新規試験法提案書

2018年改定 OECD TG 438
ニワトリ眼球を用いた眼刺激性試験（ICE法 : Isolated Chicken Eye Test）

令和元年11月

国立医薬品食品衛生研究所

新規試験法提案書
令和元年 11 月 18 日
No.

2019-032018 年改定 OECD TG 438 ニワトリ眼球を用いた眼刺激性試験（ICE 法 : Isolated Chicken Eye Test）に関する提案
令和元年 11 月 14 日に国立医薬品食品衛生研究所にて開催された新規試験法評価会議（通称：JaCVAM 評価会議）において以下の提案がなされた。
提案内容： 本試験法は、化学物質による眼刺激性を評価でき、トップダウン方式において重篤な眼の傷害を引き起こす物質ならびにボトムアップ方式において眼刺激物質として分類されない物質を識別するという用途の範囲において行政的利用は可能であると考える。
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 2018 年改定 OECD TG 438 ニワトリ眼球を用いた眼刺激性試験（ICE 法 : Isolated Chicken Eye Test）の使用を提案するものである。

大野泰雄
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
大野泰雄

（公益財団法人 木原記念横浜生命科学振興財団）： 座長

五十嵐良明（国立医薬品食品衛生研究所）

石井雄二（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）

稲若邦文（日本化学工業協会）

井上智彰（日本免疫毒性学会）

今井教安（日本動物実験代替法学会）

岩瀬裕美子（日本製薬工業協会）

久保文宏（独立行政法人 医薬品医療機器総合機構）

杉山真理子（日本化粧品工業連合会）*

中村るりこ（独立行政法人 製品評価技術基盤機構）

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）

西村次平（独立行政法人 医薬品医療機器総合機構）

沼澤（日本毒性学会）

聡

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）

廣田衞彦（日本化粧品工業連合会）**

増村健一（日本環境変異原学会）

横関博雄（日本皮膚免疫アレルギー学会）
任期：平成 30 年 4 月 1 日～令和 2 年 3 月 31 日*：平成 30 年 4 月 1 日～平成 31 年 3 月 31 日**：平成 31 年 4 月 1 日～令和 2 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

池田三恵（独立行政法人 医薬品医療機器総合機構）

石井孝司（国立感染症研究所）

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）

奥田晴宏

（国立医薬品食品衛生研究所）

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

（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）

聡

高木篤也

（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）

高畑正浩（厚生労働省 医薬・生活衛生局 医薬品審査管理課）

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）

野坂佳伸（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）

広瀬明彦（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）

笛木修（独立行政法人 医薬品医療機器総合機構）

渕岡学（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）

本間正充（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）

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 2018 revision ofOECD Test Guideline 438: Isolated Chicken Eye Test Method for Eye Irritation
or Serious Eye Damage
At a meeting held on 14 November 2019 at the National Institute of Health Sciences (NIHS)
in Kanagawa, Japan, the Japanese Center for the Validation of Alternative Methods (JaCVAM)
Regulatory Acceptance Board unanimously endorsed the following statement:
Proposal: We propose the Isolated Chicken Eye Test Method to be useful in a regulatory
context as part of a top-down approach to identifying chemicals that induce serious
eye damage (Category 1) or as part of a bottom-up approach to identifying
chemicals that do not require classification for eye irritation or serious eye damage
under the United Nations Globally Harmonized System of Classification andLabelling of Chemicals (UN GHS).
This statement was prepared to acknowledge that the results of a review and study by the
JaCVAM Regulatory Acceptance Board have confirmed the usefulness of this assay.
Based on the above, we propose the 2018 revision of OECD TG 438 as a useful means for
estimating eye irritation by regulatory agencies.

Yasuo Ohno
Chairperson
JaCVAM Regulatory Acceptance Board

Yoko Hirabayashi
Chairperson
JaCVAM Steering Committee

November 18, 2019v

The JaCVAM Regulatory Acceptance Board was established by the JaCVAM Steering
Committee and is composed of nominees from industry and academia.
This statement was endorsed by the following members of the JaCVAM Regulatory
Acceptance Board:
Mr. Yasuo Ohno (Kihara Memorial Yokohama Foundation for the Advancement ofLife Sciences): Chairperson
Ms. Yoko Hirabayashi (Center for Biological Safety and Research: CBSR, National
Institute of Health Sciences: NIHS)
Mr. Morihiko Hirota (Japan Cosmetic Industry Association)**Mr. Yoshiaki Ikarashi (NIHS)
Mr. Noriyasu Imai (Japanese Society for Alternatives to Animal Experiments)
Mr. Kunifumi Inawaka (Japan Chemical Industry Association)
Mr. Tomoaki Inoue (Japanese Society of Immunotoxicology)
Mr. Yuji Ishii (CBSR, NIHS)
Ms. Yumiko Iwase (Japan Pharmaceutical Manufacturers Association)
Mr. Fumihiro Kubo (Pharmaceuticals and Medical Devices Agency)
Mr. Kenichi Masumura (Japanese Environmental Mutagen Society)
Ms. Ruriko Nakamura (National Institute of Technology and Evaluation)
Mr. Akiyoshi Nishikawa (CBSR, NIHS/ Saiseikai Utsunomiya Hospital)
Mr. Jihei Nishimura (Pharmaceuticals and Medical Devices Agency)
Mr. Satoshi Numazawa (Japanese Society of Toxicology)
Ms. Mariko Sugiyama (Japan Cosmetic Industry Association)*
Mr. Hiroo Yokozeki (Japanese Society for Cutaneous Immunology and Allergy)
Term: From 1st April 2018 to 31st March 2020*: From 1st April 2018 to 31st March 2019**: From 1st April 2019 to 31st March 2020

vi

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

Ms. Yoko Hirabayashi (CBSR, NIHS): Chairperson
Mr. Manabu Fuchioka (Ministry of Health, Labour and Welfare)
Mr. Osamu Fueki (Pharmaceuticals and Medical Devices Agency)
Mr. Akihiko Hirose (Division of Risk Assessment, CBSR, NIHS)
Mr. Masamitsu Honma (Division of Genetics and Mutagenesis, CBSR, NIHS)
Ms. Mie Ikeda (Pharmaceuticals and Medical Devices Agency)
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. Yoshinobu Nosaka (Ministry of Health, Labour and Welfare)
Ms. Kumiko Ogawa (Division of Pathology, CBSR, NIHS)
Mr. Haruhiro Okuda (NIHS)
Mr. Atsuya Takagi (Animal Management Section of the Division of Toxicology,
CBSR, NIHS)
Mr. Masahiro Takahata (Ministry of Health, Labour and Welfare)
Mr. Masaaki Tsukano (Ministry of Health, Labour and Welfare)
Mr. Takao Ashikaga (Division of Risk Assessment, CBSR, NIHS): Secretary
Mr. Hajime Kojima (Division of Risk Assessment, CBSR, NIHS): Secretaryvii

2018年改定 OECD TG 438 ニワトリ眼球を用いた眼刺激性試験（ICE法 : Isolated Chicken Eye Test）

目

次

評価会議報告書 ------------------------------------------------------------------------------------ 1
評価報告書 ------------------------------------------------------------------------------------------ 7
OECD GUIDELINE FOR THE TESTING OF CHEMICALS 438, Isolated chicken eye test
method for identifying I) chemicals inducing serious eye damage and II) chemicals not requiringclassification for eye irritation or serious eye damage -----------------------------------------------19

評価会議報告書
2018 年改定 OECD TG 438
ニワトリ眼球を用いた眼刺激性試験（ICE 法 : Isolated Chicken Eye Test）

JaCVAM 評価会議

令和元年（2019 年）9 月 30 日

11

JaCVAM 評価会議
大野泰雄

（公益財団法人 木原記念横浜生命科学振興財団）： 座長

五十嵐良明（国立医薬品食品衛生研究所）

石井雄二（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）

稲若邦文（日本化学工業協会）

井上智彰（日本免疫毒性学会）

今井教安（日本動物実験代替法学会）

岩瀬裕美子（日本製薬工業協会）

久保文宏（独立行政法人 医薬品医療機器総合機構）

杉山真理子（日本化粧品工業連合会）*

中村るりこ（独立行政法人 製品評価技術基盤機構）

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）

西村次平（独立行政法人 医薬品医療機器総合機構）

沼澤（日本毒性学会）

聡

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）

廣田衞彦（日本化粧品工業連合会）**

増村健一（日本環境変異原学会）

横関博雄（日本皮膚免疫アレルギー学会）
任期：平成 30 年 4 月 1 日～令和 2 年 3 月 31 日*：平成 30 年 4 月 1 日～平成 31 年 3 月 31 日**：平成 31 年 4 月 1 日～令和 2 年 3 月 31 日

22

ニワトリ眼球を用いた眼刺激性試験（ICE 法 : Isolated Chicken Eye Test）は、ニワトリから摘出した眼球に被験物質を曝露して生じる角膜の変化をもとに in vivo の眼刺激性を予測する試験であり、ウサギを用いた Draize 眼刺激性試験法（Draize 法）の代替法である。眼に重篤な損傷を引き起こす化学物質（UN GHS : United Nations Globally Harmonized System of Classificationand Labeling of Chemicals 1）区分 1 物質）をトップダウン方式により検出する方法として、2009
年に OECD TG 438 として採択された 2)。2013 年に採択された改定 TG 438 3)では、トップダウン方式により UN GHS 区分 1 物質を、ボトムアップ方式により UN GHS 区分に該当しない物質を判定する場合の基準が定められており、JaCVAM 評価会議において妥当性が確認されている 4)。今回、ICE 法の再評価が行われ、2018 年改定 TG 438 5)が採択された。本改定では、ボトムアップ方式による UN GHS 区分に該当しない物質の判定基準が変更され、偽陽性率が改善された。また、トップダウン方式による区分 1 の判定において、これまで偽陰性率の高かった洗浄剤と界面活性剤の評価性能を向上させるため、病理組織学的検査の併用が追加された。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「2018 年改定
OECD TG 438 ニワトリ眼球を用いた眼刺激性試験（ICE 法 : Isolated Chicken Eye Test）」6) を用いて、本試験法の妥当性について検討した。
１．試験法の定義
名称：2018 年改定 OECD TG 438 ニワトリ眼球を用いた眼刺激性試験（ICE 法 : Isolated Chicken
Eye Test）
代替する対象毒性試験：Draize 眼刺激性試験法
試験法の概略：ICE 法では、ニワトリから摘出した眼球に被験物質を曝露し、その結果、眼球の角膜に生じる変性を、角膜の腫脹、混濁度およびフルオレセイン染色性の変化としてとらえ、個別のスコアに変換して得られる総合評価をもとに in vivo での眼刺激性を予測する。本試験では、被験物質群および陽性対照群は 3 眼球以上、媒体対照群および陰性対照群は 1 眼球以上を使用する。角膜の腫脹は、光学的厚度計を装着した細隙灯顕微鏡を用いて角膜の厚さを曝露後 240 分間測定し、経時的な変化率として定量的に求める。角膜の混濁度は、細隙灯顕微鏡にて角膜混濁度の経時的な変化を曝露後 240 分間観察し、結果を評点化する。フルオレセイン染色性は、細隙灯顕微鏡にて曝露 30 分後の角膜表面のフルオレ
セイン染色性を観察し、評点化する。得られた結果を項目毎に眼刺激性の最も弱いクラス
I から最も強いクラス IV の 4 段階に分類し、それらの分類結果を総合して、被験物質の眼刺激性を判定する。
2018 年改定点：
1）UN GHS 区分に該当しない物質のボトムアップ方式における判定基準に「1 項目がクラスⅠに分類され、2 項目がクラスⅡに分類される」場合が加わった。この基準の下に、新たな物質を加えて再構築したデータにおいて正確性が再評価された結果、TG 438（2013）の判定基準と比較して、偽陰性率は低く抑えられたまま、偽陽性率が改善された。

33

2）トップダウン方式による UN GHS 区分１物質の判定に、病理組織学的検査が追加された。2<pH<11.5 の洗浄剤および界面活性剤について、トップダウン方式において偽陰性率が高いことから、標準的な ICE 法で陰性結果が得られた際には、病理組織学的検査を併用して判定することを推奨する。ICE 法に用いた眼球の病理組織標本を作製して、角膜上皮のびらん、空胞化および壊死、角膜間質の核濃縮の程度および線維の乱れ、ならびに角膜
内皮の壊死の有無を基準に刺激性を評点化する。評点を区分 1 物質の評価基準に照らし、合致した場合に UN GHS 区分 1 と判定し、合致しない場合には予測不可とする。
3）ICE 法および病理組織学的検査における習熟度確認物質一覧が更新された。
２．評価に用いた資料および評価内容の科学的妥当性
本試験法は OECD にて TG 438（2009）、改定 TG 438（2013）を経て、改定 TG 438（2018）
として採択された。今回、改定 TG 438（2018）においてボトムアップ方式の判定基準を改定するにあたり、新たな物質を加えて再構築したデータ（184 物質）において再評価が行われた 5)。
その結果、Draize 法との比較では、ICE 法の正確度は 88%（161/184）、感度 97%（98/101）、特異度は 76%（63/83）、偽陰性率は 3 %（3/101）および偽陽性率は 24%（20/83）を示し、
TG 438（2013）の判定基準と比較して、偽陰性率は低く抑えられたまま、偽陽性率は改善された。 また、トップダウン方式において偽陰性率が高かった洗浄剤および界面活性剤について、検出感度を向上させるため、病理組織学的検査の併用について検証が行われた 7)8)。これ
らの結果を受けて、2<pH<11.5 の洗浄剤および界面活性剤 29 物質について JaCVAM 眼刺激性試験資料編纂委員会が解析した結果、in vivo の判定との比較において、病理組織学的検査を併用することにより、感度が 33%から 71%に向上し、正確度も 52%から 76%に上昇した。
評価会議では、評価資料は適切であり、TG 438 改定における評価方法の変更は科学的に妥当であると考える。

３．本試験法の有用性と適用限界
ICE 法は生きた動物を用いないため代替法として有用性がある。2018 年の改定 TG 438 に基づいて正確性が向上したことから、UN GHS の眼刺激性分類においてトップダウン方式における区分 1 物質の同定およびボトムアップ方式における UN GHS 区分に該当しない物質の同定に ICE 法を適用することは可能であると考える。なお、ボトムアップ方式で陽性結果が得られた場合、他の適切な試験法による確認が必要とされている。また、トップダウン方式
においては、2<pH<11.5 の洗浄剤および界面活性剤については、陰性結果が得られた場合、病理組織学的検査を併用することが推奨される。
４．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

4
4

社会的受け入れ性：
本試験法は、食用として屠殺されたニワトリの眼球を用いるため、Draize 法よりも社会的受け入れ性は高い。今回の改定においては、動物福祉の観点からの変更がないことから、本試験法の社会的受け入れ性は、改定前と変わらない。
行政上の利用性：
本試験法は、化学物質による眼刺激性を評価でき、トップダウン方式において UN GHS 区
分 1 物質（重篤な眼の傷害を引き起こす物質）ならびにボトムアップ方式において UN GHS
区分に該当しない物質（眼刺激物質として分類されない）を識別するという用途の範囲において行政的利用は可能であると考える。

参考文献
1) United nations (UN) (2017). Globally Harmonized System of Classification and Labeling ofChemicals (GHS), Seventh revised edition, UN New York and Geneva, 2017. Available at:[https://www.unece.org/fileadmin/DAM/trans/danger/publi/ghs/ghs_rev07/English/03e_part3.pdf]2) OECD Guidelines for The Testing of Chemicals, Isolated Chicken Eye Test for Identifying

Ocular Corrosives and Severe Irritants, TG 438 (Adopted: 7 September 2009)
3) OECD Guidelines for The Testing of Chemicals, Isolated Chicken Eye Test Method for

Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring
Classification for Eye Irritation or Serious Eye Damage, TG 438 (Adopted: 26 July 2013)
4) JaCVAM評価会議：眼刺激性試験代替法の評価会議報告書 2013年改訂 OECD TG 438 ニワトリ眼球を用いた眼刺激性試験（ICE 法: Isolated Chicken Eye Test）（2014年10月28日）
5) OECD Guidelines for The Testing of Chemicals, Isolated Chicken Eye Test Method for

Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring
Classification for Eye Irritation or Serious Eye Damage, TG 438 (Adopted: 25 June 2018)6) JaCVAM資料編纂委員会：眼刺激性試験代替法評価報告書 2018年改定 OECD TG 438 ニワトリ眼球を用いた眼刺激性試験（ICE 法: Isolated Chicken Eye Test）（2019年9月26日）
7) Cazelle E., Eskes C., Hermann M., et al. (2014). Suitability of Histopathology as an Additional

Endpoint to the Isolated Chicken Eye Test for classification of non-extreme pH detergent.
Toxicology In Vitro 28, 657-666.

8) Cazelle E., Eskes C., Hermann M., et al. (2015). Suitability of the Isolated Chicken Eye Test
for Classification of Extreme pH Detergents and Cleaning Products. Toxicology In Vitro 29,
609-616.

