新規試験法提案書

眼刺激性試験代替法

SkinEthic™ HCE TTT法

令和 6 年 2 月

国立医薬品食品衛生研究所

新規試験法提案書
令和 6 年 2 月 26 日
No.

2023-02

眼刺激性試験代替法SkinEthic™™ HCE TTT法
法に 関する提案
令和 5 年 12 月 12 日に国立医薬品食品衛生研究所にて開催された新規試験法評価会議（通称：JaCVAM 評価会議）において以下の提案がなされた。
提案内容： 本試験法は、技術移転性、施設内再現性、施設間再現性の高い試験法である。予測性に関しては、適用範囲に留意すれば、UN GHS 区分 1、区分 2 への分類および区分に該当しない場合の判定を可能とすると考えられた。ただし、UN GHS 区分 2A と 2B を区別することはできない。また、固体の UN GHS 区分 2 を区分に該当しないと判断す
る偽陰性率が 28.9％と高く、30%以下という OECＤが定めた基準に近い値であることから、固体の UN GHS 区分 2 の分類は慎重に評価されるべきである。
この提案書は、眼刺激性試験資料編纂委員会によりまとめられた文書を用いて、JaCVAM 評価
会議が評価および検討した結果、その有用性が確認されたことから作成された。
以上の理由により、行政当局の安全性評価方法として眼刺激性試験代替法 SkinEthic™ HCE TTT法の使用を提案するものである。

西川秋佳
JaCVAM 評価会議

平林容子
議長

JaCVAM 運営委員会

委員長

i

JaCVAM 評価会議

西 川 秋 佳 （国立医薬品食品衛生研究所 安全性生物試験研究センター 病理部／
名古屋徳洲会総合病院）：座長
小 島 幸 一 （一般財団法人 食品薬品安全センター）
中村るりこ （独立行政法人 製品評価技術基盤機構）
西 村 次 平 （独立行政法人 医薬品医療機器総合機構）
平 林 容 子 （国立医薬品食品衛生研究所 安全性生物試験研究センター）
松 本 一 彦 （名古屋市立大学大学院）
任期：令和 4 年 4 月 1 日～令和 6 年 3 月 31 日

ii

JaCVAM 運営委員会
平 林 容 子 （国立医薬品食品衛生研究所 安全性生物試験研究センター）：委員長
石 井 孝 司 （国立感染症研究所）
稲 角 嘉 彦 （厚生労働省 医薬・生活衛生局 医薬品審査管理課 化学物質安全対策室）
小川久美子 （国立医薬品食品衛生研究所 安全性生物試験研究センター 病理部）
諫 田 泰 成 （国立医薬品食品衛生研究所 安全性生物試験研究センター 薬理部）
北嶋聡 （国立医薬品食品衛生研究所 安全性生物試験研究センター 毒性部）

杉 山 圭 一 （国立医薬品食品衛生研究所 安全性生物試験研究センター 変異遺伝部）
髙 橋 祐 次 （国立医薬品食品衛生研究所 安全性生物試験研究センター 毒性部 動物管理室）
束 野 正 明 （厚生労働省 医薬・生活衛生局 医薬品審査管理課 化学物質安全対策室）
林亜紀子 （厚生労働省 医薬・生活衛生局 医薬品審査管理課）

本 間 正 充 （国立医薬品食品衛生研究所）
真 木 一 茂 （独立行政法人 医薬品医療機器総合機構）
増 村 健 一 （国立医薬品食品衛生研究所 安全性生物試験研究センター 安全性予測評価部）
横 田 雅 彦 （独立行政法人 医薬品医療機器総合機構）
足利太可雄 （国立医薬品食品衛生研究所 安全性生物試験研究センター 安全性予測評価部第二室）：事務局

iii

JaCVAM statement on the SkinEthic™ HCE TTT, an alternative method forevaluating eye irritation
At a meeting held on 12 December, 2023 at National Institute of Health Sciences (NIHS) in
Tokyo, Japan, the Japanese Center for the Validation of Alternative Methods (JaCVAM)
Regulatory Acceptance Board unanimously endorsed the following statement:
Proposal: This test method has high technological transferability and within- andbetween-laboratory reproducibility. Regarding predictive capacity, we believedthat consideration of the applicability domain would enable the classification of a
substance as United Nations Globally Harmonized System of Classification andLabeling of Chemicals (UN GHS) category 1 or 2 or determine no category.
However, it was impossible to distinguish between UN GHS categories 2A and2B. Moreover, regarding a solid, as the false negative rate for discriminating UN
GHS category 2 and no category is high (28.9%; close to the standard set by theOECD of ≤30%), the classification of UN GHS category 2 for solids should be
performed with caution.
This statement was released following a review prepared by the eye irritation test JaCVAM
Editorial Committee to acknowledge that the results of the review and study by the JaCVAM
Regulatory Acceptance Board have confirmed the usefulness of this assay.
Based on the above, we proposed the SkinEthic™ HCE TTT method as a useful means forassessing eye irritation potential during safety assessments by regulatory agencies.

Nishikawa Akiyoshi
Chairperson,
JaCVAM Regulatory Acceptance Board.

Hirabayashi Yoko
Chairperson,
JaCVAM Steering Committee.

February 26, 2024v

The JaCVAM Regulatory Acceptance Board was established by the JaCVAM Steering
Committee, and is composed of nominees from the industry and academia.
This statement was endorsed by the following members of the JaCVAM Regulatory
Acceptance Board:
Nishikawa Akiyoshi (Division of Pathology, Center for Biological Safety and Research:CBSR,NIHS / Nagoya Tokushukai General Hospital) : Chairperson
Hirabayashi Yoko (CBSR, NIHS)
Kojima Koichi (Food and Drug Safety Center)
Matsumoto Kazuhiko (Nagoya City University)
Nakamura Ruriko (National Institute of Technology and Evaluation)
Nishimura Jihei (Pharmaceuticals and Medical Devices Agency)
Term: From 1st April 2022 to 31st March 2024

vi

This statement was endorsed by the following members of the JaCVAM steering Committee
after receiving the report from JaCVAM Regulatory Acceptance Board:

Hirabayashi Yoko (CBSR, NIHS): Chairperson
Hayashi Akiko (Ministry of Health, Labour and Welfare)
Honma Masamitsu (NIHS)
Inazumi Yoshihiko (Ministry of Health, Labour and Welfare)
Ishii Koji (National Institute of Infectious Diseases)
Kanda Yasunari (Division of Pharmacology, CBSR, NIHS)
Kitajima Satoshi (Division of Cellular and Molecular Toxicology, CBSR, NIHS)
Maki Kazushige (Pharmaceuticals and Medical Devices Agency)
Masumura Kenichi (Division of Risk Assessment, CBSR, NIHS)
Ogawa Kumiko (Division of Pathology, CBSR, NIHS)
Sugiyama Keiichi (Division of Genetics and Mutagenesis, CBSR, NIHS)
Taquahashi Yuhji (Animal Management Section of the Division of Toxicology, CBSR, NIHS)
Tsukano Masaaki (Ministry of Health, Labour and Welfare)
Yokota Masahiko (Pharmaceuticals and Medical Devices Agency)
Ashikaga Takao (Division of Risk Assessment, CBSR, NIHS): Secretaryvii

眼刺激性試験代替法SkinEthic™ HCE TTT法
法 に関する提案

添付資料一覧
添付資料 1
評価会議報告書 ----------------------------------------------------------------------------------------- 1
添付資料 2
評価報告書 ----------------------------------------------------------------------------------------------- 5
添付資料 3
OECD GUIDELINE FOR TESTING OF CHEMICALS 492B
RECONSTRUCTED HUMAN CORNEA-LIKE EPITHELIUM (RHCE) TESTMETHOD FOR EYE HAZARD IDENTIFICATION-------------------------------------------21

添付資料 1

評価会議報告書

眼刺激性試験代替法 SkinEthic™ HCE TTT 法

JaCVAM 評価会議

令和 5 年（2023 年）12 月 12 日

1

JaCVAM 評価会議

西 川 秋 佳 （国立医薬品食品衛生研究所 安全性生物試験研究センター 病理部／
名古屋徳洲会総合病院）：座長
小 島 幸 一 （一般財団法人 食品薬品安全センター）
中村るりこ （独立行政法人 製品評価技術基盤機構）
西 村 次 平 （独立行政法人 医薬品医療機器総合機構）
平 林 容 子 （国立医薬品食品衛生研究所 安全性生物試験研究センター）
松 本 一 彦 （名古屋市立大学大学院）
任期：令和 4 年 4 月 1 日～令和 6 年 3 月 31 日

2

JaCVAM 評価会議は、眼刺激性試験資料編纂委員会により作成された「SkinEthic™ HCE
TTT 法評価報告書」1)をもとに本試験法の科学的妥当性、社会的および行政的な受け入れ性について検討した。
1. 試験法の定義および科学的妥当性
名称：SkinEthic™ Human Corneal Epithelium (HCE) Time-to-Toxicity (TTT)法
代替する対象毒性試験：Draize 眼刺激性試験 2)
科学的妥当性：
当該試験法は、再構築ヒト角膜様上皮モデル（Reconstructed human Cornea-like Epithelium:
RhCE）である SkinEthic™ HCE 用い、細胞毒性を指標として眼刺激性を評価する試験法である。経済協力開発機構（Organisation for Economic Co-operation and Development: OECD）試
験法ガイドライン（Test Guideline: TG）492B に収載されている 3)。
液体または固体の化学物質毎に異なる濃度および培養時間にて処理することにより生じる細胞生存率を用い、国際連合化学品の分類および表示に関する世界調和システム（United
Nations Globally Harmonized System of Classification and Labelling of Chemicals: UN GHS）による UN GHS 区分１、区分 2 への分類および区分に該当しない場合の判定ができる方法として開発されており、科学的には妥当である。
2. 目的とする物質又は製品の毒性を評価する試験法としての、社会的受け入れ性および行
政上の利用の可能性
社会的受け入れ性：
本試験法は RhCE に対する化学物質の細胞毒性を指標に用いて眼刺激性を評価する試験法であり、生きた動物を用いないという点で、3Rs の精神に合致している。また、SkinEthic™
HCE TTT 法に用いるキットの入手は容易であり、短時間で実施でき、特殊な機材や試薬を必要とせず、必要な手技も複雑なものでない。したがって、入手したキットの品質基準が許容範囲にあり、かつ実施する試験施設の技術習得がガイドラインの熟達度確認物質で確認できており、基本的な細胞培養の技術と設備を有する施設であれば実施可能である。技術移転性は高い上に、原料だけでなく、製剤においても UN GHS 区分１、区分 2 への分類および区分に該当しない場合の判定に用いることができる。以上より、本試験法の社会的受け入れ性は高い。
行政上の利用性：
本試験法は、技術移転性、施設内再現性、施設間再現性の高い試験法である。予測性に関しては、適用範囲に留意すれば、UN GHS 区分 1、区分 2 への分類および区分に該当しない場合の判定を可能とすると考えられた。ただし、UN GHS 区分 2A と 2B を区別することはできない。また、固体の UN GHS 区分 2 を区分に該当しないと判断する偽陰性率が 28.9％と高く、30%以下という OECＤが定めた基準に近い値であることから、固体の UN GHS 区
分 2 の分類は慎重に評価されるべきである。

3

参考文献（最終確認日：2023 年 9 月 20 日）
1) JaCVAM 眼刺激性試験資料編纂委員会：眼刺激性試験代替法SkinEthicTM HCE TTT法評価
報告書（2023年3月20日）
2) OECD (2023) OECD Guidelines for the Testing of Chemicals No. 405. Acute Eye Irritation/
Corrosion, Organisation for Economic Cooperation and Development, Paris.
Available at: https://www.oecd-ilibrary.org/docserver/9789264185333-en.pdf?expires=1695282151&id=id&accname=guest&checksum=3130BC4DE84C35C7D73325F595BDA84B
3) OECD (2022) OECD Guidelines for the testing of Chemicals No. 492B. Reconstructed humanCornea-like Epithelium (RhCE) test method for Eye Hazard Identification, Organisation for
Economic Cooperation and Development, Paris.
Available at: https://www.oecd-ilibrary.org/environment/test-no-492b-reconstructed-humancornea-like-epithelium-rhce-test-method-for-eye-hazard-identification_0d603916-en

4添付資料 2

評価報告書

眼刺激性試験代替法 SkinEthic™ HCE TTT 法

眼刺激性試験資料編纂委員会

令和 5 年（2023 年）12 月 12 日

15

眼刺激性試験資料編纂委員会
山 本 直 樹 （委員長：藤田医科大学）
小島

肇 （国立医薬品食品衛生研究所）

佐々木正治 （アレクシオンファーマ合同会社）
竹 内 小 苗 （P&G イノベーション合同会社）
波多野浩太 （ホーユー株式会社）原

範 子 （藤田医科大学）

山 下 晴 洋 （アステラス製薬株式会社）

6

2

略語
CAS:

Chemical Abstracts Services

GHS:

Globally Harmonized System of Classification and Labelling of Chemicals

HCE:

Human Corneal Epithelium

HPLC:

High Performance Liquid Chromatography

IATA：

Integrated Approaches to Testing and Assessment

JaCVAM:

Japanese Center foe the Validation of Alternative Methods

MTT:

3- (4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium BromideOECD:

Organization for Economic Co-operation and Development

PBS:

Phosphate-Buffered Saline

PRP:

Peer Review Panel

RhCE:

Reconstructed human Cornea-like Epithelium

TG:

Test Guideline

TTT:

Time-to-Toxicity

UN:

United Nations

UPLC:

Ultra Performance Liquid Chromatography

VRM:

Validated Reference Method

3

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要旨
SkinEthic™ Human Corneal Epithelium (HCE) Time-to-Toxicity (TTT)法は、L’Oréal 社に
よ っ て 開 発 さ れ 、 再 構 築 ヒ ト 角 膜 様 上 皮 モ デ ル (Reconstructed human Cornea-like
Epithelium: RhCE)である SkinEthic™ HCE 用いて、液体および固体の化学物質における国際連合化学品の分類および表示に関する世界調和システム（United Nations Globally
Harmonized System of Classification and Labelling of Chemicals: UN GHS）による UN GHS 区
分 1、UN GHS 区分 2 への分類および区分に該当しない場合の判定ができる方法として開発された眼刺激性評価試験法である。
SkinEthic™ HCE TTT 法は、バリデーション研究の後、科学的妥当性を評価するために第三者評価委員会（Peer Review Panel：PRP)による検証がなされた。その後、経済協力開発機構（Organization for Economic Co-operation and Development: OECD)による専門家会
議での議論を経て、UN GHS 区分 1、UN GHS 区分 2 への分類および区分に該当しない場合の判定が可能な試験法と判断され、OECD 試験法ガイドライン（Test Guideline：
TG)492B として 2022 年 6 月に採択された。
本バリデーション研究は、本試験法に用いられる RhCE である SkinEthic™ HCE を販売している企業の親会社である L’Oréal 社がスポンサーとなり行われた。そのため、バリデーション実行委員会の独立性について、PRP で慎重に議論された。その結果、PRP
はバリデーション報告書の記載に不備は多いが、バリデーションの結果は受け入れると結論している。いずれの施設の記録も十分であり、データも適切であることを PRP が確認していることから、バリデーション結果に大きな問題はないと本委員会も考える。
本試験法の技術移転性は高いと判断された。再現性に関しては、液体被験物質（TimeTo-Toxicity Test Method On Liquids：TTL）について、3 施設でのコード化された 20 物質
によるバリデーション研究の結果から、施設内再現性は 85-95％であり、施設間再現性
は 90％であった。固体被験物質（Time-To-Toxicity Test Method On Solids：TTS）について、3 施設でのコード化された 20 物質によるバリデーション研究の結果から、施設内再現
性は 100％であり、施設間再現性も 100％であったことから、SkinEthic™ HCE TTT 法の再現性は高いと考えられた。また、本試験法の正確度については、UN

