新規試験法提案書

眼刺激性試験代替法 Vitrigel®-EIT法

令和 4 年 6 月

国立医薬品食品衛生研究所

新規試験法提案書
令和 4 年 6 月 30 日
No.

2022-01

眼刺激性試験代替法Vitrigel®-EIT法
法 に関する提案
令和 4 年 5 月 10 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： Vitrigel®- EIT 法は適用除外を理解した上で使用し、陰性の結果が得られた場合、UN
GHS 区分に該当しない眼刺激性物質を検出する方法として用いることができる。な
お、本試験法の利用にあたっては、適用範囲外を十分に配慮した上で使用されるべきである。
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 Vitrigel®-EIT 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 病理部/済生会宇都宮病院）：座長
板垣

宏 （ITACS コンサルティング）

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 2 年 4 月 1 日～令和 4 年 3 月 31 日

西 川 秋 佳 （国立医薬品食品衛生研究所 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）
大久保貴之 （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）

合 田 幸 広 （国立医薬品食品衛生研究所）
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）

笛 木

修 （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
小島肇 （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 Vitrigel®-EIT, an alternative method for evaluating
ocular irritation
At a meeting held on 10 May, 2022 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: Vitrigel®-EIT (Eye Irritancy test) method can be used for eye irritation test to
identify chemical substances that do not require classification and labelling under
the United Nations Globally Harmonized System of Classification and Labellingof Chemicals (UN GHS) category, if negative results are obtained after
understanding exemptions. Furthermore, thorough consideration must be given to
the applicability domain when using this test.
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 Vitrigel®-EIT method as a useful means for assessing
eye irritation potential during safety assessments by regulatory agencies.

Akiyoshi Nishikawa
Chairperson,
JaCVAM Regulatory Acceptance Board

Yoko Hirabayashi
Chairperson,
JaCVAM Steering Committee

June 30, 2022v

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:
Mr. Akiyoshi Nishikawa (Division of Pathology, National Institute of Health Sciences:
NIHS / Saiseikai Utsunomiya Hospital) : Chairperson
Ms. Yoko Hirabayashi (Center for Biological Safety and Research: CBSR, NIHS)
Mr. Hiroshi Itagaki (ITACS Consulting)
Mr. Kazuhiko Matsumoto (Nagoya City University)
Ms. Ruriko Nakamura (National Institute of Technology and Evaluation)
Mr. Jihei Nishimura (Pharmaceuticals and Medical Devices Agency)
Term: From 1st April 2020 to 31st March 2022

Mr. Akiyoshi Nishikawa (Division of Pathology, NIHS / Nagoya Tokushukai General
Hospital) : Chairperson
Ms. Yoko Hirabayashi (CBSR, NIHS)
Mr. Koichi Kojima (Food and Drug Safety Center)
Mr. Kazuhiko Matsumoto (Nagoya City University)
Ms. Ruriko Nakamura (National Institute of Technology and Evaluation)
Mr. Jihei Nishimura (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:

Ms. Yoko Hirabayashi (CBSR, NIHS): Chairperson
Mr. Osamu Fueki (Pharmaceuticals and Medical Devices Agency)
Mr. Yukihiro Goda (NIHS)
Ms. Akiko Hayashi (Ministry of Health, Labour and Welfare)
Mr. Koji Ishii (National Institute of Infectious Diseases)
Mr. Yasunari Kanda (Division of Pharmacology, CBSR, NIHS)
Mr. Satoshi Kitajima (Division of Toxicology, CBSR, NIHS)
Mr. Kenichi Masumura (Division of Risk Assessment, CBSR, NIHS)
Ms. Kumiko Ogawa (Division of Pathology, CBSR, NIHS)
Mr. Takayuki Okubo (Ministry of Health, Labour and Welfare)
Mr. Keiichi Sugiyama (Division of Genetics and Mutagenesis, CBSR, NIHS)
Mr. Yuhji Taquahashi (Animal Management Section of the Division of Toxicology, CBSR, NIHS)
Mr. Masaaki Tsukano (Ministry of Health, Labour and Welfare)
Mr. Masahiko Yokota (Pharmaceuticals and Medical Devices Agency)
Mr. Takao Ashikaga (Division of Risk Assessment, CBSR, NIHS): Secretary
Mr. Hajime Kojima (Division of Risk Assessment, CBSR, NIHS): Secretaryvii

眼刺激性試験代替法Vitrigel®-EIT法
法 に関する提案目次

評 価 会 議 報 告 書 -- ---------- ------- --------- ------- --------- ------- --------- ------- --------- ------- ------ 1
評 価 報 告 書 --- ------- --------- ------- --------- ------- --------- ------- --------- ------- --------- ------- ----- 7
Vitrigel®-Eye Irritancy Test Method for Identifying Chemicals Not Requiring
Classification and Labelling for Eye Irritation or Serious Eye Damage -----------------31

評価会議報告書

眼刺激性試験代替法 Vitrigel®-EIT 法

JaCVAM 評価会議

令和 4 年（2022 年）5 月 11 日

1

JaCVAM 評価会議
西 川 秋 佳 （国立医薬品食品衛生研究所 病理部/済生会宇都宮病院）： 座長
板垣宏 （ITACS コンサルティング）

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 2 年 4 月 1 日～令和 4 年 3 月 31 日

西 川 秋 佳 （国立医薬品食品衛生研究所 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

略語
EIT：

Eye Irritancy Test (眼刺激性試験)

GHS：

Globally Harmonized System of Classification and Labelling of Chemicals(化学品の分類および表示に関する世界調和システム)

JaCVAM：Japanese Center for the Validation of Alternative Methods(日本動物実験代替法評価センター)
OECD：

Organisation for Economic Co-operation and Development (経済協力開発機構)

TEER：

Trans Epithelial Electrical Resistance（経上皮電気抵抗）

TG：

Test Guideline (試験法ガイドライン)

UN：

United Nations (国際連合)

3

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「眼刺激性試験代替法 Vitrigel®-EIT 法評価書」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について検討した。
1. 試験法の定義
名称：眼刺激性試験代替法 Vitrigel®-EIT（Eye Irritancy Test）法
代替する対象毒性試験：ウサギを用いる眼刺激性試験 2)
試験法の概略：本試験法では、ウサギ眼の代わりにコラーゲン Vitrigel®3)膜上で培養したヒト角膜上皮組織シート型培養モデルの経上皮バリア機能を反映する TEER 値の経時変化を用い、眼刺激性を評価する。
試験法の科学的妥当性：
本試験法であるVitrigel®-EIT法 は、ヒト角膜上皮組織シート型培養モデルに被験物質を曝露した際の経上皮バリア機能の経時変化を指標として、被験物質の眼刺激性の有無を判定する試験法である4)。
経上皮バリア機能を反映する TEER 値の経時変化から、被験物質の UN GHS 区分における区分に該当しないと判定する試験法であり、科学的にも妥当である。バリデーション研
究の結果、本試験法はバリデーション実行委員会の定めた再現性の基準を満たしていた 5)。
また、追加検証の結果、適用除外を理解した上で使用すれば、感度も十分であると確認された 6)。
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、通常の培養技術を習熟した施設であれば実施できる試験法である。OECD
TG4946)に承認された HCE-T 細胞は理研バイオリソース研究センターから提供を受けることができ、Vitrigel®は市販されており、簡便に短時間で UN GHS 区分に該当しない眼刺激性物質を評価できる。また、生きた動物を用いないという点で、3Rs の精神に合致しており、社会的受け入れ性は高い。
行政上の利用性：
Vitrigel®- EIT 法の適用除外を理解した上で使用し、陰性の結果が得られた場合、UN GHS
区分に該当しない眼刺激性物質を検出する方法として用いることができる。なお、本試験法の利用にあたっては、適用範囲を十分に配慮した上で使用されるべきである。

4

参考文献
1) JaCVAM

眼刺激性試験資料編纂委員会：眼刺激性試験代替法 Vitrigel®-EIT 法評価書

（2022 年 4 月 8 日）
2) OECD (2021) Test Guideline 405, Acute Eye Irritation/Corrosion
3) Takezawa,T., Ozaki, K., Nitani, A., Takabayashi, C., Shimo-Oka, T., Collagen vitrigel: A novel
scaffold that can facilitate a three-dimensional culture for reconstructing organoids. Cell Transplant,
13 (2004):463–473.
4) Takezawa, T., Nishikawa, K., and Wang, P. C., “Development of a human corneal epithelium modelutilizing a collagen vitrigel membrane and the changes of its barrier function induced by exposing
eye irritant chemicals,” Toxicology In Vitro 25 (2011a): 1237–1241.
5) VITRIGEL-EIT Validation Management Team (2017) Validation Study of the Vitrigel-EIT method
as an alternative to in vivo eye irritation testing Study Report, Version 2.0.
6) OECD (2021) Test Guideline 494, Vitrigel-Eye Irritancy Test Method for Identifying Chemicals
not requiring Classification and Labelling for Eye Irritation or Serious Eye Damage5評価報告書

眼刺激性試験代替法 Vitrigel®-EIT 法

眼刺激性試験資料編纂委員会

令和 4 年（2022 年）4 月 8 日

7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/金沢医科大学）

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）8

原範 子 （藤田医科大学）

山下

晴 洋 （大正製薬株式会社）

略語
CAS：

Chemical Abstracts Services

EIT：

Eye Irritancy Test

IATA：

Integrated Approaches to Testing and Assessment

GHS：

Globally Harmonized System of Classification and Labelling of Chemicals

JaCVAM： Japanese Center for the Validation of Alternative Methods
OECD：

Organization for Economic Co-operation and Development

RhCE：

Reconstructed human Cornea-like Epithelium

TEER：

Trans Epithelial Electrical ResistanceTG：

Test GuidelineUN：

United Nations9要旨
Vitrigel®-EIT（Eye Irritancy Test）法は、コラーゲンVitrigel® 膜上で培養したヒト角膜上皮組
織シート型培養モデルの経上皮バリア機能を指標として、TEER（Trans Epithelial Electrical
Resistance）を測定し、その物質の眼刺激性を評価する試験法である。経済協力開発機構（OECD：Organization for Economic Co-operation and Development) の試験法ガイドライン（TG：
Test Guideline）494としてボトムアップ方式で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（UN GHS: United Nations Globally Harmonized System of Classification andLabelling of Chemicals）区分に該当しない物質を検出する方法として採択されている。本報
告書では、Vitrigel®-EIT法のバリデーション研究報告書、第三者評価報告書、関連論文などをもとに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。
Vitrigel®-EIT 法の信頼性・正確性を確認するため、36 物質を用いて 3 施設でバリデー
ション研究が行われた。この研究において Vitrigel®- EIT 法の施設内再現性はそれぞれの施設で 80％、90％、100％、また、施設間再現性は 92％であり、再現性はバリデーション実
行委員会の定めた基準を満たした。一方、正確性は、感度 75~83％、特異度 42％、正確度
64~69％であった。この値は行政的利用を目指す試験法にとって十分なものではなく、開発者は予測性を改良するため、適用除外を「培養液に 2.5％になるよう混合した調製物において、pH が5 以下となる酸性の物質および調製後 3 分以内の短時間に相分離を示す物質」と定めた。
これにより、開発者が改良プロトコルを用いて実施した 158 物質のデータ（バリデーショ
ン結果を含む）から、適用除外を考慮した 107 物質のボトムアップ方式による感度は 96％、特異度は 67％、正確度は 81％となった。
以上より、本委員会は、Vitrigel®- EIT 法は適用除外を理解した上で使用すれば、ボトムアップ方式で UN GHS 区分に該当しない物質を検出する方法として用いることができると
結論した。

10

1. まえおき
Vitrigel®-EIT 法は、竹澤らにより開発されたコラーゲン Vitrigel® 膜 1)上で培養したヒト角膜上皮組織シート型培養モデルの経上皮バリア機能の経時変化を指標に用い、眼刺激性を評価する試験法である。本試験法は、ボトムアップ方式により UN GHS 区分に該当しない物質を検出することができる 2-5)。
3 次元角膜様モデルを用いた眼刺激性試験として、EpiOcularTM 眼刺激性試験、SkinEthicTM
ヒト角膜上皮モデル眼刺激性試験および LabCyte CORNEA-MODEL24 眼刺激性試験がTG492 に収載されているが 6)、いずれも細胞毒性を指標としている。本試験法は、これらと異なり経上皮バリア機能を反映する TEER 値の経時変化を指標として、簡便に短時間で眼刺激性を評価できる方法として国立研究開発法人農業生物資源研究所（現国立研究開

発法人農業・食品産業技術総合研究機構）および関東化学株式会社の共同研究において開発され 2,4,5)、JaCVAM によるバリデーション研究3) および第三者評価7) を経て 2019 年に TG
494 に収載された8)。その後、開発者より TG494 の適応範囲に関する改定の提案があり（Appendix 1）、JaCVAM および OECD における検討を経て、2021 年に OECD TG494 改定案が作成された 9)。本試験法は、作用機構に基づいたものであり、OECD ガイダンス文書 263に示す IATA（Integrated Approaches to Testing and Assessment）の構成要件として 10)、有用であると考えられる。
本報告書では、Vitrigel®-EIT 法のバリデーション研究報告書、第三者評価報告書、関連論
文などをもとに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
Vitrigel®-EIT 法は、UN GHS 区分に該当しない物質（単一物質および混合物）を検出するために用いる試験法である。
3．
．試験法の原理
Vitrigel®-EIT法 は、ヒト角膜上皮組織シート型培養モデルに被験物質を曝露した際の経上皮バリア機能の経時変化を指標として、被験物質の眼刺激性の有無を判定する試験法である。
経上皮バリア機能を反映する TEER 値の経時変化から、被験物質の UN GHS 区分における区分に該当しないと判定する。
4．
．試験手順
Vitrigel® -EIT 法の手順を以下に示す。詳細は、TG 4949) およびプロトコル11) を参照する。
使用する細胞は HCE-T 細胞（理研バイオリソース研究センター RCB2280）を購入して用いる。 コラーゲン Vitirigel® 膜チャンバーおよび TEER 測定装置は関東化学株式会社より購
入できる。