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評価報告書
評価報告書
2018 年改定 OECD TG 438
2018 年改定 OECD TG 438
ニワトリ眼球を用いた眼刺激性試験
ニワトリ眼球を用いた眼刺激性試験（ICE 法 : Isolated Chicken Eye Test）（ICE 法 : Isolated Chicken Eye Test）

眼刺激性試験資料編纂委員会
眼刺激性試験資料編纂委員会

令和元年（2019 年）9 月 26 日
令和元年（2019 年）9 月 26 日

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1 / 127眼刺激性試験資料編纂委員会
山 本 直 樹 （藤田医科大学）
小 島

肇 （国立医薬品食品衛生研究所）

佐々木正治 （アッヴィ合同会社）
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）森 村 智 美 （一般財団法人食品薬品安全センター）

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要旨
ニワトリ眼球を用いた眼刺激性試験（ICE 法 : Isolated Chicken Eye Test）は、ニワトリから摘出した眼球に被験物質を曝露して生じる角膜の変化をもとに、 in vivo の眼刺激性を予測する試験であり、OECD TG 438 として試験法が標準化されている。2013 年に採択された改定 TG 438（2013）には、トップダウン方式により UN GHS 区分 1 物質を、ボト
ムアップ方式により UN GHS 区分に該当しない物質を判定する場合の基準が定められており、既に JaCVAM 評価会議において妥当性が確認されている。本報告では、2018 年 6月に採択された改定 TG 438（2018）について改正点の概要を説明し、バリデーション研
究報告書、第三者評価報告書および関連論文などをもとに、JaCVAM 眼刺激性試験資料編纂委員会としての意見をまとめた。
改定 TG 438（2018）では、ボトムアップ方式による区分に該当しない物質の判定基準が変更され、偽陽性率が改善された。また、トップダウン方式による UN GHS 区分 1 の判定において、これまで偽陰性率の高かった界面活性剤の評価を向上させるため、病理
組織学的検査の併用が検証された。この併用により、2<pH<11.5 の洗浄剤および界面活性剤（29 物質）の感度は 33%から 71%に向上し、偽陰性率は 67%から 29%に抑制された。
また、12 の界面活性剤を用いた 3 施設間の再現性は 83%であった。
以上より、本委員会は、ボトムアップ方式による判定基準の変更は妥当であり、また、
2<pH<11.5 の洗浄剤および界面活性剤については、トップダウン方式により UN GHS 区
分 1 を検出する際、病理組織学的検査を補助的に用いることができると結論した。
略語
BCOP: Bovine Corneal Opacity and Permeability
BRD: Background Review Document
CM: Cytosensor Microphysiometer
CV: Coefficient of Variation
EURL ECVAM: European Union Reference Laboratory for Alternatives to Animal Testing
GHS: Globally Harmonized System of Classification and Labeling of Chemicals
ICCVAM: Interagency Coordinating Committee on the Validation of Alternative Methods
ICE: Isolated Chicken Eye
JaCVAM: Japanese Center for the Validation of Alternative Methods
NICEATM: NTP Interagency Center for the Evaluation of Alternative Toxicological Methods
OECD: Organization for Economic Co-operation and Development
SSD: Streamlined Summary Document
TG: Test Guideline
UN: United Nations

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1．改定の背景
ICE 法は、ウサギを用いた Draize 眼刺激性試験法（Draize 法）の代替法であり、トッ
プダウン方式により眼に重篤な損傷を引き起こす化学物質（UN GHS 1) 区分 1 物質）を検出する方法として、2009 年に OECD で TG 438（2009）として採択された。その後、眼刺激性に関する UN GHS 分類 について、in vivo と in vitro、双方のデータベースが再評価され、トップダウン方式における区分 1 物質の検出のみならず、ボトムアップ方式で UN
GHS 区分 1 物質及び眼刺激性を引き起こす化学物質（UN GHS 1) 区分 2 物質）のいずれの区分にも該当しない物質も検出することが可能であると判断され、2013 年に改定
TG 438（2013）として採択された。
今回、ICE 法の再評価が行われ、 改定 TG 438（2018）2)が採択された。主な改正点は、
ボトムアップ方式による区分に該当しない物質検出のための判定基準の変更と、トップダウン方式による UN GHS 区分 1 を識別するエンドポイントとして 2<pH<11.5 の洗浄剤
および界面活性剤についての病理組織学的検査の併用である。本資料編纂委員会は、この TG 438（2018）の改定点について検討した。以下にその 結果を報告する。
2．標準的な ICE 法の概要
ICE 法では、ニワトリから摘出した眼球に被験物質を曝露し、その結果、眼球の角膜に生じる変性を角膜の腫脹、混濁度およびフルオレセイン染色性の変化としてとらえ、
個別のスコアに変換して得られる総合評価をもとに in vivo での眼刺激性を予測する。
試験では、被験物質群および陽性対照群は 3 眼球以上、媒体対照群および陰性対照群
は 1 眼球以上を使用する。角膜の腫脹は、光学的厚度計を装着した細隙灯顕微鏡を用いて角膜の厚さを被験物質曝露後 240 分間測定し、経時的な変化率として定量的に求める。
角膜の混濁度は、細隙灯顕微鏡にて角膜混濁度の経時的な変化を曝露後 240 分間観察し、結果を評点化する。フルオレセイン染色性は、細隙灯顕微鏡にて曝露 30 分後の角膜表面のフルオレセイン染色性を観察し、評点化する。得られた結果を項目毎に眼刺激性の最も弱いクラス I から最も強いクラス IV の 4 段階に分類し、それらの分類結果を総合して、被験物質の眼刺激性を判定する。
3．改定点
3−1．ボトムアップ方式による UN GHS 区分に該当しない物質判定基準の変更
TG 438（2013）に制定された UN GHS 区分に該当しない物質のボトムアップ方式における判定基準に「1 項目がクラスⅠに分類され、2 項目がクラスⅡに分類される」場合が加わった（表 1）。
表 1.

被験物質を UN GHS 区分に該当しないと評価するための判定基準 2)

UN GHS 区分
区分外

3 評価項目の組み合わせ
3 項目ともクラスⅠに分類される
2 項目がクラスⅠに分類され、1 項目がクラスⅡに分類される
1 項目がクラスⅠに分類され、2 項目がクラスⅡに分類される

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区分外：UN GHS による眼に対する重篤な損傷性および眼刺激性のいずれの区分にも該当しない。「用語」参照。
上記の基準の下に、新たな物質を加えて再構築したデータにおいて正確性が再評価された。その結果、表 2 に示すように、Draize 法との比較では、ICE 法の正確度は 88%（161/184）、感度 97%（98/101）、特異度は 76%（63/83）、偽陰性率は 3 %（3/101）および偽陽性率は 24%（20/83）を示した。TG 438（2013）の判定基準と比較して、偽陰性率は低く抑えられたまま、偽陽性率は改善された。なお、ボトムアップ方式で陽性結果が得られた場合、他の適切な試験法による確認が必要とされている。また、固体で陰性結果が得られた場合、3 眼球を用いて 2 回目の実験を行い、陰性結果を承認または棄却することが推奨されている。

表 2.

ボトムアップ方式における ICE 法の正確性－UN GHS 区分法での区分に該当しない物質の同定 2)

ボトムアップ
方式

No.

判定基準
（2018）
判定基準（2013）

正確度
%

No.

感度

偽陰性率

特異度

偽陽性率

%

No.

%

No.

%

No.

%

No.

184

88 161/184 97

98/101

3

3/101

76

63/83

24

20/83

152

82 125/152 99

72/73

1

1/73

67

53/79

33

26/79

なお、トップダウン方式においても、新たな物質が加わったデータにおいて区分 1 物
質を検出する場合の正確性が再評価され、これらの予測性は、更新前と同程度であった。

3−2．トップダウン方式による UN GHS 区分１物質の判定
3−2−1．病理組織学的検査の追加
病理組織学的検査を行う場合、ICE 法に用いた眼球（被験物質を曝露した 3 眼球全て）の病理組織標本を作製して評価する。10%中性緩衝ホルマリン、4 %グルタールアルデヒ
ドまたは 2.5%グルタールアルデヒドと 2 %ホルムアルデヒド混合液で固定した眼球（角膜）を常法に従い脱水、パラフィン包埋、薄切し、HE 染色あるいは PAS 染色等を施した病理組織標本を作製する。角膜上皮のびらん、空胞化および壊死、角膜実質（固有層）の核濃縮の程度および線維の乱れ、ならびに角膜内皮の壊死の有無を基準に刺激性を評点化する（表 3）。

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表 3.

ニワトリ眼球の病理組織標本のための半定量的評点化システム 2)

指標上皮：びらん上皮：空胞化上皮を上、中、下の各層に分けてス
コア化
上皮：壊死

実質：核濃縮上部または下部領域
実質の線維異常
内皮：壊死

判定
ごく軽度
軽度
中等度
重度
ごく軽度
軽度

スコア
1/2
1
2
3
1/2
1

中等度
重度
正常範囲
ごく軽度
軽度
中等度

2
3
1/2
1
2

重度

3

正常範囲
軽度
中等度

－
1
2

説明
数個の細胞から最表層 1 層までの欠損
3 層以下の欠損50%までの上皮層の欠損
基底膜に達する上皮層の欠損
空胞化細胞が 1 個～数個散在性にみられる
空胞化細胞の集簇または小空胞を有する細胞が単一層にみられる上皮の 50%まで空胞化細胞が占める上皮の 50～100%を空胞化細胞が占める
細胞質が膨化した細胞 10 個未満
細胞質が膨化した細胞 10～20 個
細胞質が膨化した細胞 20～40 個
細胞質が膨化した細胞が多数認められるが上皮層の 50%未満を占める場合
細胞質が膨化した細胞が上皮層の 50～100%を占める場合5 個未満の細胞の核濃縮5～10 個の細胞の核濃縮10 個を超える細胞の核濃縮

あり
あり

P
P

不規則な線維内皮は単層のため、グレード分けはしない

表 3 の刺激性のスコアを区分 1 物質の評価基準（表 4）に照らし、合致した場合にUN GHS 区分１と判定し、合致しない場合には予測不可とする。
表 4.

UN GHS 区分 1 物質の同定のため、ICE 法に加えて実施することが推奨される病理
組織学的検査の評価基準 2)

組織層

眼に重篤な損傷を引き起こす可能性（UN GHS区分1）の評価基準
・少なくとも 3 眼中 2 眼で中等度（スコア 2）以上のびらんを示す
・3 眼中 2 眼で角膜上皮の中層ないし下層におけるごく軽度の空胞化（スコア 1/2）を伴う

角膜上皮

・3 眼中 1 眼で中程度（スコア 2）以上のびらんを示した場合、少なくともその他の 2 眼中 1 眼で角膜上皮の中層ないし下層におけるごく軽度の空胞化（スコア 1/2）を伴う・少なくとも 3 眼中 2 眼で中等度（スコア 2）以上の壊死を示す

さらに、表 3 のうち、角膜の空胞化については、洗浄剤および界面活性剤による空胞化の好発部位を考慮し、形態的な背景値に対して表 5 の鑑別基準が示されている。

6 / 1212表 5.

背景値として認められる小さな空胞と真の空胞化を鑑別するための基準 3)空胞の大きさ

発生部位

真の空胞化であるか?
（Yes/No）

上皮細胞の核の大きさの1/3 またはそれ以上

上皮細胞内 (不特定)Yes

基底膜に接する部位

No

上皮細胞内の基底膜側から数えて 1 列目の核の層よりも上Yes

基底膜と 1 列目の核の層との間追加情報を基に判断*上皮細胞の核の 1/3よりも小さい

*段階的評価：1) 他 2 つの眼球標本を観察し、2 つとも作用が認められない場合は真の空胞化ではな
いと判断し、2 つのうち 1 つの眼球ではっきりした作用が認められた場合は真の空胞化と判断する。
2) 他 2 つの眼球標本を観察してもはっきりしない場合、例えば固形物による局所的な作用の可能性を考慮するといった標準的な ICE 法での観察結果も参考にする。
これら上皮細胞の空胞化の他、界面活性剤による組織変化の特徴として、曝露および培養によって角膜上皮細胞と基底膜との接着性が低下したことにより生じた空隙や、刺激物質によって脆弱となった細胞-細胞間に病理標本作製過程で間隙が生じることが知られている。
なお、組織標本の評価は検査担当者の見解の基に成り立っていることから、病理組織学的検査は、ICE 法の組織標本観察に習熟した病理専門家が行った上で、OECD GLP
No.16 4) に準じて、別の病理学専門家によるピアレビューを実施することを定めている。
3−2−2．病理組織学的検査の追加結果
TG 438（2013）の検証 5)において偽陰性率が高かった洗浄剤および界面活性剤について、検出感度を向上させるため、病理組織学的検査の併用が検証された。検証では、まず洗浄剤（1 種類以上の界面活性剤を 3 %超含む洗浄用混合物、界面活性剤単体の希釈液は含まない）のうち、2<pH<11.5 の 30 物質 6)、pH≤2 の 9 物質および 11.5≤pH の 9 物
質 7) について病理組織学的検査を併用し、重篤な眼の損傷を引き起こす可能性（UNGHS 区分 1）を評価した。その結果、2<pH<11.5 の洗浄剤では、病理組織学的検査の併用により感度が向上した（0%から 75%、n=8）6)。一方、pH≤2 または 11.5≤pH の洗浄剤
では、病理組織学的検査の併用により、偽陽性率が高くなり（17％から 67％、n=12）、
その結果、特異性が 83%から 33%に低下した 7)。
これらの結果を受け、2<pH<11.5 の洗浄剤および界面活性剤に限り病理組織学的検査を併用することが検討され、最終的に 30 物質について検証された 8)。このうちの 1 物質がアルカリ洗剤（pH=12.0）であったことから、本委員会では、それを除く 29 物質につい7 / 1213

て解析した（表 6）。
表 6.

最終評価に用いた 29 物質の内訳 8)
区分 1

区分 2

区分外

界面活性剤

13

2

3

洗浄剤 (2 < pH < 11.5)

8

2

1

区分外：区分１および区分２のいずれにも該当しない。
その結果、表 7 に示すように、in vivo の判定との比較において、病理組織学的検査を併用することにより、感度が 33%から 71%に向上し、正確度も 52%から 76%に上昇した。
表 7.

トップダウン方式において病理組織学的検査を併用し 2<pH<11.5 の洗浄剤および界面活性剤を判定した場合の正確性の検証結果 2)

病理組織学的
検査

No.

併用あり
（2018)
併用なし（2013）

正確度

感度

偽陰性率

特異度

偽陽性率

%

No.

%

No.

%

No.

%

No.

%

No.

29

76

22/29

71

15/21

29

6/21

87

7/8

13

1/8

29

52

15/29

33

7/21

67

14/21

100

8/8

0

0/8

さらに、UN GHS 区分 1 の 6 物質、区分 2A の 3 物質、区分に該当しない 3 物質の計 12
物質の界面活性剤について、3 施設で UN GHS 区分１物質であるか否かを判定した場合の再現性を確認した結果 8)、施設間再現性は 83%（10/12）となり、良好であった。
以上、2<pH<11.5 の洗浄剤および界面活性剤については、トップダウン方式において偽陰性率が高いことから、標準的な ICE 法で陰性結果が得られた際には、病理組織学的検査を併用して判定することを推奨する。
3−3．その他
3−3−1．習熟度確認物質の変更：
TG 438（2013）では、ICE 法の習熟度確認物質として 13 物質が推奨された。本改定版
では、このうち 4 物質が変更（濃度のみの変更を含む）され、表 8 に示す 13 物質になった。

8 / 1214

表 8.