GHS 区分 1 物

質、区分 2 物質、および区分に該当しない物質がそれぞれ 79.2％、69.2％および 74.9％の割合で正しく判定され、本試験法の予測性は OECD 専門家会議が提案した採用基準を満たしていた。以上より、SkinEthic™ HCE TTT 法は、技術移転性、施設内再現性、施設間再現性は高い試験法である。ただし、UN GHS 区分 2 の固体物質を区分に該当しない物質と判定する偽陰性率が 28.9％と高く、30％以下という基準に近い値であることから、安全性評価上懸念を持つとの見解で一致した。
以上より、SkinEthic™ HCE TTT 法は、OECD TG492B に準拠して実施した場合、UN
GHS 区分 1、UN GHS 区分 2 への分類および区分に該当しない場合の判定を可能とする試験法である。ただし、固体物質の UN GHS 区分 2 の分類は慎重に評価されるべきであると本資料編纂委員会は考える。

8

4

1．
． 緒言
OECD は TG として、既に複数の眼刺激性試験代替法を採択している 1)。UN GHS 区
分 1 の分類および UN GHS 区分に該当しない場合を判定試験法としてウシ摘出角膜の混濁および透過性試験法（Bovine Corneal Opacity and Permeability test method: BCOP 法、
TG437）、ニワトリ摘出眼球を用いた眼刺激性試験（Isolated Chicken Eye Test: ICE 法、
TG438）、in vitro 短時間暴露法（Short Time Exposure: STE 法、TG491）、in vitro マクロモ
レキュラ試験法（TG496）があり、UN GHS 区分 1 を検出する試験法としてフルオレセイ
ン漏出試験法（TG460）、UN GHS 区分に該当しない場合を判定する試験法として再構築
ヒト角膜様上皮モデル法（Reconstructed human Cornea-like Epithelium: RhCE 法、TG492）2)
および Vitrigel-Eye Irritancy Test（Vitrigel-EIT, TG494）がある。ただし、いずれの試験法においても、UN GHS 区分 2 の化学物質の検出をすることはできず、in vivo ドレイズ試
験の代替法ではない。
この度、UN GHS 区分 1、UN GHS 区分 2 への分類および区分に該当しない場合の判定が可能となる試験法として L’Oréal 社によって開発された SkinEthic™ Human Corneal
Epithelium（HCE）Time-to-Toxicity（TTT）法は、RhCE である SkinEthic™ HCE 用いて液体および固体に対する化学物質の眼刺激性の評価を行う試験法である 3,4) 。SkinEthic™
HCE TTT 法は、L’Oréal 社がスポンサーとなり、L’Oréal R&I と Adriaens Consulting BVBAの主導で実施されたバリデーション研究の後、科学的妥当性を評価するために第三者評
価委員会（Peer Review Panel: PRP）による検証がなされた。その後、OECD よる専門家会
議での議論を経て、UN GHS 区分 1、UN GHS 区分 2 への分類および区分に該当しない場
合の判定が可能な試験法と判断され、OECD TG492B として 2022 年 6 月に採択された 5)。

本報告書は、バリデーション研究論文 6)、第三者評価報告書 7)、その他関連論文などをもとに本試験法の概要を説明し、JaCVAM 眼刺激性試験資料編纂委員会の意見をまとめたものである。
2．
． 試験法の位置づけ
SkinEthic™ HCE TTT 法は、化学物質（単一物質および混合物）の眼刺激性を UN GHS
において UN GHS 区分 1、UN GHS 区分 2 への分類および区分に該当しない場合の判定に用いることができる試験法である。
3．
．原理
本試験法は、SkinEthic™ HCE を用いて細胞毒性を指標として眼刺激性を評価する試験法である。角膜混濁などの細胞傷害は種々の作用機序に伴って発生する可能性があるが、細胞毒性は in vivo で見られる化学物質の眼刺激性を決定するうえで重要な役割を果たす。また、化学物質の眼刺激性は主に初期損傷の程度によって決定され、細胞死の程度とその後の反応および最終的な結果の程度と相関する。

59

4. 試験手順
SkinEthic™ HCE TTT 法の手順を以下に示す 8,9)。SkinEthic ™ HCE は EPISKIN 社（フランス）および我が国では株式会社ニコダームリサーチより購入できる。
4-1. RhCE 組織の機能的条件
RhCE 組織の機能的条件は、以下のとおりである。
陰性対照吸光度：陰性対照において 1.0 < OD ≤ 2.5（使用時の基準であり、製造者の出荷基準は異なる場合もある）
バリア機能：50 µL ドデシル硫酸ナトリウム（Sodium Lauryl Sulphate：SDS）の 30 分
間曝露において 1.0 ≤ IC50（mg/mL）≤ 3.2（IC50：細胞生存率を 50％低下させるのに必要な濃度）
形態：少なくとも 4 層の上皮細胞があり、角化していない
再現性：陽性および陰性対照の結果が背景データをもとに設定した許容範囲内にある
これらは SkinEthic ™ HCE 製造者の出荷基準として採用される。一方、SkinEthic ™
HCE の使用者は毎回、生存率と再現性を確認する必要がある。
4-2. 被験物質の適用
37C 以下においてピペットで扱えるものは液体として、それ以外は固体として試験を行う。RhCE 組織は、一晩、標準培養条件（37±2C、5±1％ CO2、≥95％ 湿度）で培養し、更に新たな培地を用いて標準培養条件で 30 分間以上培養する。
液体物質の場合（SkinEthic ™ HCE Time-to-Toxicity for Liquids chemicals: TTL）、評価対
象物質そのもの、および蒸留水で 20％（w/v）に希釈したものを用いる。SkinEthic ™ HCEに Phosphate-Buffered Saline （PBS）10±2 µL を適用し被験物質 80±2 µL を加える。評価対
象物質そのものは室温で 5±0.25 分間、20％(w/v)希釈調整液は室温で 16±1 分間および標準培養条件で 120±2 分間培養する。
その後、PBS 25mL で洗浄して被験物質を除去し、10±1 分間室温で培地に浸漬する。同時に行う陽性対照には、酢酸メチル（Chemical
Abstracts Services : CAS No. 79-20-9）が推奨され、適用・培養・後処理は対象評価物質の条件に準じる。同時に行う陰性対照には PBS が推奨され、80±2 µL のみを適用する。培養・後処理は対象評価物質の条件に準じる。
固体物質の場合（SkinEthic ™ HCE Time-to-Toxicity for Solids chemicals: TTS）、評価対
象物質そのものを用いる。被験物質 80±2 mg を均一に適用した後に蒸留水 80±2 µL を加え、標準培養条件で 30±2 分間および 120±5 分間培養する。その後、PBS 25 mL で洗浄して被験物質を除去し、30±2 分間室温で培地に浸漬する。同時に行う陽性対照として、乳酸 1％（w/v）水溶液（CAS No. 50-21-5）が推奨され、80±2 µL を適用した後に蒸留水
80±2 µL を加える。同時に行う陰性対照には PBS が推奨され、80±2 µL のみを適用する。
陽性・陰性対照とも培養・後処理は対象評価物質の条件に準じる。
それぞれの曝露条件で少なくとも 2 つの RhCE 組織を用いる。

106

4-3. 細胞生存率の算出
細胞生存率算出には 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide（MTT）
還元法を用いる。培地を除去し、1 mg/mL MTT 溶液 0.3 mL 中で 180±15 分間標準培養
条件で反応させ、その後 1.5 mL イソプロパノール（または同様な溶媒）で青色のホルマ
ザンを抽出する。液体被験物質の場合は、RhCE 組織の上部・底部両方から抽出する。
固体被験物質および無色透明でない液体被験物質の場合は、組織に残存する被験物質の混入を最小限に抑えるため、ホルマザンの抽出は RhCE 組織の底部のみから行う。液体
被験物質でも、洗浄が困難な場合には、底部のみから抽出を行う。同時対照物質に対しての抽出方法は被験物質と同様に行う。抽出したホルマザンの定量は OD570nm 測定または High Performance Liquid Chromatography（HPLC）/ Ultra Performance Liquid Chromatography（UPLC）で行う。
被 験 物 質 が MTT 還 元 物 質 の 場 合 、 あ る い は ホ ル マ ザ ン と 同 じ よ う な 波 長（570nm±30nm）に吸収を持つ着色物質の場合、細胞生存率の補正を行う必要がある。その手順については TG 本文の説明（33‐40 項）および Annex 3 のフローチャートを参照
する 5)。
4-4. 試験成立の承認基準
以下の条件をすべて満たした場合、試験の成立を承認する。
1) 陰性対照の平均 OD が 1.0 < OD ≤ 2.5 であること。
2) 液体被験物質では、陽性対照物質の平均細胞生存率が 5 分間の曝露時間で 50％以下、16 分および 120 分間の曝露時間で 50％を超えること、固体被験物質では、陽性対照物質は平均細胞生存率が 30 分間の曝露時間で 40％を超えること、120 分
間の曝露時間で 20％ < ％生存率 ≤ 70％であること。いずれも背景データの範囲内にあること。
3）被験物質および陰性・陽性対照のそれぞれにおいて 2 つの RhCE 組織の細胞生存率の差が 20％未満であること。3 つの RhCE 組織用いた場合は細胞生存率の標準偏差が 18％以下であること。
なお、複数の被験物質を同時に試験した場合は、この条件を満たさない被験物質のみ不成立とする。
4-5. 刺激性の判定
液体被験物質では、すべての曝露時間（5 分、16 分および 120 分）で平均細胞生存率が50％を超えた場合、被験物質は UN GHS 区分に該当しない、すべての曝露時間で平均細胞生存率が 50％以下の場合は UN GHS 区分 1 と判断される。それら以外の場合は UN
GHS 区分 2 と判断される。たとえば、5 分間（原体）と 16 分間（20％希釈液）の曝露条件
では平均細胞生存率が 50％を超えていても、120 分間（20％希釈液）で 50％以下である
物質は UN GHS 区分 2 となる。

711

固体被験物質では、平均細胞生存率が 30 分間（原体）の曝露時間で 40％を超え、かつ120 分間（原体）の曝露時間で 60％を超えた場合、被験物質は UN GHS 区分に該当しないと判断され、平均細胞生存率が 30 分間（原体）の曝露時間で 40％以下、かつ 120 分間（原体）の曝露時間で 60％以下の場合は UN GHS 区分 1 と判断される。それら以外の場
合は UN GHS 区分 2 と判断される。
なお、平均細胞生存率がカットオフ値近辺の場合（±5％）は 2 回目の試験を検討する。
1 回目と 2 回目の試験で結果が一致しない場合は 3 回目の試験を検討する。
5. バリデーション研究5-1. バリデーション研究の透明性
本バリデーション研究は、本試験法に用いられる RhCE である SkinEthic™を販売している企業の親会社である L’Oréal 社がスポンサーとなり行われた。そのため、バリデーション実行委員会の独立性が PRP でも慎重に議論された 7)。バリデーション実行委員
会の中の第三者機関としては、コンサルティング企業である Adriaens Consulting BVBA
のみが関与し、コード化やデータ解析を実施しているが、被験物質の選択は L’Oréal 社
が実施している。さらに、統計学者の報告書もバリデーションの採用基準も計画にはな
く、バリデーション報告書にも不明確な記載が多かった。これらの事実から、PRP はOECD GD 34 で推奨されている試験マネジメントや被験物質の選択に関する独立性については不明確であるとしている。
このような状況を PRP が慎重に検討して明らかにし、以下の 15 項目について確認した 7)。その結果、PRP はバリデーション報告書の記載に不備は多いが、バリデーションの結果は受け入れると結論している。
1)

バリデーション研究と試験法の目的（Study objective and test method purpose)2)

既存の試験法との比較による必要性と有用性（Need and benefits in comparison toexisting test methods)3)

性能標準（OECD GD216）10)に記載された要件に叶う試験法との比較（Comparison of
the test method with the essential test method components as described in the PerformanceStandards）

4)

生物学的および作用機序的な関連性 （Biological and mechanistic relevance)

5)

試験法プロトコル（Test method protocol）

6)

バリデーション研究の管理と実行の適正（Appropriateness of the validation study
management and conduct)

7)

バリデーション研究の目的に沿った適切な被験物質の選択（Adequacy of chemicals
selection for the validation study objective)8)

関連性評価に使われた参照データの質（Quality of the reference data used for theevaluation of relevance)

9)

経験のない施設へのトレーニングと技術移転（Training of naive laboratories andtransferability）

12810) データの信頼性保証（Use of quality assurance system（s）during data generation）
11) 施設内および施設間再現性（Within- and between-laboratory reproducibility)12) 予測性（Predictive capacity)13) 適用範囲と限界（Applicability domain and limitations）
14) データと文書の完成度（Completeness of data and documentation)15) 他の関連事項（Other relevant aspects)
なお、予測性に関しては OECD の専門家会議で定められた採用基準をもとに評価された。
本委員会では、バリデーション研究を遂行する場合には、開発者でなく、第三者機関が主導し、透明性や利益相反に考慮するべきであると考える。ただし、バリデーション
報告書の記載に不備は多いが、いずれの施設の記録も十分であり、データも適切である
ことを PRP が確認しており、バリデーション結果に大きな問題はないと本委員会も考えた。
5-2. トレーニングと技術移転
SkinEthic™ HCE TTT 法のバリデーション研究にはリードラボの EPISKIN 社と本試験法の経験がない 2 施設（EUROSAFE 社、VITROSCREEN 社）を加えた合計 3 施設が参加した。すべての施設が開発者から 3~4 日間のトレーニングを受けた。
技術移転性として、コード化された 3 物質、陰性対照物質および陽性対照物質を用い、少なくとも 2 回の実験が実施された。液体被験物質の場合（TTL）において、1 回目で 1
物質の 5 分間処理の判定結果が 2 施設で食い違ったが、細胞毒性 50％の基準前後のバ
ラツキによるものと判断された。しかし、すべての参加施設で 2 回目は正確に予測できていた。固体被験物質の場合（TTS）では、3 施設の判定結果はすべて同じであった。
以上の結果から、本試験法の技術移転性は高いと判断されている。なお、本試験法を実施する試験施設の技術習得を確かめるための習熟度確認物質の一覧は Appendix 1（液体は Appendix 1-1、固体は Appendix 1-2）にある。
5-3. 施設内および施設間再現性 3,4)
TTL に関しては、コード化された 20 物質（新規 14 物質を含む）を用いたバリデーシ
ョン研究が実施され、合計 60 回（20 物質×3 回）の試験が行われた。施設内再現性は 85、90 および 95％であり、20 物質の施設間再現性は 90％であった。TTS に関しても、コード化された 20 物質（新規 11 物質を含む）を用いたバリデーション研究において、施設内再現性は 100％であり、施設間再現性も 100％であった。これらの結果から、SkinEthic™
HCE TTT 法の再現性は高いと考えられた。