11

4-1. モデルの構築
コラーゲンVitrigel® 膜チャンバーを 12wellプレートにセットし、ウェル内に培養液を 1.5mLを加え、チャンバー内に1.2×105cells/mLに調製した細胞懸濁液を 0.5mLずつ分注しCO2イン
キュベーター（37℃, 5％CO2-Air）内に静置する。播種後 2日目に、チャンバー内の培養液を除き気相‐液相界面培養を開始する。播種後 6日目のモデルのチャンバー内に培養液を0.5mL入れ、温度を 28℃±2℃に調節する。TEER 測定用電極をチャンバーにセットし TEER
値を測定する。TEER値がモデルの成立基準を満たしているかを確認する。
4-2. 被験物質の調製培養液で 2.5％(w/v) になるように被験物質を溶解または懸濁し、調製物を作製する。
調製物を pH 試験紙（測定範囲に pH 1～11 が含まれるもの）または pH メーターにて測定
し、pH が 5 以下となる酸性の物質は試験に適応できない。また、調製物の作製後 0 および3 分に UV/VIS 分光光度計にて 660 nm の吸光度を測定し、絶対値で 0.1 を超える差がみられた物質は短時間に相分離を示す物質として試験に適応できない。被験物質が吸光度の測定を阻害する場合には、異なった波長を用いることもできる。
4-3. 被験物質の適用および測定成立基準を満たすモデルに TEER 測定用電極を設置した後、被験物質 2.5％(w/v)水溶液または懸濁液をモデルに曝露する。被験物質の適用時のモデル温度は、28℃±2℃とする。
1 種類の被験物質について 3 個のモデルを用いて試験を行い、TEER値を 10 秒毎に 3 分間記録し、それぞれのTEERの初期値に対する変化率の平均値から、3 つの指標（Time lag、Intensity、Plateau level）を求める。これら指標の経時変化を自動解析し、眼刺激性の有無を判定する。
4-4. 判定
計算手順
3 つの指標は以下の式から算出される（図 1 参照）
Time lag (sec) ：t1
Intensity (％/sec)：− [P2 − P1]/[t2 − t1]
Plateau level (％)：100 − P2
t1 (sec)：TEER 値の変化速度が 0 ≥ dP/dT > − 0.03％/sec の範囲内にある最大の曝露時間。
t2 (sec)：t1 後、TEER 値の変化速度が dP/dT ≤ − 0.03％/sec の条件を満たした後、 0 ≥dP (P3 − P2)/dT (t3 − t2) > − 0.03％/sec の条件を満たした最初の曝露時間。
t3 (sec)：t2 + 30 sec
P1, P2, P3 (％)：曝露時間 t1, t2, t3 の時 TEER 値の t0 時の TEER 値に対する百分率。

12

図 1．TEER 測定値の推移
上記の指標をもとにした予測モデルを表１に示す。
表１．予測モデル基準

予測Time lag ≤ 180 (sec) or Intensity ≥ 0.05 (％/sec) or Plateau level > 5.0 (％)

不可（I）(1)Time lag > 180 (sec) and Intensity < 0.05 (％/sec) and Plateau level ≤ 5.0 (％)

区分外（NI）(2)

注釈：(1) 不可の場合には、IATA9) の既存データや情報をもとに、被験物質におけるすべの眼刺激性作用機構の妥当性を考慮する必要がある。
(2) 区分外とは、UN GHS 区分に該当しない化学物質を指す。
4-5. 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。
21) 初期の TEER 値が 140～220Ω・cm の範囲にあるモデルである。
2) 陰性対照（生理食塩水：Saline）のPlateau level（PL）が 5 ％以下である。
3) 陽性対照（塩化ベンザルコニウム：BAC）のPL が40％以上である。
4) 参照試料（エタノール：EtOH）のPL が10％以上である。
5) 被験物質の 3 つの指標の平均標準偏差が 15％以下である。
5．
．バリデーション研究開発者とは異なる 3 施設（株式会社ダイセル：Daicel、株式会社ボゾリサーチセンター：BRC、一般財団法人食品薬品安全センター：FDSC）の協力を得て、Vitrigel®- EIT 法のバリデーショ
ン研究が行われた 3)。これら 3 施設はバリデーション研究の前に開発者から技術実習を受けた
後、技術移転性を確認した。試験はすべての被験物質について 3 施設でそれぞれ 3 回実施された。バリデーション結果は Appendix2 に示した。

13

5-1．試験法の信頼性
5-1-1. 技術移転性
技術移転性については、プレバリデーション研究（phase 0）にて、3 施設による 5 物質（BAC、2-Propanol、Glycerol、n-Hexanol、Silicon dioxide n-hydrate）および陽性対照物質（EtOH）を用いた試験が行われ、プロトコルの確認がなされた 3)。その結果、EtOH の受け入れ PL 基準（20~30％）
が厳しく、1 施設で基準を満たせなかった。バリデーション実行委員会で議論した結果、この基準値を 10~30％に広げ、この物質を自家製モデルの適正を評価する参照試料とし、陽性対照
を BAC とすることになった。
5-1-2. 施設内再現性
Phase I バリデーション研究において、10 物質を用いて施設内再現性が評価され、UN GHS 区
分判定の施設内再現性は 80％、90％、100％とバリデーション実行委員会の基準（80％以上）を満たした。バリデーション実行委員会で議論した結果、EtOH の受け入れ PL 基準値を 10~40％に広げ、適用温度を 18℃~30℃から 22℃~30℃に上げることが合意された。
5-1-3. 施設間再現性
Phase II バリデーション研究において、10 物質を用いて施設間再現性が評価された。その結
果、施設間再現性は 80％以上であるバリデーション実行委員会の基準を満たした。ただし、UN GHS 区分１にあたるイミダゾール（No.2-1）を 2 施設が陰性と判断した。バリデーション実
行委員会はこの結果を重く受け止め、Phase II の結果を不採用とし、プロトコルの改善を開発者に依頼した。その結果、開発者より、被験物質適用温度を 28℃±2℃とする改訂が提案された。このプロトコルを用いて、3 施設がイミダゾールで陽性結果を得たことを確認した上で 36
物質を用いて Phase III バリデーション研究を実施した。その結果、施設間再現性は 92％であり、バリデーション実行委員会の定めた基準（80％以上）を満たした。施設内・施設間再現性を高めるためのプロトコルの変遷を Appendix 2 の図 1 に示す。Phase II 終了後、バリデーション
実行委員会で議論した結果、EtOH の受け入れ PL 基準値は≧10％となった。
5-2．試験法の正確性
Phase III バリデーション研究の結果をもとに、3 施設で行われたデータを用いて正確性が検証された。その結果、感度 75~83％、特異度 42％、正確度 64~69％となった。この値は行政的利用を目指す試験法にとって十分なものではないとバリデーション実行委員会は考えた。
6. 追加検証
開発者は、Appendix1 に示した経緯を経て、OECD 専門家の意見をもとに 158 物質まで追加検討を行い（Appendix 3）、表 2 に示すように、感度 88％（61/69 物質）、特異度 63％（56/89 物質）
および正確度 74％（117/158 物質）を得ている。なお、特異度は、phase III 結果と大きく異なる
が、バリデーション研究で選択された適用物質は感度の検出を優先していることから、選ばれた陰性物質数の偏りにより、結果として特異度が低くなったと考える。
本追加検討において偽陰性を示した 10 物質のうち 5 物質が酸性であり、調製物の pH は 5よりも低い値であった。また、調製液に不溶で短時間に相分離する物質も偽陰性となる可能性が示唆された。これらの点を考慮し、pH が 5 以下となる 12 物質および調製後 3 分以内の短14

時間に相分離を示す 39 物質を適用除外とした結果、107 物質においてボトムアップ方式での正確性は感度 96％（51/53 物質）、特異度 67％（36/54 物質）、正確度 81％（87/107 物質）へと改善した（表 3）。液体の物質は、感度 100％（34/34 物質）、特異度 71％（22/31 物質）、正確度 86％（56/65 物質）、固体の物質は、感度 89％（17/19 物質）、特異度 61％（14/23 物質）、正確度 74％（31/42 物質）であった。
なお、2 つの偽陰性物質は Camphene と 1,4-Dibutoxybenzene である。いずれも US GHS 区分
2B に分類される。Drivers of classification12) によると角膜に対する障害は認められず、結膜に対する障害のみ認められていることから、弱い刺激性であるため偽陰性になったものと開発者は考えた。
この適用除外は、バリデーション研究報告書および第三者評価報告書とは異なるが、改定 TG
検討の際に OECD にて合意されたものである。
表 2. 158 物質を用いた開発者によるボトムアップ方式での解析結果
Vitrigel®-EIT 法

UN GHS

Total

I

NI

Cat.1, 2A, 2B

61

8

69

No Category

33

56

89

94

64

158

Total
感度: 88％ (61/69)
特異度: 63％ (56/89)
正確度: 74％ (117/158)

表 3．適用除外 39 物質を除く 107 物質を用いた開発者によるボトムアップ方式での解析結果
Vitrigel®-EIT 法

UN GHS
Total

Total

I

NI

Cat.1, 2A, 2B

51

2

53

No Category

18

36

54

69

38

107

感度: 96％ (51/53)
特異度: 67％ (36/54)
正確度: 81％ (87/107)15

7．
．試験法の適用範囲および留意点
TG 494 は、試験法の適正と正確性の確保を考慮して Vitrigel®-EIT 法の適用に以下の制限を設けている。
1) バリデーション研究および追加検証において混合物（単一物質の水溶液を除く）、気体（ガス）およびエアゾール様物質は被験物質として用いられていない。したがって、本試験法
でこれら物質を評価する場合には、物性や規格要件を把握し、科学的に妥当な結果が得られるかどうかを前もって検討する必要がある。
2) UN GHS 区分 1、区分 2（2A・2B）物質の検出には用いることはできない。
3) 培養液で 2.5％(w/v)になるように溶解または懸濁した調製物において、pHが 5 以下となる物質および調製後 3 分以内に相分離を示す物質は適用できない。
還元作用や着色のある被験物質ではMTT（3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide）等を用いた細胞毒性評価に支障をきたすが、本試験法の指標であるTEERではそのよ
うな被験物質でも評価できる特徴をもつ。
8. 本委員会の結論
Vitrigel®-EIT 法のバリデーション研究の結果、本試験法はバリデーション実行委員会の定めた再現性の基準を満たしていた。また、追加検証の結果、適用除外を理解した上で使用すれば、正確性も十分であると確認された。よって、Vitrigel®- EIT 法 はボトムアップ方式で UN
GHS 区分に該当しない物質を検出する方法として用いることができると本委員会は考える。
謝辞
当該試験法の眼刺激性試験評価報告書を作成するにあたり、国立研究開発法人 農業・食品産業技術総合研究機構（農研機構）の竹澤俊明氏、関東化学株式会社の山口宏之氏、国立医薬品食品衛生研究所の小島肇氏による情報提供等のご協力に深く感謝申し上げます。

16

9. 参考文献

1) Takezawa,T., Ozaki, K., Nitani, A., Takabayashi, C., Shimo-Oka, T., Collagen vitrigel: A novel
scaffold that can facilitate a three-dimensional culture for reconstructing organoids. Cell Transplant,
13 (2004):463–473.

2) Takezawa, T., Nishikawa, K., and Wang, P. C., Development of a human corneal epithelium modelutilizing a collagen vitrigel membrane and the changes of its barrier function induced by exposing
eye irritant chemicals, Toxicology In Vitro 25 (2011): 1237–1241.

3) VITRIGEL-EITValidation Management Team(2017) Validation Study of the Vitrigel-EIT method
as an alternative to in vivo eye irritation testing Study Report, Version 2.0

4) Yamaguchi, H., Kojima, H., and Takezawa, T., Vitrigel-eye irritancy test method using HCE-T cells,
Toxicological Sciences 135 (2013): 347–355.

5) Yamaguchi, H., Kojima, H., and Takezawa, T., Predictive performance of the Vitrigel-eye irritancy
test method using 118 chemicals, Journal of Applied Toxicology 36 (2016): 1025–1037.

6) OECD (2018) Test Guideline 492. Reconstructed human Cornea-like Epithelium (RhCE) test
method for identifying chemicals not requiring classification and labelling for eye irritation orserious eye damage.

7) Vitrigel-Eye Irritation Test (EIT) method Report of the Peer Review Panel (2017)8) OECD (2019) Test Guideline 494, Vitrigel-Eye Irritancy Test Method for Identifying Chemicals not
requiring Classification and Labelling for Eye Irritation or Serious Eye Damage
9) OECD (2021) Test Guideline 494, Vitrigel-Eye Irritancy Test Method for Identifying Chemicals not
requiring Classification and Labelling for Eye Irritation or Serious Eye Damage10) 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)11) Standard Protocol for the Vitrigel-EIT method, Version 1.80e. (2015)12) Barroso, J., Pfannenbecker, U., Adriaens, E., Alépée, N., Cluzel, M., De Smedt, A., Hibatallah, J.,
Klaric, M., Mewes, K.R., Millet, M., Templier, M., and McNamee, P., 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), Archives of toxicology 91 (2017): 521–547.