ICE 法の習熟度確認物質 2)
化学物質CAS 番号

分類

物理的性状

In Vivo
UN GHS 区分

ICE
UN GHS 区分

Benzalkonium chloride(10%)

8001-54-5

オニウム
化合物類

液体

区分 1

区分 1

Chlorhexidine

55-56-1

アミン類
アミジン類

固体

区分 1

区分 1

Sodium hydroxide (10%)

1310-73-2

塩基

液体

区分 1

区分 1

Imidazole288-32-4

ヘテロサイクリック化合物類

固体

区分 1

区分 1

Trichloroacetic acid (30%)

76-03-9カルボン酸類

液体

区分 1

区分 1

2,6- Dichlorobenzoyl
chloride4659-45-4

アシルハライド
類

液体

区分 2A

予測不可

Ammonium nitrate

6484-52-2

無機塩

固体

区分 2B

予測不可

Sodium hydroxide (1%)

1310-73-2

塩基

液体

区分 2B

予測不可

Dimethyl sulfoxide

67-68-5

有機硫黄
化合物

液体

区分外

区分外

Ethyl trimethyl acetate

3938-95-2

エステル

液体

区分外

区分外

Methylcyclopentane

96-37-7

炭化水素
（環状）

液体

区分外

区分外

n-Hexane

110-54-3

炭化水素
（鎖状）

液体

区分外

区分外

Triacetin102-76-1

脂質

液体

区分外

区分外

区分外：表１参照。
予測不可：エンドポイントのスコアの組み合わせが区分 1 および区分外のどちらにも該当しないため、ICE における区分が判定できない
3−3−2．病理組織学的検査における習熟度確認物質
今回、新たに ICE 法の病理組織学的検査における習熟度確認物質として、表 9 に示す 6物質（同一物質の濃度違いを含む）が推奨された。

9 / 1215

表 9．ICE 法の病理組織学的検査の習熟度確認物質 2)
UN GHS 区分

界面活性剤 物理的の種類
性状

In Vivo

標準的ICE

ICE 病理組織学的検査*Benzalkonium chloride
8001-54-5
(5%)

カチオン

液体

区分 1

区分 1

区分 1
（びらん）

Benzensulphonylchloride

98-09-9

アニオン

液体

区分 1

区分 1

区分 1
(壊死および空胞化)

Cetylpiridinium
bromide (10%)

140-72-7

カチオン

液体

区分 1

予測不可

区分 1
（空胞化）

Cetylpiridinium
bromide (1%)

140-72-7

カチオン

液体

区分 2A

予測不可

予測不可

N-Lauroyl sarcosine Na137-16-6
salt (10%)

アニオン

液体

区分 2A

予測不可

予測不可

Cetylpiridinium
bromide (0.1%)

カチオン

液体

区分外

予測不可

予測不可

化学物質

CAS 番号

140-72-7*表 3 ～ 表 5 の基準に従って判定し、合致することを確認する。
区分外：区分に該当しない
4．結論
JaCVAM 眼刺激性試験資料編纂委員会は、TG 438（2018）に基づいた正確性と再現性の結果から、UN GHS の眼刺激性分類においてトップダウ ン方式における区分 1 物質の同定およびボトムアップ方式における区分に該当しない物質の同定に ICE 法を適用することは可能であると考えた。また、2<pH<11.5 の洗浄剤および界面活性剤については、トップダウン方式において陰性結果が得られた場合、病理組織学的検査を併用することを推奨する。

10 / 1216用語（TG 438 ANNEX 1, DEFINITIONS）2)
トップダウン方式：
TG 438（2018）では、眼に重篤な損傷を引き起こす疑いのある化学物質に適用する段階的な評価方式を指し、眼に重篤な損傷を引き起こす化学物質（陽性結果）と、それ以外の化学物質（陰性結果）を 識別することから始める。
ボトムアップ方式：
TG 438（2018）では、眼刺激性あるいは重篤な眼の損傷性はないと予測される化学物
質に適用される段階的な評価方式を指し、眼刺激性物質に区分されない化学物質と、それ以外の化学物質を識別することから始める。
区分 1 物質:UN GHS 分類体系 2)下、眼球表面へ適用することにより、眼球の組織損傷や重篤な視力低下を引き起こす化学物質。いわゆる「重篤な眼の損傷」や「眼に対する非可逆的な作用」を示し、その損傷は適用後 21 日を経ても十分には回復しない。
区分 2 物質:UN GHS 分類体系下、眼球表面へ適用することにより、眼球の変化をもたらす化学物質。
「眼刺激性」や「眼に対する可逆的な作用」を示し、その損傷は適用後 21 日以内に完全に回復する。
区分外または UN GHS 区分に該当しない物質:
UN GHS 分類体系下、GHS 区分 1 あるいは 2（2A または 2B）への分類を要求される
ような刺激性を有しておらず、眼刺激性物質として区分されない化学物質。

引用文献
1) United nations (UN) (2017). Globally Harmonized System of Classification and Labeling ofChemicals (GHS), Seventh revised edition, UN New York and Geneva, 2017. Available at:[https://www.unece.org/fileadmin/DAM/trans/danger/publi/ghs/ghs_rev07/English/03e_part3.p
df]2) OECD Guidelines for The Testing of Chemicals, Isolated Chicken Eye Test Method for
Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring
Classification for Eye Irritation or Serious Eye Damage, TG 438 (Adopted 25 June 2018)
3) OECD (2017) Guidance Document on "The Bovine Corneal Opacity and Permeability (BCOP)
and the Isolated Chicken Eye (ICE) Test Methods: Collection of Tissues for Histological
Evaluation and Collection of Data on Non-Severe Irritants. Series on Testing and Assessment
No. 160, ENV/JM/MONO(2011)45/REV1. Available at: [https://www.oecd.org/ chemicalsafety/testing/series-testing-assessment-publications-number.htm]
4) OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring, Number
16 Advisory Document of the Working Group on Good Laboratory Practice -Guidance on the
GLP Requirements for Peer Review of Histopathology, JT03362564 26-Sep-2014
5) OECD Guidelines for The Testing of Chemicals, Isolated Chicken Eye Test Method for

11 / 1217

Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring
Classification for Eye Irritation or Serious Eye Damage, TG 438 (Adopted: 26 July 2013)
6) Cazelle E., Eskes C., Hermann M., et al. (2014). Suitability of Histopathology as an Additional
Endpoint to the Isolated Chicken Eye Test for classification of non-extreme pH detergent.
Toxicology In Vitro 28, 657-666.
7) Cazelle E., Eskes C., Hermann M., et al. (2015). Suitability of the Isolated Chicken Eye Test
for Classification of Extreme pH Detergents and Cleaning Products. Toxicology In Vitro 29,
609-616.
8) OECD (2019). Streamlined Summary Document Supporting OECD Test Guideline 438 on the
Isolated Chicken Eye for Eye Irritation/Corrosion. Series on Testing and Assessment No 188.
ENV Publications, OECD, Paris.

12 / 1218

OECD/OCDE

438

Adopted:25 June 2018

OECD GUIDELINE FOR THE TESTING OF
CHEMICALS
Isolated chicken eye test method for identifying I) chemicals inducing
serious eye damage and II) chemicals not requiring classification for eye
irritation or serious eye damage
INTRODUCTION
1.
The Isolated Chicken Eye (ICE) test method was evaluated by theInteragency Coordinating Committee on the Validation of Alternative Methods(ICCVAM), in conjunction with the European Centre for the Validation of
Alternative Methods (ECVAM) and the Japanese Centre for the Validation of
Alternative Methods (JaCVAM), in 2006 and 2010 (1) (2) (3). In the original
evaluation, the ICE was endorsed as a scientifically valid test method for use as ascreening test to identify chemicals (substances and mixtures) inducing serious
eye damage (Category 1) as defined by the United Nations (UN) Globally
Harmonized System of Classification and Labelling of Chemicals (GHS) (1) (2)(4). A re-evaluation of the in vitro and in vivo dataset used in the validation study
concluded that the ICE test method could also be used to identify chemicals not
requiring classification for eye irritation and serious eye damage as defined by the
UN GHS which led to the revised version of TG 438 adopted in 2013 (4) (5).
Since then, the Decision Criteria used to identify chemicals not requiringclassification according to the UN GHS Classification System, has been revised
based on the latest acceptance standards (5) (6) (7) (8). Furthermore,
histopathology has been shown to be a useful additional endpoint to identify UN
GHS Category 1 non-extreme pH (2 < Ph < 11.5) detergents and surfactants (9)(10). This Test Guideline (adopted in 2009 and updated in 2013 and in 2018)
includes the latest recommended uses and limitations of the ICE test method
based on these evaluations.
2.
It is currently generally accepted that, in the foreseeable future, no single
in vitro eye irritation test will be able to fully replace the in vivo Draize eye test to
predict across the full range of irritation for different chemical classes. However,strategic combinations of alternative test methods within a (tiered) testing strategy
and/or Integrated Approaches to Testing and Assessment (IATA) may be able to© OECD, (2018)
You are free to use this material subject to the terms and conditions available at http://www.oecd.org/termsandconditions/.
In accordance with the decision of the Council on a delegation of authority to amend Annex I of the decision of the council on the
Mutual Acceptance of Data in the assessment of chemicals [C(2018)49], this Guideline was amended by the OECD’s Joint Meetingof the Chemicals Committee and the Working Party on Chemicals, Pesticides and Biotechnology by written procedure on 25 June
2018.

19

22││

438
438

OECD/OCDE
OECD/OCDE

replacereplacethe
theDraize
Draizeeye
eyetesttest(7)(11).
(7)(11).The
TheTop-Down
Top-Downapproach
approachisisdesigned
designedtotobebe
usedusedwhen,when,based
basedononexisting
existinginformation,
information,aachemicalchemicalisisexpected
expectedtotohave
havehighhighirritancy
irritancypotential,
potential,while
whilethetheBottom-Up
Bottom-Upapproach
approachisisdesigned
designedtotobebeused
usedwhen,
when,
based
basedononexisting
existinginformation,
information,aachemicalchemicalisisexpected
expectednot
nottotocause
causesufficientsufficienteyeeyeirritation
irritationtotorequire
requireaaclassification
classification(7)(11).
(7)(11).The
TheICE
ICEtesttestmethod
methodisisananininvitro
vitrotest
test
methodmethodthat
thatcancanbebeused,
used,under
undercertain
certaincircumstances
circumstancesandandwithwithspecificspecificlimitations
limitations
asasdescribed
describedininparagraphs
paragraphs77toto11
11for
foreyeeyehazard
hazardclassification
classificationand
andlabellinglabellingofof
chemicals.
chemicals.While
Whileititisisnot
notconsidered
consideredvalid
validasasaastand-alone
stand-alonereplacement
replacementfor
forthetheininvivo
vivorabbit
rabbiteyeeyetest,test,thetheICE
ICEtesttestmethod
methodisisrecommended
recommendedasasananinitial
initialstep
stepwithin
withinaa testing
testing strategy
strategy such
such asas thethe Top-Down
Top-Down approach
approach recommended
recommended within
within thetheOECDOECD GD
GD 263263 (7)(7) toto identify
identify chemicals
chemicals inducing
inducing serious
serious eyeeye damage,damage, i.e.,
i.e.,chemicals
chemicalstotobebeclassifiedclassifiedasasUN
UNGHS
GHSCategory
Category11without
withoutfurther
furthertesting
testing(4).
(4).TheThe
ICE
ICEtesttestmethod
methodisisalso
alsorecommended
recommendedtotoidentify
identifychemicals
chemicalsthat
thatdodonot
notrequire
requireclassification
classificationfor
foreyeeyeirritation
irritationororserious
seriouseye
eyedamagedamageasasdefined
definedby
bythe
theUN
UNGHS
GHS
(No
(NoCategory)Category)(4),(4),andandmaymaytherefore
thereforebebeused
usedasasananinitial
initialstep
stepwithin
withinaaBottomBottomUp
Uptestingtestingstrategy
strategyapproach
approach(OECD(OECDGD
GD263
263(7).
(7).However,
However,aachemicalchemicalthat
thatisisnot
notpredicted
predicted asas causing
causing serious
serious eyeeye damage
damage oror asas not
not classifiedclassified forfor eyeeyeirritation/serious
irritation/seriouseye
eyedamagedamagewith
withthetheICE
ICEtesttestmethod
methodwould
wouldrequire
requireadditional
additional
information
informationtotoestablish
establishaadefinitive
definitiveclassification.
classification.Choice
Choiceofofthe
themostmostappropriate
appropriate
test
testmethod(s)method(s)andanduseuseofofthis
thisTest
TestGuideline
Guidelineshould
shouldbebeseen
seenininthe
thecontext
contextofofthe
theOECDOECD Guidance
Guidance Document
Document on
on anan Integrated
Integrated Approach
Approach on
on Testing
Testing andandAssessment
Assessment forfor Serious
Serious Eye
EyeDamage
DamageandandEyeEyeirritation
irritation(7).
(7).Furthermore,
Furthermore,thetheappropriate
appropriateregulatory
regulatoryauthorities
authoritiesshould
shouldbebeconsulted
consultedbefore
beforeusing
usingthetheICE
ICEtesttest
methodmethodininaaBottom-Up
Bottom-Upapproach
approachfor
forclassification
classificationschemes
schemesother
otherthan
thanthe
theUN
UN
GHS.
GHS.
3.3.
The
Thepurpose
purposeofofthis
thisTest
TestGuideline
Guidelineisistotodescribe
describethe
theprocedures
proceduresused
usedtoto
evaluateevaluatethe
theeyeeyehazard
hazardpotential
potentialofofaatest
testchemicalchemicalasasmeasured
measuredby
byitsitsability
abilitytoto
induceinduceorornot
notinduce
inducetoxicity
toxicityininthe
theenucleatedenucleatedeyes
eyesofofchicken.
chicken.Toxic
Toxiceffects
effectstoto
thethe cornea
cornea areare measured
measured byby (i)(i) aa qualitative
qualitative assessment
assessment ofof opacity,
opacity, (ii)(ii) aaqualitative
qualitative assessment
assessment ofof damage
damage toto epithelium
epithelium based
based onon application
application ofof
fluorescein
fluoresceintotothe
theeyeeye(fluorescein(fluoresceinretention),
retention),(iii)(iii)aaquantitative
quantitativemeasurement
measurementofof
increased
increasedthickness
thickness(swelling),(swelling),and
and(iv)(iv)aaqualitative
qualitativeevaluation
evaluationofofmacroscopic
macroscopic
morphologicalmorphologicaldamage
damagetotothe
thesurface
surfaceofofthe
thetreatedtreatedeyes.
eyes.The
Thecorneal
cornealopacity,
opacity,swelling,
swelling, andand damage
damage assessments
assessments following
following exposure
exposure toto aa test
test chemicalchemical areareassessedassessedindividuallyindividuallyand
andthenthencombined
combinedtotoderive
deriveananEye
EyeIrritancy
IrritancyClassification.
Classification.
Furthermore,
Furthermore,histopathologicalhistopathologicalobservations
observationsmay
mayalsoalsobebeused
usedasasananadditional
additional
endpoint
endpoint toto potentiallypotentially improve
improve thethe prediction
prediction ofof UN
UN GHSGHS Category
Category 11 nonnonextreme
extremepH
pH(2(2<<pH
pH<<11.5)
11.5)detergentsdetergentsand
andsurfactants
surfactants(see(seeparagraphs
paragraphs88andand56).
56).
4.4.

Definitions
Definitionsare
areprovided
providedininAnnex
Annex1.1.

INITIALINITIALCONSIDERATIONSCONSIDERATIONSAND
ANDLIMITATIONSLIMITATIONS
5.5.
This
ThisTest
Test Guideline
Guidelineisis based
basedononthe
theprotocol
protocol suggested
suggestedininthe
theOECDOECD
Guidance
GuidanceDocument
Document160
160(12),(12),which
whichwaswasoriginallyoriginallyadopted
adoptedinin2011
2011andandfurther
further
updatedupdatedinin2017
2017andand2018.
2018.The
Theprotocol
protocolisisbased
basedononinformation
informationobtained
obtainedfrom
frompublishedpublishedprotocols
protocols(13)(13)(14)(14)(15)(15)(16)(16)(17).
(17).

©©OECD
OECD2018
2018

20

OECD/OCDE

ch is designed to be
xpected to have highned to be used when,o cause sufficient eye
hod is an in vitro testth specific limitations
ation and labelling of
eplacement for the in
an initial step within
mmended within theus eye damage, i.e.,
urther testing (4). Thes that do not require
ined by the UN GHS
tep within a Bottoma chemical that is notclassified for eye
uld require additional
the most appropriate
in the context of thech on Testing and
7). Furthermore, there using the ICE test
es other than the UN

e procedures used to
sured by its ability token. Toxic effects to
nt of opacity, (ii) a
on application of
ative measurement ofation of macroscopic
The corneal opacity,
a test chemical areitancy Classification.
used as an additional
HS Category 1 nonaragraphs 8 and 56).

gested in the OECDd in 2011 and further
mation obtained from438 │ 3

6.
A wide range of chemicals has been tested in the evaluation underlying
this Test Guideline and the overall database currently amounts to 184 test
chemicals including 75 substances and 109 mixtures (5). The Test Guideline isapplicable to solids, liquids, emulsions and gels. The liquids may be aqueous or
non-aqueous; solids may be soluble or insoluble in water. Gases and aerosols
have not been assessed yet in a validation study.
7.
The ICE test method can be used to identify chemicals inducing serious
eye damage, i.e., chemicals to be classified as UN GHS Category 1 (4). Whenused for this purpose, the identified limitations for the ICE test method are basedon the high false positive rates for alcohols and the high false negative rates forsolids and surfactants (1) (3) (18). Moreover, test chemicals inducing persistent
non severe effects in vivo may also risk underprediction (22). However, false
negative rates in this context (UN GHS Category 1 identified as not being UN
GHS Category 1) are not critical since all test chemicals that come out negative
would be subsequently tested with other adequately validated in vitro test(s), or asa last option in rabbits, depending on regulatory requirements, using a sequential
testing strategy in a weight-of-evidence approach. Furthermore, histopathology
was found to be a useful additional endpoint to decrease the false negative rates
when used to identify UN GHS Category 1 non-extreme pH (2 < pH < 11.5)
detergents shown to induce mainly persistent non severe effects in vivo (andsurfactants (9) (10) (19). Regarding solids, it should be noted that these may leadto variable and extreme exposure conditions in the in vivo Draize eye irritation
test, which may result in irrelevant predictions of their true irritation potential(20). Investigators could consider using this test method for all types ofchemicals, whereby a positive result should be accepted as indicative of serious
eye damage, i.e., UN GHS Category 1 classification without further testing.
However, positive results obtained with alcohols should be interpreted cautiously
due to risk of over-prediction.
8.
When used to identify chemicals inducing serious eye damage (UN GHS
Category 1), the ICE test method (without use of histopathology) was found tohave an overall accuracy of 83% (142/172), a false positive rate of 7% (9/127)
and a false negative rate of 47% (21/45) when compared to in vivo rabbit eye test
method data classified according to the UN GHS classification system (4) (5).
When histopathology is considered as an additional endpoint to identify UN GHS
Category 1 non-extreme pH (2 < pH < 11.5) detergents and surfactants, the false
negative rate of the ICE test method and its accuracy are improved (from 64% to
27% false negatives (n=22) and from 53% to 77% accuracy (n=30)), whilst an
acceptable false positive rate is maintained (from 0% to 12.5% false positives(n=8)) (10).
9.
The ICE test method can also be used to identify chemicals that do not
require classification for eye irritation or serious eye damage under the UN GHS
classification system (4). The test method can be used for all types of chemicals,
whereby a negative result could be accepted for not classifying a chemical for eye
irritation and serious eye damage. However, on the basis of one result from thevalidation database, anti-fouling organic solvent-containing paints may be underpredicted (5).