913

5-4 試験法の予測性 1,2,6)
表 1 に SkinEthic™ HCE TTT 法の正確性を示す。SkinEthic™ HCE TTT 法の正確性は、加重算出した。たとえば、1 種類の被験物質について 3 回の試験結果がある場合で、2回は Cat.1、1 回は Cat.2 と判定された場合、Cat.1 および Cat.2 の予測はそれぞれ 0.66
（2/3）および 0.33（1/3）とした。
TTLでは、UN GHS区分1物質、UN GHS区分2物質および区分に該当しない物質が、そ
れぞれ、85.4％、79.8％および79.2％の割合で、また、TTSでは、それぞれ、74.7％、55.3％
および71.7％の割合で正しく判定された。TTLとTTSを合わせたTTTの正確度について
は、UN GHS区分1物質、UN GHS区分2物質および区分に該当しない物質が、それぞれ79.2％、69.2％および74.9％の割合で正しく判定され、本試験法の予測性はOECD専門家
会議が承認した採用基準を満たしていた。詳細な結果はAnnex 3および4に示している。
この基準はSkinEthic™ HCE TTTの開発者の提案を受け、peer review report, 3.12 予測性(Evaluation criterion 12)11)での検討をもとにOECD専門家会議が承認したものであるが、本委員会で再度慎重に議論した。特に、UN GHS区分2の固体物質を区分に該当しない物
質と判定する偽陰性率が28.9％と高く、OECDが定めた30％以下という採用基準に近い値であることから、安全性評価上懸念を持つとの見解で一致した。
SkinEthic™ HCE TTT 法は、OECD TG492B に準拠して実施した場合、UN GHS 区分
1、UN GHS 区分 2 への分類および区分に該当しない場合の判定を可能とする試験法である。ただし、固体物質の UN GHS 区分 2 の分類は慎重に評価されるべきであるとの意見でまとまった。

14

10

表1

SkinEthic™ HCE TTT 法の正確性*

UN GHS Cat.

SkinEthic™ HCE TTL (％)(# of Chemical)

Cat. 1

Cat.2

No Cat.

Cat 1 (21)

85.4

14.6

0.0

Cat. 2 (25)

20.2

79.8

0.0

No Cat. (24)

0.0

20.8

79.2

UN GHS Cat.

SkinEthic™ HCE TTS (％)(# of Chemical)

Cat. 1

Cat.2

No Cat.

Cat 1 (29)

74.7

25.3

0.0

Cat. 2 (19)

15.8

55.3

28.9

No Cat. (33)

3.0

25.3

71.7

UN GHS Cat.

SkinEthic™ HCE TTT (％)(# of Chemical)

Cat. 1

Cat.2

No Cat.

Cat 1 (50)

79.2

20.8

0

Cat. 2 (44)

18.3

69.2

12.5

No Cat. (57)

1.8

23.4

74.9

*: 値は加重算出した。網掛け箇所が正しく予測できた割合。
# of Chemical：物質数、Cat.1：区分 1、Cat.2：区分 2、No Cat.：区分に該当しない
表 2 に SkinEthic™ HCE TTT 法とその他の RhCE 法の UN GHS 区分に該当しない物質に対する正確性を示す。EpiOcular™ Eye Irritation Test (EpiOcular), SkinEthic™ Human
Corneal Epithelium Eye Irritation Test (SkinEthic),

LabCyte CORNEA-MODEL24 Eye

Irritation Test (LabCyte)の正確度、感度および特異度は 80％～88％、95％～100％および63％～73％であった。SkinEthic™ HCE TTT 法の US GHS 区分に該当しない物質に対する正確度、感度および特異度は、
それぞれ、
87％、
94％および 75％であった。
SkinEthic™
HCE TTT 法の正確度、感度、特異度はその他の RhCE 法と同等であった。

11

15

表2

UN GHS 区分に該当しない物質に対しての正確性の比較
正確度 ％

感度 ％

特異度％

(# of Chemical)(# of Chemical)(# of Chemical)*

EpiOcular

80 (112)96 (57)63 (55)

SkinEthic*

84 (200)

95 (97)72 (103)

LabCyte**88 (139)100 (76)

73 (63)

SkinEthic™ HCE

87 (151)

94 (94)75 (57)

TTT***
Round to the first decimal place when necessary, EpiOcular=EpiOcular™ Eye Irritation Test,
SkinEthic=SkinEthic™ Human Corneal Epithelium Eye Irritation Test, LabCyte=LabCyte
CORNEA-MODEL24 Eye Irritation Test, #=number*TG 4922)**Peer Review report, December 201811)***TEST GUIDELINE 492B より算出
．試験法の適用範囲および留意点
6．
SkinEthic™ HCE TTT は、UN GHS 区分 1、UN GHS 区分 2 への分類および区分に該当しない場合の判定に用いることができるが、UN GHS 区分 2A と 2B を区別することはできない。UN GHS 区分 2A と 2B の区別が必要となる場合は、他の方法やアプローチで対応する必要がある 12)。
本試験法は、プロトコルで定められた濃度に溶解又は均一懸濁した液体の評価が可能である単一物質、混合物、固体、液体、半固体およびワックスに適用することができる。液体は水性と非水性、固体は可溶性と不溶性のいずれも適用可能であるが、水溶性の低い固体（< 0.014 mg/mL）についてはバリデーション研究において予測性が低かったことから注意が必要である（例： 区分 1 の 9 物質のうち 5 つが区分 2 に分類された）。
水溶性の低い液体では、20％（w/v）希釈水溶液で 16 分または 120 分間曝露する際に安定した混合液の状態を保持できない可能性があり、予測性への影響が考えられる。しかしながら、バリデーション試験では水への溶解性が 0.1 g/L 未満の液体 16 物質において予測性に大きな問題はみられなかった。ガスとエアロゾルについてはバリデーション研
究で評価されておらず本試験法には適用できない。多成分の物質やその混合物について
は、技術的には適応可能であるものの、その適応性については限られた情報しか得られていない 1)。特定の製剤において本試験が適応できないことを示す根拠がある場合、その製剤は本試験法に適用できない。混合物や試験が困難な化学物質（例：不安定な化学物質）、あるいは適応範囲に明らかに含まれていない物質の試験を検討する場合は、その試験結果が科学的に意味のある結果をもたらすかどうか事前に考慮すべきである。
ホルマザン色素と同じ範囲の光吸収スペクトルを持つ物質や MTT を直接還元する
物質は組織生存率の測定に干渉する可能性があり、補正のために適合させた対照物質の使用が必要となる。必要となる対照物質は、被験物質によって生じる干渉の種類および各ホルマザン色素の定量化に使用する手順によって異なる（TG492B の 33-40 項参照）6)。

16

12

7. 結論
SkinEthic™ HCE TTT は、技術移転性、施設内再現性および施設間再現性の高い試験法である。予測性に関しては、OECD 専門家会議が定めた採用基準は満たしていた。
バリデーション報告書の記載に不備は多いが、いずれの施設の記録も十分であり、データも適切であることを PRP が確認しており、バリデーション結果に大きな問題はな
いと本委員会は考える。
よって、SkinEthic™ HCE TTT 法は、OECD TG492B に準拠して実施した場合、UN GHS
区分 1、UN GHS 区分 2 への分類および区分に該当しない場合の判定を可能とする試験法である。ただし、固体物質の UN GHS 区分 2 の分類は慎重に評価されるべきであると本資料編纂委員会は考える。

1317

8. 参考文献
1） OECD Test Guidelines for Chemicals, Available at:https://www.oecd.org/env/ehs/testing/oecdguidelinesforthetestingofchemicals.htm (2022 年
6 月現在)
2） OECD (2018) Test Guideline 492. Reconstructed human Cornea-like Epithelium (RhCE)
test method for identifying chemicals not requiring classification and labelling for eye
irritation or serious eye damage.
3） Alépée N, Leblanc V, Grandidier MH, Teluob S, Viricel A, Adriaens E, Michaut V.(2021)
SkinEthic HCE Time-to-Toxicity on solids: A test method for distinguishing chemicals
inducing serious eye damage, eye irritation and not requiring classification and labelling.,
Toxicol In Vitro.,75:105203. doi: 10.1016/j.tiv.2021.105203.
4） Alépée N, Leblanc V, Grandidier MH, Teluob S, Tagliati V, Adriaens E, Michaut V.(2020)
Development of the SkinEthic HCE Time-to-Toxicity test method for identifying liquid
chemicals not requiring classification and labelling and liquids inducing serious eye damage andeye irritation.,Toxicol In Vitro., 269:104960. doi: 10.1016/j.tiv.2020.104960.
5） OECD (2022) Test Guideline 492B. Reconstructed human Cornea-like Epithelium (RhCE)
test method for Eye Hazard6） Alépée N, Grandidier MH, Teluob S, Amaral F, Caviola E, De Servi B, Martin S, Meloni M,
Nardelli L, Pasdelou C, Tagliati V, Viricel A, Adriaens E, Michaut V. (2022) Validation of
the SkinEthic HCE Time-to-Toxicity test method for eye hazard classification of chemicals
according to UN GHS.Toxicol In Vitro. 80:105319. doi: .1016/j.tiv.2022.105319.
7） Barroso, J., Eskes, C., Germolec, D., Kim, B-H., Kojima, H. (2021). Peer Review Panel
Report on the scientific validity of the SkinEthic™ Human Corneal Epithelium (HCE) TimeTo-Toxicity Test (TTT) on liquids (TTL) and on solids (TTS). January, 12, 2021.
8） SkinEthic™ HCE Time-to-Toxicity on Liquids SOP, Version 1. (June 16, 2020). In Vitro
Eye Irritation Time-To-Toxicity Test Method On Liquids (TTL): SkinEthic™ Human
Corneal Epithelium Model (HCE). Available at: [www.episkin.com].
9） SkinEthic™ HCE Time-to-Toxicity on Solids SOP, Version 1. (June 16, 2020). In Vitro Eye
Irritation Time-To-Toxicity Test Method On Solids (TTS): SkinEthic™ Human Corneal
Epithelium Model (HCE). Available at: [www.episkin.com].
10） OECD (2017) Performance Standards for the Assessment of proposed Similar or
Modified In Vitro Reconstructed Human Cornea-like Epithelium (RHCE) Test Methods for
Eye Hazard11） OECD (2018) PEER REVIEW REPORT OF THE VALIDATION OF THE LABCYTECORNEA-MODEL24 EYE IRRITATION TEST FOR INCLUSION IN TEST GUIDELINE 492,
SERIES ON TESTING AND ASSESMENT No. 282
12） OECD (2019） Guidance Document on an Integrated Approach on Testing and Assessment(IATA) for Serious Eye Damage and Eye Irritation Series on Testing & Assessment No. 263(Second Edition)

18

14

Appendix 1
SkinEthic™ HCE TTL (Table 2A) および SkinEthic™ HCE TTS (Table 2B)のの習熟度確認物質リスト
Appendix 1-1

SkinEthic™ HCE TTL リスト
CASRN

物質名

有機官能グループ1

物性

生存率1

生存率2

生存率3

分
5分

分
16分

120 分

％) 2,3(％％) 2,3(％％) 2,3(％

VRM
予測性

In Vivo カテゴリー1
N.N--Diethylethanolamine

100-37-8

Alcohol. Aliphatic Amine. tertiaryL

2.9±1.7

1.0±1.3

0.6±0.5

Cat 1

Acetic acid (10％)

64-19-7

Carboxylic acidL

4.2±0.425.9±12.7

2.8±0.3

Cat 1

KetoneL

21.1±5.490.8±13.787.6±12.3

Cat 2

In Vivo カテゴリー2
2-Butanone

78-93-3Acetone67-64-1

KetoneL

6.4±2.697.1±3.999.1±3.5

Cat 2

112-02-7

Ammonium salt. Alkylammonium
salt

L

58.0±10.762.1±9.2

2.4±0.8

Cat 2

Hexadecyltrimethylamm
oniu m chloride (2％)

In Vivo 非カテゴリー
1.3-Diisopropylbenzene

99-62-7

Cyclic. Phenyl. AromaticL

100.5±8.5

94.5±6.3

96.5±9.3

No CatDodecane112-40-3

Methyl. MethyleneL

98.3±6.9

103.1±5.6

98.9±5.1

No Cat

生存率1

生存率2

30 分

120 分

(％
％) 2,3(％％) 2,3

Appendix 1-2

SkinEthic™ HCE TTSの
のリスト

物質名

CASRN

有機官能グループ

1

物性

VRM
予測性

In Vivo カテゴリー1
1-Naphthalene acetic acid
Na salt
1.2-Benzisothiazol-3(2H)one

61-31-4

Benzyl. Carboxylic aromatic.
NaphthaleneS

1.6±0.7

1.6±0.4

Cat 1

2634-33-5

Benzothiazole/ BenzoisothiazoleS

4.0±1.0

4.0±0.4

Cat 1

In Vivo カテゴリー2
4-Carboxybenzaldehyde

619-66-9S80.1±12.3

5.0±1.4

Cat 2

2-Hydroxy-1.4naphthoquinone

Aldehyde. Aryl. Carboxylicacid

83-72-7

Enol. Naphthoquinone

S

82.1±8.9

4.5±1.5

Cat 2

Ammonium nitrate

6484-52-2

Amine. Nitrate. Ammonium

S

61.0±4.8

1.8±0.4

Cat 2

In Vivo 非カテゴリー
Magnesium carbonate
Anthracene

56378-72-4

Magnesium. Carbonate

S

98.3±7.1

93.4±11.7

No Cat120-12-7

Anthracene. Carboxylic
aromatic

S

97.7±6.6

98.1±4.7

No Cat

略号: CASRN = Chemical Abstracts Service Registry Number
VRM = Validated Reference Method, SkinEthic™ HCE TTTQSAR Toolbox 分析に従い分類された (version 3.2; https://www.oecd.org/
chemicalsafety/risk-assessment/oecd-qsar-toolbox.htm)。
2 SkinEthic™ HCE TTT バリデーション研究を基に得られた。