17Appendix 1

Vitrigel®-EIT 法 TG494 の改定にいたる経緯

Vitrigel®-EIT 法は、ボトムアップ方式にて UN GHS 区分に該当しない物質の検出をする眼刺激性試験代替法としてバリデーション研究が行われた。3 施設で行われた 36 物質を用いたバリデーション研究の結果、予測性は、感度 75～83％、特異度 42％、正確度 64～69％となった。
さらに、開発者のデータを追加した計 93 物質の結果では、感度 83%、特異度 70%、正確度 78%となった。この結果をもとに、以下に示す適用除外条件が提案され、この適用除外条件に当てはまる 17 物質を除いた 76 物質を評価したところ、感度 93％、特異度 69％、正確度 83％となった。
＜適用除外条件＞
1． 培養液で 2.5% (w/v) になるように溶解または懸濁した調製物において、pH が 5 以下となる
物質
2．logP 値 2.5 以上、および、密度 0.95 g/ cm3 以下または 1.10 g/cm3 以上の固体
上述の適用除外条件を取り入れた Vitrigel®-EIT 法は、第三者評価を経て OECD の TG として提案された。しかしながら、条件 2 については、当てはまる固体が少なく評価できないという判断で OECD 専門家会議により却下された。結果として、固体物質がすべて適用除外になった。
その場合の予測性は、この適用除外条件に当てはまる 42 物質を除いた 51 物質を評価したとこ
ろ、感度 96％、特異度 74％、正確度 84％となった。以下に示す適用除外条件下で、2019 年 6月 18 日に Vitrigel®-EIT 法は TG494 として OECD で採択された。
＜適用除外条件＞
1． 培養液で 2.5％(w/v)になるように溶解または懸濁した調製物において、pH が 5 以下となる物質
2．固体物質TG は採択されたものの、適用可能物質が約 55％(51/93)という少ない状態を改善するため、開発者らは直ちに追加実験の立案に入った。解決すべき問題点は、固体物質を適用するための条件設定であった。
固体物質を適用するための条件設定に関しては、JaCVAM 眼刺激性試験資料編纂委員会の意見も参考に日本薬局方の濁度測定法を用い、被験物質の濃度が 20％変化する指標として、調製直後と 3 分後の吸光度の差が 0.1 を超える場合に培養液中での相分離が起きている、すなわち懸濁液の分散安定性が低いと判断した。
＜適用除外条件＞
1． 培養液で 2.5％ (w/v)になるように溶解または懸濁した調製物において、pH が 5 以下となる
物質
2． 調製後 3 分以内に相分離を示す物質
あらたな適用除外条件を用いて、158 物質（液体 94、固体 64）の追加検討を行った、そのうち、
51 物質（22 の固体のうち、pH5 以下 7 物質、相分離 15 物質；29 の液体のうち、pH5 以下 5 物
質、相分離 24 物質）が適用除外となった。残りの 107 物質の適用除外を含む 158 物質の結果と比較すると、感度が 88％から 96％、特異度は 63％から 67％、正確度が 74％から 81％と改善さ18

れた。予測性値は以前の TG と大きな変化はないが、適用可能物質が拡大し、適用範囲が科学的に適正となった、この改定案に国際的な合意が得られ、2021 年 6 月 14 日の改定版に反映された（報告書の表 2 および表 3 参照）、107 物質における固体・液体毎の予測性は、TG494 の Table
１に記載されている。表 3 および Table 1 に示す偽陰性物質はいずれも固体の UN GHS 区分で2B であった。
なお、以前の TG では許容されていた液体で相分離を示す 24 物質に関しては、偽陰性が 2 物
質（UN GHS 区分 1、区分 2B）含まれており、改定により改善が認められた、ちなみに、固体において相分離を示した 15 物質に関しては、偽陰性が 2 物質（いずれも UN GHS 区分 1）認めら
れた。

19

20
Cat. 1

sulfaten,n-Dimethylguanidine

propanediol

3-Methoxy-1,2-

1-10 Gluconolactone

1-9

1-8 Toluene

1-790-80-2

623-39-2

108-88-3

598-65-2

No Cat.

0.69 (I)

0.19 (I)0.04 (NI)190 (NI) -0.04 (NI)190 (NI) -0.05 (NI)190 (NI) -0.01 (NI)190 (NI) -0.05 (NI)0 (I)6484-52-2

1-6 Ammonium nitrate

67-64-1

1-5 3-Chloropropionitrile

1-4 Acetone

0.16 (I)190 (NI) -0.06 (NI)30 (I)

Cat.2A or 10 (I)2B
542-76-7
90 (I)

1-3 3,3-Dithiodipropionic acid 1119-62-6

108-93-0

1-2 Cyclohexanol-

190 (NI) -0.03 (NI)20 (I)

Reference control

0.33 (I)190 (NI) 0.00 (NI)0 (I)288-32-4

FDSC
Time lag Intensity

Positive control

Negative control

Test chemical

1-1 Imidazole

No.

CASRN UN GHS

Appendix 2 表１. Phase I バリデーション結果

0 (NI)

0 (NI)

0 (NI)

2 (NI)

21 (I)

20 (I)

10 (I)

0 (NI)

25 (I)

0 (NI)

3 (NI)

59 (I)

0 (NI)

level

Plateau

NI

NI

NI

NI

I

I

I

NI

I

NI

NG

I

NI

0.40 (I)

0.13 (I)

0.12 (I)

0.23 (I)0.02 (NI)0.53 (I)

0.67 (I)

0.20 (I)

0.12 (I)0 (I)0.28 (I)190 (NI) -0.12 (NI)150 (I)

0 (I)

0 (I)

10 (I)

0 (I)

190 (NI) -0.07 (NI)0 (I)160 (I)

0 (I)

0 (I)

190 (NI) -0.03 (NI)Result Time lag Intensity

BRC

8 (I)

0 (NI)

1 (NI)

21 (I)

27 (I)

36 (I)

21 (I)

0 (NI)

48 (I)

4 (NI)

21 (I)

42 (I)

0 (NI)

level

Plateau

I

NI

I

I

I

I

I

NI

I

I

I

I

NI

level

Plateau

0.54 (I)1.05 (I)

0.21 (I)

0.17 (I)

(NI)

-0.05

0.34 (I)

0.11 (I)

0.17 (I)

0.42 (I)

10 (I)

190 (NI)

0.18 (I)

(NI)

-0.11

190 (NI) 0.00 (NI)

0 (I)

0 (I)

10 (I)

0 (I)

190 (NI)

0 (I)

140 (I)

0 (I)

0 (I)

10 (I)

0 (NI)

0 (NI)

27 (I)

52 (I)

39 (I)

30 (I)

0 (NI)

51 (I)

7 (I)

30 (I)

75 (I)

190 (NI) -0.04 (NI) 0 (NI)Result Time lag Intensity

Daicel

I

NI

NI

I

I

I

I

NI

I

I

I

I

NI

ResultAppendix 2 表 2．Phase II バリデーション結果
No.

Test chemical

CASRN

UN GHS

FDSC

BRC

Daicel

NI

I

NI

I

I

I

I

I

I

2-1

Imidazole288-32-4

2-2

Cyclohexanol

108-93-0

2-3

Sodium dodecyl sulfate

151-21-3

2-4

Sodium salicylate

54-21-7

I

I

I

2-5

Cyclopentanol

96-41-3

I

I

I

2-6

2-Methyl-1-pentanol

105-30-6

I

I

I

2-7

α-Hexylcinnamaldehyde

101-86-0

NI

NI

NI

2-8

n,n-Dimethylguanidine sulfate598-65-2

I

I

I

2-9

Toluene108-88-3

I

NI

NI

2-10

Gluconolactone

90-80-2

I

I

I

Cat. 1

Cat. 2A & 2B

No Category

Appendix 2 図１．プロトコルの変遷

21

Appendix 2 表 3．Phase III バリデーション結果

22

No.

Test chemical

CASRN

3-1

2,5-Dimethyl-2,5-hexanediol

110-03-2

3-2

2-Benzyl-4-chlorophenol

3-3Physical

UN

state

GHS

FDSC

BRC

Daicel

Solid

I

I

I

120-32-1

Solid

I

I

I

2,2-Dimethyl butanoic acid595-379

Liquid

I

I

I

3-4

Captan133-06-2

Solid

NI

NI

NI

3-5

Tetra-n-Octylammonium bromide

14866-33-2

Solid

I

I

NI

3-6

Butanol

71-36-3

Liquid

I

I

I

3-7

3-(2-Aminoethylaminopropyl)
trimethoxysilane

1760-24-3

Liquid

I

I

I

3-8

Sodium dodecyl sulfate

151-21-3

Solid

I

I

I

3-9

m-Phenylenediamine

108-45-2

Solid

I

I

I

3-10

Tetraethylene glycol

17831-71-9

Liquid

I

I

I

3-11

Imidazole288-32-4

Solid

I

I

I

3-12

Sodium salicylate

54-21-7

Solid

I

I

I

3-13

gamma-Butyrolactone

96-48-0Liquid

I

I

I

3-14

Methyl acetate79-20-9Liquid

I

I

I

3-15

Myristyl alcohol

112-72-1

Solid

NI

NI

NI

3-16

2,6-Dichlorobenzoyl chloride

4659-45-4

Liquid*

NI

I

NI

3-17

Dibenzyl phosphate

1623-08-1

Solid

I

I

I

3-18

1-(2-Propoxy-1-methylethoxy)-2-propanol

29911-27-1

Liquid

I

I

I

3-19

Camphene

79-92-5

Solid

I

NI

NI

3-20

Ethyl-2-methylacetoacetate609-14-3

Liquid

I

I

I

3-21

Propylene glycol propyl ether

1569-01-3

Liquid

I

I

I

3-22

2-Methyl-1-pentanol

105-30-6

Liquid

I

I

I

3-23

α-Hexylcinnamaldehyde

101-86-0

Liquid

NI

NI

NI

3-24

Cyclopentanol

96-41-3

Liquid

I

I

I

3-25

Methyl amyl ketone

110-43-0

Liquid

I

I

I

3-26

2-(n-Dodecylthio)ethanol

1462-55-1

Liquid

NI

NI

NI

3-27

iso-Octylthioglycolate

25103-09-7

Liquid

NI

NI

NI

3-28

2,4-Difluoronitrobenzene

446-35-5

Liquid

I

I

I

3-29

tetra-Aminopyrimidine sulfate

5392-28-9

Solid

NI

NI

NI

3-30

2,4-Pentanediol

625-69-4

Liquid

I

I

I

3-31

iso-Octyl acrylate

29590-42-9

Liquid

NI

NI

NI

3-32

Silicon dioxide n-hydrate

7699-41-4

Solid

NI

NI

NI

3-33

Potassium tetrafluorobroate

14075-53-7

Solid

I

I

I

Cat. 1

Cat. 2A
or 2B

No Cat.

3-34

n,n-Dimethylguanidine sulfate598-65-2

Solid

I

I

I

3-35

Toluene108-88-3

Liquid

I

I

I

3-36

Gluconolactone

90-80-2

Solid

I

I

I

*: pH of 2.5％ solution ≤ 5.0

Appendix 2 表 4-1. Phase III

FDSC および BRC の正確性
Vitrigel®-EIT 法

UN GHS

Total

I

NI

Cat.1, 2A, 2B

20

4

24

No Category

7

5

12

27

9

36

Total

感度: 83.3％ (20/24)
特異度: 41.7％ (5/12)
正確度: 69.4％ (25/36)

Appendix 2 表 4-2. Phase III

Daicel の正確性
Vitrigel®-EIT 法

UN GHS
Total

Total

I

NI

Cat.1, 2A, 2B

18

6

24

No Category

7

5

12

25

11

36

感度: 75.0％ (18/24)
特異度: 41.7％ (5/12)
正確度: 63.9％ (23/36)

23

Appendix 3 開発者による追加検討（158 物質）

24

25

140-72-7

110-86-1

Cycloalkane;Isocyanate

Aryl;Ether moiety;Quaternary ammonium salts;Surfactants - Cationic

Acrylate;Alkene moiety ;Carboxylic acid ester;Ether moiety

Alcohol;Alkyl halide

Alkane, branched with quaternary carbon;Alkyl (hetero)arenes;Alkyl-, alkenyland alkynyl (hetero)arenes;Aryl;Benzyl;Ether moiety;Quaternary ammonium
salts;Surfactants - Cationic;tert-Butyl

Alcohol;Cycloalkane

Aryl;Pyridine/Pyridinium ion;Surfactants - Cationic

3 Cyclohexyl isocyanate

4 Domiphen bromide (10%)

5 Tetraethylene glycol

6 8-Chloro-1-n-octanol

7 Benzethonium chloride (10%)

8 Cyclohexanol

9 Cetylpyridinium bromide (10%)

111-76-2
9002-93-1

Alcohol;Aliphatic amine, tertiary;Amine, tertiary

Alcohol;Ether moiety

Alcohol;Alkane, branched with quaternary carbon;Alkyl (hetero)arenes;Alkyl-,
alkenyl- and alkynyl (hetero)arenes;Aryl;Ether moiety;tert-Butyl

12 Diethylethanolamine

13 Butyl cellosolve

14 Triton X-100

Alcohol

Alcohol;Alkane, branched with tertiary carbon

25 1-Octanol

26 2-Ethyl-1-hexanol

KetoneAlcohol

24 n -Hexanol

29 Acetone

Acyl halide;Aryl;Aryl halide

23 2,6-Dichlorobenzoyl chloride

KetoneAcetoxy;Carboxylic acid ester;Ether moiety

22 2-Ethoxyethyl acetate

Alcohol

Alcohol;Aryl;Benzyl

21 Benzyl alcohol

27 Methyl ethyl ketone

Quaternary ammonium salts;Surfactants - Cationic

20 Stearyltrimethylammonium chloride (10%)

28 Ethanol

Aryl;Pyridine/Pyridinium ion;Surfactants - Cationic

19 Cetylpyridinium chloride (10%)

167-64-1

64-17-5

78-93-3

104-76-7

111-87-5

111-27-3

4659-45-4

111-15-9

100-51-6

112-03-8

LiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquid

2A

2A

2A

2A

2A

2A

2A

2

2

1

1

1760-24-3

Liquid168439-51-0 Liquid

6004-24-6

1
1

Liquid

1

1

1

1

1

1

1

1

9004-82-4

LiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquid

1

1

1

1

1

1

GHS

27028-82-6 Liquid

100-37-8

50-21-5Aryl;Pyridine/Pyridinium ion

Alcohol;Carboxylic acid

10 Pyridine

108-93-0

121-54-0

11 Lactic acid

Triethanolamine polyoxyethylene (3) lauryl ether
Alcohol;Aliphatic amine, tertiary;Amine, tertiary;Ether moiety
15
sulfate, 40%
Sodium polyethylene (3) lauryl ether sulfate,
16
Alkene moiety ;Sulfate
25.5%
Polyoxyethylene (10) polyoxypropylene (1.5)
17Alcohol;Ether moiety
lauryl—mystyl ether
Aliphatic amine, primary;Aliphatic amine, secondary;Alkoxysilane;Amine,
18 3-(2-Aminoethylamino)propyltrimethoxysilane
primary;Amine, secondary;Silane

LiquidLiquidLiquidLiquidLiquidForm

23144-52-7 Liquid

17831-71-9

538-71-6

3173-53-3

n/a

111-86-4

Aliphatic amine, primary;Amine, primary

CAS No.