© OECD 2018
© OECD 2018

21

42││

438
438

OECD/OCDE
OECD/OCDE

10.
replaceWhen
the Draize
used toeye
identify
test (7)(11).
chemicals
Thethat
Top-Down
do not requireapproachclassification
is designed
for toeyebe
irritation
used when,and basedseriousoneye
existing
damage,information,
the ICE testa method
chemicalhasis an
expectedoverall toaccuracy
have highof88%
irritancy(161/184),
potential,
a falsewhile
positive
the Bottom-Up
rate of 24%
approach(20/83),isanddesigneda false tonegativebe usedrate
when,
of
3%
based
(3/101),on existing
when information,compared toa inchemicalvivo rabbit
is expected
eye testnotmethod
to cause
data
sufficientclassified
eyeaccordingirritation to therequireUN aGHS
classification(4) (5). When(7)(11).
test Thechemicals
ICE test
withinmethodcertainis anclasses
in vitro(i.e.,
test
anti-foulingmethod thatorganic
can be solvent
used, undercontaining
certainpaints)
circumstances
are excluded
and with
fromspecificthe database,limitations
theaccuracy
as describedof thein ICE
paragraphs
test method
7 to 11is 88%
for eye(159/181),
hazard classification
the false positiveand labellingrate 24%of(20/83),chemicals.
andWhile
the false
it isnegativenot considered
rate ofvalid
2% (2/99)
as a stand-alone
for the UNreplacement
GHS classification
for the insystemvivo rabbit(4). eye test, the ICE test method is recommended as an initial step withina testing strategy such as the Top-Down approach recommended within the11.
is notchemicals
recommended
for serious
the identificationof test
OECDTheGDICE
263test(7) method
to identify
inducing
eye damage,i.e.,chemicals
shouldbe classified
as irritating
to 1eyes(i.e.,further
UN GHS
Category
2chemicalsthat
to beclassified
as UN GHS
Category
without
testing(4). The
orICE
Category
2A) oristestthat should
be classified
as mildly
totest method
alsochemicals
recommended
to identify
chemicalsthat doirritating
not requireeyes(UN GHS2B) ordue
to thenumberbyoftheUNclassification
forCategory
eye irritation
seriouseyeconsiderable
damage as defined
UNGHS
GHS
Category
1 chemicals
2A ora 2B
and(No Category)(4), andunderclassified
may therefore as
be UN
usedGHS
as anCategory
initial step2,within
BottomUN
GHS
No
Categorychemicals
overclassified
as
UN
GHS
Category
2,
2A
Up testing strategy approach (OECD GD 263 (7). However, a chemical that is ornot
2B.
For thisaspurpose,
informationand if orneeded,additional
testing
predicted
causingfurther
seriouseye damage
as notclassified
forwitheye
anothersuitable method
may bewith
required.
irritation/serious
eye damage
the ICE test method would require additional
information to establish a definitive classification. Choice of the most appropriate
12.
All procedureschickeneyes should
follow
applicable
test method(s)and usewithof thisTest Guideline
shouldbe seenin the geographicalcontext of theregulations
and the test
facility’sonproceduresfor handlingof humanor animalOECD GuidanceDocumentan Integrated
Approachon Testingandderivedmaterials,
which
include,
but
are
not
limitedto,tissuesandtissuefluids.
Assessment for Serious Eye Damage and Eye irritation (7). Furthermore, theUniversal
laboratory
precautions
areshould
recommended(21). before using the ICE test
appropriate
regulatory
authorities
be consulted
method in a Bottom-Up approach for classification schemes other than the UN
13.
GHS. Whilst the ICE test method does not directly address conjunctival andiridial injuries as evaluated in the rabbit ocular irritancy test method, it addresses
cornealeffects
whichof are
driverclassificationin vivo used
whento3.
Thepurposethis theTestmajor
Guideline
is toofdescribe
the proceduresconsidering
GHSpotential
Classification.
In chemical
this respect,
it shouldby
beits
noted
thattoevaluate thetheeyeUN
hazardof a test
as measured
abilityeffects
on
the
iris
are
of
lesserimportanceforclassificationofchemicals
induce or not induce toxicity in the enucleated eyes of chicken. Toxic effects toaccordingto UN(8) (22).
the reversibility
the cornea
are GHSmeasuredby Also,(i) aalthoughqualitative
assessment of corneal
opacity,lesions(ii) acannotbe evaluated
per ofse in
the ICEmethod, based
it has on
beenapplication
shown thatof
qualitative
assessment
damageto testepithelium
histopathologicalcan retention),
help in identifying
test chemicals
causingof
fluorescein to theobservationseye (fluorescein(iii) a quantitative
measurement
irreversible
effects not(swelling),
linked withandinitiallevel injury
such as thosecaused byincreased thickness(iv)higha qualitativeevaluationof macroscopic
non-extreme
pH damage(2 < pH <detergents(9). treatedFinally,eyes.
the ICEmethoddoesmorphologicalto11.5)the surfaceof theThetestcornealopacity,
not
allow
for
an
assessmentofthepotentialforsystemic
toxicity
associatedwithswelling, and damage assessments following exposure to a test chemicalareocularexposure.
assessed individually and then combined to derive an Eye Irritancy Classification.
Furthermore, histopathological observations may also be used as an additional
14.
ThistoTest
Guideline
will bethe
updatedperiodicallynew Category
information
endpoint
potentially
improvepredictionof UNasGHS
1 andnondataare pHconsidered.
further
histopathology
data may8 andbecomeextreme(2 < pH <For
11.5)example,
detergentsand surfactants(see paragraphs
56).
available for test chemicals other than non-extreme pH detergents and surfactants.
Tothis possibility,
usersinareencouraged
to preserve eyes and prepare
4. evaluate
Definitions
are provided
Annex
1.
histopathology specimens that can be used to develop a database and decision
criteriathat may further improve
the LIMITATIONS
accuracy of this test method. The OECD has
INITIALCONSIDERATIONSAND
developed Guidance Document 160 to be considered when using the ICE andBCOP
inThisvitroTest
oculartoxicity test
methods,
includessuggested
detailed procedures
on5.
Guideline
is basedon whichthe protocolin the OECDthecollection
and processingof histopathology
specimensfor evaluation(12).
Guidance
Document
160 (12),which was originallyadoptedin 2011 andfurther
updated in 2017 and 2018. The protocol is based on information obtained frompublished protocols (13) (14) (15) (16) (17).

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OECD/OCDE

438 │ 5ch is designed to DEMONSTRATIONbe
OF PROFICIENCY
xpected to have highned to be used when,15.
For any laboratory initially establishing the standard ICE test method, theo cause sufficient eye
proficiency chemicals provided in Annex 2 should be used. A laboratory can use
hod is an in vitro test
these chemicals to demonstrate their technical competence in performing theth specific limitations
standard ICE test method prior to submitting ICE data for regulatory hazard
ation and labelling ofclassification purposes. For any laboratory willing to establish ICE histopathology
eplacement for the infor the regulatory hazard classification of non-extreme pH detergents andan initial step withinsurfactants, the ICE Atlas and recommendations provided within the revised
mmended within theOECD GD 160 should be used (12). Consolidated training, transferability andus eye damage, i.e.,
proficiency appraisal are recommended to ensure harmonized, consistent andurther testing (4). The
reproducible histopathological observations. Furthermore, an internal pathologys that do not require
peer review should be conducted in accordance with current recommendations
ined by the UN GHS(23) and according to the OECD advisory document n. 16 on GLP requirements
tep within a Bottomfor peer review of histopathology (24), and as described in paragraph 50. Sucha chemical that is not
peer review process allows to verify and improve the accuracy and quality ofclassified for eye
pathology diagnoses and interpretations. Finally, the proficiency chemicals
uld require additional
provided in Annex 3 should be used for a laboratory to demonstrate technicalthe most appropriatecompetence in scoring the ICE histopathology effects, prior to submitting ICE
in the context of thehistopathology data for the regulatory hazard classification of non-extreme pH
ch on Testing anddetergents and surfactants.
7). Furthermore, there using the ICE test
PRINCIPLE OF THE TESTes other than the UN
16.
The ICE test method is an organotypic model that provides short-term
maintenance of the chicken eye in vitro. In this test method, damage by the test
e procedures used tochemical is assessed by determination of corneal swelling, opacity, andsured by its ability to
fluorescein retention. Furthermore, histopathology can be used to increase theken. Toxic effects tosensitivity of the method for identifying UN GHS Category 1 non-extreme pH
nt of opacity, (ii) a(2 < pH < 11.5) detergents and surfactants (10). Whilst measurement of corneal
on application ofswelling provides for a quantitative assessment, corneal opacity, fluorescein
ative measurement ofretention and histopathological changes each involve a qualitative assessment.
ation of macroscopic
Each measurement is either converted into a quantitative score used to assign an
The corneal opacity,
ICE Class (I to IV), or assigned a qualitative categorization that is used to assign
a test chemical are
an in vitro ocular hazard classification, either as UN GHS Category 1 or as UN
itancy Classification.
GHS No Category (see Decision Criteria). However, no prediction can be madeused as an additional
for chemicals not identified as UN GHS Category 1 or as UN GHS No Category
HS Category 1 nonwith the ICE test method (see paragraph 11); in these cases, the “No prediction
aragraphs 8 and 56).
can be made” result of the ICE test would require additional information forclassification purposes [see (7) for guidance].

Source and Age of Chicken Eyes

gested in the OECDd in 2011 and further
mation obtained from

17.
Historically, eyes collected from slaughterhouse chickens killed forhuman consumption have been used for this assay, eliminating the need forlaboratory animals. Only the eyes of healthy animals considered suitable for entry
into the human food chain are used.
18.
Although a controlled study to evaluate the optimum chicken age has notbeen conducted, the age and weight of the chickens used historically in this test
method are that of spring chickens traditionally processed by a poultry© OECD 2018
© OECD 2018

23

62││

438
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OECD/OCDE
OECD/OCDE

slaughterhousereplace the Draize(i.e., approximately
eye test (7)(11).
7 weeks
The Top-Downold, 1.5 - 2.5
approachkg).
is designed to be
used when, based on existing information, a chemical is expected to have highirritancy potential,
while theis designed to be used when,
Collection
and TransportofBottom-Up
Eyes to theapproach
Laboratory
based on existing information, a chemical is expected not to cause sufficient eye
19.
Heads should be removed immediately after humane stunning of theirritation to require a classification (7)(11). The ICE test method is an in vitro test
chickens and incision of the neck for bleeding. Humane stunning methods include
method that can be used, under certain circumstances and with specific limitations
electrical stunning and controlled atmosphere stunning, as long as it can be shownas described in paragraphs 7 to 11 for eye hazard classification and labelling of
not to adversely impact the quality of the chicken eyes (see paragraph 21). A local
chemicals. While it is not considered valid as a stand-alone replacement for the insource of chickens close to the laboratory should be located so that their heads canvivo rabbit eye test, the ICE test method is recommended as an initial step withinbe transferred from the slaughterhouse to the laboratory quickly enough toa testing strategy such as the Top-Down approach recommended within theminimize deterioration and/or bacterial contamination. The time interval between
OECD GD 263 (7) to identify chemicals inducing serious eye damage, i.e.,collection of the chicken heads and placing the eyes in the superfusion chamber
chemicals to be classified as UN GHS Category 1 without further testing (4). The
following enucleation should be minimized (typically within two hours) to assure
ICE test method is also recommended to identify chemicals that do not require
meeting assay acceptance criteria. All eyes used in the assay should be from theclassification for eye irritation or serious eye damage as defined by the UN GHS
same group of eyes collected on a specific day.
(No Category) (4), and may therefore be used as an initial step within a BottomUp testing strategy approach (OECD GD 263 (7). However, a chemical that is not
20.
Since eyes are dissected in the laboratory, the intact heads are transported
predicted as causing serious eye damage or as not classified for eye
from the slaughterhouse at ambient temperature (typically between 18oC andirritation/serious eye damage with the ICE test method would require additional
25oC) in plastic boxes humidified with tissues moistened with isotonic saline.
information to establish a definitive classification. Choice of the most appropriate
test method(s) and use of this Test Guideline should be seen in the context of theSelection Criteria and Number of Eyes Used in the ICE
OECD Guidance Document on an Integrated Approach on Testing and21.
Eyes thathave highbaseline
fluorescein0.5) or corneal
Assessment
for Serious
Eye
Damageand Eye staining
irritation(i.e.,(7). >Furthermore,theopacityscore regulatory(i.e., > 0.5)authorities
after they areenucleated
are rejected.
appropriate
shouldbe consulted
before using the ICE test
method in a Bottom-Up approach for classification schemes other than the UN
22.
GHS. Each treatment group and concurrent positive control consists of at least
three eyes. The negative control group or the solvent control (if using a solvent
othersaline)consistsof atTest
leastGuideline
one eye. is to describe the procedures used to
3. thanThepurposeof thisevaluate the eye hazard potential of a test chemical as measured by its ability to23.
of solidmaterials
leading to aeyes
GHSofNo
Categoryoutcome,induceInorthe
notcaseinducetoxicityin the enucleatedchicken.
Toxic
effects ato
secondrun ofarethreeeyes is byrecommended
to confirm
or discard
the negative
the cornea
measured(i) a qualitative
assessmentof opacity,(ii) aoutcome.
qualitative assessment of damage to epithelium based on application offluorescein to the eye (fluorescein retention), (iii) a quantitative measurement of
PROCEDURE
increased thickness (swelling), and (iv) a qualitative evaluation of macroscopic
morphological damage to the surface of the treated eyes. The corneal opacity,swelling, andofdamage
Preparation
the Eyesassessments following exposure to a test chemical areassessed individually and then combined to derive an Eye Irritancy Classification.
24.
The eyelids are carefully excised, taking care not to damage the cornea.
Furthermore, histopathological observations may also be used as an additional
Corneal integrity is quickly assessed with a drop of 2% (w/v) sodium fluorescein
endpoint to potentially improve the prediction of UN GHS Category 1 nonapplied to the corneal surface for a few seconds, and then rinsed with isotonic
extreme pH (2 < pH < 11.5) detergents and surfactants (see paragraphs 8 and 56).
saline. Fluorescein-treated eyes are then examined with a slit-lamp microscope toensure that the cornea is undamaged (i.e., fluorescein retention and corneal
4.
Definitions are provided in Annex 1.
opacity scores ≤ 0.5).

INITIALCONSIDERATIONS
AND LIMITATIONS
25.
If undamaged, the eye is further dissected from the skull, taking care not

to damage the cornea. The eyeball is pulled from the orbit by holding the5.
This Test Guideline is based on the protocol suggested in the OECD
nictitating membrane firmly with surgical forceps, and the eye muscles are cut
Guidance Document 160 (12), which was originally adopted in 2011 and further
with a bent, blunt-tipped scissor. It is important to avoid causing corneal damage
updated in 2017 and 2018. The protocol is based on information obtained fromdue to excessive pressure (i.e., compression artefacts).
published protocols (13) (14) (15) (16) (17).
26.
When the eye is removed from the orbit, a visible portion of the optic
nerve should be left attached. Once removed from the orbit, the eye is placed on©©OECD
OECD2018
2018

24

OECD/OCDE

ch is designed to be
xpected to have highned to be used when,o cause sufficient eye
hod is an in vitro testth specific limitations
ation and labelling of
eplacement for the in
an initial step within
mmended within theus eye damage, i.e.,
urther testing (4). Thes that do not require
ined by the UN GHS
tep within a Bottoma chemical that is notclassified for eye
uld require additional
the most appropriate
in the context of thech on Testing and
7). Furthermore, there using the ICE test
es other than the UN

e procedures used to
sured by its ability token. Toxic effects to
nt of opacity, (ii) a
on application of
ative measurement ofation of macroscopic
The corneal opacity,
a test chemical areitancy Classification.
used as an additional
HS Category 1 nonaragraphs 8 and 56).

438 │ 7an absorbent pad and the nictitating membrane and other connective tissue are cut
away.
27.
The enucleated eye is mounted in a clamp (stainless steel or suitable
alternative) with the cornea positioned vertically, and avoiding too much pressure
on the eye by the clamp (due to the relatively firm sclera of the chicken eye-ball,
only slight pressure is needed to fix the eye properly). The clamp is then transferred
to a chamber of the superfusion apparatus (25). The clamps should be positioned in
the superfusion apparatus such that the entire cornea is supplied with the isotonic
saline drip (3-4 drops per minute or 0.1 to 0.15 mL/min). The chambers of thesuperfusion apparatus should be temperature controlled at 32 ± 1.5°C. Annex 4
provides a diagram of a typical superfusion apparatus and the eye clamps, which
can be obtained commercially or constructed. The apparatus can be modified tomeet the needs of an individual laboratory (e.g., to accommodate a different numberof eyes).
28.
After being placed in the superfusion apparatus, the eyes are again
examined with a slit-lamp microscope (e.g., Haag-Streit BP900) to ensure thatthey have not been damaged during the dissection procedure. Corneal thickness
should also be measured at this time at the corneal apex using the depth
measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein
retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs ofdamage should be replaced. For eyes that are not rejected based on any of thesecriteria, individual eyes with a corneal thickness deviating more than 10% fromthe mean value for all eyes are to be rejected. For the Haag-Streit slit lamp BP900
fitted with depth-measuring device no. 1, the slit-width setting should be 9½
equalling 0.095 mm. Alternatively the slit-lamp BQ900 from Haag-Streit may be
used as long as it can be mounted with the depth measuring device and a slit
width of 0.095 can be applied (see also paragraph 53). Users should be aware that
slit-lamp microscopes could yield different corneal thickness measurements if theslit-width setting is different.
29.
Once all eyes have been examined and approved, the eyes are incubatedfor approximately 45 to 60 minutes to equilibrate them to the test system prior to
dosing. Following the equilibration period, a zero reference measurement isrecorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0).
The fluorescein score determined at dissection is used as the baseline
measurement for that endpoint.