1 有機機能グループ OECD

3 液体の場合, the SkinEthic™ HCE TTL は 3 曝露回数を用いる。

原体 5 分、20％水溶液 16 分および 120分である. 固体の場合, the SkinEthic™ HCE TTS は 2 曝露回数を用いる。 原体 30 分および 120 分であ
る。

15

19

添付資料 3

OECD/OCDE

492B

Adopted:30 June 2022

OECD GUIDELINE FOR THE TESTING OF CHEMICALS

Reconstructed Human Cornea-like Epithelium (RHCE) Test Method for Eye Hazard
Identification

INTRODUCTION
1.
Serious eye damage refers to the production of tissue damage in the eye, or serious physicaldecay of vision, which is not fully reversible, occurring after exposure of the eye to a test chemical, as
defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals(UN GHS) (1). Also according to UN GHS, eye irritation refers to the production of changes in the eye,which are fully reversible, occurring after exposure of the eye to a test chemical. Test chemicals inducing
serious eye damage are classified as UN GHS Category 1, while those inducing eye irritation are classified
as UN GHS Category 2. Test chemicals not classified for eye irritation or serious eye damage are definedas those that do not meet the requirements for classification as UN GHS Category 1 or 2 (2A or 2B) i.e.,they are referred to as UN GHS No Category (No Cat).
2.
The assessment of serious eye damage/eye irritation has initially involved the use of laboratory
animals (OECD Test Guideline (TG) 405; adopted in 1981 and revised in 1987, 2002, 2012 and 2017) (2).
In vitro or ex vivo test methods have been adopted as OECD Test Guidelines (TGs) 437 (4), 438 (5), 460(6), 491 (7), 492 (8), 494 (9) and 496 (10) to identify either chemicals for serious eye damage potential
and/or to identify chemicals not requiring classification for eye hazard potential. The choice of the mostappropriate test method to be used should be considered in the context of the OECD Guidance Document
on an Integrated Approaches on Testing and Assessment (IATA) for Serious Eye Damage and Eyeirritation (3).
3.
This TG describes an in vitro procedure allowing the identification on its own of chemicals(substances and mixtures) not requiring classification (No Cat), requiring classification for eye irritation(Cat 2) and requiring classification for serious eye damage (Cat 1) according to the UN GHS ocular hazard
categories (1).
4.
This TG describes a validated test method, namely the SkinEthic™ Human Corneal Epithelium(HCE) Time-to-Toxicity (TTT) test using a commercially available reconstructed Human Cornea-like
Epithelium (HCE). The RhCE is designed to closely mimic the histological, morphological, biochemical andphysiological properties of the human corneal epithelium. A validation study for assessing the three UN
GHS ocular hazard categories has been conducted (11)(12)(13) on this method, referred to in the following
text as the Validated Reference Method (VRM). From the validation study and its independent peer review(14) it was concluded that the SkinEthic™ HCE TTT is able to correctly identify chemicals (both substancesand mixtures) by discriminating the three UN GHS categories for serious eye damage/eye irritation, i.e.
UN GHS Cat. 1, Cat. 2 and No Cat chemicals (1), and the test method was recommended as a full
replacement to the in vivo Draize acute eye irritation test for classification of chemicals. It is recognized© OECD, (2022)
You are free to use this material subject to the terms and conditions available at http://www.oecd.org/termsandconditions/.

21

OECD/OCDE

492B

that the use of this test guideline is subject to national and international regulatory considerations andconditions. The Guidance Document No. 263 on IATA should be consulted for further testing with otheradequate in vitro tests in a weight-of-evidence approach, if deemed necessary (3). Annexes II-V provide a
synopsis of the important elements of the test method, as well as flowcharts providing guidance for specificsituations.
5.
The purpose of this TG is to describe the procedure used to evaluate the eye hazard potential ofa test chemical based on its ability to induce cytotoxicity in a RhCE tissue construct, as measured by
reduction of a vital dye (MTT [3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue
tetrazolium bromide; CAS RN 298-93-1]) hereafter designated as the tetrazolium dye (TD) (15) (seeparagraph 23). The viability of the RhCE tissue following exposure to a test chemical is determined incomparison to tissues treated with the negative control substance (%viability) for the two or three exposure
times, and is then used to predict the eye hazard potential of the test chemical.
6.

Definitions are provided in Annex I.

INITIAL CONSIDERATIONS AND LIMITATIONS
7.
The SkinEthic™ HCE tissue construct is a three-dimensional model produced using cells from thespecies of interest which mimics the in vivo corneal epithelium(17). The test method directly measures
cytotoxicity resulting from penetration of the chemical through the corneal epithelium and production of celland tissue damage following chemical exposure, which is used to predict the eye hazard identification ofa test chemical. Cell damage can occur by several modes of action (see paragraph 16), but cytotoxicity
plays an important, if not the primary, mechanistic role in determining the overall serious eye damage/eye
irritation response of a chemical, manifested in vivo mainly by corneal opacity, iritis, conjunctival redness
and/or conjunctival chemosis, regardless of the physicochemical processes underlying tissue damage.
8.
A total of 151 chemicals covering a variety of chemical types, chemical classes, chemical
structures, as well as molecular weights, LogP and other physical-chemical properties have been tested
in the validation study underlying this TG. The validation database covered 134 different organic functionalgroups (11)(12)(13) and all key in vivo drivers of classification (26)(27). The majority of these chemicals
represented mono-constituent substances (a total of 151 substances, of which 16 were tested in dilution),but several multi-constituent substances (including surfactants or polymers) were also included in the
study. In terms of physical state and according to UN GHS Categories (1), the 151 tested chemicals were
distributed as follows: 70 liquids, comprising 21 Cat. 1, 25 Cat. 2 (incl. 16 Cat. 2A and 9 Cat. 2B) and 24
No Cat, and 81 solids, comprising 29 Cat. 1, 19 Cat.2, and 33 No Cat. (11)(12)(13).
9.
The SkinEthic™ HCE TTT is not intended to discriminate between UN GHS Category 2A (eyeirritation, effects fully reversible within 21 days) and UN GHS Category 2B (mild eye irritation, effects fully
reversible within 7 days). This differentiation needs to be addressed by other methods or approaches, if
discrimination is deemed necessary (3).
10.
This TG is applicable to substances and mixtures, to solids, liquids, semi-solids and waxes. The
liquids may be aqueous or non-aqueous, solids may be soluble or insoluble in water. Caution should be
used when testing solid chemicals with poor water solubility (< 0.014 mg/mL) as they were frequently
underpredicted by SkinEthicTM HCE TTS in the validation study (e.g., 5 out of 9 Cat.1 chemicals were
underpredicted to be Cat.2). Gases and aerosols have not been assessed in a validation study. While it isconceivable that these can be tested using RhCE technology, the current TG does not allow testing ofgases and aerosols. Limited information is currently available on the applicability of the test method tomulti-constituent substances/mixtures (11). The test method is nevertheless technically applicable to thetesting of multi-constituent substances and mixtures. In cases where evidence demonstrates the non© OECD, (2022)

22

OECD/OCDE

492B

applicability of the TG to specific formulations, the TG should not be used for those formulations. Whenconsidering testing of difficult-to-test chemicals (e.g. unstable substances), or test chemicals or mixtures
not clearly within the applicability domain described in this Guideline, upfront consideration should be given
to whether the results of such testing will yield results that are meaningful scientifically.
11.
Test chemicals absorbing light in the same range as formazan dye (FD, naturally or after
treatment) and test chemicals able to directly reduce the vital dye MTT (to FD) may interfere with the tissue
viability measurements and need the use of adapted controls for corrections. The type of adapted controlsthat may be required will vary depending on the type of interference produced by the test chemical and theprocedure used to quantify each FD (see paragraphs 33-38).
12.
The validation study carried out in three laboratories demonstrated that SkinEthic™ HCE TTT istransferable to laboratories considered to be naïve in the conduct of the assays and also is reproducible
within- and between laboratories (13)(14). The within-laboratory reproducibility (WLR) for SkinEthicTM was
85-95% for TTL (20 chemicals) and 100% for TTS (20 chemicals). The between-laboratory reproducibility(BLR) was 90% for TTL and 100% for TTS.
13.
The SkinEthic™ HCE TTT test can be used to identify chemicals that do not require classificationfor eye irritation or serious eye damage according to the UN GHS classification system (1). Considering
the data obtained (13)(14) (Table 1), the SkinEthic™ HCE TTT test has a balanced accuracy of 74.4%(based on 151 chemicals) with correct predictions of 79% for Cat 1 (based on 50 chemicals), 69% for Cat
2 (based on 44 chemicals) and 75% for No Cat (based on 57 chemicals), when compared to reference invivo rabbit eye test data (OECD TG 405) (2)(16) and classified according to the UN GHS classification
system (1). The performance for SkinEthicTM HCE TTL and TTS is different (Annex V).

Table 1. The weighted1 performance of the SkinEthic™ HCE TTT using the 3x3 matrix showing
correct, under- and over- predictions per UN GHS category
UN GHS categories
Cat 1 (N=50)
Cat 2 (N=44)
No Cat (N=57)

SkinEthic HCE TTT - Predicted categories(n/N%)Cat 1 (n)Cat 2 (n)No Cat (n)79.2% (39.60)
20.8% (10.40)
0% (0.00)
18.3% (8.06)
69.2% (30.46)
12.5% (5.48)
1.8% (1.00)
23.3% (13.33)
74.9% (42.67)

14.
The term "test chemical" is used in this TG to refer to what is being tested and is not related to theapplicability of the RhCE test method to the testing of substances and/or mixtures.

PRINCIPLE OF THE TEST15.
The test chemical is applied topically to a minimum of two three-dimensional RhCE tissue
constructs and tissue viability is measured following exposure and a post-soak incubation period. The
SkinEthic™ HCE tissues are reconstructed from human immortalized corneal epithelial cells, which havebeen cultured for several days to form a stratified, highly differentiated squamous epithelium
morphologically similar to that found in the human cornea. The SkinEthic™ HCE tissue construct consists
1

In the weighted calculation, each chemical has an equal weight of 1 in the performance regardless of the numberof test runs (i.e., in 3 overall test runs, if chemical A is tested Cat.1 two times and Cat.2 one time, a fractional weightof 0.66 (2/3) and 0.33 (1/3) is assigned to the number of predictions (n) for Cat.1 and Cat.2, respectively (11).

© OECD, (2022)

23

OECD/OCDE

492B

of at least 4 viable layers of cells including columnar basal cells, transitional wing cells and superficial
squamous cells similar to that of the normal human corneal epithelium (17)(18).
16.
Chemical-induced serious eye damage/eye irritation, manifested in vivo mainly by corneal opacity,
iritis, conjunctival redness and/or conjunctival chemosis, is the result of a cascade of events beginning withpenetration of the chemical through the cornea and/or conjunctiva and production of damage to the cells.
Cell damage can occur by several modes of action, including: cell membrane lysis (e.g., by surfactants,
organic solvents); coagulation of macromolecules (particularly proteins) (e.g., by surfactants, organic
solvents, alkalis and acids); saponification of lipids (e.g., by alkalis); and alkylation or other covalentinteractions with macromolecules (e.g., by bleaches, peroxides and alkylators) (19)(20)(21). However, it
has been shown that cytotoxicity plays an important, if not the primary, mechanistic role in determining theoverall serious eye damage/eye irritation response of a chemical regardless of the physicochemical
processes underlying tissue damage (22). Moreover, the serious eye damage/eye irritation potential of achemical is principally determined by the extent of initial injury (20), which correlates with the extent of cell
death (22) and with the extent of the subsequent responses and eventual outcomes (23)(24). Thus, slightirritants generally only affect the superficial conjunctival and corneal epithelium, the mild and moderate
irritants damage principally the epithelium and superficial stroma and the severe irritants damage theepithelium, deep stroma and at times the endothelium (22)(25). The measurement of viability of the RhCE
tissue construct after topical exposure to a test chemical to identify chemicals not requiring classification
for serious eye damage/eye irritancy (UN GHS No Category) is based on the assumption that all chemicals
inducing serious eye damage or eye irritation will induce cytotoxicity in the corneal epithelium and/or
conjunctiva.
17.
RhCE tissue viability is classically measured by enzymatic conversion of MTT for the SkinEthic™
HCE TTT by the viable cells of the tissue into a formazan dye (FD), which is quantitatively measured after
extraction from tissues (15).
18.
The SkinEthic™ HCE TTT Test is based on two protocols, one for liquids (SkinEthic™ HCE TTL)
and one for solids (SkinEthic™ HCE TTS). The SkinEthic™ HCE TTT uses three exposure times for TTL
and two exposure times for TTS (see paragraphs 29-30). The SkinEthicTM HCE TTL and TTS protocols
make use of different prediction models. For SkinEthic™ HCE TTL, a chemical that results in a mean
viability below or equal to 50% within all time treatments will be classified as a Cat 1, and one that results
in a mean viability strictly above 50% as a No Cat. Any other combination of mean viability values will leadto a Cat 2 classification (see paragraph 43). For SkinEthic™ HCE TTS, a chemical that results in a mean
viability below or equal to 40% after 30 minutes. exposure and below or equal to 60% after 120 minutes.
exposure will be classified as a Cat 1. A mean viability strictly above these cut-offs within the two timetreatments will classify the chemical as a No Cat. Any other combination of mean viability values willclassify the chemical as a Cat 2 (see paragraph 43).

DEMONSTRATION OF PROFICIENCY
19.
Prior to routine use of the test method for regulatory purposes, laboratories should demonstratetechnical proficiency by correctly predicting the fourteen proficiency chemicals listed in Table 2. Thesechemicals were selected from the chemicals used in the validation studies of the SkinEthic™ HCE TTT(13)(14). The selection includes, to the extent possible, chemicals that: (i) cover different physical states;(ii) cover the range of in vivo serious eye damage/eye irritation responses based on high quality results
obtained in the reference in vivo rabbit eye test (OECD TG 405) (2)(16) and the UN GHS classification
system (i.e., Categories 1, 2, or No Category) (1); (iii) cover the key in vivo drivers of classification (26)(27);(iv) are representative of the chemical classes used in the validation study (13); (v) cover a good and wide
representation of organic functional groups (11)(12)(13); (vi) have chemical structures that are well-defined(11)(12)(13); (vii) produced reproducible results in the RhCE test method during the validation study; (viii)© OECD, (2022)

24

OECD/OCDE

492B

were correctly predicted by the RhCE test method during the validation study; (ix) cover the range of invitro responses based on high quality of the test method data; (x) are commercially available; and (xi) are
not associated with prohibitive acquisition and/or disposal costs. In situations where a listed chemical isunavailable or cannot be used for other justified reasons, another chemical fulfilling the criteria describedabove, e.g. from the chemicals used 2in the validation of the SkinEthic™ HCE TTT, could be used. Suchdeviations should however be justified.

