identification Sensitivity 34/34 100 33/34 97.1 Specificity 11/13 84.6 12/13 92.3 UN GHS Prediction n % n % Category Cat 1 11/15 73.3 11/15 73.3 Cat 1 No pre 4/15 26.7 4/15 26.7 No Cat 0/15 0 0/15 0 Potency Cat 1 7/19 36.8 7/19 36.8 identification Cat 2 No pre 12/19 63.2 11/19 57.9 No Cat 0/19 0 1/19 5.3 Cat 1 1/13 7.7 1/13 7.7 No Cat No pre 1/13 7.7 0/13 0 No Cat 11/13 84.6 12/13 92.3 *

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Proposed A and B approaches are considered to be efficient strategies for evaluating eye irritation potency, with great predictive capacity.

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Predictive capacity rate of the A and B approaches had higher than that of standalone in vitro test method.

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Especially, the proposed B approach is considered as an useful test method for distinguishing the negative chemicals for Bottom-Up approach.

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Figure 1

Figure 1