Application of the Test Chemical
30.
Immediately following the zero reference measurements, the eye (in its
holder) is removed from the superfusion apparatus, placed in a horizontal
position, and the test chemical is applied to the cornea.

gested in the OECDd in 2011 and further
mation obtained from31.
Liquid test chemicals are typically tested undiluted, but may be diluted if
deemed necessary (e.g., as part of the study design). The preferred solvent for
dilution of test chemicals is physiological (isotonic) saline. However, alternativesolvents may also be used under controlled conditions, but the appropriateness ofsolvents other than physiological saline should be demonstrated.

© OECD 2018
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82││

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OECD/OCDE
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32.
replaceLiquid
the Draize
test chemicals
eye test are(7)(11).
applied
ThetoTop-Down
the cornea suchapproachthat theis designed
entire surface
to beofusedthe when,corneabasedis evenly
on existing
coveredinformation,with the testa chemicalchemical; istheexpected
standardtovolume
have highis
0.03
irritancy
mL. potential, while the Bottom-Up approach is designed to be used when,based on existing information, a chemical is expected not to cause sufficient eye
33.
If possible,
test chemicals
shouldbeICE
groundfinely isasan
possibleairritation
to requiresolida classification(7)(11).
The
test asmethodin vitrointest
mortarcomparable
grinding
tool. Theandpowder
is applied
to themethodand
thatpestle,
can beorused,under certain
circumstanceswith specificlimitations
corneasuch thatthe surface7 iswith the testandchemical;theof
as describedin paragraphs
to uniformly
11 for eye covered
hazard classification
labelling
standard
amountis it0.03
g. considered valid as a stand-alone replacement for the inchemicals.
While
is notvivo rabbit eye test, the ICE test method is recommended as an initial step within
34.
Thestrategy
test chemical(liquidor solid) isapproach
applied for
10 seconds within
and thena testingsuch asthe Top-Down
recommended
therinsed
from
the
eye
with
isotonic
saline(approximately
20
mL)
at
ambient
OECD GD 263 (7) to identify chemicals inducing serious eye damage,i.e.,temperature.
eye (in itsas holder)is Category
subsequently
returnedto thesuperfusion
chemicals toThebe classified
UN GHS
1 withoutfurther
testing(4). The
apparatus
the original
position.toInidentify
case of chemicals
need, additionalmay
ICE test inmethodis alsouprightrecommended
that dorinsing
not require
beclassification
used after the
10-secapplicationand eyeat subsequent
points(e.g.,
for eyeirritationor serious
damage as time
definedby theUNuponGHS
discoveryof residuesof maytest chemical
the cornea).
In general
the amountof(No Category)(4), and
therefore on
be usedas an initial
step within
a Bottomsaline
additionallyusedforrinsingis
notcritical,buttheobservationofadherence
Up testing strategy approach (OECD GD 263 (7). However, a chemical that is notofpredictedchemical tocornea isimportant.
as thecausing
seriouseye damage or as not classified for eye
irritation/serious eye damage with the ICE test method would require additional
ControlChemicals
information
to establish a definitive classification. Choice of the most appropriate
test
method(s)and negativeuse of thisorTest
Guideline should
be seenthe context
of the35.
Concurrent
solvent/vehicle
controls
andinpositive
controls
OECD
Guidance
DocumentonanIntegrated
Approachon
Testingandshould be included in each experiment.
Assessment for Serious Eye Damage and Eye irritation (7). Furthermore, the36.
Whenregulatory
testing liquids
at 100%or solids,
physiologicalisappropriate
authorities
shouldbe consulted
before(isotonic)
using thesaline
ICE test
used
as thecontrolin the ICE test
methodto thandetectthenonmethodin aconcurrent
Bottom-Upnegative
approachfor classification
schemesotherUNspecific
GHS. changes in the test system, and to ensure that the assay conditions do notinappropriately result in an irritant response.
3.
The purposeof this liquids,
Test Guideline
is to solvent/vehicle
describe the procedures
usedisto37.
When
testing diluted
a concurrent
control group
evaluatetheeyehazardpotentialofatest
chemicalasmeasuredbyitsabilityincluded in the test method to detect non-specific changes in the test system, andto
not induce
toxicityin the doenucleatedeyes of chicken.
toinduce
ensureorthat
the assay
conditions
not inappropriately
resultToxic
in aneffects
irritanttothecorneaaremeasuredby(i)aqualitative
assessmentof
opacity,(ii) aresponse. As stated in paragraph 31, only a solvent/vehicle that has been
qualitative
assessmentofdamagetoepithelium
basedon
applicationofdemonstrated to have no adverse effects on the test system can be used.
fluorescein to the eye (fluorescein retention), (iii) a quantitative measurement of38.
A knownocularirritant andis included
as a concurrent
positive
control inincreased
thickness(swelling),(iv) a qualitativeevaluationof macroscopic
eachexperiment damage
to verifytothat
appropriate
induced.
the ICEtest
morphologicaltheansurfaceof theresponse
treated iseyes.
The Ascornealopacity,method
is
being
used
in
this
Test
Guideline
toidentify
chemicals
inducing
swelling, and damage assessments following exposure to a test chemical areseriousdamage, andthe thenpositive
controlshouldbe aIrritancy
referencechemicalassessedeye
individuallycombined
to derive
an EyeClassification.
inducing
responses
that fulfil theobservations
criteria for classification
UN as
GHS
Furthermore,
histopathologicalmay also beasused
an Category
additional
1endpointin this test
However,ensurethat variability
the Category
positive control
to method.
potentially
improveto thepredictionof UN inGHS
1 nonresponse
acrossassessed,and
thesurfactantsmagnitude(seeof paragraphs
the severe 8response
extreme pH(2 <time
pH <can11.5)bedetergentsand 56).
should not be excessive. Sufficient in vitro data for the positive control shouldbesuch thata statisticallydefined
4. generated
Definitions
are provided
in Annex
1. acceptable range for the positive
control can be calculated. If adequate historical ICE test method data are notavailable
for a particular positive
studies may need to be conducted to
INITIALCONSIDERATIONS
ANDcontrol,
LIMITATIONS
provide this information.
5.
This TestofGuideline
is based
the test
protocolsuggestedin acetic
the OECD
39.
Examplespositive controls
foron
liquidchemicalsare 10%acid
Guidance
Document
160
(12),
which
was
originallyadoptedin2011
andor 5% benzalkonium chloride, while examples of positive controls for solidfurther
test
updated
in
2017
and
2018.
The
protocol
is
based
on
information
obtainedfromchemicals are sodium hydroxide or imidazole.
published protocols (13) (14) (15) (16) (17).
40.
Benchmark chemicals are useful for evaluating the ocular irritancy
potential of unknown chemicals of a specific chemical or product class, or for©©OECD
OECD2018
2018

26

OECD/OCDE

ch is designed to be
xpected to have highned to be used when,o cause sufficient eye
hod is an in vitro testth specific limitations
ation and labelling of
eplacement for the in
an initial step within
mmended within theus eye damage, i.e.,
urther testing (4). Thes that do not require
ined by the UN GHS
tep within a Bottoma chemical that is notclassified for eye
uld require additional
the most appropriate
in the context of thech on Testing and
7). Furthermore, there using the ICE test
es other than the UN

e procedures used to
sured by its ability token. Toxic effects to
nt of opacity, (ii) a
on application of
ative measurement ofation of macroscopic
The corneal opacity,
a test chemical areitancy Classification.
used as an additional
HS Category 1 nonaragraphs 8 and 56).


gested in the OECDd in 2011 and further
mation obtained from438 │ 9evaluating the relative irritancy potential of an ocular irritant within a specificrange of irritant responses.

Endpoints Measured
41.
Treated corneas are evaluated prior to treatment and at 30, 75, 120, 180,
and 240 minutes (±5 minutes) after the post-treatment rinse. These time points
provide an adequate number of measurements over the four-hour observation
period, while leaving sufficient time between measurements for the requisite
observations to be made for all eyes.
42.
The endpoints evaluated are corneal opacity, swelling, fluorescein
retention, and morphological effects (e.g., pitting or loosening of the epithelium).
All of the endpoints, with the exception of fluorescein retention (which isdetermined only prior to treatment and 30 minutes after test chemical exposure)are determined at each of the above time points.
43.
Photographs are advisable to document corneal opacity, fluorescein
retention, morphological effects and, if conducted, histopathology.
44.
After the final examination at four hours, users are encouraged to preserveeyes in an appropriate fixative (e.g., neutral buffered formalin) for possible
histopathological examination in particular for non-extreme pH (2 < pH < 11.5)
detergents and surfactants (see paragraphs 7, 14 and 56). If histopathology isconducted, eyes should be fixed, trimmed, embedded in paraffin wax, sectioned
and stained according to the procedures described for the collection andprocessing of histopathology specimens within the OECD GD 160 (12).
45.
Corneal swelling is determined from corneal thickness measurements
made with an optical pachymeter on a slit-lamp microscope. It is expressed as apercentage and is calculated from corneal thickness measurements according to
the following formula:corneal thickness at time t  corneal thickness at time  0   100
corneal thickness at time  0


46.
The mean percentage of corneal swelling for all test eyes is calculated forall observation time points. Based on the highest mean score for corneal swelling,
as observed at any time point, an ICE Class is assigned for each test chemical (seeparagraph 53).
47.
Corneal opacity is evaluated by using the area of the cornea that is mostdensely opacified for scoring according to the observations described inTable 1. The mean corneal opacity value for all test eyes is calculated for allobservation time points. Based on the highest mean score for corneal opacity, as
observed at any time point, an ICE class is assigned for each test chemical (seeparagraph 53).

© OECD 2018
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10
2│

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OECD/OCDE
OECD/OCDE

replace the Draize eyeTable
test (7)(11).
The opacity
Top-Down
approach is designed to be
1. Corneal
scoresused when, based on existing information, a chemical is expected to have highirritancy
is designed to be used when,
Score potential, while the Bottom-Up approach
Observation
based on existing information, a chemical is expected not to cause sufficient eye0
No opacity
irritation to require a classification (7)(11). The ICE test method is an in vitro test
0.5
opacityunder certain circumstances and with specific limitations
method thatVerycanfaint
be used,asdescribed
inparagraphs
7 to 11 for eye hazard classification and labelling of1
Scattered or diffuse areas; details of the iris are clearly visible
chemicals. While it is not considered valid as a stand-alone replacement for the in2vivo rabbit Easily
discernible
translucent
area; detailsof the iris are asslightly
obscuredeye test,
the ICE
test method
is recommended
an initial
step withinsuch opacity;
as the noTop-Down
approachtheis
3a testing strategy
Severe corneal
specific detailsof the irisrecommended
are visible; size withinof the pupilOECD GDbarely
263 discernible(7) to identify chemicals inducing serious eye damage, i.e.,chemicals to be classified as UN GHS Category 1 without further testing (4). The
4Complete corneal opacity; iris invisible
ICE test method
is also recommended to identify chemicals that do not requireclassification for eye irritation or serious eye damage as defined by the UN GHS
48.
Fluoresceinevaluated
the as30 anminutepoint(No Category)(4),retention
and may istherefore
beatusedinitialobservation
step withintime
a Bottomonly
accordingto thescores shown
Table
mean fluorescein
Up testing
strategy
approach(OECDinGD
263 2.
(7).TheHowever,a chemical retention
that is notvalueof all testeyes is thencalculated
the 30-minute
observation
timeforpoint,
predicted
as causing
seriouseye fordamageor as notclassified
eyeandused to assign eye
an ICE
class withfor each(seewould
paragraph53).additional
irritation/serious
damagethe test
ICEchemical
test method
requireinformation to establish a definitive classification. Choice of the most appropriate
test method(s) and useof thisTest Guideline
should scores
be seen in the context of theTable
2. Fluorescein
retention
OECD Guidance Document on an Integrated Approach on Testing andAssessment for Serious Eye Damage and Eye irritation (7). Furthermore, the
Score
Observation
appropriate regulatory authorities should be consulted before using the ICE test
0method in No
fluorescein retention
a Bottom-Up
approach for classification schemes other than the UN
GHS.
0.5
Very minor single cell staining
1

Single cell staining scattered throughout the treated area of the cornea

3.
The purpose of this Test Guideline is to describe the procedures used to2evaluate the
Focal
confluentdense single
eyeorhazard
potentialof acellteststaining
chemical as measured by its ability to
inducetoxicity
inthe
thecornea
enucleatedof chicken. Toxic effects to
3induce or not
Confluent
large
areas ofretainingeyes
fluoresceinthe cornea are measured by (i) a qualitative assessment of opacity, (ii) a
qualitative
assessmentof damageto epithelium
based
on application
49.
Morphologicaleffects
include “pitting”
of corneal
epithelium,“loosening”offluorescein
to
the
eye
(fluorescein
retention),(iii)
aquantitative
measurementof epithelium, “roughening” of the corneal surface and “sticking” of the testof
increasedtothickness(swelling),(iv) acanqualitativeof macroscopic
chemicalthe cornea.
These andfindings
vary in evaluation
severity andmay occurmorphologicaldamagetothesurfaceofthetreatedeyes.
Thecornealsimultaneously. The classification of these findings is subjective accordingopacity,
to theswelling, andof damage
assessments following exposure to a test chemical areinterpretation
the investigator.
assessed individually and then combined to derive an Eye Irritancy Classification.
Furthermore,
histopathologicalobservations
also be used scoringas an additional50.
If histopathology
is conducted,
the maysemi-quantitative
systemendpointtopotentially
improvetheprediction
of
UN
GHS
Category
1 nondescribed in Table 3 should be used. It is critical to distinguish, for example
extreme
pH
(2
<
pH
<
11.5)
detergents
andsurfactants(seeparagraphs
8
and
56).
regarding epithelial vacuolation effects, the treatment-related effects from

histopathological artefacts and/or background morphology. For this purpose the4. presented
Definitions
are provided
Annex GD
1. 160 should be carefully consulted
Atlas
in Annex
II of theinOECD(12). Furthermore, original slides (rather than photomicrographs) need to be used
INITIALCONSIDERATIONS
AND LIMITATIONS
as someeffects require a three-dimensional
evaluation of the tissues. Only effects
that are observed should be scored. No assumptions should be made (e.g., if the
5. layerThis
Guideline isis based
protocolOECD
topof Testthe epithelium
missingonitthewill
not besuggested
possible into thescore
for
Guidance
Document
160
(12),
which
was
originallyadoptedin2011
andfurther
vacuolation in that layer). Furthermore, effects/changes close to the limbus should
The protocol
based on However,information
obtained frombeupdatedscoredinif2017
the andtissue2018.
architecture
was ispreserved.
effects/changes
publishedprotocols
(13)
(14)
(15)
(16)
(17).
occurring within the limbus should not be scored due to effects not linked to thechemical exposure. An internal pathology peer review system should be
conducted in accordance with current recommendations (23) and according to the©©OECD
OECD2018
2018

28

OECD/OCDE

ch is designed to be
xpected to have highned to be used when,o cause sufficient eye
hod is an in vitro testth specific limitations
ation and labelling of
eplacement for the in
an initial step within
mmended within theus eye damage, i.e.,
urther testing (4). Thes that do not require
ined by the UN GHS
tep within a Bottoma chemical that is notclassified for eye
uld require additional
the most appropriate
in the context of thech on Testing and
7). Furthermore, there using the ICE test
es other than the UN

438 │ 11

OECD advisory document n. 16 on GLP requirements for peer review ofhistopathology (24). In this process, a pathologist (with expertise on the tissues tobe evaluated)peer-reviews a number of slides and pathology data (e.g., 1 out of 3eyes) to assist the study pathologist in refining pathology diagnoses andinterpretations. Such peer review process allows to verify and improve theaccuracy and quality of pathology diagnoses and interpretations. Finally,
consolidated training, transferability and proficiency appraisal are recommended
to ensure consistent histopathological observations.

e procedures used to
sured by its ability token. Toxic effects to
nt of opacity, (ii) a
on application of
ative measurement ofation of macroscopic
The corneal opacity,
a test chemical areitancy Classification.
used as an additional
HS Category 1 nonaragraphs 8 and 56).

gested in the OECDd in 2011 and further
mation obtained from© OECD 2018
© OECD 2018