Table 2: List of proficiency chemicals for SkinEthic™ HCE TTL (Table 2A) and for SkinEthic™ HCETTS (Table 2B)
Table 2A. List of Proficiency Chemicals for SkinEthic™ HCE TTL
ChemicalName

CASRN

1

OrganicFunctionalGroup

Physicalstate

Viability 1
5 min.
2,3(%)

Viability 2
16 min.

Viability
3

2,3

(%)

120 min.

VRM
Prediction

2,3

N.N--Diethylethanolamine
Acetic acid (10%)

100-378
64-19-7

2-Butanone
AcetoneHexadecyltrimethylammonium
chloride (2%)

78-93-3
67-64-1
112-027

1.3-Diisopropylbenzene
Dodecane

99-62-7
112-403

In VivoCategory1
Alcohol. Aliphatic Amine. tertiary
Carboxylic acid
In VivoCategory2
KetoneKetoneAmmonium salt.
Alkylammonium salt
In VivoNoCategory
Cyclic. Phenyl. Aromatic
Methyl. Methylene(%)
L

2.9±1.7

1.0±1.3

0.6±0.5Cat 1

L

4.2±0.425.9±12.7

2.8±0.3

Cat 1

L
L
L

21.1±5.4
6.4±2.658.0±10.790.8±13.7
97.1±3.962.1±9.2

87.6±12.3
99.1±3.5
2.4±0.8

Cat 2
Cat 2
Cat 2

L
L

100.5±8.5
98.3±6.9

94.5±6.3
103.1±5.6

96.5±9.3
98.9±5.1

No CatNo CatTable 2B. List of Proficiency Chemicals for SkinEthic™ HCE TTS
ChemicalName

CASRN

Viability 1
30 min.

Viability 2
120 min.

VRM

(%)

(%)

Prediction

S

1.6±0.7

1.6±0.4Cat 1

S

4.0±1.0

4.0±0.4

Cat 1

S80.1±12.3

5.0±1.4

Cat 2

S
S

82.1±8.961.0±4.8

4.5±1.5
1.8±0.4

In VivoNoCategory
Magnesium. Carbonate
S

98.3±7.1

93.4±11.7

No Cat

97.7±6.6

98.1±4.7

No Cat1

OrganicFunctionalGroup

Physicalstate

2,3

1-Naphthalene acetic acid Na
salt
1.2-Benzisothiazol-3(2H)-one

61-31-4

4-Carboxybenzaldehyde

619-669
83-72-7648452-2

2-Hydroxy-1.4-naphthoquinone
Ammonium nitrate
Magnesium carbonate
Anthracene263433-55637872-4
120-127

In VivoCategory1
Benzyl. Carboxylic aromatic.
Naphthalene
Benzothiazole/ Benzoisothiazole
In VivoCategory2
Aldehyde. Aryl. Carboxylic acid
Enol. Naphthoquinone
Amine. Nitrate. Ammonium

Anthracene. Carboxylic
aromatic

S

2,3

-

Cat 2
Cat 2

1. 3-Diisopropylbenzene
2. 2-Benzisothiazol-3(2H)-one
Abbreviations: CASRN = Chemical Abstracts Service Registry Number; VRM = Validated Reference Method, SkinEthic™ HCE TTT.

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1
Organic functional group assigned according to an OECD QSAR Toolbox analysis (version 3.2;https://www.oecd.org/chemicalsafety/riskassessment/oecd-qsar-toolbox.htm).
2
Based on results obtained with SkinEthic™ HCE TTT in the validation study (13)(14).
3
For liquids, the SkinEthic™ HCE TTL uses three exposure-times : Test chemicals are applied neat for 5 minutes (viability 1), and diluted at
20% (w/v) for 16 minutes (viability 2) and 120 minutes (viability 3). For solids, the SkinEthic™ HCE TTS uses two exposure-times: Test chemicals
are applied neat for 30 minutes (viability 1) and 120 minutes (viability 2) (see paragraph 29).

20.
As part of the proficiency testing, it is recommended that users verify the barrier properties of thetissues after receipt as specified by the RhCE tissue construct producer (see paragraphs 24, 25 and 27).
This is particularly important if tissues are shipped over long distance / time periods. Once a test method
has been successfully established and proficiency in its use has been acquired and demonstrated, suchverification will not be necessary on a routine basis. However, when using a test method routinely, it isrecommended to continue to assess the barrier properties at regular intervals.

PROCEDURE
21.
The test method currently covered by this TG is the scientifically validated SkinEthic™ HCE TTT
Test (14). The Standard Operating Procedures (SOP) for both SkinEthic™ HCE TTL and TTS are available
and should be employed when implementing and using the test method in a laboratory (28)(29). The
following paragraphs and Annex II describe the main components and procedures of the RhCE test
method.

RhCE TEST METHOD Components
GENERAL CONDITIONS
22.
Relevant human-derived cells should be used to reconstruct the cornea-like epithelium threedimensional tissue, which should be composed of progressively stratified but not cornified cells. The RhCE
tissue construct is prepared in inserts with a porous synthetic membrane through which nutrients can passto the cells. Multiple layers of viable, non-keratinized epithelial cells should be present in the reconstructed
cornea-like epithelium. The RhCE tissue construct should have the epithelial surface in direct contact with
air so as to allow for direct topical exposure of test chemicals in a fashion similar to how the corneal
epithelium would be exposed in vivo. The RhCE tissue construct should form a functional barrier with
sufficient robustness to resist rapid penetration of cytotoxic benchmark substances, e.g., sodium dodecyl
sulphate (SDS). For example, the barrier function should be demonstrated and may be assessed bydetermination of the concentration at which a benchmark substance reduces the viability of the tissues by
50% (IC50) following a fixed exposure time e.g., 30 minutes-treatment with 50 μL SDS (see paragraph 27).
The containment properties of the RhCE tissue construct should prevent the passage of test chemicalaround the edge of the viable tissue, which could lead to poor modelling of corneal exposure. The humanderived cells used to establish the RhCE tissue construct should be free of contamination by bacteria,
viruses, mycoplasma, and fungi. The sterility of the tissue construct should be checked by the supplier forabsence of contamination by fungi and bacteria.

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FUNCTIONAL CONDITIONS
Viability
23.
The assay used for quantifying tissue viability is the tetrazolium dye (MTT) assay (15). Viable cellsof the RhCE tissue construct reduce the vital dye MTT into a blue MTT formazan precipitate, which is thenextracted from the tissue using isopropanol (or a similar solvent). The extracted formazan dye may bequantified using either a standard absorbance (Optical Density (OD)) measurement or an HPLC/UPLCspectrophotometry procedure (28)(29). The OD of the blank solution alone (which is the extraction solvent
for MTT assay) should be sufficiently small, i.e., OD < 0.1. Users of the RhCE tissue construct should
ensure that each batch of the RhCE tissue construct used meets defined criteria for the negative control.
Acceptability ranges for the negative control OD values for the SkinEthic™ HCE TTT are given in Table 3.
For HPLC/UPLC-spectrophotometry, the negative control OD ranges provided in Table 3 should be used
as acceptance criterion for the negative control. It should be documented in the test report that the tissues
treated with the negative control substance are stable in culture (provide similar tissue viability
measurements) for the duration of the test exposure period. A similar procedure should be followed by thetissue producer as part of the quality control tissue batch release, but in this case different acceptancecriteria than those specified in Table 3 may apply. An acceptability range (upper and lower limit) for thenegative control OD values (in the QC test method conditions) should be established by the RhCE tissue
construct developer/supplier.

Table 3. Acceptability ranges for negative control OD values (for the test method users)
Test Method
SkinEthic™
HCE
TTT(HCE/S) - VRM(for both TTL and TTS
protocols)

Lower acceptance limit> 1.0

Upper acceptance limit≤ 2.5

Barrier function
24.
The RhCE tissue construct should be sufficiently thick and robust to resist the rapid penetration ofcytotoxic benchmark substances, as estimated e.g. by IC50 (SDS) (Table 4). The barrier function of eachbatch of the RhCE tissue construct used should be demonstrated by the RhCE tissue constructdeveloper/vendor upon supply of the tissues to the end user (see paragraph 27).

Morphology
25.
Histological examination of the RhCE tissue construct should demonstrate human cornea-likeepithelium structure (including at least four layers of viable epithelial cells). For the VRM, appropriate
morphology has been established by the supplier and therefore does not need to be demonstrated again
by a test method user for each tissue batch used.

Reproducibility
26.
The results of the positive and negative controls of the test method should demonstrate
reproducibility over time.

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Quality control (QC)
27.
The RhCE tissue construct should only be used if the developer/supplier demonstrates that eachbatch of the RhCE tissue construct used meets defined production release criteria, the most relevant
among which are those for viability (paragraph 23) and barrier function. An acceptability range (upper andlower limits) for the barrier functions as measured by the IC 50 should be established by the RhCE tissue
construct supplier. The IC50 acceptability range used as QC batch release criterion by the supplier of the
RhCE tissue constructs used in the test method is given in Table 4. Data demonstrating compliance withall production release criteria should be provided by the RhCE tissue construct supplier to the test method
users so that they are able to include this information in the test report. Only results produced with tissues
fulfilling all of these production release criteria can be accepted for reliable prediction of chemicals for eye
hazard identification in accordance with UN GHS.

Table 4. QC batch release criteria of the SkinEthic™ HCE tissue construct
Test Method
SkinEthic™ HCE TTT(HCE/S) - VRM(30 minutes treatment with 50 µL SDS)

Lower
acceptance limit
IC50 = 1.0 mg/mL

Upper acceptance
limit
IC50 =3.2 mg/mL

Application of the Test Chemical and Control Substances
28.
At least two tissue replicates for each time-treatment point should be used for each test chemicaland each control substance in each run. Two different treatment protocols are used, one for liquid test
chemicals and one for solid test chemicals (28)(29). For the SkinEthic™ HCE TTT, the tissue constructsurface should be moistened with calcium and magnesium-free Dulbecco's Phosphate Buffered Saline(Ca2+/Mg2+-free DPBS) for liquids’ protocol before application of test chemicals or with water for solids’
protocol, to mimic the wet conditions of the human eye. The treatment of the tissues is initiated with
exposure to the test chemical(s) and control substances. For any treatment protocols, a sufficient amountof test chemical or control substance should be applied to uniformly cover the epithelial surface.
29.
Test chemicals that can be pipetted at 37°C or lower (using a positive displacement pipette, if
needed) are treated as liquids in the test method, otherwise they should be treated as solids (seeparagraph 30). Specific procedures for liquids that do not form a solution or a stable suspension are
included in the SOP (28). In the test method, liquid test chemicals are evenly spread over the tissue surface(i.e. 160±2 μL/cm2 application) (Annex II). SkinEthic™ HCE tissues are topically exposed for three different
treatment times: Test chemicals are applied neat for 5 minutes, and diluted at 20% (w/v) in distilled waterfor 16 minutes and 120 minutes at the pre-defined conditions of the method (28). At the end of the exposure
period, the liquid test chemical and the control substances should be carefully removed from the tissue
surface by extensive rinsing with Ca2+/Mg2+-free DPBS at room temperature. This rinsing step should be
followed by a 10 minute post-exposure immersion in fresh medium at room temperature (to remove any
test chemical absorbed into the tissue) prior to performing the MTT assay (Annex II)(28).
30.
Test chemicals that cannot be pipetted (for example, highly viscous liquids) are treated as solids
in the test method. The amount of test chemical applied should be sufficient to cover the entire surface ofthe tissue, i.e. a minimum of 160±2 mg/cm2 application should be used (Annex II). Whenever possible,
solids should be tested as a fine powder. SkinEthic™ HCE tissues are topically exposed to test chemicals
for 30 and 120 minutes at standard culture conditions (see Annex II)(29). At the end of the exposure period,
the solid test chemical and the control substances should be carefully removed from the tissue surface byextensive rinsing with Ca2+/Mg2+-free DPBS at room temperature. This rinsing step should be followed by© OECD, (2022)

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a 30 minute post-exposure immersion in fresh medium at room temperature (to remove any test chemical
absorbed into the tissue), prior to performing the TD assay (Annex II)(29).
31.
Concurrent negative and positive controls should be included in each run to demonstrate that theviability (determined with the negative control) and the sensitivity (determined with the positive control) of
the tissues are within acceptance ranges defined based on historical data. The concurrent negative control
also provides the baseline (100% tissue viability) to calculate the relative percent viability of the tissues
treated with the test chemical (%Viabilitytest). The recommended positive control substance to be used with
the SkinEthic™ HCE TTT is neat methyl acetate (CAS RN. 79-20-9, commercially available) for liquids’
protocol, and lactic acid at 1% (W/V) in water (CAS RN. 50-21-5, commercially available) for solids’
protocol. The recommended negative control substance to be used is Ca2+/Mg2+-free DPBS for both liquids
and solids protocols. These were the control substances used in the validation studies of the SkinEthic™
HCE TTT and are those for which most historical data exist. The use of suitable alternative positive or
negative control substances should be scientifically and adequately justified. Negative and positive controls
should be tested with the same protocol(s) as the one(s) used for the test chemicals included in the run(i.e. for liquids and/or solids). This application should be followed by the treatment exposure, rinsing, anda post-exposure immersion, as described for controls run concurrently to liquid test chemicals (seeparagraph 29) or for controls run concurrently to solid test chemicals (see paragraph 30), prior toperforming the MTT assay (see paragraph 32) (26)(27). One single set of negative and positive controls is
sufficient for all test chemicals of the same physical state (liquids or solids) included in the same run.