(n/a)

Organic functional groups

1 n ‐Octylamine

Chemical2 Basasol Orange 52L

No.

1)

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

NI

I

I

≥1

≥2

≥1

≥1

≥1

≥1

≥1

>0

≥1

≥1

≥1

≥1

>0

≥1

≥3

≥3

≥3

≥3

≥3

≥3

≥1

≥3

≥3

n/a

Judgment by
COVitigelmean
EIT

≥2

≥1

≥2

≥2

≥2

≥2

≥2

≥2

≥2

≥2

≥2

≥2

Conj
mean

Cut-off values

≥1

≥1

≥1

≥1

≥1

≥1

> 1.5

≥1

> 1.5

> 1.5

IR
mean

Drivers of classification 2)
Severity

6/6; 5/6;
5/6
4/4; 4/4;
4/4

3/4; 3/4

4/4; 3/4

2/3; 2/3;
2/3

4/4; 4/4

6/6; 6/6

1/4

2/3

3/3; 3/3;
2/3
3/3; 3/3;
3/3
3/3; 3/3;
2/3

3/3; 2/3

7

21

7

21

21

21

21

21

Unknown

21

6/6; 6/6;
5/6
5/6

21

21

21

21

21

21

21

21

21

21

7

7

7

7

7

21

21

21

21

7

7

21

Conj IR
pers pers

Unknown

CO persCut-off time

Persistence

4/6

3/3

2/3

6/6; 4/6

3/4

2/3

3/3; 3/3

6/6

2/3

2/2

animals

Numberof

4/4; 1/4; 1/4

1/6

3/4

3/4; 1/4

4/4

6/6; 2/6

at least 1/3

3/3

at least 2/3

3/3; 2/3

3/3; 3/3

1/6

5/6; 5/6

2/3

at least 1/3

=4

=4

=4

=4

=4

=4

2/3

3/3

3/3

5/6

3/3

1/3

2/6

-0.04

-0.03

-0.03

2.24

2.32

1.93

0.86

-0.03

-0.02

0.30

0.06

0.01

-0.06

-0.06

-0.06

-0.03

0.01

-0.01

-0.01

0.00

0.16

=4

0.32

3.07

0.05

0.57

2.16

3.03

2.55

-0.04

-0.03

-0.04

2.17

2.20

1.63

0.14

-0.03

-0.03

0.32

0.06

0.01

-0.06

-0.06

-0.06

-0.03

0.01

-0.01

-0.01

0.00

0.15

0.08

0.32

3.06

0.04

0.54

1.97

2.63

0.36

3min

0min

0.08

2/3

5/6

3/3

2/2

4/4

A660 at

A660 at

1/4

=4

=4

=4

=4

=4

animals

Specific Observations
Number
CO=4 ofat least 4/6(max 5); at
least 3/6
(max 4); 3/6

at least 2/3

3/3

at least 5/6

at least 2/3

2/2

Number ofanimals

0.00

0.00

0.00

0.07

0.12

0.30

0.73

0.00

0.01

0.02

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.01

0.01

0.03

0.19

0.40

2.19

minutes.
|ΔA660 0-

7

7

7

7

7

7

3

7

7

7

7

10

6

7

7

7

8

10

3

7

7

7

9

7

7

7

7

4

12

✔

✔

✔

✔

✔

✔

✔

✔

✔

Absolute
differences
between the
ExcludedabsorbanceExcluded by pre-test
pH ofvalues of test
by pre-test 2(Briefly
2.5%chemical
1(Acidicphasesolution
preparation
chemicals) separating
at 0 and 3
chemicals)

26

Aldehyde

Alcohol;Alkane, branched with secondary carbon;Ether moiety

Alcohol;Alkane, branched with secondary carbon;Ether moiety

Alkyl halide;Nitrile

Carboxylic acid ester;Ketone

Alcohol;Aliphatic amine, primary;Amine, primary

Alcohol;Carboxylic acid

Alkane, branched with secondary carbon;Alkyl halide

Alkyl (hetero)arenes;Alkyl-, alkenyl- and alkynyl (hetero)arenes;Aryl

Carboxylic acid ester;tert-Butyl

Alkene moiety ;Alkenyl (hetero)arenes;Alkyl-, alkenyl- and alkynyl(hetero)arenes;Aryl

39 n -Butanal

40 Propasol solvent P

41 Di(propylene glycol) propyl ether

42 3-Chloropropionitrile

43 Ethyl-2-methylacetoacetate

44 Monoethanolamine (10%)

45 Glycolic acid (10%)

46 1,2,3-Trichloropropane

47 Xylene

48 Ethyl trimethyl acetate

49 Styrene

96-37-7

LiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquid3970-62-5

Alcohol;Alkane, branched with quaternary carbon;Alkane, branched withsecondary carbon;tert-Butyl

69 2,2-Dimethyl-3-pentanol

LiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquid110-52-1

LiquidLiquidLiquid25103-09-7 Liquid8032-32-4

110-27-0

110-54-3

629-03-8

63 Hexane

Alkyl halide

Alkanes;Hydrocarbons

62 Methyl cyclopentane

75-26-3

Alkyl halide

Alkanes;Cycloalkane;Hydrocarbons

61 Isopropyl bromide

112-40-3

68 1,6-Dibromohexane

Alkane, branched with secondary carbon;Alkyl halide;Isopropyl

60 Dodecane

95-63-6

108-10-1

67 1,4-Dibromobutane

Alkanes;Hydrocarbons

59 1,2,4-Trimethylbenzene

Alkane, branched with tertiary carbon;Carboxylic acid ester;Isopropyl;Thiol

Alkyl (hetero)arenes;Alkyl-, alkenyl- and alkynyl (hetero)arenes;Aryl

58 Methyl isobutyl ketone

107-83-5
592-42-7

66 iso -Octylthioglycolate

Alkane, branched with tertiary carbon;Isopropyl;Ketone

57 1,5-Hexadiene

Alkane, branched with secondary carbon;Carboxylic acid ester;Isopropyl

Alkene moiety ;Alkenes;Allyl;Hydrocarbons

56 2-Methylpentane

110-43-0

Alkanes;Hydrocarbons

Alkane, branched with tertiary carbon;Alkanes;Hydrocarbons;Isopropyl

55 Methyl amyl ketone562-49-2

111-83-1

110-27-0

108-83-8

446-35-5

1330-20-7

96-18-4

79-14-1

141-43-5

609-14-3

542-76-7

65 Petroleum ether

Ketone54 3, 3-Dimethylpentane

LiquidLiquid29911-27-1 Liquid

1569-01-3

141-10-6

105-30-6

79-20-978-83-1

71-36-3

64 iso -Propyl myristate

Alkyl halide

Alkane, branched with quaternary carbon;Alkanes;Hydrocarbons

53 n -Octyl bromide

Alkane, branched with secondary carbon;Carboxylic acid ester;Isopropyl

Aldehyde;Alkane, branched with tertiary carbon;Isopropyl

38 Isobutanal

52 Isopropyl myristate

Alkene moiety ;Allyl;Conjugated system;Ketone;Terpenes

37 Pseudoionone

Aryl;Aryl halide;Nitrobenzene

100-42-5

Alcohol;Alkane, branched with tertiary carbon

36 2-Methyl-1-pentanol

Alkane, branched with tertiary carbon;Isopropyl;Ketone

3938-95-2

Acetoxy;Carboxylic acid ester

35 Methyl acetate

50 2,4-Difuroronitrobenzene

123-72-8

Alcohol;Alkane, branched with tertiary carbon;Isopropyl

34 Isobutyl alcohol

51 Diisobutyl ketone

78-84-2

Alcohol

33 Butanol

96-41-3

Alcohol;Cycloalkane

96-48-0

67-63-0

32 Cyclopentanol

Alcohol;Alkane, branched with secondary carbon;Isopropyl

Lactons;Oxolane

30 Isopropyl alcohol

31 Butyrolactone

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

2B

2B

2B

2B

2B

2B

2B

2B

2B

2B

2A

2A

2A

2A

2A

2A

I

I

Ni

NI

NI

NI

NI

NI

NI

I

NI

NI

I

NI

NI

I

NI

NI

NI

NI

I

NI

I

I

I

NI

I

I

I

I

I

I

I

NI

I

I

I

I

I

I

>0

=0

=0

=0

=0

=0

=0

=0

=0

=0

=0

>0

=0

=0

>0

=0

=0

=0

=0

=0

>0

>0

=0

>0

≥1

≥1

≥1

≥1

≥1

≥1

≥1

≥1

≥1

≥1

≥1

≥1

≥1

≥2

≥2

≥2

≥2

≥2

≥2

≥2

≥2

≥2

≥2

≥2

≥1

3/4

7

7

CO 1/3

Conj 1/4

CO 1/6

Conj 1/4

2/3

2/3

3/3

2/3

3/3; 3/3

5/6

2/3; 2/3

2/3; 2/3

2/3

2/3

3/4; 3/4

4/4; 4/4

7

7

7

3/3; 3/3;
at least Unknown
2/3

3/3; 3/3

3/3; 3/3

7

7

7

7

7

7

4/4

1/6; 1/6

4/4; 1/4

4/4; 1/4

1/3; 1/3

2/3; 1/3

=4

1/3

2.24

3.03

3.09

2.15

0.08

1.07

0.55

1.69

0.19

0.75

1.88

1.24

0.28

0.55

1.82

0.82

0.40

1.82

1.51

1.50

1.97

1.49

1.69

1.39

-0.02

-0.04

-0.04

-0.04

0.00

-0.03

0.02

0.06

2.90

1.32

-0.06

-0.05

-0.02

0.12

-0.02

-0.03

2.20

2.99

3.02

2.08

0.00

0.99

0.45

1.54

0.04

0.59

1.71

1.06

0.10

0.31

1.52

0.49

0.05

1.43

1.00

0.89

1.33

0.85

0.54

0.11

-0.02

-0.04

-0.04

-0.05

0.00

-0.03

0.00

0.00

2.78

0.66

-0.06

-0.05

-0.02

0.12

-0.03

-0.03

0.04

0.04

0.06

0.07

0.08

0.09

0.11

0.14

0.15

0.16

0.17

0.18

0.18

0.24

0.29

0.33

0.35

0.39

0.51

0.61

0.63

0.64

1.16

1.28

0.00

0.00

0.00

0.00

0.00

0.01

0.03

0.06

0.12

0.66

0.00

0.00

0.00

0.00

0.00

0.00

7

7

7

7

7

7

7

7

8

7

7

7

7

7

7

7

8

7

7

7

7

7

7

7

4

9

7

5

7

8

7

6

7

7

7

7

8

7

7

7

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

27

Polyoxyethylene hydrogenated castoroil(60E.O.)

123-86-4
56-81-5

Alcohol;Ether moiety

Acetal;Alcohol;Alkane, branched with secondary carbon;Alkene moiety;Allyl;Carboxylic acid ester;Ether moiety;Oxolane;Saturated heterocyclic
fragment

Alcohol;Alkane, branched with secondary carbon;Dihydroxyl derivatives

Alcohol;Alkane, branched with secondary carbon;Dihydroxyl derivatives

Alkoxy moiety;Aryl;Aryl halide;Ether moiety

Acetoxy;Carboxylic acid ester

Alcohol;Alkane, branched with secondary carbon;Glycerol and derivatives

81 Plyoxyethylene (160) sorbitan triisostearate

82 Tween20

83 2,4-Pentanediol

84 Propylene glycol

85 4-Bromophenetole

86 Butyl acetate133-06-2
2743-38-6
58-33-3
120-32-1

Alkene moiety ;Alkyl halide;Allyl;Cycloalkene
moiety;Imide;Pyrrolidine;Pyrrolidone/Pyrrolidinedione;Succinimide/Succinimide, thio;Sulfenamide;Tetrahydrophthalimide;Unsaturated carbocyclic fragment

Aryl;Carboxylic acid;Carboxylic acid ester

Alkane, branched with secondary carbon;Amine, tertiary;Ammonium
salt;Aryl;Phenothiazine

Aryl;Aryl halide;Phenol;Precursors quinoid compounds

96 Captan

97 (-)-Dibenzoyl-L-tartaric acid98 Promethazine hydrochloride

140-66-9

108-45-2
9016-45-9

Alkane, branched with quaternary carbon;Alkyl (hetero)arenes;Alkyl-, alkenyland alkynyl (hetero)arenes;Aryl;Phenol;tert-Butyl

Aryl;Carboxylic acid;Phenol

Amine, secondary;Aryl;Imidazole

Aromatic perhalogencarbons;Aryl;Aryl halide;Phenol;Sulfonate ester

Amine, primary;Aniline;Aryl;Phenylenediamine, meta-

Alcohol;Alkyl (hetero)arenes;Alkyl-, alkenyl- and alkynyl(hetero)arenes;Aryl;Ether moiety

101 4‐tert ‐Octylphenol

102 Sodium salicylate

103 Imidazole

104 Tetrabromophenol blue

105 m -Phenylenediamine

106 Nonylphenyl-polyethylene glycol

4430-25-5

288-32-4

54-21-7

107-64-2

Quaternary ammonium salts;Surfactants - Cationic

100 Distearyldimethylammonium chloride

99 2-Benzyl-4-chlorophenol

102-36-3

Aryl;Aryl halide;Isocyanate

2530-83-8

95 3,4-Dichlorophenyl isocyanate

Liquid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Liquid104376-75-2 Liquid