29

12
2│

438

OECD/OCDE
OECD/OCDE

Table
3. Semi-quantitative
histopathologicalscoring systemused forisisolated
chickenreplacethe Draize eye test
(7)(11). The Top-Down
approachdesignedto beeyeswhen,that were
fixed,
in paraffin
wax,issectioned
used
based
ontrimmed,
existing embedded
information,a chemical
expectedand
to stained
have highirritancy potential, while the Bottom-Up approach is designed to be used when,based onParameter
existing information, a Observation
chemical is expected
not to cause
sufficient eye
Score
Description*
irritation to require a classification (7)(11). The ICE test method is an in vitro test
methoderosionthat can be used, under certain
circumstancesandspecific
Epithelium:
Very slight½
Fewwithsinglecells uplimitations
to the entire
single superficial
as described in paragraphs 7 to 11 for eye hazard classification
andlayerlabelling ofchemicals. While it is not consideredSlightvalid as a stand-alone
replacement
for the in
1
Up to 3layers are gone
vivo rabbit eye test, the ICE test method is recommended as an initial step within
Moderate approach2Up
to 50 % of the epitheliallayer
a testing strategy such as the Top-Down
recommended
within theis gone*OECD GD 263 (7) to identify chemicals inducing serious eye damage, i.e.,chemicals to be classified as UN GHS
Category 13 without
further
SevereEpithelial
layertestingis gone(4).
up Theto thebasementthat
membrane
ICE test method is also recommended to identify chemicals
do not requireclassification for eye irritation or serious
eye damage
asdefined by the UN GHS
Epithelium: vacuolation
Very slight½
Single to few scattered cells(No Category) (4), and may therefore be used as an initial step within a BottomSeparately scored for the top, mid,Groupsa chemicalof vacuolated
Up testing strategy approach (OECDSlight
GD 263 (7).1 However,that cells
is notorand lower parts of the epithelium**
single string of cells with small
predicted as causing serious eye damage or asvacuoles
not classified for eye
irritation/serious eye damage with the ICE test method would require additional
Up of
to the50%mostof theepithelium
information to establish a definitiveModerateclassification.2 Choice
appropriateof thevacuolated
test method(s) and use of this Test Guideline should beconsists
seen incontextcells*of theOECD Guidance Document on Severe
an Integrated
Approachon
Testingand350 – 100% of the epithelium
consists
cells theAssessment for Serious Eye Damage and Eye irritation(7).of vacuolated
Furthermore,
appropriate regulatory
authorities
shouldbeconsulted
before
using
the
ICE
test
Epithelium:necrosis***Normal< 10 necrotic cells†
method in a Bottom-Up approach for classification schemes other than the UN
GHS.
Very slight½
10 – 20 necrotic cells††

Slight20 – 40 necrotic cells
3.
The purpose of this Test Guideline
is to 1describe
the procedures used toevaluate the eye hazard potential ofatestchemicalasmeasured
its but
ability
toof
Moderate
2
Many necroticby
cells
< 50%
the
epithelial
layer
induce or not induce toxicity in the enucleated eyes of chicken. Toxic effects tothe cornea are measured by (i) Severe
a qualitative3 assessmentof opacity, (ii) a
50 – 100% of the epithelial layer
qualitative assessment of damage to epithelium isbasedonapplication of
necrotic.
fluorescein to the††; eye(fluorescein
retention),(iii)
aquantitative
measurementof
Stroma:pyknoticthickness
nuclei †††(swelling), andNormal<evaluation
5 pyknotic nuclei
increased(iv) a qualitativeof macroscopic
In topor bottom region
morphologicaldamage to the surfaceof the treatedeyes.
The corneal
Slight1
5-10 pyknotic
nuclei opacity,swelling, and damage assessments following exposure to a test chemical are
Moderate> 10Irritancy
pyknotic nuclei
assessed individually and then combined
to derive2 an EyeClassification.
†††
Furthermore,
histopathologicalobservations
may
also
be
used
as
an additional
Stromal disorder of fibres
Present
P
Irregular appearanceof theendpoint to potentially improve the prediction of UN
GHS Category 1 nonfibres.
extreme pH (2 < pH < 11.5) detergents and surfactants (see paragraphs 8 and 56).
Endothelium:necrosis

4.

Present

Definitions are provided in Annex 1.

P

The endothelium consists of only
one layer, so a grade is not
relevant

Notes:CONSIDERATIONS
Annex II of the OECD GD AND
160 (12)LIMITATIONS
displays an Atlas with typical photomicrographs of
INITIAL

untreated as well as treated Isolated Chicken Eyes illustrating the various possible histopathologicaleffects described above.
This Test Guideline is based on the protocol suggested in the OECD
* 5.
Over the entire cornea except in case of test chemicals (e.g. some solid chemicals) causing
Guidance
Document(12), which
was originally2011 andfurther
localized effects
despite of 160
the homogenous
applicationof the testadopted
chemical inas required
withintheupdatedin 2017
The protocol
based
on localized
information
from
OECD
TG 438.
In thisandcase 2018.
the evaluation
should beisbasedon theeffectsobtainedat the site(s)ofexposure.
publishedprotocols
(13)
(14)
(15)
(16)
(17).
**
Top, mid and lower parts represent equal one third parts of the epithelial layer each. If the top
layer is missing, the mid layer does not become the ‘new’ top layer, but is still the mid layer (seeAnnex II of the OECD GD 160 for more details (12)).

©©OECD
OECD2018
2018

30

OECD/OCDE

ch is designed to be
xpected to have highned to be used when,o cause sufficient eye
hod is an in vitro testth specific limitations
ation and labelling of
eplacement for the in
an initial step within
mmended within theus eye damage, i.e.,
urther testing (4). Thes that do not require
ined by the UN GHS
tep within a Bottoma chemical that is notclassified for eye
uld require additional
the most appropriate
in the context of thech on Testing and
7). Furthermore, there using the ICE test
es other than the UN

438 │ 13***

Only necrosis of attached cells/tissues.
Necrotic cells are counted across the entire length of the cornea (there is no need for a specificfixed length to report cell counts because the entire length of the cornea is consistent on each slideas there is almost no variation in the size of the chicken eyes used and in the size of the samples
evaluated microscopically). The scoring system uses absolute cell counts from ‘normal’ to ‘slight’,
versus a percentage for ‘moderate’ and ‘severe’. This is due to the way the evaluation is performedby the examiner: necrotic cells are seen as individual items. If there are more, they are usuallyscattered. Therefore the examiner counts them to get an impression of the amount of necrosis. This
is in contrast to erosion, for which the first effect the examiner notices is that a part of the epithelium
is missing, so it makes sense to use an estimated percentage of loss.
††
The ICE test method already includes a precise measurement of the thickness of the cornea using
a slit lamp microscope. Therefore, swelling of the stroma is not separately scored during thesubsequent histopathological evaluation.
†††
The stromal effects that are scored consist of (1) pyknotic nuclei, which originate from thescoring system used by Maurer (2001) based on his observations in corneas of rabbits after in vivo
exposure (described as keratocyte loss/necrosis), and of (2) disorder of fibres. Regarding (1), thepresence of pyknotic nuclei is observed only occasionally and the development of pyknotic nuclei is
proposed to be dependent on the depth of injury and/or the inflammation process of the cornea (invivo). Furthermore, due to the elongated form of the stromal fibroblasts, normal nuclei could be
misleadingly considered as pyknotic nuclei depending on the section orientation of cells . Regarding(2), the observation and scoring of disorder of fibres may be difficult because the stromal fibres
already show a “natural” disorder. The processing of the cornea for microscopy can also contributeto an artificial disorder of stromal fibres. In both cases (pyknotic nuclei and disorder of fibres), theseobservations coincide with severe corneal effects already observed by the slit-lamp microscopeobservations, and with effects observed in the mid and/or lower epithelial layer.
†

52.
The OECD TG 438 requires test chemicals to be homogenously
distributed on the surface of the treated eyes. Based on such exposure, test
chemicals usually cause homogenous effects in the cornea of the isolated chicken
eyes, and the mean of histopathological effects over the entire slide should bescored. However, some test chemicals may cause focal or multifocal effects
confined to certain spots despite their homogenous application (e.g., as for somesolid test chemicals). If (multi)focal effects are observed during the performanceof the ICE test method, the histopathologist should be informed and thehistopathological scoring should be conducted based on the localized adverse
effects observed where exposure to the test chemical occurred. Furthermore, if
doubts remain (e.g. a discrepancy between the ICE results and thehistopathological observations is noticed), additional slices may be prepared onother parts of the cornea to ensure the localized effects are present in the observed
section.

e procedures used to
sured by its ability token. Toxic effects to
nt of opacity, (ii) a
on application of
ative measurement ofation of macroscopic
The corneal opacity,
a test chemical areitancy Classification.
used as an additional
DATA AND REPORTING
HS Category 1 nonaragraphs 8 and 56).

Data Evaluation

gested in the OECDd in 2011 and further
mation obtained from53.
Results from corneal opacity, swelling and fluorescein retention should be
evaluated separately to generate an ICE class for each endpoint. The ICE classes
for each endpoint are then combined to predict the In Vitro Classification of eachtest chemical. Similarly, histopathology evaluation, if applicable, should be
conducted separately and considered according to paragraphs 55 and 56.

Decision Criteria
54.
Once each endpoint has been evaluated, ICE classes can be assigned
based on a predetermined range. Interpretation of corneal swelling (Table 4),
opacity (Table 5), and fluorescein retention (Table 6) using four ICE classes is© OECD 2018
© OECD 2018

31

14
2│

438

OECD/OCDE
OECD/OCDE

done
replaceaccordingthe Draize
to theeye
scalestest shown(7)(11).below.
The Top-Down
It is important
approachto noteisthat
designedthe corneal
to beswelling
used when,scoresbased
shown
on existingin Tableinformation,
4 are only applicablea chemical ifisthickness
expected is
to measured
have highwithirritancya Haag-Streit
potential, BP900
while theslit-lamp
Bottom-Up
microscope
approach(orisalternatively
designed to abeHaag-Streit
used when,
BQ900
based onslit-lamp
existingmicroscope)
information,witha chemical
depth-measuring
is expected
devicenot tono.
cause
1 and
sufficientslit-widtheyesetting
irritation
at to9½,
requireequallinga classification
0.095 mm.
(7)(11).
Users
Theshould
ICE testbemethod
awareisthat
an inslit-lamp
vitro test
microscopes
method that could
can beyield
used,different
under certain
cornealcircumstances
thickness measurements
and with specific
if the slit-width
limitations
settingas describedis different.
in paragraphs 7 to 11 for eye hazard classification and labelling ofchemicals. While it is not considered valid as a stand-alone replacement for the in
4. ICE
classification
for cornealasswelling
vivo rabbitTableeye test,
the ICE
test methodcriteria
is recommended
an initial step withina testing strategy such as the Top-Down approach recommended within theOECD GD 263Mean(7) Corneal
to identify
chemicals
inducing serious eye damage,
Swelling(%)*
ICE Class i.e.,chemicals to be classified 0astoUN
GHS
Category
1
without
further
testing(4). The
5I
ICE test method is also recommended to identify chemicals that do not require>5 to 12or serious eye damage as defined by the UN
II
classification for eye irritation
GHS(No Category)>12
(4),
and
may
therefore
be
used
as
an
initialstep
withina
Bottomto 18 (>75 min after treatment)
II
Up testing strategy
approach
(OECD
GD
263
(7).
However,achemicalthatis not>12 to 18 (=75 min after treatment)III
predicted as causing serious eye damage or as not classified for eye>18 to with
26
III
irritation/serious eye damage
the ICE test method would require additional
to 32 (>75
min after treatment)III
information to>26
establisha definitiveclassification. Choice of the most appropriate
test method(s)>26
and
use
of
this
Test
Guideline
should
be
seen
in
the
context
to 32 (=75 min after treatment)
IV of theOECD Guidance Document on an Integrated Approach on Testing and>32
IV
Assessment for Serious Eye
Damage and Eye irritation (7). Furthermore,theappropriate
regulatory
authorities
shouldbeconsulted
before
using
the
ICE
test
Note: Highest mean score observed at any time point.
method in a Bottom-Up approach for classification schemes other than the UN
GHS.
3.
The purpose
Guideline criteria
is to describe
the procedures used toTableof5. thisICETestclassificationfor opacity.
evaluate the eye hazard potential of a test chemical as measured by its ability to*
induce or not Maximum
induce toxicity
in the Score
enucleatedeyes of chicken. Toxic
effects to
Mean Opacity
ICE Classthe cornea are measured by (i) a qualitative assessment of opacity, (ii) aI
qualitative assessment 0.0-0.5
of damage to epithelium based on applicationof
0.6-1.5
II
fluorescein to the eye (fluorescein
retention), (iii) a quantitative measurementofincreased thickness (swelling),
and
(iv)
a
qualitativeevaluationofmacroscopic
1.6-2.5
IIImorphological damage to the surface of the treated eyes. The corneal opacity,
2.6-4.0IV
swelling, and damage assessments following exposure to a test chemical areassessedindividually
andobserved
then combined
derivean EyeIrritancy
Classification.
Note:*Maximummean scoreat any timetopoint(basedon opacity
scoresas defined inTable1). *Based onscores as defined in Table
2.
Furthermore,
histopathologicalobservations
may also be used as an additional
endpoint to potentially improve the prediction of UN GHS Category 1 nonTable
criteria
mean fluorescein
retention.
extremepH6.
(2ICE< pHclassification< 11.5) detergents
andforsurfactants(see paragraphs
8 and 56).
4.

Definitions
providedin Annex
1.
Mean
Fluoresceinare
Retention
Score
at 30 minutes
post-treatment*

ICE Class

0.0-0.5I

0.6-1.5II

INITIAL CONSIDERATIONS AND LIMITATIONS

5.
This Test Guideline is1.6-2.5
based on the protocol suggested in the OECDIII
Guidance Document 160 (12),2.6-3.0
which was originally adopted in 2011 and furtherIV
updated in 2017 and 2018. The protocol is based on information obtained from
Note:
Based onscores as definedin Table
publishedprotocols(13) (14)(15)2.(16) (17).

©©OECD
OECD2018
2018

32

OECD/OCDE

438 │ 15ch is designed to be
55.
The in vitro classification for a test chemical is assessed by reading the
xpected to have highUN GHS classification that corresponds to the combination of categories obtained
ned to be used when,for corneal swelling, corneal opacity, and fluorescein retention as described ino cause sufficient eye
Table 7.
hod is an in vitro test
Table 7. Overall in vitro classificationsth specific limitations
ation and labelling of
eplacement for the in
UN GHS Classification
Combinations of the 3 Endpoints
an initial step within
No Category
3xI
mmended within the
2 x I, 1 x II
us eye damage, i.e.,
urther testing (4). The
2 x II, 1 x I
s that do not require
Other combinations
ined by the UN GHS No prediction can be madetep within a BottomCategory 1
3 x IVa chemical that is not
2 x IV, 1 x III
classified for eye
uld require additional
2 x IV, 1 x II*
the most appropriate
2 x IV, 1 x I*
in the context of theCorneal opacity = 3 at 30 min (in at least 2 eyes)
ch on Testing and
7). Furthermore, theCorneal opacity = 4 at any time point (in at least 2 eyes)
re using the ICE test
Severe loosening of the epithelium (in at least 1 eye)es other than the UN
Note: Combinations less likely to occur.

e procedures used to
sured by its ability token. Toxic effects to
nt of opacity, (ii) a
on application of
ative measurement ofation of macroscopic
The corneal opacity,
a test chemical areitancy Classification.
used as an additional
HS Category 1 nonaragraphs 8 and 56).

56.
If histopathology is used for non-extreme pH (2 < pH < 11.5) detergentsand surfactants, the decision criteria shown in Table 8 should be used. In addition,in case stromal pyknotic nuclei scores ≥ slight (score 1) in at least 2 out of 3 eyes
are observed; or any endothelium effects are observed in at least 2 out of 3 eyes,
such effects should be noted as observations to give indication on the severity ofeffects.
Table 8. Histopathology decision criteria to be used in addition to thestandard validated ICE test method for the identification of UN GHS
Category 1 non-extreme pH (2<pH<11.5) detergents and surfactants
Tissuelayer
Epithelium

Effects triggering eye serious damage (GHS Category 1) identification- erosion = moderate (score 2) in at least 2 out of 3 eyes
- and/or, any vacuolation (= very slight, score ½) observed in the mid and/or lower parts
in at least 2 out of 3 eyesgested in the OECDd in 2011 and further
mation obtained from- or, if erosion = moderate (score 2) in 1 out of 3 eyes + vacuolation = very slight in mid
and/or low part (score ½) is observed in at least another eye out of the 3 eyes
- and/or, necrosis = moderate (score 2) observed in at least 2 out of 3 eyes

57.
Furthermore, the prediction model shown in table 9 should be used. The
ICE histopathology criteria and the prediction model described in Tables 8 and 9,© OECD 2018
© OECD 2018

33

16
2│

438

OECD/OCDE
OECD/OCDE

respectively
replace the are
Draizeapplicableeye test
only(7)(11).
to identify
The Top-Down
UN GHS Category
approach1 is
non-extreme
designed topHbe(2used< pHwhen,< 11.5)based
detergentson existingand surfactants.
information, a chemical is expected to have highirritancy potential, while the Bottom-Up approach is designed to be used when,Tablemodel for identificationof non-extreme
pH (2<pH<11.5)
based 9.
onPrediction
existing information,a chemical is expected
not to cause
sufficient eye
detergentsand asurfactants
based
on ICE
irritation
to requireclassification(7)(11).
The histopathology
ICE test methodevaluations
is an in vitro test
method that can be used, under certain circumstances and with specific limitations
as describedin paragraphs
7 to 11 for eye
hazardclassificationofICE histopathology
criteria
described
inStandard
ICE
UN andGHS labellingClassification
chemicals. While it is not consideredTable
valid8as a stand-alone replacement for the invivo rabbit eye test, the ICE test method is recommended as an initial step withina testing strategy such as the Top-Down
approach recommended
within the
No prediction can be
Criteria met
UN GHS Category 1
OECD
GD
263
(7)
to
identify
chemicals
inducing
seriouseyedamage,i.e.,
madechemicals to be classified as UN GHS Category 1 without further testing (4). The
No prediction can be
Criteria not met
ICE test method is also recommended
to identify chemicals that
do not require
madeclassification for eye irritation or serious eye damage as defined by the UN GHS(No Category) (4), and may therefore be used as an initial step within a BottomUp testing strategy approach (OECD GD 263 (7). However, a chemical that is not
Study Acceptance Criteria
predicted as causing serious eye damage or as not classified for eye
58.
A test iseyeconsidered
acceptable
if method
the concurrent
negativeor
irritation/serious
damage with
the ICE test
would require
additional
vehicle/solventthe concurrent
positive controls
aretheidentified
as GHS
information to controls
establishanda definitiveclassification.
Choice ofmost appropriate
Non-Classified
respectively.
test method(s) andand GHSuse ofCategory
this Test1,Guideline
should be seen in the context of theOECD Guidance Document on an Integrated Approach on Testing andAssessment
Test
Report for Serious Eye Damage and Eye irritation (7). Furthermore, theappropriate regulatory authorities should be consulted before using the ICE test
59.
The test report should include the following information, if relevant to themethod in a Bottom-Up approach for classification schemes other than the UN
conduct of the study:
GHS.