Tissue Viability Measurements
32.
The MTT assay is a standardised quantitative method (15) that should be used to measure tissue
viability under this TG. It is compatible with use in a three-dimensional tissue construct. The MTT assay is
performed immediately following the post-soak procedure. The RhCE tissue construct sample is placed in
0.3 mL of MTT solution at 1 mg/mL for 180±15 minutes at standard culture conditions. The vital dye MTT
is reduced into a blue MTT formazan precipitate by the viable cells of the RhCE tissue construct. The
precipitated blue MTT formazan product is then extracted from the tissue using an appropriate volume ofisopropanol (or a similar solvent) (28)(29). Tissues tested with liquid test chemicals should be extracted
from both the top and the bottom of the tissues, while tissues tested with solid test chemicals or coloured
liquids should be extracted from the bottom of the tissue only (to minimise any potential contamination of
the isopropanol extraction solution with any test chemical that may have remained on the tissue). Tissues
tested with liquid test chemicals that are not readily washed off may also be extracted from the bottom of
the tissue only. The concurrently tested negative and positive control substances should be treatedsimilarly to the tested chemical. The extracted MTT formazan may be quantified either by a standard
absorbance (OD) measurement at 570 nm using a filter band pass of maximum ± 30 nm or by using an
HPLC/UPLC-spectrophotometry procedure (see paragraph 40).
33.
Optical properties of the test chemical or its chemical action on MTT may interfere with themeasurement of formazan dye (FD) leading to a false estimate of tissue viability, i.e., under-prediction ofeye irritation. Test chemicals may interfere with the measurement of FD by direct reduction of the MTT intocoloured FD (blue MTT formazan) and/or by colour interference if the test chemical absorbs, naturally or
due to treatment procedures, in the same OD range as FD (i.e., MTT formazan: around 570 nm). Potentialof chemicals to directly reduce TD and/or interfere with colour (only necessary for coloured test chemicals)
should be checked before testing. In case of FD interference, additional controls should be used to correct
for potential interference from such test chemicals (see paragraphs 34-36 and Annexes III). This isespecially important when a specific test chemical is not completely removed from the RhCE tissue
construct by rinsing or when it penetrates the cornea-like epithelium and is therefore present in the RhCE
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as FD (naturally or after treatment), which are not compatible with the standard absorbance (OD)measurement of FD due to strong interference, i.e., strong absorption at 570±30 nm (with MTT formazan),an HPLC/UPLC-spectrophotometry procedure to measure FD may be employed (see paragraphs 39 and40) (28). A detailed description of how to detect and correct for direct MTT reduction and interferences bycolouring agents is available in the test method SOPs (28)(29). Illustrative flowcharts providing guidanceon how to identify and handle direct MTT-reducers and/or colour interfering chemicals are also provided in
Annexes III.
34.
To identify potential interference by test chemicals absorbing light in the same range as FD(naturally or after treatment) and decide on the need for additional controls, the test chemical is added towater and incubated for an appropriate time at room temperature (Annex II) (28)(29). If a coloured solution
is obtained when mixing the test chemical with water (see Annex III), the test chemical is presumed tointerfere with the standard absorbance (OD) measurement of FD; in such a case, further colorant controls
should be performed or, alternatively, an HPLC/UPLC-spectrophotometry procedure should be used,which do not require these controls (see paragraphs 39 and 40 and Annexes III and IV). When performing
the standard absorbance (OD) measurement, each interfering test chemical should be applied on at least
two viable tissue replicates, which undergo the entire testing procedure but are incubated with medium
instead of MTT solution during the MTT incubation step, to generate a non-specific colour in living tissues(NSCliving) control (28)(29). The NSCliving control needs to be performed concurrently to the testing of thecoloured test chemical and, in case of multiple testing, an independent NSC living control needs to be
conducted with each test performed (in each run) due to the inherent biological variability of living tissues.
True tissue viability is calculated as: the percent tissue viability obtained with living tissues exposed to theinterfering test chemical and incubated with the MTT (%Viabilitytest) minus the percent non-specific colour
obtained with living tissues exposed to the interfering test chemical and incubated with medium without
MTT, run concurrently to the test being corrected (%NSC living), i.e., True tissue viability = [%Viabilitytest] [%NSCliving].
35.
To identify direct MTT reducers, each test chemical should be added to freshly prepared MTT
solution. An appropriate amount of test chemical is added to a MTT solution and the mixture is incubatedfor approximately 3 hours at standard culture conditions (see Annex III) (28)(29). If the MTT mixture
containing the test chemical (or suspension for insoluble test chemicals) turns blue/purple, the test
chemical is presumed to directly reduce the MTT and a further functional check on non-viable RhCE tissue
constructs should be performed, independently of using the standard absorbance (OD) measurement oran HPLC/UPLC- spectrophotometry procedure. This additional functional check employs killed tissues thatpossess only residual metabolic activity but absorb and retain the test chemical in a similar way as viabletissues. Killed tissues of SkinEthic™ HCE TTT are prepared by prolonged incubation (e.g., at least 24±1
hours) in water followed by storage at low temperature ("water-killed"). Each MTT reducing test chemical
is applied on at least two killed tissue replicates, which undergo the entire testing procedure, to generatea non-specific MTT reduction (NSMTT) control (28)(29). A single NSMTT control is sufficient per test
chemical regardless of the number of independent tests/runs performed. True tissue viability is calculated
as: the percent tissue viability obtained with living tissues exposed to the MTT reducer (%Viabilitytest) minus
the percent non-specific TD reduction obtained with the killed tissues exposed to the same reducer,
calculated relative to the negative control run concurrently to the test being corrected (%NSMTT), i.e. true
tissue viability = [%Viabilitytest] - [%NSMTT].
36.
Test chemicals that are identified as producing both colour interference (see paragraph 34) anddirect MTT reduction (see paragraph 35) will also require a third set of controls when performing thestandard absorbance (OD) measurement, apart from the NSMTT and NSC living controls described in theprevious paragraphs. This is usually the case with darkly coloured test chemicals absorbing light in therange of 570±30 nm for MTT formazan (e.g., blue, purple, black) because their intrinsic colour impedes
the assessment of their capacity to directly reduce MTT as described in paragraph 35. This forces the useof NSMTT controls, by default, together with the NSC living controls. Test chemicals for which both NSMTT© OECD, (2022)

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and NSCliving controls are performed may be absorbed and retained by both living and killed tissues.
Therefore, in this case, the NSMTT control may not only correct for potential direct MTT reduction by thetest chemical, but also for colour interference arising from the absorption and retention of the test chemical
by killed tissues. This could lead to double correction for colour interference since the NSCliving control
already corrects for colour interference arising from the absorption and retention of the test chemical byliving tissues. To avoid a possible double correction for colour interference, a third control for non-specificcolour in killed tissues (NSCkilled) needs to be performed (Annex III) (28)(29). In this additional control, thetest chemical is applied on at least two killed tissue replicates, which undergo the entire testing procedure
but are incubated with medium instead of MTT solution during the MTT incubation step. A single NSC killedcontrol is sufficient per test chemical regardless of the number of independent tests/runs performed, butshould be performed concurrently to the NSMTT control and with the same tissue batch. True tissue
viability is calculated as: the percent tissue viability obtained with living tissues exposed to the test chemical(%Viabilitytest) minus %NSMTT minus %NSCliving plus the percent non-specific colour obtained with killed
tissues exposed to the interfering test chemical and incubated with medium without MTT, calculated
relative to the negative control ran concurrently to the test being corrected (%NSC killed), i.e., True tissue
viability = [%Viabilitytest] - [%NSMTT] - [%NSCliving] + [%NSCkilled].
37.
It is important to note that non-specific MTT reduction and non-specific colour interferences may
increase the OD (when performing standard absorbance measurements) of the sample above the linearity
range of the spectrophotometer and that non-specific MTT reduction can also increase the FD peak area
(when performing HPLC/UPLC- spectrophotometry measurements) of the sample above the linearity
range of the spectrophotometer. On this basis, when using RhCEs, it is important for each laboratory to
determine the OD/peak area linearity range of their spectrophotometer with MTT formazan (CAS RN.
57360-69-7) which is commercially available.
38.
The standard absorbance (OD) measurement using a spectrophotometer is appropriate to assess
direct MTT-reducers and colour interfering test chemicals, when the observed interference with themeasurement of FD is not strong (i.e., the ODs of the samples obtained with the test chemical without any
correction for direct MTT reduction and/or colour interference are within the linear range of thespectrophotometer). Nevertheless, results for test chemicals producing %NSMTT and/or %NSCliving ≥ 50%
for liquids protocol or 60% for solids protocol of the negative control should be taken with caution. Standard
absorbance (OD) can however not be measured when the interference with the measurement of FD isstrong (i.e., leading to uncorrected ODs of the test samples falling outside of the linear range of thespectrophotometer). Coloured test chemicals or test chemicals that become coloured in contact with waterthat interfere strongly with the standard absorbance (OD) measurement of FD may still be assessed using
HPLC/UPLC-spectrophotometry (Annex III). This is because the HPLC/UPLC system allows for the
separation of the FD from the chemical before its quantification. For this reason, NSC living or NSCkilledcontrols are never required when using HPLC/UPLC-spectrophotometry, independently of the chemical
being tested. NSMTT control should nevertheless be used if the test chemical is suspected to directlyreduce MTT (following the procedure described in paragraph 35). NSMTT control should also be used withtest chemicals having a colour (intrinsic or appearing when in water) that impedes the assessment of theircapacity to directly reduce MTT as described in paragraph 35. When using HPLC/UPLCspectrophotometry to measure FD, the percent tissue viability is calculated as percent FD peak area
obtained with living tissues exposed to the test chemical relative to the FD peak obtained with theconcurrent negative control. For test chemicals able to directly reduce MTT, true tissue viability is
calculated as: %Viabilitytest minus %NSMTT as described in the last sentence of paragraph 35.
39.
Finally, it should be noted that direct MTT-reducers that are also colour interfering, which areretained in the tissues after treatment and reduce MTT so strongly that they lead to ODs (using standard
OD measurement) or peak areas (using UPLC/HPLC- spectrophotometry) of the tested samples that falloutside of the linearity range of the spectrophotometer cannot be assessed with the RhCE test method,
although these are expected to occur in only very rare situations.

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40.
HPLC/UPLC-spectrophotometry may be used with all types of test chemicals (coloured, noncoloured, MTT-reducers and non-MTT reducers) for measurement of FD. Due to the diversity of
HPLC/UPLC-spectrophotometry systems, it is not feasible for each user to establish the exact same
system conditions. As such, qualification of the HPLC/UPLC-spectrophotometry system should bedemonstrated before its use to quantify MTT from samples by meeting the acceptance criteria for a set ofstandard qualification parameters based on those described in the U.S. Food and Drug Administration
guidance for industry on bioanalytical method validation (30)(31). These key parameters and theiracceptance criteria are shown in Annex IV. Once the acceptance criteria defined in Annex IV have beenmet, the HPLC/UPLC-spectrophotometry system is considered qualified and ready to measure FD underthe experimental conditions described in this TG.

Acceptance Criteria
41.
For each run using RhCE tissue batches that met the quality control (see paragraph 27), tissues
treated with the negative control substance should exhibit OD reflecting the quality of the tissues that
followed shipment, receipt steps and all protocol processes and should not be outside the historicallyestablished boundaries described in Table 3(see paragraph23). For the SkinEthic™ HCE TTL protocol,
tissues treated with the positive control substance, i.e., methyl acetate, should show a mean tissue viability≤ 50% at the time exposure of 5 minutes and > 50% at both times 16 and 120 minutes, relative to thenegative control. For the SkinEthic™ HCE TTS protocol, tissues treated with the positive control
substance, i.e., lactic acid at 1% (W/V) in water, should show a mean tissue viability > 40% relative to thenegative control at the time exposure of 30 minutes and 20%< %Viability ≤ 70% after 120 minutes
treatment, thus reflecting the ability of the tissues to respond to an irritant test chemical under the conditionsof the test method (28)(29).
42.
The variability between tissue replicates of test chemicals and control substances should fallwithin the accepted limits (i.e., the difference of viability between two tissue replicates should be less than
20% or the standard deviation (SD) between three tissue replicates should not exceed 18%). If either thenegative control or positive control included in a run is outside of the accepted ranges, the run is considered"non-qualified" and should be repeated. If the variability between tissue replicates of a test chemical isoutside of the accepted range, the test must be considered "non-qualified" and the test chemical should
be re-tested.

Interpretation of Results and Prediction Model
43.
The OD values/peak areas obtained with the replicate samples for each test chemical should be
used to calculate the mean percent tissue viability (mean between tissue replicates) normalised to thenegative control, which is set at 100%. The percentage tissue viability cut-off values used to identify andclassify test chemicals for ocular hazard categories according to UN GHS classification; i.e. No Category(not classified), Category 2 (eye irritation) and Category 1 (serious eye damage) are given in Table 5.
Results should thus be interpreted as follows:

The test chemical is identified as a chemical not requiring classification and labelling according to
UN GHS (No Category) if the mean percent tissue viability after exposure and a post-soak
incubation is more than (>) the established percentage tissue viability cut-off value within all-timetreatments.



The test chemical is identified as a chemical inducing serious eye damage according to UN GHS(Category 1) if the mean percent tissue viability after exposure and a post-soak incubation is lessthan or equal to (≤) the established percentage tissue viability cut-off value within all-timetreatments.

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The test chemical is identified as a chemical inducing eye irritation according to UN GHS (Category2) if the combination of mean percent tissue viability within all-time treatment fall outside of thecriteria established to identify the test chemical as a No Cat or a Cat 1, as shown in Table 5.

Table 5. Prediction Model according to UN GHS classification
SkinEthic HCE TTL(for the liquids protocol)
SkinEthic HCE TTS(for thesolids protocol)

No Category
Mean tissue viability> 50% within all-timetreatments
Mean tissue viability> 40% after 30
minutesand> 60% after 120
minutesCategory 2
Any other combinationof values1
Any other combinationof values1

Category 1
Mean tissue viability≤ 50% within all-timetreatments
Mean tissue viability≤ 40% after 30
minutesand≤ 60% after 120
minutes

1Any combination of values other than those defined for No Cat or Cat 1.

44.
A single test composed of at least two tissue replicates should be sufficient for a test chemical
when the result is unequivocal. However, in cases of borderline results, such as non-concordant replicate
measurements and/or mean percent tissue viability equal to established percentage tissue viability cut-off
value ±5% within the time treatments, a second test should be considered, as well as a third one in caseof discordant results between the first two tests.

DATA AND REPORTING
Data
45.
Data from individual replicate tissues in a run (e.g., OD values/FD peak areas and calculated
percent tissue viability data for the test chemical and controls, and the final RhCE test method prediction)
should be reported in tabular form for each test chemical, including data from repeat tests, as appropriate.
In addition, mean percent tissue viability and difference of viability between two tissue replicates (if n=2
replicate tissues) or SD (if n≥3 replicate tissues) for each individual test chemical and control should bereported. Any observed interferences of a test chemical with the measurement of FD through direct MTT
reduction and/or coloured interference should be reported for each tested chemical.

Test report
46.

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Test chemical
Mono-constituent substanceChemical identification, such as IUPAC or CAS name(s), CAS registry number(s), SMILES or InChI
code, structural formula, and/or other identifiers;Physical state, volatility, pH, LogP, molecular weight, chemical class, and additional relevant
physicochemical properties relevant to the conduct of the study, to the extent available;

Purity, chemical identity of impurities as appropriate and practically feasible, etc.;Treatment prior to testing, if applicable (e.g., warming, grinding);

Storage conditions and stability to the extent available.

Multi-constituent substance, UVCB and mixtureCharacterisation as far as possible by e.g., chemical identity (see above), purity, quantitative
occurrence and relevant physicochemical properties (see above) of the constituents, to the extent
possiblePhysical state and additional relevant physicochemical properties relevant to the conduct of thestudy, to the extent possible;

Purity, chemical identity of impurities as appropriate and practically feasible, etc.;Treatment prior to testing, if applicable (e.g., warming, grinding);

Storage conditions and stability to the extent possible.