Alcohol;Ether moiety

Alkoxysilane;Epoxide;Ether moiety;Saturated heterocyclic fragment;Silane

67-68-5116998-28-8 Liquid

94 3-Glycidoxypropyltrimethoxysilane

Sulfoxide

LiquidLiquid102-71-6

9005-65-6

LiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquid

623-39-2

588-96-5

57-55-6

625-69-4

123-54-6

25322-68-3 Liquid

Polyoxyethylene (13) (mono—, di, tri-)93
styrenated phenyl ether

Alcohol;Ether moiety

91 Polyethylene (14) tribenzylated phenyl ether

92 Dimethyl sulfoxide

90 Tween80

89 Triethanolamine

88 3-Methoxy-1.2-propanediol

Alcohol;Alkane, branched with secondary carbon;Dihydroxyl derivatives;Ether
moiety;Glycerol and derivatives
Alcohol;Aliphatic amine, primary;Aliphatic amine, secondary;Aliphatic amine,
tertiary;Amine, primary;Amine, secondary;Amine, tertiary;Dihydroxyl
derivatives
Acetal;Alcohol;Alkane, branched with secondary carbon;Alkene moiety;Allyl;Carboxylic acid ester;Ether moiety;Oxolane;Saturated heterocyclic
fragment

9005-64-5

Diketone;Ketone

87 Glycerol

54392-28-8 Liquid

Alcohol;Dihydroxyl derivatives;Ether moiety

80 2,4-Pentanedione

Liquid79 Polyethylene glycol 400

51-03-6

Alkyl (hetero)arenes;Alkyl-, alkenyl- and alkynyl(hetero)arenes;Aryl;Benzodioxole;Benzyl;Ether moiety

LiquidLiquid78 Piperonyl butoxide9002-92-0

99-62-761788-85-0 Liquid

LiquidAlcohol;Ether moiety

Alkane, branched with tertiary carbon;Alkyl (hetero)arenes;Alkyl-, alkenyl- andalkynyl (hetero)arenes;Aryl;Isopropyl

108-94-1

77 Polyoxyethylene 23 lauryl ether (10%)

76 1,3-Di-isopropylbenzene

75

Acylal; Alcohol; Allyl; Ether

Alkane, branched with tertiary carbon;Alkyl (hetero)arenes;Alkyl-, alkenyl- and
135-98-8 Liquidalkynyl (hetero)arenes;Aryl
Alkane, branched with tertiary carbon;Amine, tertiary;Aromatic
21245-02-3 Liquidamine;Aryl;Carboxylic acid ester

73 sec -Butylbenzene

74 2-Ethylhexyl p -dimethyl-amino benzoate

Cycloketone;Ketone

LiquidLiquid72 Cyclohexanone

111-24-0

Alkene moiety ;Alkenes;Allyl;Hydrocarbons

71 1,9-Decadiene

1647-16-1

Alkyl halide

70 1,5-Dibromopentane

1

1

1

1

1

1

1

1

1

1

1

1

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

I

I

I

I

I

I

I

I

I

I

NI

NI

I

NI

NI

NI

NI

I

NI

I

I

NI

NI

I

NI

NI

I

I

NI

NI

NI

NI

NI

NI

I

NI

NI

=0

≥1

≥1

≥3

≥3

≥1

≥3

≥3

≥3

≥3

≥3

≥1

=0

>0

=0

>0

=0

=0

>0

=0

=0

=0

=0

>0

=0

=0

=0

=0

=0

=0

=0

>0

=0

≥2

≥2

≥2

≥2

≥2

≥1

≥1

> 1.5

> 1.5

≥1

21

21

21

Unknown

21

21

6/6; 6/6;
6/6

3/3

3/3; 2/3

3/3; 3/3

21

Unknown

21

21

6/6; 6/6 Unknown

3/3

6/6

3/3

2/3

3/3; 3/3;
3/3

2/3

Conj 2/6

CO 1/321

21

at least 4/6(max 5)at least 2/3

2/3

3/3

6/6

3/3

3/3; 2/3; 1/3

3/3

=4

=4

=4

=4

=4

=4

=4

=4

=4

5/6

3/3

2/3

3/3

1/6

3/3

6/6

3/3

3/3

-0.02

-0.01

3.80

-0.03

0.00

3.48

2.90

3.07

2.63

3.13

2.89

3.62

0.19

-0.06

-0.05

-0.06

-0.07

-0.05

-0.03

-0.04

-0.01

3.58

-0.02

-0.05

-0.01

-0.07

0.04

0.03

3.24

-0.04

3.06

2.63

2.69

3.52

0.39

0.76

2.50

-0.02

-0.01

3.80

-0.03

-0.01

3.47

2.89

3.05

2.60

3.11

2.65

3.03

0.19

-0.06

-0.05

-0.06

-0.07

-0.05

-0.03

-0.04

-0.01

3.58

-0.02

-0.05

-0.01

-0.07

0.04

0.03

3.23

-0.05

3.05

2.62

2.68

3.51

0.37

0.74

2.46

0.00

0.00

0.00

0.00

0.00

0.00

0.02

0.02

0.02

0.02

0.24

0.59

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.01

0.01

0.01

0.01

0.01

0.01

0.02

0.03

0.04

7

8

6

9

7

7

7

7

6

7

7

7

7

7

7

7

7

9

7

7

7

7

7

8

7

7

6

7

7

7

7

7

7

7

7

7

7

✔

✔

28

Amine, primary;Aniline;Aryl;Phenol;Precursors quinoid compounds

Amine, primary;Aniline;Aryl;Dianilines;Sulfone

Amine, primary;Aniline;Aryl;Phenol;Sulfone

Inorganic salt

Carboxylic acid;Quaternary ammonium salts

Aliphatic amine, tertiary;Amine, tertiary;Bridged-ring heterocycles;Saturated
heterocyclic fragment

Aliphatic Amine, tertiary; Carboxylic acid

Amidine;Ammonium salt

Alcohol;Lactons

Ether moiety;Saturated heterocyclic fragment;Trioxane

Inorganic salt

Aryl;Hydrazine derivatives;Pyrazolidine;Pyrazolidinedione/Pyrazolidone

129 2,3‐Indolinedione

130 2‐Aminophenol

131 4,4'-Sulfonylbisbenzenamide

132 Potassium tetrafluoroborate

133 Betaine monohydrate

134 Hexamethylenetetramine

135 EDTA,di-potassium

136 N ,N -Dimethylguanidine sulfate

137 Gluconolactone

138 1,3,5-Trioxane

139 Sodium hydrogen sulfite

140 1-Phenyl-3-pyrazolidinone

33089-61-1
53112-28-0

Alkyl (hetero)arenes;Alkyl-, alkenyl- and alkynyl (hetero)arenes;Amidine;Aryl

Alkyl (hetero)arenes;Alkyl-, alkenyl- and alkynyl (hetero)arenes;Amine,secondary;Aromatic amine;Aryl;Pyrimidine

Aryl;Aryl halide;Ether moiety;Ketoxime derivatives;Pyrimidine

145 Amitraz

146 Pyrimethanil

147 Fluoxastrobin

361377-29-9 Solid

Solid

Solid

161326-34-7 Solid

Amidine;Aryl;Hydrazine derivatives;Sulfide

144 Fenamidone

NC

NC

NC

NC

NC

NC
153719-23-4 Solid

Aryl;Aryl halide;Ether moiety;Guanidine;N-Nitro;Thiazole/Isothiazole

131807-57-3 Solid

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

2B

2B

2B

2B

2B

2B

2A

2A

2A

2A

NC

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

2
2

Solid

110-85-0

92-43-3

7631-90-5

110-88-3

90-80-2

598-65-2

25102-12-9

14075-53-7

80-08-0

95-55-6

91-65-5

120-12-7

92-84-2

Solid
Solid

143 Thiamethoxam

142 Famoxadone

Aliphatic amine, secondary;Amine, secondary;Piperazine;Saturated
heterocyclic fragment
Aryl;Carbamate;Ether moiety;Hydrazine derivatives;Imide;Oxazolidine
derivatives

100-97-0

Anthracene ;Aryl;Fused carbocyclic aromatic;Naphtalene

128 Anthracene

141 Piperazine

590-47-6

Amine, secondary;Aryl;Phenothiazine

127 Phenothiazine494-19-9

94-13-3

120-14-9Aryl;Carboxylic acid ester;Phenol

Aldehyde;Alkoxy moiety;Aryl;Ether moiety

124 3,4‐Dimethoxybenzaldehyde

83-67-0
68610-92-4

Amine, secondary;Aryl

Alcohol; Ammonium salt; Ether

123 Hydroxyethylcellulose ethoxylate, quaternized

125 Propylparaben

Amine, tertiary;Aryl;Dihydropurinedione;Fused heterocyclicaromatic;Imidazole;Imide;Urea derivatives

122 Theobromine

21645-51-2

1450-85-7

99-76-36484-52-2

3926-62-3

104-36-9

79-92-596568-04-6

1119-62-6

143-18-0

105-34-0

1623-08-1

814-71-1

143-07-7

18472-87-2

126 Iminodibenzyl

Aldimine;Alkene moiety ;Cycloalkene moiety

Aryl;Carboxylic acid ester;Phenol

119 Methyl p ‐hydroxybenzoate

Inorganic salt

Organic salts

118 Ammonium nitrate

120 2‐Mercaptopyrimidine

Alkyl halide;Carboxylic acid

117 Sodium monochloroacetate

121 Aluminium hydroxide

Alkoxy moiety;Aryl;Ether moiety

116 1,4-Dibutoxybenzene

Alkene moiety ;Alkenes;Bicycloheptane ;Bridged-ringcarbocycles;Cycloalkane;Hydrocarbons;Terpenes

Aryl;Aryl halide;Carboxylic acid ester;Ketone;Pyridine/Pyridinium ion

115 Camphene

Ethyl 2,6-dichloro-5-fluoro-beta-oxo-3114
pyridinepropanoate

Carboxylic acid ester;Nitrile

111 Methyl cyanoacetate

Alkene moiety ;Allyl;Carboxylic acid;Surfactants - AnionicAryl;Benzyl;Phosphate ester

110 Dibenzyl phosphate

Carboxylic acid;Disulfide

Carboxylic acid;Thiol

109 Calcium thioglycolate

113 3,3'-Dithiodipropionic acid

Carboxylic acid;Surfactants - Anionic

108 Dodecanoic acid

112 Potassium oleate

Aromatic perhalogencarbons;Aryl;Aryl halide;Ether moiety;Heterocyclic spirorings;Lactons;Phenol;Xanthene/ Xanthone

107 Acid red 92
I

NI

NI

NI

NI

NI

NI

I

I

I

I

NI

I

I

I

I

I

NI

I

I

NI

NI

NI

NI

I

NI

NI

NI

NI

I

I

I

NI

NI

I

NI

I

I

I

I

I

≥1

>0

=0

=0

=0

=0

=0

=0

>0

>0

>0

=0

=0

>0

=0

>0

=0

=0

=0

=0

=0

=0

>0

>0

=0

=0

=0

=0

=0

=0

≥1

≥1

≥1

≥3

≥1

≥2

≥2

≥2

≥2

≥2

≥2

≥2

≥1

CO 1/3

CO 1/3;
Conj 1/3

Conj 1/3

3/3

3/3

3/3

3/3

2/3

3/3

3/3; 3/3

3/3; 3/3

3/3

3/3

7

7

7

21

21

7

7

1/3

1/3

2/3

3/3; 1/3

3/3

at least 2/3

=4

=4

3/3

3/3

2.61

2.95

3.16

2.71

1.23

2.79

0.03

2.69

0.00

0.00

-0.01

-0.01

-0.01

-0.02

0.02

0.04

2.73

2.95

2.95

2.84

2.74

3.69

3.43

2.73

0.27

3.51

2.19

2.72

2.13

-0.05

0.02

3.17

2.40

1.89

2.58

0.51

0.00

2.75

2.11

3.19

2.65

2.54

2.95

3.08

2.66

0.98

2.76

0.03

2.66

0.00

0.00

-0.01

-0.01

-0.01

-0.02

0.01

0.03

2.70

2.92

2.92

2.79

2.68

3.62

3.35

2.59

0.09

3.28

1.74

1.89

0.92

-0.05

0.01

3.16

2.39

1.49

1.60

0.51

0.00

2.73

2.07

3.19

2.65

0.07

0.01

0.08

0.05

0.24

0.02

0.00

0.03

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.01

0.02

0.03

0.03

0.05

0.05

0.07

0.08

0.14

0.18

0.23

0.45

0.83

1.21

0.00

0.00

0.00

0.01

0.41

0.97

0.01

0.00

0.02

0.04

0.00

0.01

7

7

7

7

7

7

14

5

5

7

6

7

5

7

7

7

7

7

7

7

7

7

7

7

7

7

7

7

7

8

7

7

7

5

4

9

7

3

10

6

8

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

29

Aryl;Aryl halide;Phenol

79-94-7

101-20-2
103-85-5
118-82-1

Alcohol;Aliphatic amine, secondary;Amine, secondary;Aryl;Phenol

Aryl;Hydrazine derivatives;Pyrazolidine;Pyrazolidinedione/Pyrazolidone

Aryl;Aryl halide;Urea derivatives

Aryl;Thiourea derivatives

Alkyl (hetero)arenes;Alkyl-, alkenyl- and alkynyl(hetero)arenes;Aryl;Phenol;tert-Butyl

Alkane, branched with tertiary carbon;Nitroaliphatic compounds

Aryl;Ketone;Phenol

152 L-Phenylephrine

153 1-Phenyl-3-pyrazolidone

154 3,4,4'-Trichlorocarbanilide

155 N-Phenylthiourea

156 4,4'-Methylenebis(2,6-di-tert-butylphenol)157 2,3-Dimethyl-2,3-dinitrobutane

158 3',5'-Dihydroxyacetophenone

Analyzed by OECD QSAR Toolbox 4.4.1, 2) Barroso et al., Arch Toxicol. 9, 521-547 (2017).