Test
andTheControlChemicals
3.
purpose of this Test Guideline is to describe the procedures used toevaluatethe eye identification,
hazard potentialof aastest
chemicalas measured
its ability
 Chemical
such
IUPAC
or CAS
name(s),byCAS
registryto
inducenumber(s),or not induce
toxicityorin InChI
the enucleatedeyes of chicken.
effects
SMILES
code, structural
formula,Toxic
and/orothertothe cornea
are measured by (i) a qualitative assessment of opacity, (ii) a
identifiers;
qualitativeof damageepithelium substancebased onorapplication
 Purityassessment
and compositionof theto test/control
mixture (inoffluorescein
to
the
eye
(fluorescein
retention),(iii)
aquantitative
measurementof
percentage(s) by weight), to the extent this information is available;
increased
thickness(swelling),
and
(iv)
a
qualitativeevaluationofmacroscopic
 In case of multi-constituent and UVCB: characterization as far as possible
morphologicaldamage tothe surfaceof thepurity,
treatedquantitativeeyes. The occurrence
corneal opacity,
by e.g., chemical
identity(see above),andswelling,and damage
assessments
following
exposureto aconstituents,
test chemicalarerelevant
physicochemical
properties(see above)of theto theassessedindividuallyextentavailable;and then combined to derive an Eye Irritancy Classification.
Furthermore,
histopathologicalobservations
may also
used as pH,
an additional
 Physicochemical
properties
such as physicalstate,bevolatility,
stability,
endpointtopotentially
improvetheprediction
of
UN
GHS
Categorychemical class water solubility relevant to the conduct of the study; 1 nonextreme
pH (2 < pH
< 11.5)detergentsand surfactants(see paragraphs
8 and(e.g.
56).
 Treatmentof thetest/control
chemicalprior to testing,
if applicablewarming, grinding);
4.
Definitions are provided in Annex 1.
 Storage conditions and stability to the extent available;

INITIALCONSIDERATIONSLIMITATIONS
Information
Concerning theAND
Sponsorand the Test Facility
5.  Name
This Test
Guideline
is basedon testthe facility
protocoland
suggestedin the whereOECD
and addressof thesponsor,study director;
Guidance
Document
which was originally adopted in 2011 and further
applicable,
the 160
study(12),pathologist;
updatedin 2017 andon2018.
The protocol
is basedobtainedfrom Identification
the sourceof the eyes
(e.g.,ontheinformation
facility fromwhich they
publishedprotocols
(13)
(14)
(15)
(16)
(17).
were collected);©©OECD
OECD2018
2018

34

OECD/OCDE

ch is designed to be
xpected to have highned to be used when,o cause sufficient eye
hod is an in vitro testth specific limitations
ation and labelling of
eplacement for the in
an initial step within
mmended within theus eye damage, i.e.,
urther testing (4). Thes that do not require
ined by the UN GHS
tep within a Bottoma chemical that is notclassified for eye
uld require additional
the most appropriate
in the context of thech on Testing and
7). Furthermore, there using the ICE test
es other than the UN

Test Method Conditions

e procedures used to
sured by its ability token. Toxic effects to
nt of opacity, (ii) a
on application of
ative measurement ofation of macroscopic
The corneal opacity,
a test chemical areitancy Classification.
used as an additional
HS Category 1 nonaragraphs 8 and 56).

Test Procedure







438 │ 17

Description of test system used;
Slit-lamp microscope and pachymeter used (e.g., model) and the
instrument settings used;
Reference to historical negative and positive control results and, if
applicable, historical data demonstrating acceptable concurrent
benchmark control ranges;
The procedure used to ensure the integrity (i.e., accuracy and reliability)of the test method over time (e.g., periodic testing of proficiency
chemicals)).
The procedure used for tissues fixation in case histopathology is
performed.

Eyes Collection and Preparationgested in the OECDd in 2011 and further
mation obtained from

















Age and weight of the donor animal and if available, other specificcharacteristics of the animals from which the eyes were collected (e.g.
sex, strain);
Storage and transport conditions of eyes (e.g., date and time of eyecollection, time interval between collection of chicken heads and placing
the enucleated eyes in superfusion chamber);Preparation & mounting of the eyes including statements regarding their
quality, temperature of eye chambers, and criteria for selection of eyes
used for testing.

Number of replicates used;
Identity of the negative and positive controls used (if applicable, also thesolvent and benchmark controls);
Test chemical dose, application and exposure time used;
Observation time points (pre- and post- treatment);
Description of evaluation and decision criteria used including forhistopathology if applicable;
Peer-review system used for histopathological observations, if applicable;
Description of study acceptance criteria used;
Description of any modifications of the test procedure.
Furthermore, if not included in the e.g. standard operating procedure(SOP), when available, the following information shall be included:
Description of consolidated training and transferability;
Fixative, dehydration and clarifying agents, and protocols used;
Embedding material, infiltration solvents, and concentrations used;
Thickness of tissue sections;
Stain (in report) and the associated staining protocol used;
Information on instruments used;© OECD 2018
© OECD 2018

35

18
2│

438

OECD/OCDE
OECD/OCDE

replace the Draize eye test (7)(11). The Top-Down approach is designed to be
Resultsused when, based on existing information, a chemical is expected to have high Tabulation
cornealopacityand isfluorescein
retention
irritancy
potential,ofwhile
the swelling,
Bottom-Up
approachdesigned to
be usedscores
when,
obtainedeach individual
eye andat each notobservation
time point,
based onexistingforinformation,a chemical
is expected
to cause sufficienteyeincludingthe ameanscores at (7)(11).
each observation
timemethodof all tested
irritation
to requireclassification
The ICE test
is an eyes;in vitro test Description
any undermorphologicaleffects observed;
methodthat can beofused,certain circumstances
and with specific limitations
The
highestmean
cornealswelling,
opacityand fluorescein
retentionof
as described in paragraphs 7 to 11 for eye hazard
classificationand labellingscoresobserved(fromany timevalid
point),
its relatingreplacement
ICE class.; for the inchemicals.
While
it is notconsidered
as anda stand-alone
 rabbit
Tabulation
histopathologicalobservations
vivo
eye test,ofthe
ICE test method semi-quantitativeis recommended asscoring
an initial
step within
andderivedconclusions
ifapplicable;
a testing strategy such as the Top-Down approach recommended within the IfGDapplicable,
use of localized
effects
for histopathologicalOECD
263 (7) indication
to identifyof chemicals
inducing
seriouseye damage, i.e.,scoring;chemicals
to be classified as UN GHS Category 1 without further testing (4). The testDescription
any recommended
other effects observed;
ICE
method isofalso
to identify chemicals that do not requireclassificationfor eyeirritation
serious eye damage as defined by the UN GHS
 The derived
in vitro
GHSorclassification;(No Category)(4), andmay therefore
used asancontrol
initial step
If appropriate,photographsof thebetreatedandeyes within a BottomUptesting
strategy approach(OECD
263 (7).orHowever,a chemical
not
If applicable,
optional
digitalGDimages
digital slide
scansthatof isthepredicted
as causingspecimens;
serious eye damage or as not classified for eyehistopathology
irritation/serious eye damage with the ICE test method would require additional
informationof
to theestablisha definitive classification. Choice of the most appropriate
Discussion
Results.
test method(s) and use of this Test Guideline should be seen in the context of theOECD Guidance Document on an Integrated Approach on Testing andConclusion.
Assessment for Serious Eye Damage and Eye irritation (7). Furthermore, theappropriate regulatory authorities should be consulted before using the ICE test
method in a Bottom-Up approach for classification schemes other than the UN
Literature
GHS.
(1) ICCVAM(2007).
Test Method
Report- Indescribe
Vitro Ocular
Toxicity used
Test Methods
3.
The purposeof thisEvaluation
Test Guidelineis tothe procedures
tofor Identifying
OcularSevere potential
Irritants andInteragency
Coordinating
Committee
evaluate theeye hazardof a Corrosives.
test chemical
as measured
by its ability
toon theinduce
Validationof
Alternative
Methods(ICCVAM)
andthe
National
Toxicology
Programor not induce toxicity in the enucleated eyes of chicken. Toxic effects to(NTP)theInteragency
for bythe(i)Evaluationof assessment
Alternative ofToxicologicalcornea are Center
measureda qualitative
opacity, (ii) Methodsa(NICEATM).
NIH
Publication
No.:
07-4517.
Availableat:qualitative assessment of damage to epithelium based on applicationof[http://iccvam.niehs.nih.gov/methods/ocutox/ivocutox/ocu_tmer.htm]
fluorescein to the eye (fluorescein retention), (iii) a quantitative measurement of
increased thickness (swelling), and (iv) a qualitative evaluation of macroscopic(2) ESAC(2007). Statement
the surface
conclusion
the ICCVAM
study onmorphologicaldamage onto theof theoftreatedeyes. Theretrospective
corneal opacity,
organotypicin vitro
screening tests
to identify
potentialtoocularswelling,
and assays
damageas assessments
following
exposurea testcorrosives
chemical andare severe
eye irritants.
Availableat:[http://ecvam.jrc.it/index.htm].
assessed individually and then combined to derive an Eye Irritancy Classification.
Furthermore, histopathological observations may also be used as an additional(3) ICCVAM(2010).
ICCVAM Test
MethodStatus of1InnonVitro Test
endpoint
to potentially
improve
theEvaluation
predictionReportof UN– Current
GHS Category
Methods
forIdentifying
Mild/Moderate
OcularIrritants:
The
Isolated
ChickenEye(ICE)
extreme pH (2 < pH < 11.5) detergents and surfactants (see paragraphs 8 and 56). Test
Method. Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM)andthe National
Toxicology(NTP) Interagency Center for the
4.
Definitions
are provided
in AnnexProgram1.
Evaluation of Alternative Toxicological Methods (NICEATM). NIH Publication No.: 107553A. Available
at: [http://iccvam.niehs.nih.gov/methods/ocutox/MildMod-TMER.htm]
INITIALCONSIDERATIONS
AND LIMITATIONS(4) United(UN)
Globally
HarmonizedClassification
andOECD
Labelling of
5. nations
This
Test(2017).
Guideline
is basedon the Systemprotocolofsuggested
in theChemicals(GHS),
Seventhrevisededition,
UN
New
York
and
Geneva,2017.
Availableat:
Guidance Document 160 (12), which was originally adopted in 2011 and further
https://www.unece.org/fileadmin/DAM/trans/danger/publi/ghs/ghs_rev07/English/03e_part3.pdf.
updated in 2017 and 2018. The protocol is based on information obtained frompublished protocols (13) (14) (15) (16) (17).
(5) OECD (2018). Streamlined Summary Document Supporting OECD Test Guideline 438 on
the Isolated Chicken Eye for Eye Irritation/Corrosion. Series on Testing and Assessment No
188. ENV Publications, OECD, Paris.

©©OECD
OECD2018
2018

36

OECD/OCDE

438 │ 19ch is designed to (6)be OECD (2018). Test Guideline 492. OECD Guideline for Testing of Chemicals. Reconstructed
xpected to have high human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring
ned to be used when, classification and labelling for eye irritation or serious eye damage. Organisation foro cause sufficient eye Economic Cooperation and Development, Paris, France. Available at: http://www.oecdhod is an in vitro test ilibrary.org/environment/oecd-guidelines-for-the-testing-of-chemicals-section-4-healthth specific limitations effects_20745788.
ation and labelling of
eplacement for the (7)in OECD (2018). Guidance Document on an Integrated Approach on Testing and Assessment
an initial step within for Serious Eye Damage and Eye Irritation. Series on Testing and Assessment, No. 263.
mmended within the Environment, Health and Safety Publications, Organisation for Economic Cooperation andus eye damage, i.e., Development, Paris, France.
urther testing (4). The(8) Adriaens E., Barroso J., Eskes C., Hoffmann S., McNamee P., Alépée N., Bessou-Touya S.,s that do not require
ined by the UN GHS De Smedt A, de Wever B., Pfannenbecker U., Tailhardat M., Zuang V. (2014). Draize test for
tep within a Bottom- serious eye damage / eye irritation: importance of the endpoints evaluated with regard toa chemical that is not UN GHS / EU CLP classification. Archives of Toxicology 88, 701-723.
classified for eye(9) Cazelle E., Eskes C., Hermann M., Jones P., McNamee P., Prinsen M., Taylor H.,
uld require additional
the most appropriate Wijnands M.V.W. (2014). Suitability of histopathology as an additional endpoint to thein the context of the isolated chicken eye test for classification of non-extreme pH detergent and cleaning products.
ch on Testing and Toxicology In Vitro 28, 657-666.
7). Furthermore, the(10) Reproducibility and predictive capacity of the Isolated Chicken Eye Test for there using the ICE test
es other than the UN identification of non-extreme pH detergents and surfactants causing serious eye damage.
Manuscript in preparatione procedures used (11)to Scott L, Eskes C, Hoffman S, Adriaens E, Alepee N, Bufo M, Clothier R, Facchini D,
sured by its ability to Faller C, Guest R, Hamernik K, Harbell J, Hartung T, Kamp H, Le Varlet B, Meloni M,
ken. Toxic effects to Mcnamee P, Osborn R, Pape W, Pfannenbecker U, Prinsen M, Seaman C, Spielmann H,
nt of opacity, (ii) a Stokes W, Trouba K, Vassallo M, Van den Berghe C, Van Goethem F, Vinardell P, Zuang Von application of (2010). A proposed Eye Irritation Testing Strategy to Reduce and Replace in vivo Studies
ative measurement of Using Bottom-Up and Top-Down Approaches. Toxicology In Vitro 24, 1-9.
ation of macroscopic(12) OECD (2018) Guidance Document on “The Bovine Corneal Opacity and Permeability
The corneal opacity,
a test chemical are (BCOP) and Isolated Chicken Eye (ICE) Test Methods: Collection of Tissues for Histologicalitancy Classification. Evaluation and Collection of Data on Non-Severe Irritants. Series on Testing and Assessment
Hyperlinkreferencenotvalid.Available at:used as an additional No. 160. Error!
http://www.oecd.org/env/ehs/testing/series-testing-assessment-publications-number.htm.
HS Category 1 nonaragraphs 8 and 56).
(13) Prinsen, M.K. and Koëter, B.W.M. (1993). Justification of the enucleated eye test with eyesof slaughterhouse animals as an alternative to the Draize eye irritation test with rabbits.
Fd. Chem. Toxicol. 31, 69-76.

(14) DB-ALM (INVITTOX) (2009). Protocol 80: Chicken enucleated eye test (CEET) / Isolated
gested in the OECD Chicken Eye Test, 13pp. Available at: [http://ecvam-dbalm.jrc.ec.europa.eu/].
d in 2011 and further(15) Balls, M., Botham, P.A., Bruner, L.H. and Spielmann H. (1995). The EC/HO international
mation obtained fromvalidation study on alternatives to the Draize eye irritation test. Toxicol. In Vitro 9, 871-929.