Positive and Negative Control Substances

Chemical identification, such as IUPAC or CAS name(s), CAS registry number(s), SMILES or InChI
code, structural formula, and/or other identifiers;Physical state, volatility, molecular weight, chemical class, and additional relevant physicochemical
properties relevant to the conduct of the study, to the extent available;

Purity, chemical identity of impurities as appropriate and practically feasible, etc.;Treatment prior to testing, if applicable (e.g., warming, grinding);

Storage conditions and stability to the extent available;Justification for the use of a different negative control than those referenced in Annex II, if
applicable;Justification for the use of a different positive control than those referenced in Annex II, if applicable;

Reference to historical positive and negative control results demonstrating suitable run acceptancecriteria.

Information Concerning the Sponsor and the Test FacilityName and address of the sponsor, test facility and study director.

RhCE Tissue Construct and Protocol Used (providing rationale for the choices)
Test Method Conditions


RhCE tissue construct used, including batch number;© OECD, (2022)

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

Wavelength and band pass (if applicable) used for quantifying FD, and linearity range of measuring
device (e.g., spectrophotometer);Description of the method used to quantify FDDescription of the HPLC/UPLC-spectrophotometry system used, if applicable; LLOQ, ULOQ, andresults of calibration curves and QCs using the same type of fitting and weighting as in thevalidation of the HPLC/UPLC should be included in the reporting.

Complete supporting information for the specific RhCE tissue construct used including its
performance. This should include, but is not limited to:i.

Viability quality control (supplier)ii.

Viability under test method conditions (user);iii.

Barrier function quality control (supplier);

iv.

Morphology, if available (supplier);v.

Other quality controls (QC) of the RhCE tissue construct, if available (supplier);

Reference to historical data of the RhCE tissue construct. This should include, but is not limited to:
Acceptability of the QC data with reference to historical batch data;Statement that the testing facility has demonstrated proficiency in the use of the test method before
routine use by testing of the proficiency chemicals;

Run and Test Acceptance CriteriaPositive and negative control means and acceptance ranges based on historical data;

Acceptable variability between tissue replicates for positive and negative controls;

Acceptable variability between tissue replicates for the test chemical;

Test Procedure:

Details of the test procedure used (e.g., version of the SOP);



Doses of test chemical and control substances used;

Duration and temperature of exposure, post-exposure immersion and post-exposure incubation
periods (where applicable);Description of any modifications to the test procedure;Indication of controls used for direct MTT-reducers and/or colouring test chemicals, if applicable;

Number of tissue replicates used per test chemical and controls (positive control, negative control,
NSMTT, NSCliving and NSCkilled, if applicable);

Results:


Tabulation of data from individual test chemicals and control substances for each run (including
repeat experiments where applicable) and each replicate measurement, including OD value or FD
peak area, percent tissue viability, mean percent tissue viability, difference between tissue
replicates or SD, and final prediction;



If applicable, results of controls used for direct MTT-reducers and/or coloured test chemicals,
including OD value or FD peak area, %NSMTT, %NSCliving, %NSCkilled, difference between
tissue replicates or SD, final correct percent tissue viability, and final prediction;© OECD, (2022)

35

OECD/OCDEResults obtained with the test chemical(s) and control substances in relation to the define run andtest acceptance criteria;

Description of other effects observed, e.g., colouration of the tissues by a coloured test chemical;

Discussion of the results
Conclusion© OECD, (2022)

36

492B

OECD/OCDE

492B

LITERATURE(1) United Nations (UN) (2021), Globally Harmonized System of Classification and Labelling ofChemicals (GHS). Ninth revised edition, New York and Geneva, United Nations Publications.
Available at: [https://unece.org/transport/standards/transport/dangerous-goods/ghs-rev9-2021].
(2) OECD (2017). Guideline for Testing of Chemicals No. 405: Acute Eye Irritation/Corrosion.
Organisation for Economic Cooperation and Development, Paris. Available at:[http://www.oecd.org/env/testguidelines].
(3) OECD (2018). Guidance Document on an Integrated Approach on Testing and Assessment for
Serious Eye Damage and Eye irritation. Series on Testing and Assessment No.263. ENV
Publications, Organisation for Economic Cooperation and Development, Paris.
(4) OECD (2020). Guideline for Testing of Chemicals No. 437: Bovine Corneal Opacity and Permeability
Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not
Requiring Classification for Eye Irritation or Serious Eye Damage. Organisation for Economic
Cooperation
andDevelopment, Paris. Availableat:[http://www.oecd.org/env/testguidelines].
(5) OECD (2018). Guideline for Testing of Chemicals No. 438: Isolated Chicken Eye Test Method for
Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification.
Organisation for Economic Cooperation and Development, Paris. Available at:[http://www.oecd.org/env/testguidelines].
(6) OECD (2017). Guideline for Testing of Chemicals No. 460: Fluorescein Leakage Test Method for
Identifying Ocular Corrosives and Severe Irritants. Organisation for Economic Co-operation andDevelopment, Paris. Available at: [http://www.oecd.org/env/testguidelines].
(7) OECD (2020). Guideline for Testing of Chemicals No. 491: Short Time Exposure In Vitro Test Method
for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring
Classification for Eye Irritation or Serious Eye Damage. Organisation for Economic Co-operation andDevelopment, Paris. Available at: [http://www.oecd.org/env/testguidelines].
(8) OECD (2019). Guideline for Testing of Chemicals No. 492: Short Time Exposure In Vitro Test Method
for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring
Classification for Eye Irritation or Serious Eye Damage. Organisation for Economic Co-operation andDevelopment, Paris. Available at: [http://www.oecd.org/env/testguidelines].
(9) OECD (2021). Guideline for Testing of Chemicals No. 494: Vitrigel-Eye Irritancy Test Method for
Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye
Damage. Organisation for Economic Co-operation and Development, Paris. Available at:[http://www.oecd.org/env/testguidelines].
(10) OECD (2019). Guideline for Testing of Chemicals No. 496: In vitro Macromolecular Test Method for
Identifying Chemicals Inducing Serious Eye Damage and Chemicals Not Requiring Classification for
Eye Irritation or Serious Eye Damage. Organization for Economic Co-operation and Development,
Paris. Available at: [http://www.oecd.org/env/testguidelines].
(11) Alépée, N., Leblanc, V., Grandidier, MH., Teluob, S., Tagliati, V., Adriaens, E., Michaut, V. (2020)
Development of the SkinEthic HCE Time-to-Toxicity test method for identifying liquid chemicals not
requiring classification and labelling and liquids inducing serious eye damage and eye irritation.
Toxicol. in Vitro 69, 104960.
(12) Alépée, N., Leblanc, V., Grandidier, MH., Teluob, S., Viricel, A., Adriaens, E., Michaut, V. (2021).
SkinEthic HCE Time-to-Toxicity on Solids: A test method for distinguishing chemicals inducing serious
eye damage, eye irritation and not requiring classification and labelling. Toxicol. in Vitro 75, 105203.

© OECD, (2022)

37

OECD/OCDE

492B(13) Alépée, N., Grandidier, MH., Teluob, S., Amaral, F., Caviola, E., De Servi, B., Martin, S., Meloni, M.,
Nardelli, L., Pasdelou, C., Tagliati, V., Viricel, A., Adriaens, E., Michaut, V. (2022). Validation of the
SkinEthic HCE Time-to-Toxicity test method for eye hazard classification of chemicals according to
UN GHS. Toxicol. in Vitro, 80, 105319. https://doi.org/10.1016/j.tiv.2022.105319(14) OECD (2022). Peer Review Panel Report on the scientific validity of the SkinEthic TM Human Corneal
Epithelium (HCE) Time-to-Toxicity Test (TTT) on liquids (TTL) and on solids (TTS). Series on Testingand Assessment, No. 355. Organisation for Economic Cooperation and Development, Paris.
Available at: [https://www.oecd.org/chemicalsafety/testing/series-testing-assessment-publicationsnumber.htm](15) Mosmann, T. (1983). Rapid Colorimetric Assay for Cellular Growth and Survival: Application to
Proliferation and Cytotoxicity Assays. J. Immunol. Methods 65, 55-63.
(16) Draize, J.H., Woodard, G., Calvery, H.O. (1944). Methods for the Study of Irritation and Toxicity of
Substances Applied Topically to the Skin and Mucous Membranes. Journal of Pharmacol. and Exp.
Therapeutics 82, 377-390(17) Nguyen, D.H., Beuerman, R.W., De Wever, B., Rosdy, M. (2003). Three-dimensional construct of
the human corneal epithelium for in vitro toxicology. In: Salem, H., Katz, S.A. (Eds), Alternative
Toxicological Methods, CRC Press, pp. 147-159.
(18) Meloni, M., De Servi, B., Marasco, D., Del Prete, S. (2011). Molecular mechanism of ocular surface
damage: Application to an in vitro dry eye model on human corneal epithelium. Molecular Vision 17,
113-126.
(19) Scott, L., Eskes, C., Hoffmann, S., Adriaens, E., Alépée, 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., Spielman, H., Stokes, W.,
Trouba, K., Van den Berghe, C., 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.
(20) Hackett, R.B., McDonald, T.O. (1991). Eye Irritation. In Advances in Modern Toxicology:
Dermatoxicology Marzulli F.N.and Maibach H.I. (Eds.), 4th Edition, pp. 749–815. Washington, DC,
USA: Hemisphere Publishing Corporation.
(21) Fox, D.A., Boyes, W.K. (2008). Toxic Responses of the Ocular and Visual System. In Casarett andDoull’s Toxicology: The Basic Science of Poisons Klaassen C.D.(Ed.), 7th Edition, pp. 665–697.
Withby, ON, Canada: McGraw-Hill Ryerson.
(22) Jester, J.V., Li, H.F., Petroll, W.M., Parker, R.D., Cavanagh, H.D., Carr, G.J., Smith, B., Maurer, J.K.
(1998). Area and Depth of Surfactant Induced Corneal Injury Correlates with Cell Death. Invest.
Ophthalmol. Vis. Sci. 39, 922–936.
(23) Maurer, J.K., Parker, R.D., Jester, J.V. (2002). Extent of Corneal Injury as the Mechanistic Basis for
Ocular Irritation: Key Findings and Recommendations for the Development of Alternative Assays.
Reg. Tox. Pharmacol. 36, 106-117.
(24) Jester, J.V., Li, L., Molai, A., Maurer, J.K. (2001). Extent of Corneal Injury as a Mechanistic Basis for
Alternative Eye Irritation Tests. Toxicol. In Vitro 15, 115–130.
(25) Jester, J.V., Petroll, W.M., Bean, J., Parker, R.D., Carr, G.J., Cavanagh, H.D., Maurer, J.K. (1998).
Area and Depth of Surfactant-Induced Corneal Injury Predicts Extent of Subsequent Ocular
Responses. Invest. Ophthalmol. Vis. Sci. 39, 2610–2625.
(26) Adriaens, E., Barroso, J., Eskes, C., Hoffmann, S., McNamee, P., Alépée, N., Bessou-Touya, S., De
Smedt, A., De Wever, B., Pfannenbecker, U., Tailhardat, M., Zuang, V. (2014). Retrospective
Analysis of the Draize Test for Serious Eye Damage/Eye Irritation: Importance of Understanding thein vivo Endpoints Under UN GHS/EU CLP for the Development and Evaluation of In Vitro Test

© OECD, (2022)

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OECD/OCDE

492B

Methods. Arch. Toxicol. 88, 701-723.
(27) Barroso, J., Pfannenbecker, U., Adriaens, E., Alépée, N., Cluzel, M., De Smedt, A., Hibatallah, J.,
Klaric, M., Mewes, K.R., Millet, M., Templier, M., McNamee, P. (2017). Cosmetics Europe compilationof historical serious eye damage/eye irritation in vivo data analysed by drivers of classification to
support the selection of chemicals for development and evaluation of alternative methods/strategies:
the Draize eye test Reference Database (DRD). Arch. Toxicol. 91, 521-547.
(28) SkinEthic™ HCE Time-to-Toxicity on Liquids SOP, Version 1. (June 16, 2020). In Vitro Eye Irritation
Time-To-Toxicity Test Method On Liquids (TTL): SkinEthic™ Human Corneal Epithelium Model(HCE). Available at: [www.episkin.com/eye-irritation/Time-To-Toxicity].
(29) SkinEthic™ HCE Time-to-Toxicity on Solids SOP, Version 1. (June 16, 2020). In Vitro Eye Irritation
Time-To-Toxicity Test Method On Solids (TTS): SkinEthic™ Human Corneal Epithelium Model (HCE).
Available at: [www.episkin.com/eye-irritation/Time-To-Toxicity].
(30) Alépée, N., Barroso, J., De Smedt, A., De Wever, B., Hibatallah, J., Klaric, M., Mewes, K.R., Millet,
M., Pfannenbecker, U., Tailhardat, M., Templier, M., McNamee, P. (2015). Use of HPLC/UPLCSpectrophotometry for Detection of Formazan in In Vitro Reconstructed Human Tissue (RhT)-Based
Test Methods Employing the MTT-Reduction Assay to Expand their Applicability to Strongly Coloured
Test Chemicals. Toxicol. In Vitro 29, 741-761(31) US FDA (2018). Guidance for Industry: Bioanalytical Method Validation. U.S. Department of Healthand Human Services, Food and Drug Administration. May 2018. Available at:[http://www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf].
(32) OECD (2005). Series on Testing and Assessment No. 34: Guidance Document on the Validation
and International Acceptance of New or Updated Test Methods for Hazard Assessment. Organisation
for Economic
Cooperation
andDevelopment, Paris. Available athttps://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=env/jm/mono(2005)14&docl
anguage=en© OECD, (2022)

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OECD/OCDE

492B

ANNEX 1: DEFINITIONS
Balanced Accuracy: Average of the proportion of the correct predictions of each category. The closenessof agreement between test method results and accepted reference values taking into account imbalanceof the class distribution.
Benchmark chemical: A chemical used as a standard for comparison to a test chemical. A benchmark
chemical should have the following properties: (i) consistent and reliable source(s) for its identification andcharacterisation; (ii) structural, functional and/or chemical or product class similarity to the chemical(s)being tested; (iii) known physicochemical characteristics; (iv) supporting data on known effects; and (v)known potency in the range of the desired response.
Chemical: A substance or mixture.
Concordance: See "Accuracy".
Cornea: The transparent part of the front of the eyeball that covers the iris and pupil and admits light tothe interior.
CV: Coefficient of Variation.
Dev: Deviation.
Eye irritation: Production of changes in the eye, which are fully reversible, occurring after the exposure of
the eye to a substance or mixture. Interchangeable with “Reversible effects on the eye” and with “UN GHS
Category 2” (1) with the optional Categories 2A (effects fully reversible within 21 days) and 2B (effects fully
reversible within 7 days).
Formazan dye (FD): Chromogenic product of the reduction of MTT.
Hazard: Inherent property of an agent or situation having the potential to cause adverse effects when an
organism, system or (sub) population is exposed to that agent.
HCE: SkinEthic™ Human Corneal Epithelium
HPLC: High Performance Liquid Chromatography.
IC50: Concentration at which a benchmark chemical reduces the viability of the tissues by 50% following afixed exposure time (e.g., 30 minutes-treatment with SDS).
Irreversible effects on the eye: See “Serious eye damage”.
LLOQ: Lower Limit of Quantification.
LogP: Logarithm of the octanol-water partition coefficient
Mixture: A mixture or a solution composed of two or more substances in which they do not react (1).
Mono-constituent substance: A substance, defined by its quantitative composition, in which one main
constituent is present to at least 80% (w/w).
Multi-constituent substance: A substance, defined by its quantitative composition, in which more than
one main constituent is present in a concentration ≥ 10% (w/w) and < 80% (w/w). A multi-constituent
substance is the result of a manufacturing process. The difference between mixture and multi-constituent
substance is that a mixture is obtained by blending of two or more substances without chemical reaction.
A multi-constituent substance is the result of a chemical reaction.
MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide.
(CAS RN 298-93-1)© OECD, (2022)