286-62-4

Cycloalkane;Epoxide;Fused saturated heterocycles;Saturated heterocyclic
fragment

151 1,2-Epoxycyclooctane

51863-60-6

3964-18-9

92-43-3

59-42-72736-23-4

Aryl;Aryl halide;Carboxylic acid;Sulfonamide

1)

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

Solid

231278-20-9 Solid

150 2,4-Dichloro-5-sulfamoylbenzoic Acid

4-[3-Chloro-4-(3-fluorobenzyloxy)phenylamino]- Amine, secondary;Aromatic amine;Aryl;Aryl halide;Benzyl;Ether
moiety;Fused heterocyclic aromatic;Pyrimidine;Quinazoline
6-iodoquinazoline

148 Tetrabromobisphenol A

149

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

I

NI

NI

NI

NI

I

I

I

NI

NI

NI

=0

=0

=0

=0

=0

=0

>0

>0

>0

>0

=0

CO 1/3

2.13

2.34

2.45

2.58

3.26

2.55

2.65

3.03

2.73

2.66

2.96

0.89

2.03

1.38

1.76

3.19

2.26

2.56

2.83

2.64

2.64

2.94

1.23

0.31

1.06

0.82

0.07

0.29

0.09

0.20

0.09

0.02

0.02

7

7

7

7

7

7

14

7

3

7

7

✔

✔

✔

✔

✔

✔

✔

OECD/OCDE

494

Adopted:14 June 2021

OECD GUIDELINE FOR THE TESTING OF CHEMICALS

Vitrigel®-Eye Irritancy Test Method for Identifying Chemicals Not Requiring
Classification and Labelling for Eye Irritation or Serious Eye Damage
INTRODUCTION
1.
Serious eye damage refers to the production of tissue damage in the eye, or
serious physical decay of vision, which is not fully reversible, occurring after exposure of
the eye to a substance or mixture, 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 substance or mixture. Test chemicals
that induce serious eye damage are classified as UN GHS Category 1, and those thatinduce eye irritation are classified as UN GHS Category 2, which includes subcategories
2A or 2B. Test chemicals that are neither Category 1 nor Category 2 do not requireclassification for eye irritation or serious eye damage and are referred to as UN GHS No
Category.
2.
The assessment of serious eye damage and eye irritation has historically involved
the use of laboratory animals as described in OECD Test Guideline (TG) 405, which was
adopted in 1981 and revised in several occasions (2). The choice of the most appropriate
test method and the use of this TG should be seen in the context of the OECD GuidanceDocument (GD) 263 on Integrated Approaches to Testing and Assessment (IATA) for
Serious Eye Damage and Eye irritation (3). However, this method does not address the
toxicity and reversibility aspects of ocular toxicity. Therefore, consideration would need to
be given to all possible mechanisms of ocular toxicity that may be relevant to the test
chemical, based on existing data and knowledge as outlined in GD263 (3) when selecting
the combination of tests to use for classification purposes.
3.
The Vitrigel®-Eye Irritancy Test (EIT) method is an in vitro test method that allows
the identification of test chemicals not requiring classification and labelling for eye irritationor serious eye damage (UN GHS No Category) as defined by the UN GHS (1) without
further testing (4, 5, 6), and therefore is performed in a bottom-up approach, as suggested
by Scott et al (7). However, the Vitrigel®-EIT method is not intended to identify nor
differentiate between UN GHS Category 1 and UN GHS Category 2. This differentiation
will need to be addressed by another tier of a testing strategy (3).
4.
This TG is based on a protocol developed by Yamaguchi and Takezawa (8), which
was subjected to a validation study by a validation management team (VMT) organizedby the Japanese Centre for the Validation of Alternative Methods (JaCVAM) in
cooperation with the International Collaboration on Alternative Test Methods (ICATM) (9).
The validation study was carried-out by three participating Japanese laboratories. The
validation report was evaluated by an independent peer-review panel composed of© OECD, (2021)
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international experts (10). Further, the OECD Expert Group concluded that the Vitrigel®EIT method is valid for use as an initial step in a bottom-up approach for identification oftest chemicals not requiring classification and labelling for eye irritation or serious eye
damage (UN GHS No Cat. chemicals).
5.
This TG describes a procedure for assessing the eye irritation potential of a test
chemical based on its ability to induce damage to the barrier function of the human corneal
epithelium (hCE) models used in the Vitrigel®-EIT method. In existing in vitro test methods,
the viability of cells in culture in vitro or the corneal opacity of isolated eyeballs ex vivo
have been utilized as an endpoint. It is known that chemicals that are irritating to the eye
first destroy tear film and epithelial barrier function of the eye, subsequently induce
epithelial cell death, and finally produce stromal degeneration and endothelial cell death,resulting in corneal opacity (11, 12). Therefore, the change of the epithelial barrier function
is a relevant endpoint for detecting eye irritation (13, 14). In the Vitrigel®-EIT method, timedependent changes in the Transepithelial Electrical Resistance (TEER) values areindicative of damage to the barrier function of the corneal epithelium following exposure
to a test chemical; this situation is similar to the observed damage of the rabbit cornea
following exposure to a test chemical, which is an important mode of action leading todamage of the corneal epithelium and eye irritation (4, 13).
6.
The term “test chemical” is used in this TG to refer to the chemicals being tested
and is not a reference to the applicability of the Vitrigel®-EIT method to the testing ofchemicals. The term “test chemical preparation” is used to describe the mixture of the test
chemical with a culture medium (see paragraph 27).
7.

Definitions are provided in ANNEX 1 - .

PRINCIPLE OF THE TEST8.
The Vitrigel®-EIT method is an in vitro assay using hCE models fabricated in acollagen vitrigel® membrane (CVM) chamber (5). The eye irritation potential of the test
chemical is predicted by analyzing time-dependent changes in TEER values using thescore of three indexes (see the prediction model in Table 2).
9.
The Vitrigel®-EIT method makes use of the destructive activity of the test chemical
against the barrier function of hCE models as an endpoint to assess the extent of damage
to the hCE model. The test chemical is dissolved or suspended in the culture medium
before exposure of the hCE model to prevent a delay of the reaction due to slow
dissolution. In a previous study it was observed that the TEER values of the hCE models
decreased immediately after exposing the test chemical preparations and became
constant within 3 minutes (4). Therefore, the exposure period of the hCE model to the test
chemical preparation was limited to 3 minutes.

INITIAL CONSIDERATIONS AND LIMITATIONS
10.
The Vitrigel®-EIT can be applied to test chemicals absorbing light in the samerange as formazan dye, and test chemicals able to directly reduce the tetrazolium dye(see paragraph 16). However, test chemical preparations of both solids and liquids
showing acidity (pH ≤ 5) and rapid phase separation are not in the applicability domain of
the test method as explained below. When the absolute difference of the absorbancevalues of the 2.5% weight/volume (w/v) test chemical preparation at 0 and 3 minutes isgreater than 0.1, the chemical should not be tested. See paragraph 28 for further details.

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11.
The results of the validation study showed within-laboratory reproducibility to be
80–100% at all three laboratories and a between-laboratory reproducibility of 92%. The
predictive capacity was evaluated based on validation and the developer’s in-house datafor 93 chemicals (9, 15, 16, 17). The Vitrigel®-EIT method achieved a sensitivity of 83%(50/60), a specificity of 70% (23/33), and an accuracy of 78% (73/93). The assay, as
described in this paragraph, would not be the method of choice because of its limited
predictivity (i.e., low specificity).
12.
Analysis of the false-negative reactions showed that five of the 10 false-negative
chemicals were acidic, and the 2.5% w/v preparations used for exposure had a pH level
lower than five. Typically, the TEER values of the hCE model after exposure to UN GHS
No Category chemicals changed little from their initial TEER values. The TEER values of
the hCE models increased after exposure to the five acidic test chemicals that yielded
false-negatives as previously reported (18, 19). Also, water-insoluble solids that readily
separate from the culture medium may yield false-negative results. It should be noted thatthese chemicals lead to variable and extreme exposure conditions in the in vivo Draize
eye irritation test, which may result in irrelevant predictions of their true irritation potential(2). The Vitrigel®-EIT method can be applied not only to liquids but also to solids by
performing a pre-test to exclude solid test chemical preparations showing pH ≤ 5 and/or
rapid phase separation during the testing time of three minutes (see paragraph 28).
13.
Following these considerations, 158 test chemicals comprising 94 liquids and 64
solids were tested. Among these 158 tested chemicals, 22 solids were excluded on thebasis of the pre-test: 7 showed a pH≤ 5 (2 of which also showed rapid phase separation),and 15 showed rapid phase-separation only; 29 liquids were excluded: 5 showed acidity(2 of which also showed rapid phase- separation) and 24 showed rapid phase-separation
only as well. Accounting for these exclusions, the sensitivity, specificity and accuracy of
the assay for the 107 test chemicals remaining in-domain is 96% (51/53), 67% (36/54)
and 81% (87/107), respectively. Table 1 shows the predictive capacity for the test liquids
and solids separately.
Table 1. Predictive Capacity
Liquids

SolidsSensitivity: 100% (34/34)
Specificity: 71% (22/31)
Accuracy: 86% (56/65)

Sensitivity: 89% (17/19)
Specificity: 61% (14/23)
Accuracy: 74% (31/42)

The two false negative chemicals were GHS Cat. 2B (mild eye irritants).
14.
The test method shows a high percentage of false positive results. The false
positive rates obtained with the Vitrigel®-EIT method are not critical in the context of thisTG since all test chemicals that result in “No Stand-alone Prediction Can Be Made” will
require further information and/or testing, depending on regulatory requirements,
according to the OECD GD 263 (3). The appropriate regulatory authorities should be
consulted before using the test method under classification schemes other than UN GHS.
15.
A limitation of this TG is that it does not allow discrimination between eye
irritation/reversible effects on the eye (UN GHS Category 2) and serious eye
damage/irreversible effects on the eye (UN GHS Category 1), nor between eye irritants(optional Category 2A) and mild eye irritants (optional Category 2B), as defined by UN
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(3).
16.
This TG is technically applicable to mono-constituent substances, multiconstituent substances, substances of unknown or variable composition, complex
reaction products or biological materials (UVCBs). Mixtures, gases and aerosols,
however, have not been assessed in the validation study. When considering testing of
mixtures, difficult-to-test chemicals (e.g. unstable or water reactive), or test chemicals notclearly within the applicability domain described in this TG , upfront consideration should
be given to whether the results of such testing will yield results that are meaningful
scientifically. Such considerations are not needed, when there is a regulatory requirement
for testing of the mixture. Test chemicals absorbing light in the same range as formazan
dye and test chemicals able to directly reduce the tetrazolium dye MTT can also be tested
using the Vitrigel®-EIT method.
17.
Any test chemical satisfying the criteria of the pre-test described above (i.e., test
chemical preparations showing pH > 5 and keeping dissolution or homogeneous
dispersion for at least three minutes) in a 2.5% w/v concentration in culture medium canbe tested with the Vitrigel®-EIT method. Test chemicals that do not dissolve readily canbe tested after using one of the following techniques: a) mix mechanically using a vortex
mixer, b) sonication, and/or c) heating to a maximum temperature of 70°C (SeePROCEDURE).

DEMONSTRATION OF PROFICIENCY
18.
Prior to routine use of the Vitrigel®-EIT method described in this TG, laboratories
should demonstrate technical proficiency by correctly classifying the 10 substances
recommended in Table 32 in ANNEX 2 These substances were selected to represent thefull range of responses for serious eye damage or eye irritation based on results of TG
405 on in vivo rabbit eye tests (2) and the UN GHS classification system (1). Other
selection criteria stipulated that the proficiency substances should be commerciallyavailable, with high-quality in vivo reference data and high-quality in vitro data from theVitrigel®-EIT method available (9, 10). In situations where a listed substance is unavailable
or cannot be used for other justifiable reason, it should be substituted with anotherproficiency substance for which adequate in vivo and in vitro reference data are available.

PROCEDURE
19.
The following paragraphs describe the main components and procedures of theVitrigel®-EIT method, being referred as the Validated Reference Method (VRM)(8).
Testing should be performed in accordance with Good In Vitro Method Practices (20).
Values specified in this protocol as integers are considered to be accurate to one
additional significant digit. Thus, “37oC” indicates an acceptable range from 36.5oC to
37.4oC.
Culture of hCE cells
20.

Immortalized hCE cells 1 are maintained in a culture medium comprising a 1:1

1 HCE-T cells, RCB no. 2280 obtained from RIKEN BioResource Research Center, Tsukuba, Japan. MTA with RIKEN

BRC are required to perform this Test Guideline.

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mixture of Dulbecco’s modified Eagle medium and nutrient mixture F-12 supplemented
with 5% heat-inactivated fetal bovine serum, 5 μg/mL recombinant human insulin,
10 ng/mL recombinant human epidermal growth factor, 0.5% dimethyl sulfoxide,
100 units/mL penicillin, and 100 μg/mL streptomycin. Cells are grown at 37°C in ahumidified atmosphere of 5% CO₂
2 in air. The cells should be free of contamination bybacteria, viruses, mycoplasma, and fungi except for the application of test chemical
preparations to hCE models.
Preparation of CVM chambers
21.
A collagen xerogel membrane chamber (i.e. ad-MED Vitrigel®, as used for the2VRM ) is set in the well of a 12-well plate and immersed in the culture medium by pouring
1.5 mL outside and 0.5 mL inside the chamber in the well and waiting for 10 minutes toallow the xerogel to convert into vitrigel® immediately before use. If alternative chambers
are used, hCE models prepared in the chamber should show the appropriate TEER
values.
Fabrication of a hCE model
Culture procedure
22.
The culture medium outside the chamber in the well of the 12-well plate is replacedwith 1.5 mL of the fresh medium. The medium inside the chamber is carefully removed byusing a micropipette and 0.5 mL of the cell suspension in the culture medium at a densityof 1.2 × 105 cells/mL is poured onto the CVM in the chamber and cultured for two days at
37°C. After carefully removing the inside medium by using a micropipette and changing
the outside medium to fresh medium, the cells are cultured for four more days at the airliquid interface to obtain the hCE model. On the third day of culture at the air-liquidinterface, the medium outside the chamber is changed.
Quality check23.
The hCE model should possess sufficient robustness equivalent to hCE in order
to avoid rapid disruption after chemical exposure. The barrier function of each hCE model
is checked by measuring its TEER value. First, 500 μL of fresh culture medium is pouredin the chamber of the hCE models and the temperature of the culture medium is adjusted
to 28±2°C. Next, the longer electrode of a TEER Measuring System (refer to the section“Measurement of TEER value in a hCE model”) is set into the culture media outside thechamber, and the shorter electrode is set into the culture media inside the chamber, after
which the TEER value of each hCE model (pre-exposure TEER values) is measured. OnlyhCE models with a TEER value within adequate range are acceptable for the testing ofchemicals conducted on the same day. For the VRM, hCE models with a TEER value
between 140 Ω·cm2 and 220 Ω·cm2 are acceptable for the testing.
Measurement of TEER value in a hCE model
24.
TEER values of the hCE model should be measured by using an electricalresistance meter with low-voltage and alternating current. General specifications of the
instrument are an alternating current of 50–1,000 Hz and a measuring range of at least
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0.1–3 kΩ. Photographic images of the TEER measuring system for the VRM 3 are shownin Annex 3. If an alternative apparatus is used, it should be demonstrated that it produces
the same results. The electrode unit is 23 mm in diameter and 35 mm in height. The innerelectrode is positioned inside the chamber, and the outer electrode is positioned outside
the chamber. The distance between the inner and outer electrode is fixed, because thisdistance affects the electrical resistance value obtained. Also, during resistancemeasurement, the depth to which the electrodes are submerged in the medium or buffer
solution inside and outside of the chamber is also fixed. The electrical resistance value ofthe hCE model cultured in the CVM chamber (Rmodel) and that of a blank empty CVM
chamber (Rblank) are measured. The TEER value of a hCE model is calculated asfollows:
TEER value of a hCE model (Ω·cm2) ={Rmodel (Ω) – Rblank (Ω)} × effective surface area (cm2)25.
The sensitivity of the TEER Measuring System should be checked before testing,and adequate ranges should be provided. This can be achieved by measuring theelectrical resistance of two or more solutions having different conductivities, thereby
confirming that the differences of these conductivities are within the predetermined value.
For the VRM, pre-operation check of the TEER Measuring System is performed asfollows. The individual CVM-free chamber (ad-MED Vitrigel® without a CVM) is set in two
wells of a 12-well plate, and subsequently one well is filled with 3.0 mL of 0.90% NaClaqueous solution and another well is filled with 0.45% NaCl aqueous solution at 25±5°C.
Then, the TEER values in both wells are measured using the TEER Measuring System.
The TEER measurement is functioning normally when the measured TEER values satisfy
the following conditions.

(TEER value of 0.45% NaCl aqueous solution)− (TEER value of 0.90% NaCl aqueous solution) ≥ 60 Ω·cm2

Preparation of Control Substances
26.
The Vitrigel®-EIT method uses saline as a negative control, benzalkonium chloride
as a positive control, and ethanol which induces a response in the medium range as areference control. The reference control is used to check the quality of the hCE models.
Control substance solutions are prepared in the culture medium at a concentration of 2.5%
w/v by adding 0.1–0.2 g of saline, benzalkonium chloride, or ethanol to a 15-ml tube,
pouring an appropriate volume of the culture medium into the tube, and mixing until
dispersed uniformly. In addition, benchmark substances are useful for evaluating theocular irritancy potential of unknown chemicals, or for evaluating the relative ocular
irritancy potential of a chemical within a specific range of irritant responses.

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Preparation of Test Chemicals
27.
A test chemical solution or suspension is prepared in the culture medium at aconcentration of 2.5% w/v, as the electrical resistance value of 2.5% w/v preparation
usually has little or no effect on the electrical resistance of the culture medium irrespectiveof the test chemical conductivity. The test chemical is manually mixed in the medium until
dissolved or for a maximum of one minute. If the test chemical does not dissolve readily,
one of the following techniques is used, listed here in order of preference:a) mix mechanically for a maximum of one minute using a vortex mixer,b) sonication for a maximum of 20 minutes, orc) heating to a maximum temperature of 70°C.
After mixing, the temperature of the test chemical preparation is adjusted to 28±2°C using
a hot plate, a water bath, or an air conditioner, and the solubility of the test chemical ischecked by visual inspection. The next step is taken only once the test chemical
preparation is well dissolved or homogeneously dispersed. For test chemicals that prove
to be insoluble or immiscible using the above techniques, the test chemical preparation isprepared as a homogeneous suspension by vortexing the test chemical in the medium for
up to one minute immediately before use.
28.
The pH of each 2.5% w/v test chemical preparation is measured using a universal
pH test paper covering a range from pH 1 to 11 or a pH meter. If pH of a 2.5% w/v
preparation is ≤ 5 the chemical should not be tested. Also, absorbance at 660 nm of the
2.5% w/v test chemical preparation is measured by a UV-VIS spectrophotometer at 0 and
3 minutes after the preparation. However, a different wavelength has to be used in casethe absorption of the test chemical at this wavelength disturbs the measurement. In thecase where the absolute difference measured is above 0.1, the chemical should not be
tested.
Application of the Test Chemicals and Control Substances29.
The hCE models that pass the quality check can be used for exposure to the test
chemical. The hCE model should be subjected to the chemical exposure experiment
within two hours after drawing it from a CO2 incubator. The medium inside the chamber isreplaced with 500 μL of test chemical preparation, and Rmodel values are measured atintervals of 10 seconds for a period of three minutes after exposure to the test chemical
preparation. At least three hCE models should be used for each control substance solution
and each test chemical preparation in each run. The eye irritation potential of the test
chemical is predicted using the result of one run.
30.
To ensure reproducibility, it is essential that measurements begin between two to
five seconds after adding the test chemical preparation. A minimum of a two-second wait
before beginning measurements is necessary, because the liquid around the electrode isoften unstable for up to two seconds after adding the test chemical preparation. However,it should begin before 5 seconds after adding the test chemical preparation, as the TEER
value of the hCE model changes in the presence of the test chemical for over five seconds.
31.
The temperature of the hCE models and the test chemical preparations should bemaintained at 28±2°C during the chemical exposure tests. This can be done using a hot
plate, a water bath, or an air conditioner. The temperature of the hCE model can bechecked by measuring the actual temperature of culture medium outside the hCE model.

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Prediction Model
32.
The TEER values of the hCE model after exposure to a test chemical is calculatedusing the formula given above in the section “Measurement of TEER value in a hCE
model”. The mean TEER values for all three tests are analyzed by using the following
three indexes: time lag (t1), intensity (− [P2 − P1] / [t2 − t1]), and plateau level (100 − P2).
Annex 4 provides a graph showing an analysis of a TEER profile after exposure of an hCE
model to a test chemical. The score of each index is calculated. The test chemical isidentified as not requiring classification and labelling according to UN GHS (No Category)
if the scores of the indexes are Time lag > 180 seconds and Intensity < 0.05 %/seconds
and Plateau level ≤ 5.0 %, as shown in Table 2. In this case, no further testing with anothertest method is considered necessary. If the scores of the indexes are Time lag ≤ 180
seconds or Intensity ≥ 0.05 %/seconds or Plateau level > 5.0 %, then no stand-alone
prediction can be made from this result in isolation, as shown in Table 2. This is because
in case of a true positive, the method cannot resolve between UN GHS Categories 1 and2. Furthermore, the Vitrigel®-EIT method shows a high percentage of false positive results(see paragraph 14). In both cases, further information and or testing will be required forclassification purposes according to GD 263 (3).
Table 2. Prediction Models according to UN GHS classification*
Criteria

Time lag ≤ 180 seconds
or Intensity≥0.05 %/seconds orPlateau level >5.0 %Time lag > 180 seconds
and Intensity<0.05 %/seconds andPlateau level ≤ 5.0 %Prediction
No Stand-alone Prediction
Can Be Made1

No Category2

Notes:
1No Stand-alone Prediction Can Be Made corresponds to chemicals that require further information forclassification purposes according to the IATA guidance document (3).
2No Category corresponds to chemicals that do not require classification for serious eye damage or eye
irritation according to UN GHS* Consideration would need to be given to all possible mechanisms of ocular toxicity that may be relevant to
the test chemical, based on existing data and knowledge as outlined in GD263 (3), when deriving aclassification.

Acceptance Criteria
33.

Test run is judged to be acceptable when the following four criteria are all satisfied:a) Negative control: The plateau level is ≤ 5%.
b) Positive control: The plateau level is ≥ 40%c) Reference control: The plateau level is ≥ 10%.
d) The average standard deviation of the overall TEER profile for each test
chemical is ≤ 15%.

The range of historical results for the positive control in the validation study is from 65%
to 90%.

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DATA AND REPORTING
Data
34.
TEER values obtained for each individual hCE model, the scores of each index,
and the final prediction by the Vitrigel®-EIT method should be reported.
Test Report
35.

The test report should include the following information:
Test Chemical and Control Substances•

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•Multi-constituent substance, UVCB and mixture: Characterization as far as
possible by e.g., chemical identity (see above), purity, quantitative
occurrence and relevant physicochemical properties (see above) of theconstituents, to the extent possible•

Physical state, pH, volatility, molecular weight, chemical class, andadditional relevant physicochemical properties relevant to the conduct of
the study, to the extent possible•

Purity, chemical identity of impurities as appropriate and practically feasible,
etc.

•

Treatment prior to testing, if applicable (e.g., warming)•

Storage conditions and stability to the extent possible

Test Method Conditions and Procedures•

Name and address of the sponsor, test facility and study director•

Description of the test method used•

Details of test procedure used•

Cell line used, its source, passage number and confluence of cells used fortesting•

Supplier, catalog number and lot number of a reagent•

Time and date of sub-culturing hCE cells, duration of tripsinization, dilution
ratio of the cells•

Duration of each step for preparation of hCE models•

Data of QC check for TEER measuring system•

Record of test chemical preparation (e.g. weight of test chemical, volume of
medium, mixing method and solubility of test chemical, pH of the test
chemical preparation, and absorbance values at 660 nm values at 0 and 3
minutes of the test chemical preparation)•

Temperature of the hCE models and test chemical preparation at the startof exposure test

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Lot number of a hCE model•Time of removal of the hCE model from CO2 incubator, and exposure of thehCE model to the test chemical•Time of starting TEER measurement by a TEER Measuring System•

Test chemical
recommended)•

Duration of exposure to the test chemical (if different than the one
recommended)•

Number of runs and number of hCE models used within each run (if different
than recommended)•

Description of any modifications of the test procedure•Statement that the testing facility has demonstrated proficiency in the useof the test method before routine use by testing of the proficiency chemicals

concentrations

used

(if

different

than

the

ones

Results•

For each test chemical and control substance, tabulation should be given
for preexposure TEER values and time dependent TEER values after
exposing test chemicals for three minutes for each hCE model used, scoresof three indexes, and the in vitro prediction of the test chemical.

•

Description of other effects observed

Discussion of the Results
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LITERATURE
1. United Nations UN (2019). Globally Harmonized System of Classification andLabelling of Chemicals (GHS). Eighth revised edition. New York & Geneva: United
Nations Publications. ISBN: 9789210040839. Available at: [https://doi.org/10.18356/f8fbb7cb-en].
2. OECD. (2020). Test No. 405: Acute Eye Irritation/Corrosion. Organisation for
Economic Cooperation and Development, Paris.
3. OECD (2019). Guidance Document No. 263 on Integrated Approaches to Testingand Assessment (IATA) for Serious Eye Damage and Eye Irritation (Second
Edition). Publications, Organisation for Economic Cooperation and Development,
Paris.
4. Takezawa T, et al. (2011). Development of a human corneal epithelium modelutilizing a collagen vitrigel membrane and the changes of its barrier function
induced by exposing eye irritant chemicals. Toxicol in Vitro 25, 1237–1241.
5. Yamaguchi H, et al. (2013). Vitrigel-eye irritancy test method using HCE-T cells.
Toxicol Sci 135, 347–355.
6. Yamaguchi H, et al. (2016). Predictive performance of the Vitrigel-eye irritancy test
method using 118 chemicals. J Appl Toxicol 36, 1025-1037.
7. Scott L, et al. (2010) A proposed eye irritation testing strategy to reduce andreplace in vivo studies using Bottom-Up and Top-Down approaches. Toxicol. In
Vitro 24: 1-9.
8. Yamaguchi H and Takezawa T. (2019). Standard Protocol for the Vitrigel-EIT
method, Version 1.82e. (May 20 2019). Available at: [https://www.jacvam.jp/files/list/04/04_09_Z7.pdf].
9. Kojima H, et al. (2019). Multi-laboratory Validation Study of the Vitrigel-Eye
Irritancy Test Method as an Alternative to In Vivo Eye Irritation Testing. Altern Lab
Anim. 47, 140-157.
10. OECD (2019), Report of the Validation Study and Report of the Peer Review of the
Validation of the Vitrigel Eye Irritancy Test Method in Test Guideline 494. Series
on Testing and Assessment No. 301. Organisation for Economic Cooperation andDevelopment, Paris.
11. Reim M. (2003). The Ocular Surface: Barrier Function and Mechanisms of Injury
and Repair, in Alternative Toxicological Methods (Salem H and Katz SA eds) pp.
89-108, CRC Press, Boca Raton.
12. Movahedan A, et al. (2013). Loss of Notch1 Disrupts the Barrier Repair in theCorneal Epithelium. PLoS One 8, e69113
13. Uematsu M, et al. (2007). Acute corneal epithelial change after instillation of
benzalkonium chloride evaluated using a newly developed in vivo corneal
transepithelial electric resistance measurement method. Ophthalmic Res. 39, 308–
314.
14. Meloni M, et al. (2010). Occludin gene expression as an early in vitro sign for mild
eye irritation assessment. Toxicol in Vitro 24, 276–285.