40

OECD/OCDE

492B

Negative control: A sample containing all components of a test system and treated with a substanceknown not to induce a positive response in the test system. This sample is processed with test chemicaltreated samples and other control samples and is used to determine 100% tissue viability.
Not Classified: Chemicals that are not classified for Eye irritation (UN GHS Category 2, 2A, or 2B) or
Serious eye damage (UN GHS Category 1). Interchangeable with “UN GHS No Category”.
NSCkilled: Non-Specific Colour in killed tissues. NSCliving: Non-Specific Colour in living tissues. NSMTT:
Non-Specific MTT reduction.
OD: Optical Density.
Positive control: A sample containing all components of a test system and treated with a substance known
to induce a positive response in the test system. This sample is processed with test chemical-treatedsamples and other control samples. To ensure that variability in the positive control response across time
can be assessed, the magnitude of the positive response should not be excessive.
Relevance: Description of relationship of the test to the effect of interest and whether it is meaningful anduseful for a particular purpose. It is the extent to which the test correctly measures or predicts the biologicaleffect of interest. Relevance incorporates consideration of the accuracy (concordance) of a test method(32).
Reliability: Measures of the extent that a test method can be performed reproducibly within and between
laboratories over time, when performed using the same protocol. It is assessed by calculating intra- andinter-laboratory reproducibility and intra-laboratory repeatability (32).
Replacement test: A test which is designed to substitute for a test that is in routine use and accepted forhazard identification and/or risk assessment, and which has been determined to provide equivalent or
improved protection of human or animal health or the environment, as applicable, compared to theaccepted test, for all possible testing situations and chemicals (32).
Reproducibility: The agreement among results obtained from repeated testing of the same test chemicalusing the same test protocol (See "Reliability") (32).
Reversible effects on the eye: See “Eye irritation”.
RhCE: Reconstructed human Cornea-like Epithelium.
Run: A run consists of one or more test chemicals tested concurrently with a negative control and with apositive control.
SD: Standard Deviation.
Serious eye damage: refers to the production of tissue damage in the eye, or serious physical decay ofvision, which is not fully reversible, occurring after exposure of the eye to a substance or mixture.
Interchangeable with “Irreversible effects on the eye” and with “UN GHS Category 1” (1).
Standard Operating Procedures (SOP): Formal, written procedures that describe in detail how specificroutine, and test-specific, laboratory operations should be performed. They are required by GLP.
Substance: Chemical elements and their compounds in the natural state or obtained by any production
process, including any additive necessary to preserve the stability of the product and any impurities
deriving from the process used, but excluding any solvent which may be separated without affecting thestability of the substance or changing its composition (1).
Test: A single test chemical concurrently tested in a minimum of two tissue replicates as defined in thecorresponding SOP.

© OECD, (2022)

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OECD/OCDE

492B

Tissue viability: Parameter measuring total activity of a cell population in a reconstructed tissue as theirability to reduce the vital dye MTT, which, depending on the endpoint measured and the test design used,correlates with the total number and/or vitality of living cells.
Test chemical: The term "test chemical" is used to refer to what is being tested.
TTL: SkinEthic™ HCE Time-to-Toxicity for Liquids chemicals
TTS: SkinEthic™ HCE Time-to-Toxicity for Solids chemicals
TTT: SkinEthic™ HCE Time-to-Toxicity test method
ULOQ: Upper Limit of Quantification.
United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UNGHS): A system proposing the classification of chemicals (substances and mixtures) according tostandardised types and levels of physical, health and environmental hazards, and addressing
corresponding communication elements, such as pictograms, signal words, hazard statements,
precautionary statements and safety data sheets, so that to convey information on their adverse effects
with a view to protect people (including employers, workers, transporters, consumers and emergency
responders) and the environment (1).
UN GHS Category 1: See “Serious eye damage”.
UN GHS Category 2: See “Eye irritation”.
UN GHS No Category: Chemicals that do not meet the requirements for classification as UN GHS
Category 1 or 2 (2A or 2B). Interchangeable with “Not Classified”.
UPLC: Ultra-High Performance Liquid Chromatography.
UVCB: substances of unknown or variable composition, complex reaction products or biological materials.
Valid test method: A test method considered to have sufficient relevance and reliability for a specificpurpose and which is based on scientifically sound principles. A test method is never valid in an absolutesense, but only in relation to a defined purpose (31).
Validated test method: A test method for which validation studies have been completed to determine therelevance (including accuracy) and reliability for a specific purpose. It is important to note that a validated
test method may not have sufficient performance in terms of accuracy and reliability to be found acceptable
for the proposed purpose (31).
VRM: Validated Reference Method.
Weight-of-evidence: The process of considering the strengths and weaknesses of various pieces of
information in reaching and supporting a conclusion concerning the hazard potential of a test chemical.

© OECD, (2022)

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43

492B

and

10 mg + 90 μL H2O mixed for 30±2 min at RT→ If test chemical is coloured, living adapted
controls should be performed

10 μL + 90 μL H2O mixed for 30±2 min at
Room Temperature (RT2)→ If test chemical is coloured, living adapted
controls should be performed
50-80 μL chemical + 300 μL MTT 1 mg/mL
solution for 180± 15 min at 37±2ºC, 5±1% CO2,≥95% RH→if solution turns blue/purple, water-killed
adapted controls should be performed(50-80 μL of sterile deionized water in MTT
solution is used as negative control)
10 μL Ca2+/Mg2+-free DPBS
+ 80±2 μL (160 μL/cm2)

30-80 mg chemical + 300 μL MTT 1 mg/mL
solution for 180± 15 min at 37±2ºC, 5±1% CO2,≥95% RH→if solution turns blue/purple, water-killed
adapted controls should be performed(30-80 μL of sterile deionized water in MTT
solution is used as negative control)80±2 mg (160 mg/cm2) (grounded if needed tocover the whole tissue surface)

At least 2

At least 2

SkinEthic™ HCE TTT(VRM)
TTL protocol: Liquid and viscous test
TTS protocol: Solid test chemicals
chemicals(not pipettable)(pipettable)
0.5 cm2
0.5 cm2

Room Temperature (RT): 18-28°C© OECD, (2022)

2

For details of the protocol, the latest version of the Standard Operating Procedures (SOP) should be consulted and employed when implementing the test method
in a laboratory (28-29).

1Treatment doses
application

Pre- or post-check fordirecttetrazolium
reduction

colour interference

Model surface
Numberoftissuereplicates
Pre- or post-check forProtocol1

Test Method Component

ANNEX 2: MAIN TEST METHOD COMPONENTS OF THE SKINETHIC™ HCE TTT TEST METHOD VALIDATED FOR
EYE HAZARD IDENTIFICATION

OECD/OCDE44

80 ± 2μL Ca2+/Mg2+-free DPBS tested
concurrently
80 ± 2μL Lactic acid 1% in water (w/v) tested
concurrently (CAS RN 50-21-5)
300 μL 1 mg/mL
180 min (±15 min) at 37±2ºC, 5±1% CO2,≥95% RH
1.5 mL isopropanol(extraction from bottom of insert)

80 ± 2μL Ca2+/Mg2+-free DPBS tested
concurrently
80 ± 2μL Methyl acetate tested concurrently(CAS RN 79-20-9)300 μL 1 mg/mL
180 min (±15 min) at 37±2ºC, 5±1% CO2,≥95% RH
1.5 mL isopropanol (750µL under and 750µL
over)(extraction from top and bottom of insert)
At least 2 h with shaking (~120 rpm) at RT or
at least overnight without shaking at 4-10°C
570 nm (540 - 600 nm)
without reference filter
30±2 min treatment with SDS (50 μL)
1.0 mg/mL ≤ IC50 ≤ 3.2 mg/mL
1. Mean OD of the tissue replicates treatedwith the negative control should be > 1.0 and

salt

salt
and

and

© OECD, (2022)

Acceptance Criteria

Tissue Quality Control

Extraction time
temperature
OD reading

Tetrasolium
solution
Tetrazolium
incubation
temperature
Extraction solvent

Positive control

Negative control

570 nm (540 - 600 nm)
without reference filter
30±2 min treatment with SDS (50 μL)
1.0 mg/mL ≤ IC50 ≤ 3.2 mg/mL
1. Mean OD of the tissue replicates treatedwith the negative control should be > 1.0 and

At least 2 h with shaking (~120 rpm) at RT

25 mL of Ca2+/Mg2+-free DPBS (2 mL per push)
30 min (±2 min) at RT in 4 mL culture medium+ 80 μL distilled water
Use a nylon mesh
30 min (±2 min) and 120 min (±5 min) (neat) in
culture medium at 37±2ºC, 5±1% CO2, ≥95% RH

492B

5 min (±15 sec) (neat), 16 min (±1 min) (at 20% (w/v) in distilled water) in culture medium atRT
and 120 min (±2 min) (at 20 % (w/v) in distilledwater) in culture medium at 37±2ºC, 5±1%
CO2, ≥95% RH
25 mL of Ca2+/Mg2+-free DPBS (2 mL perpush)
10 min (±1 min) at RT in 4 mL culture medium

andRinsingatroom
temperature
Post-soak immersion

Exposuretime
temperature

For viscous, use a nylon mesh

OECD/OCDE

45© OECD, (2022)≤ 2.5
2. Mean viability of the PC, expressed as %of the NC, is ≤ 50% at the time exposure of
5 minutes and > 50% at both times 16 and120 minutes.
3. The difference of viability between two
tissue replicates should be less than 20%.

492B

≤ 2.5
2. Mean viability of the PC, expressed as % of
the NC, is > 40% at the time exposure of 30
minutes and 20%< PC ≤ 70% at the time
exposure of 120 minutes.
3. The difference of viability between two
tissue replicates should be less than 20%.

OECD/OCDE26  ENV/CBC/WRPR(2022)21

ANNEX 3: ILLUSTRATIVE FLOWCHART PROVIDING GUIDANCE ON HOW TOIDENTIFY AND HANDLE DIRECT MTT-REDUCERS AND/OR COLOUR
INTERFERING CHEMICALS, BASED ON THE SKINETHIC™ HCE TTT SOP© OECD, (2022)

46

ENV/CBC/WRPR(2022)21  27

ANNEX IV: KEY PARAMETERS AND ACCEPTANCE CRITERIA FORQUALIFICATION OF AN HPLC/UPLC-SPECTROPHOTOMETRY SYSTEM FOR
MEASUREMENT OF MTT FORMAZAN EXTRACTED FROM RhCE TISSUE
CONSTRUCTS
Parameter

Accuracy

Protocol Derived from FDA Guidance(43)(45)
Analysis of isopropanol, living blank(isopropanol extract from living RhCE tissue
constructs without any treatment), dead blank(isopropanol extract from killed RhCE tissue
constructs without any treatment), and of a dye(e.g., methylene blue)
Quality Controls (i.e., MTT formazan at 1.6
μg/mL, 16 μg/mL and 160 μg/mL ) in
isopropanol (n=5)
Quality Controls in isopropanol (n=5)

Matrix EffectQuality Controls in living blank (n=5)

CarryoverAnalysis of isopropanol after an ULOQ2 standard

Reproducibility(intra-day)

3 independent calibration curves (based on 6
consecutive 1/3 dilutions of MTT formazan in
isopropanol starting at ULOQ, i.e., 200 μg/mL);
Quality Controls in isopropanol (n=5)
Day 1: 1 calibration curve and Quality Controls in
isopropanol (n=3)
Day 2: 1 calibration curve and Quality Controlsinisopropanol (n=3)
Day 3: 1 calibration curve and Quality Controlsinisopropanol (n=3)
Quality Controls in living blank (n=3) analysed
the day of the preparation and after 24 hours ofstorage at room temperature
Quality Controls in living blank (n=3) analysed
the day ofthe preparation and after several days of storageat -20°C

Selectivity

Precision

Reproducibility(inter-day)

Short Term Stabilityof MTT Formazan in
RhCE Tissue Extract
Long Term Stabilityof MTT Formazan in
RhCE Tissue Extract,
if required

AcceptanceCriteria
Areainterference ≤ 20% of
AreaLLOQ1

CV ≤ 15% or ≤ 20% for
the LLOQ%Dev ≤ 15% or ≤ 20%
for LLOQ
85% ≤ %Matrix Effect≤ 115%
Areainterference ≤ 20% of
AreaLLOQ
Calibration Curves:%Dev ≤ 15% or ≤ 20%
for LLOQ
Quality Controls:%Dev≤ 15% and CV ≤ 15%%Dev ≤ 15%%Dev ≤ 15%

1LLOQ: Lower Limit of Quantification, defined to cover 1-2% tissue viability, i.e., 0.8 μg/mL.

2ULOQ: Upper Limit of Quantification, defined to be at least two times higher than the highest expected MTT formazanconcentration in isopropanol extracts from negative controls (~70 μg/mL in the VRM), i.e., 200 μg/mL.

F© OECD, (2022)47

28  ENV/CBC/WRPR(2022)21

ANNEX V: PERFORMANCE OF THE SKINETHIC™ HCE TTT FOR THE LIQUIDSAND SOLIDS USING THE 3X3 MATRIX SHOWING CORRECT, UNDER- AND
OVER- PREDICTIONS PER UN GHS CATEGORY

Table 1: Performance of the SkinEthic™ HCE TTL protocol
47.
UN GHS categories

SkinEthic™ HCE TTL - Predicted categories
Cat 1

Cat 2

No CatCat 1 (N=21)

85.4%

14.6%

0.0%

Cat 2 (N=25)

20.2%

79.8%

0.0%

No Cat (N=24)

0.0%

20.8%

79.2%

Table 2: Performance of the SkinEthic™ HCE TTS protocol
48.
UN GHS categories© OECD, (2022)

48

SkinEthic™ HCE TTS - Predicted categories
Cat 1

Cat 2

No CatCat 1 (N=29)

74.7%

25.3%

0.0%

Cat 2 (N=19)

15.8%

55.3%

28.9%

No Cat (N=33)

3.0%

25.3%

71.7%