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15. ECETOC. (1998). Eye Irritation Reference Chemicals Data Bank. Technical Report(No. 48. (2)), Brussels, Belgium.
16. National Institute of Environmental Health Sciences. (2010). Appendix J:
NICEATM Analysis: Reduced Eye Hazard Labeling Resulting from Using Globally
Harmonized System (GHS) Instead of Current U.S. Regulatory Classification
Criteria. ICCVAM Test Method Evaluation Report: Current Validation Status of In
Vitro Test Methods Proposed for Identifying Eye Injury Hazard Potential ofChemicals and Products, National Institutes of Health Publication No. 10-7553,
National Institutes of Health (2010): http://ntp.niehs.nih.gov/iccvam/docs/ocutox_docs/invitro-2010/appj-analysis.pdf.
17. ICCVAM. (2013). Short Time Exposure (STE) Test Method Summary Review
Document. NIH. Available at: http://www.ntp.niehs.nih.gov/iccvam/docs/ocutox_
docs/STE-SRD-NICEATM508.pdf.
18. Farré R, et al. (2008). Short exposure of oesophageal mucosa to bile acids, bothin acidic and weakly acidic conditions, can impair mucosal integrity and provoke
dilated intercellular spaces. Gut 57, 1366-1374.
19. Oshima, et al. (2012). Acid modulates the squamous epithelial barrier function bymodulating the localization of claudins in the superficial layers. Lab. Invest 92,
2231.
20. OECD (2018), Guidance Document on Good In Vitro Method Practices (GIVIMP),
Series on Testing and Assessment No. 286. Organisation for Economic
Cooperation and Development, Paris.
21. OECD (2005), Guidance Document on the Validation and International Acceptanceof New or Updated Test Methods for Hazard Assessment. Environmental, Healthand Safety Publications, Series on Testing and Assessment No. 34. Organisation
for Economic Cooperation and Development, Paris.
22. Takezawa T, et al. (2004). Collagen vitrigel: a novel scaffold that can facilitate athree-dimensional culture for reconstructing organoids. Cell Transplant 13, 463–
473.
23. Takezawa T, et al. (2007). Reconstruction of hard connective tissue utilizing apressed silk sheet and type-I collagen as the scaffold for fibroblasts. Tissue
Engineering 13, 1357–1366.
24. Takezawa T, et al. (2007). A protein-permeable scaffold of a collagen vitrigel
membrane useful for reconstructing crosstalk models between two different cell
types. Cells Tissues Organs 185, 237–241.
25. Takezawa T, et al. (2007). Collagen vitrigel membrane useful for paracrine assays
in vitro and drug delivery systems in vivo,” Journal of Biotechnology 131, 76–83.
26. Takezawa T, et al. (2008). A novel culture model of rabbit corneal epithelium
utilizing a handy scaffold of collagen vitrigel membrane and its cryopreservation,”
Alternatives to Animal Testing and Experimentation 13, 176.
27. OECD (2021), Performance Standards on Vitrigel-Eye Irritancy Test Method for
Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation
or Serious Eye Damage. Series on Testing and Assessment No. 313. Organisation
for Economic Cooperation and Development, Paris.

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ANNEX 1 - DEFINITIONS
Accuracy: The closeness of agreement between test method results andaccepted reference values. It is a measure of test method performance and one
aspect of relevance. The term is often used interchangeably with “concordance” tomean the proportion of correct outcomes of a test method (21).
Applicability domain: A description of the physicochemical or other properties of
the chemicals for which a test method is applicable for use (21).
Bottom-Up Approach: A step-wise approach used for a test chemical suspectedof not requiring classification for eye irritation or serious eye damage, which starts
with the determination of chemicals not requiring classification (negative outcome)from other chemicals (positive outcome) (3).
Chemical: means a substance or mixture.
Collagen vitrigel membrane (CVM): A membrane composed of high density
collagen fibrils modelling the connective tissues in vivo and is easily handled withtweezers. Also, it possesses excellent transparency and permeability of proteinwith high molecular weight and consequently provides an ideal cell culture scaffold(22-26). The CVM chamber is prepared from a commercially available collagen
xerogel membrane chamber.
Effective surface area: The bottom surface area of the CVM chamber.
Eye irritation: The production of changes in the eye, which are fully reversible,
occurring after the exposure of the eye to a substance or mixture (1).
False negative rate: The proportion of all positive chemicals falsely identified bya test method as negative. It is one indicator of test method performance (21).
False positive rate: The proportion of all negative (non-active) chemicals that are
falsely identified as positive. It is one indicator of test method performance (21).
Hazard: The potential for an adverse health or ecological effect. The adverse effectis manifested only if there is an exposure of sufficient level (21).
hCE: human corneal epithelium.
Mixture: A mixture or a solution composed of two or more substances in which
they do not react.
MoA: mode of action.
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 amanufacturing 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.
Negative control: A sample containing all components of a test system andtreated with a substance known not to induce a positive response in the test
system. This sample is processed with test chemical-treated samples and othercontrol samples and is used to check the durability of the hCE models.

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Performance standards: Standards, based on a validated test method, thatprovide a basis for evaluating the comparability of a proposed test method that ismechanistically and functionally similar. Included are (i) essential test method
components; (ii) a minimum list of reference chemicals selected from among thechemicals used to demonstrate the acceptable performance of the validated test
method; and (iii) the comparable levels of accuracy and reliability, based on what
was obtained for the validated test method, that the proposed test method should
demonstrate when evaluated using the minimum list of reference chemicals (21).
Positive control: A sample containing all components of a test system and treatedwith a substance known to induce a positive response. To ensure that variability inthe positive control response across time can be assessed, the magnitude of thepositive response should not be excessive.
Reference control: A sample containing all components of a test system andtreated with a substance known to induce a middle class response in the system.
This sample is processed with test chemical-treated samples and other control
samples and is used to check the quality of the hCE models.
Relevance: Description of relationship of the test to the effect of interest andwhether it is meaningful and useful for a particular purpose. It is the extent to which
the test correctly measures or predicts the biological effect of interest. Relevance
incorporates consideration of the accuracy (concordance) of a test method (21).
Reliability: Measures of the extent that a test method can be performed
reproducibly within and between laboratories over time, when performed using thesame protocol. It is assessed by calculating intra- and inter-laboratory
reproducibility and intra-laboratory repeatability (21).
Run: A run consists of one or more test chemicals tested concurrently with anegative control, a positive control and a reference control.
Sensitivity: The proportion of all positive/active substances that are correctlyclassified by the test. It is a measure of accuracy for a test method that produces
categorical results and is an important consideration in assessing the relevance ofa test method (21).
Serious eye damage: The production of tissue damage in the eye, or serious
physical decay of vision, which is not fully reversible, occurring after exposure of
the eye to a substance or mixture (1).
Specificity: The proportion of all negative/inactive substances that are correctlyclassified by the test. It is a measure of accuracy for a test method that produces
categorical results and is an important consideration in assessing the relevance ofa test method (21).
Substance: Chemical elements and their compounds in the natural state or
obtained by any production process, inducing 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 the stability of thesubstance or changing its composition (1).
Test chemical: The term "test chemical" is used to refer to what is being tested.
Tiered testing strategy: A stepwise testing strategy where all existing information
on a test chemical is reviewed, in a specified order, using a weight of evidence
process at each tier to determine if sufficient information is available for a hazard
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of a test chemical can be assigned based on the existing information, no additional
testing is required. If the irritancy potential of a test chemical cannot be assigned
based on the existing information, a stepwise sequential animal testing procedure
is performed until an unequivocal classification can be made (21).
Top-Down Approach: step-wise approach used for a test chemical suspected of
causing serious eye damage, which starts with the determination of chemicals
inducing serious eye damage (positive outcome) from other chemicals (negative
outcome) (3).
Transepithelial electrical resistance (TEER): The electrical resistance of anepithelium or epithelial cell layers. It is considered a suitable means (index) for
evaluating the integrity of the tight junction of corneal epithelium.
United Nations Globally Harmonized System of Classification and Labellingof Chemicals (UN GHS): A system proposing the classification of chemicals(substances and mixtures) according to standardized 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: Serious eye damage/irreversible effects on the eye (1).
UN GHS Category 2: Eye irritation/reversible effects on the eye (1).
UN GHS Category 2A: Irritating to eyes (1).
UN GHS Category 2B: Mildly irritating to eyes (1).
UN GHS No Category: Chemicals that are not classified as UN GHS Category 1
or 2 (2A or 2B) (1).
UVCB: Substances of unknown or variable composition, complex reaction
products or biological materials.
Vitrigel: The ad-MED Vitrigel® collagen xerogel membrane chamber was used for
the VRM. The definition of vitrigel is a gel in a stable state produced by rehydration
after the vitrification of a traditional hydrogel (22, 23, 24, 25, 26). The literal elementfor “VITRIGEL” is a trademark of National Agriculture and Food Research
Organization (NARO) and registered in Japan, USA, EU and China.
VRM: Validated Reference Method.

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ANNEX 2 - PROFICIENCY CHEMICALS FOR THE VITRIGEL®-EYE IRRITANCYTEST METHOD
Prior to routine use of a test method that adheres to this Test Guideline,
laboratories should demonstrate technical proficiency by correctly identifying theeye hazard classification of the 10 chemicals recommended in Table 3. The
Vitrigel®-Eye Irritancy Test Method outcomes provided represent examples of theresults observed during its validation study (9, 10). The selection includes, insofar
as possible, chemicals that(i) cover the full range of in vivo serious eye damage/eye irritation responses
based on the UN GHS classification system (i.e., Categories 1, 2A, 2B or
No Category),(ii) are based on high quality results obtained in the reference in vivo rabbit eye
test (OECD TG 405), (2)(iv) cover a broad range of the chemical classes and organic functionalgroups, representative of those used in the validation study, (10)(v)

cover the range of in vitro responses based on high quality Vitrigel®-EIT
data,(vi) produced correct and reproducible predictions in the VRM,(vii) are commercially available, and(viii) are not prohibitively expensive either to acquire or dispose of.
In situations where a listed chemical is unavailable or cannot be used for otherjustifiable reason, it should be substituted with another chemical that fulfills thecriteria described above, e.g. from the chemicals used in the validation of theVitrigel®-Eye Irritancy Test Method or listed as a reference chemical within the
Performance Standards (27).
Table 3. Recommended chemicals for demonstrating technical proficiency with theVitrigel®-Eye Irritancy Test Method

1)

CASRN and physicochemical properties

Chemical Name
In vivo UN GHS Category 11
1
3-(2-Aminoethylamino)
propyl]trimethoxysilane
2
Tetraethylene
glycol diacrylate
3
Sodium salicylate
In vivo UN GHS Category 2A1
4
Cyclopentanol
5
Methyl cyanoacetate
In vivo UN GHS Category 2B1
6Ethyl-2-methylacetoacetate
7
Ammonium nitrate
In vivo UN GHS No Category1
8
iso-Octylthioglycolate
9Tetrabromobisphenol A
10
4,4'-Sulfonylbisbenzenamide

CASRN

Organic Functional Group

Physical StatepH

1760-24-3

Silicon compound

Liquid1017831-71-9

Acrylate, Ester

Liquid7

54-21-7

Organic salts

Solid

7

96-41-3
105-34-0

Alcohols
Esters, Nitrile compounds

LiquidSolid

77609-14-3
6484-52-2

EstersInorganic salts

LiquidSolid

7825103-09-7
79-94-7
80-08-0Thiocompound, Ester
Aryl; Aryl halide Phenol
Dianilines, Sulfone

Liquid
Solid
Solid

7
7
7

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Test results

Chemical Name
In vivo UN GHS Category 11

Time lag (seconds)2
Mean± Min.
Max
SD

Intensity (%/seconds)2
Mean± SD Min.
Max

Plateau level (%)2
Mean± Min.
Max
SD

1

3-(2-Aminoethylamino)
propyltrimethoxysilane

0±0

0

0

0.36 ± 0.05

0.33

0.41

64 ± 860

73

2

Tetraethylene
glycol diacrylate

7 ± 12020

0.23 ± 0.02

0.20

0.24

40 ± 535

43

3Sodium salicylate

0±0

0

0

0.48 ± 0.11

0.35

0.54

41 ± 338

43

In vivo UN GHS Category 2A1
4

Cyclopentanol

0±0

0

0

0.29 ± 0.01

0.28

0.30

52 ± 2

51

55

5

Methyl cyanoacetate

10 ± 100200.10 ± 0.06

0.06

0.17

18 ± 111130

In vivo UN GHS Category 2B1
6Ethyl-2methylacetoacetate

3±6

0

10

0.22 ± 0.03

0.19

0.25

41 ± 634

46

7

Ammonium nitrate

0±0

0

0

0.69 ± 0.12

0.58

0.82

29 ± 17

18

49

>180

>180

>180

-0.01 ± 0.01

-0.02

0.00

1±1

0

2

In vivo UN GHS No Category1
8

iso-Octylthioglycolate

Prediction

No Stand-alone
Prediction
Can Be MadeNo Stand-alone
Prediction
Can Be MadeNo Stand-alone
Prediction
Can Be MadeNo Stand-alone
Prediction
Can Be MadeNo Stand-alone
Prediction
Can Be MadeNo Stand-alone
Prediction
Can Be MadeNo Stand-alone
Prediction
Can Be Made
No Category

Tetrabromobisphenol
No Category
>180
>180
>180
-0.02 ± 0.01
-0.02
-0.01
0±0
0
0
A
4,4'-Sulfonylbis
No Category
10
>180
>180
>180
-0.02 ± 0.01
-0.03
-0.01
0±0
0
0
benzenamide
Abbreviations: CASRN, Chemical Abstracts Service Registry Number; UN GHS, United Nations Globally Harmonized System ofClassification and Labelling of Chemicals; VRM, Validated Reference Method.
9

1

Based on results from the in vivo rabbit eye test (OECD TG 405) (2) and using the UN GHS. (1)Based on results obtained with validation Study of the Vitrigel®-EIT method (9,10). Data for chemical No. 5, 7, 9 and 10 are basedon results obtained with the developer’s in-house data. Each score was calculated from the data of three runs. Three hCE models
were used for each run.

2

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ANNEX 3 - PHOTOGRAPHIC IMAGES OF THE TEER MEASURING SYSTEMThe TEER measuring system (A) and the electrode unit (B).

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ANNEX 4 - GRAPH SHOWING AN ANALYSIS OF A TEER PROFILE AFTEREXPOSURE OF A MODEL TO A TEST CHEMICAL

Notes:
t1 (second); The maximum time at which a profile is maintained at 0 ≥ dP/dT > -0.03%/second.
t2 (second); The initial time at which the profile is maintained at 0 ≥ dP (P3 - P2)/dT (t3 - t2) >−0.03%/second after the profile is maintained at dP/dT ≤ -0.03%/second.
t3 (second); t2 + 30 seconds because the plateau level is evaluated by the profile for 30 seconds.
P1 (%); The percentage of TEER value at t1 against the TEER value at 0 second.
P2 (%); The percentage of TEER value at t2 against the TEER value at 0 second.
P3 (%); The percentage of TEER value at t3 against the TEER value at 0 second.
dP/dT; The derivative of P with respect to t.

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