© OECD 2018
© OECD 2018

37

20
2│

438

OECD/OCDE
OECD/OCDE(16) Prinsen,
replaceM.K.
the(1996).
Draize The
eye chicken
test (7)(11).
enucleated
The Top-Down
eye test (CEET):
approach
A ispractical
designed(pre)screen
to be forthe assessment
used when,ofbased
eye irritation/corrosion
on existing information,
potentiala chemicalof test materials.
is expected
Food
to haveChem.
highToxicol.
34, 291-296.
irritancy potential, while the Bottom-Up approach is designed to be used when,based on existing information, a chemical is expected not to cause sufficient eye(17) Chamberlain,M.,
Gad, S.C.,
Gautheron,(7)(11).
P. and The
Prinsen,(1997).isIRAG
irritation torequirea classification
ICE M.K.
test method
an in Working
vitro test Group
I: Organotypicmodelsforused,the assessment/predictionof ocular
Foodlimitations
Chem. Toxicol.
method thatcan beunder certain circumstances
andirritation.
with specific35, 23-37.
as described in paragraphs 7 to 11 for eye hazard classification and labelling ofchemicals. While it is not considered valid as a stand-alone replacement for the in(18) ICCVAM.
(2006).
document:
Current Status
In Vitro
Methods
vivo rabbit
eye Backgroundtest, the ICE review
test method
is recommended
as anofinitial
stepTest
withinfor Identifying
OcularCorrosives
andSevereIrritants:
Isolated
ChickenEye
Test
Method.
a testing strategy such as the Top-Down approach recommended within the
NIH OECD
Publication
No.:
06-4513.
Research
Triangle
Park:
National
Toxicology
Program.
GD 263 (7) to identify chemicals inducing serious eye damage, i.e.,
Available
at: [http://iccvam.niehs.nih.gov/methods/ocutox/ivocutox/ocu_brd_ice.htm]
chemicals
to be classified as UN GHS Category 1 without further testing (4). The
ICE test method is also recommended to identify chemicals that do not require(19) CazelleE., Eskes forC., eyeHermannM., orJonesP., McNamee
P.,asPrinsen
Wijnands
classification
irritation
seriouseye damage
definedM.,
byTaylor
the UNH.,
GHS
M.V.W.
Suitability
the Isolated
Test
for Classification
Extreme pH(No(2015).
Category)(4), andofmay
thereforeChicken
be usedEye
as aninitialstep within a of
BottomDetergentsand Cleaning
Products. (OECD
Toxicology
609-616.
Up testing
strategy approach
GD In
263Vitro(7). 29,
However,a chemical that is notpredicted as causing serious eye damage or as not classified for eye(20) Prinsen,
M.K. (2006).eye
ThedamageDraize with
Eye Test
and inalternatives;a left-handedmarriage?
irritation/serious
the ICE
testvitro
methodwould require
additional
Toxicology
in
Vitro
20,78-81.
information to establish a definitive classification. Choice of the most appropriate
test method(s) and use of this Test Guideline should be seen in the context of the(21) Siegel,
J.D., Guidance
Rhinehart, Document
E., Jackson,onM., anChiarello,
L., andthe Healthcare
Infection
OECD
Integrated
Approachon TestingandControl
Practices
Advisory
Committee(2007).
Guideline
for
Isolation
Precautions:
Preventing
Assessment for Serious Eye Damage and Eye irritation (7). Furthermore, theTransmission
Infectious
AgentsHealthcarebefore
Settings.
at:appropriateofregulatory
authorities
shouldinbe consulted
using the Available
ICE test[http://www.cdc.gov/ncidod/dhqp/pdf/isolation2007.pdf].
method in a Bottom-Up approach for classification schemes other than the UN
GHS.
(22) Barroso J., Pfannenbecker U., Adriaens E., Alépée N., Cluzel M., De Smedt A., HibatallahJ., Klaric
Mewes
K.R.,ofMillet
M., Guideline
Templier M.,
P. (2017).
Cosmetics
3. M.,The
purposethis Test
is toMcNamee
describe theprocedures
used toEurope
compilation
ofthe
historicalseriouseye damage/eye
irritation as
in measured
vivo data analysedby driversofevaluate
eye hazard
potentialof a test chemical
by its ability
toclassification
tosupporttheselectionofchemicalsfordevelopment
andevaluationofinduce or not induce toxicity in the enucleated eyes of chicken. Toxic effects toalternative
methods/strategies:
theDraizeeye
test
Reference
Database(DRD).
Archivesofthe cornea are measured by (i) a qualitative assessment of opacity, (ii) a
Toxicology
91, 521-547.
qualitative
assessment of damage to epithelium based on application offluorescein to the eye (fluorescein retention), (iii) a quantitative measurement of(23) Morton
D., Sellers
R.S., (swelling),
Barale-ThomasE., Bolon
B., George
C., Hardisty
J.F., Irizarry A.,
increased
thicknessand (iv)
a qualitativeevaluationof macroscopic
McKay
J.S.,
Odin
M.,
Teranishi
M.
(2010).
Recommendations
for
Pathology
Peer Review.
morphological damage to the surface of the treated eyes. The corneal opacity,
Toxicol.
Pathol.and
38, 1118-1127.
swelling,
damage assessments following exposure to a test chemical areassessed individually and then combined to derive an Eye Irritancy Classification.
(24) OECD(2014). Advisory
document n.observations
16. Guidancemay
on thelaboratory(GLP)
Furthermore,
histopathologicalalsogood
be usedas anpracticeadditional
Requirements
for
Peer
ReviewofHistopathology.
OECD
Seriesonprinciplesof
good
endpoint to potentially improve the prediction of UN GHS Category 1 nonlaboratory
practicecompliancemonitoring.
documentof the working
group onextremepH (2and< pH< 11.5) detergents
andAdvisory
surfactants(see paragraphs
8 and 56).
GLP. ENV/JM/MONO(2014)30. Organisation for Economic Cooperation and Development,
Paris.4.Available
at:Definitions
are provided in Annex 1.
http://www.oecd.org/chemicalsafety/testing/oecdseriesonprinciplesofgoodlaboratorypracticegl
pandcompliancemonitoring.htm.
INITIALCONSIDERATIONS AND LIMITATIONS(25) Burton,York
and R.S.isLawrence
The in vitro
assessment
severe5. A.B.G.,
This M.
Test
Guideline
based on(1981).
the protocolsuggestedin theofOECD
irritants.
Fd.
Cosmet.
Toxicol.
19,471-480.
Guidance Document 160 (12), which was originally adopted in 2011 and further

updated in 2017 and 2018. The protocol is based on information obtained from(26) OECD(2017).protocols
Test Guideline
405.
Guideline for Testing of Chemicals. Acute eyepublished(13) (14)(15)OECD(16) (17).
irritation/corrosion. Organisation for Economic Cooperation and Development, Paris, France.
Available at: http://www.oecd-ilibrary.org/environment/oecd-guidelines-for-the-testing-ofchemicals-section-4-health-effects_20745788.
©©OECD
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OECD/OCDE

438 │ 21ch is designed to be
ANNEX 1: DEFINITIONS
xpected to have highned to be used when,
Accuracy: The closeness of agreement between test method results and acceptedo cause sufficient eye
reference values. It is a measure of test method performance and one aspect of
hod is an in vitro test
“relevance.” The term is often used interchangeably with “concordance”, to meanth specific limitations
the proportion of correct outcomes of a test method.
ation and labelling of
Benchmark chemical: A chemical used as a standard for comparison to a test
eplacement for the inchemical. A benchmark chemical should have the following properties; (i), a
an initial step withinconsistent and reliable source(s); (ii), structural and functional similarity to themmended within theclass of chemicals being tested; (iii), known physical/chemical characteristics; (iv)us eye damage, i.e.,
supporting data on known effects; and (v), known potency in the range of theurther testing (4). The
desired response.
s that do not require
ined by the UN GHS
Bottom-Up Approach: step-wise approach used for a chemical suspected of not
tep within a Bottomrequiring classification for eye irritation or serious eye damage, which starts witha chemical that is not
the determination of chemicals not requiring classification (negative outcome)classified for eye
from other chemicals (positive outcome).
uld require additional
Cornea: The transparent part of the front of the eyeball that covers the iris andthe most appropriate
pupil and admits light to the interior.
in the context of thech on Testing andCorneal opacity: Measurement of the extent of opaqueness of the cornea
7). Furthermore, thefollowing exposure to a test chemical. Increased corneal opacity is indicative ofre using the ICE test
damage to the cornea.
es other than the UN
Corneal swelling: An objective measurement in the ICE test of the extent ofdistension of the cornea following exposure to a test chemical. It is expressed as apercentage and is calculated from baseline (pre-dose) corneal thickness
e procedures used tomeasurements and the thickness recorded at regular intervals after exposure to thesured by its ability totest material in the ICE test. The degree of corneal swelling is indicative ofken. Toxic effects todamage to the cornea.
nt of opacity, (ii) a
on application of
Detergents: a mixture (excluding dilutions of single surfactant) containing one or
ative measurement of
more surfactants at a final concentration of > 3%, intended for washing andation of macroscopic
cleaning processes. Detergents may be in any form (liquid, powder, paste, bar,
The corneal opacity,
cake, moulded piece, shape, etc.) and marketed for or used in household, ora test chemical are
institutional or industrial purposes.
itancy Classification.
Eye Irritation: Production of changes in the eye following the application of test
used as an additional
chemical to the anterior surface of the eye, which are fully reversible within 21
HS Category 1 nondays of application. Interchangeable with "Reversible effects on the Eye" andaragraphs 8 and 56).
with "UN GHS Category 2" (4).
False negative rate: The proportion of all positive chemicals falsely identified bya test method as negative. It is one indicator of test method performance.

gested in the OECDd in 2011 and further
mation obtained from

False positive rate: The proportion of all negative chemicals that are falsely
identified by a test method as positive. It is one indicator of test method
performance.
Fluorescein retention: A subjective measurement in the ICE test of the extent offluorescein sodium that is retained by epithelial cells in the cornea following
exposure to a test chemical. The degree of fluorescein retention is indicative of
damage to the corneal epithelium.

© OECD 2018
© OECD 2018

39

22
2│

438

OECD/OCDE
OECD/OCDE

Hazard:replace theInherent
Draizepropertyeye testof(7)(11).
an agentTheor situation
Top-Down
havingapproachthe potential
is designed
to cause
to beadverseused when,effects
basedwhenonanexistingorganism,
information,system ora(sub)chemicalpopulation
is expected
is exposed
to haveto thathigh
agent.
irritancy potential, while the Bottom-Up approach is designed to be used when,based on existing information, a chemical is expected not to cause sufficient eye
IATA: Integrated Approach on Testing and Assessment.
irritation to require a classification (7)(11). The ICE test method is an in vitro test
Irreversible
effects
on thesee circumstances"Serious eye anddamage"and "UN
GHS
method that canbe used,undereye:certainwith specificlimitations
Category
1". in paragraphs 7 to 11 for eye hazard classification and labelling of
as describedchemicals.
it isconsideredvalid asofa twostand-alone
the inMixture:A Whilemixtureor nota solution
composedor more replacement
substances inforwhichvivo
rabbit
eye
test,
the
ICE
test
methodisrecommendedasaninitialstep
withinthey do not react (4).
a testing strategy such as the Top-Down approach recommended within the
Negative
control:
An tountreatedcomponents
of a test
OECD GD
263 (7)
identify replicate
chemicalscontaining
inducing allseriouseye damage,i.e.,
system.
Thisto sample
is processedwith Category
test chemical-treatedsamplesand(4).
otherchemicals
be classified
as UN GHS
1 without further
testing
The
control
samples
to determine
whether the tosolvent
interactswith theICE test
methodis also recommended
identify
chemicals
thattest
do system.
not requireclassificationforeyeirritationor
seriouseyedamageasdefined
by
the
UNGHS
GHS
Not Classified: Test chemicals that are not classified for eye irritation (UN(No
Category)
(4),
and
may
therefore
be
used
as
an
initialstep
withina
BottomCategory 2) or serious damage to eye (UN GHS Category 1). Interchangeable
Up testing
strategy
approach (OECD GD 263 (7). However, a chemical that is not
with“UN GHSNo Category”.
predicted as causing serious eye damage or as not classified for eye
Positive
control: eye
A replicate
containing
components
of a require
test system
andirritation/serious
damage with
the ICEalltest
method would
additional
treatedwith ato chemical
to induce
a positive
response.
To ensurethat
information
establish aknown
definitiveclassification.
Choiceof the mostappropriate
variability
in thecontrol
response acrosscanin betest method(s)andpositive
use of thisTest Guideline
should time
be seentheassessed,
context ofthe
themagnitudeof the severe
response on
shouldbe excessive.
OECD GuidanceDocument
an notIntegrated
Approach on Testing andAssessment Measuresfor Seriousof Eyeand aEyetheReliability:
the Damage
extent that
testirritation
method (7).
canFurthermore,
be performed
appropriate
regulatory
authorities
shouldbeconsulted
before
using
the
ICE
test
reproducibly within and between laboratories over time, when performed using
methoda Bottom-Up
schemesthan the UN
thesameinprotocol.
It is approachassessed forby classification
calculating intraandotherinter-laboratory
GHS.
reproducibility and intra-laboratory repeatability.
Reversible
on ofthethisEye:
seeGuideline"Eye Irritation"and "UNthe
GHS
Category used
2". to3.
Theeffects
purposeTest
is to describe
procedures
evaluatetheeyehazardpotentialofatest
chemicalasmeasuredbyitsability
Serious eye damage: Production of tissue damage in the eye, or serious physicalto
inducenot induce
toxicityin the enucleatedeyes of tochicken.
Toxicsurface
effectsofto
decay
ofor
vision,following
applicationof a test chemical
the anterior
thecorneaaremeasuredby(i)aqualitative
assessmentof
opacity,(ii) athe eye, which is not fully reversible within 21 days of application.
qualitative
assessmentofdamagetoepithelium
basedon
application
Interchangeable with "Irreversible effects on the eye" and with "UN GHSof
fluoresceinto the eye (fluorescein retention), (iii) a quantitative measurement of
Category1" (4).
increased thickness (swelling), and (iv) a qualitative evaluation of macroscopic
Slit-lamp
microscope:to directly
eye underthemorphologicaldamageAn
to instrument
the surfaceusedof thetreated examineeyes. Thethecorneal
opacity,magnificationofabinocular
microscopebycreatingastereoscopic,
erect
image.
swelling, and damage assessments following exposure to a test chemical are
Inassessed
the ICEindividually
test method,and
thistheninstrument
used
to view
theIrritancy
anterior Classification.
structures ofcombinedis toderivean Eyethechicken
eye
as
well
as
to
objectively
measurecornealthicknesswitha depthFurthermore, histopathological observations may also be used as an additional
measuring
deviceattachment.
endpoint to potentially improve the prediction of UN GHS Category 1 nonextreme pH (2 <control:
pH < 11.5)
detergentssample
and surfactants(seeanda 56).
Solvent/vehicle
An untreatedcontaining
all paragraphs
components8 oftestsystem, including the solvent or vehicle that is processed with the test chemical4.
Definitions
are provided
in Annex
1.
treatedandother control
samples
to establish
the baseline response for the
samples treated with the test chemical dissolved in the same solvent or vehicle.
INITIALAND
LIMITATIONSWhenCONSIDERATIONS
tested with a concurrent
negative
control, this sample also demonstrates
whether the solvent or vehicle interacts with the test system.
5.
This Test Guideline is based on the protocol suggested in the OECD
Substance:
Chemical 160
elements
and their
compounds
in thein natural
state
orGuidance Document(12), which
was originallyadopted2011 andfurtherobtained
includingany additivenecessary
to preserve
updated byin any2017productionand 2018.process,The protocol
is basedon information
obtainedfromthestabilityprotocolsof the productand(15)any (16)
impurities
published(13) (14)(17). deriving from the process used, but
excluding any solvent which may be separated without affecting the stability ofthe substance or changing its composition (4).

©©OECD
OECD2018
2018

40

OECD/OCDE

ch is designed to be
xpected to have highned to be used when,o cause sufficient eye
hod is an in vitro testth specific limitations
ation and labelling of
eplacement for the in
an initial step within
mmended within theus eye damage, i.e.,
urther testing (4). Thes that do not require
ined by the UN GHS
tep within a Bottoma chemical that is notclassified for eye
uld require additional
the most appropriate
in the context of thech on Testing and
7). Furthermore, there using the ICE test
es other than the UN

e procedures used to
sured by its ability token. Toxic effects to
nt of opacity, (ii) a
on application of
ative measurement ofation of macroscopic
The corneal opacity,
a test chemical areitancy Classification.
used as an additional
HS Category 1 nonaragraphs 8 and 56).

gested in the OECDd in 2011 and further
mation obtained from438 │ 23

Surfactants: Also called surface-active agent, this is a substance and/or its
dilution (in an appropriate solvent/vehicle), which consists of one or morehydrophilic and one or more hydrophobic groups, that is capable of reducing thesurface tension of a liquid and of forming spreading or adsorption monolayers at
the water-air interface, and/or of forming emulsions and/or microemulsions and/or
micelles, and/or of adsorption at water-solid interfaces.
Top-Down Approach: step-wise approach used for a 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).
Test chemical: Chemical (substance or mixture) assessed in the test method.
Tiered testing strategy: A stepwise testing strategy where all existing
information on a test chemical is reviewed, in a specified order, using a weightof-evidence process at each tier to determine if sufficient information is available
for a hazard classification decision, prior to progression to the next tier. If theirritancy potential 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 step-wisesequential animal testing procedure is performed until an unequivocal
classification can be made.
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 (4).
UN GHS Category 1: see "Serious damage to eyes" and/or "Irreversible effects
on the eye".
UN GHS Category 2: see "Eye Irritation" and/or "Reversible effects to the eye".
UN No Category: Test chemicals that do not meet the requirements forclassification as UN GHS Category 1 or 2 (2A or 2B). Interchangeable with “Notclassified”.
Validated test method: A test method for which validation studies have been
completed to determine the relevance (including accuracy) and reliability for a
specific purpose. It is important to note that a validated test method may not havesufficient performance in terms of accuracy and reliability to be found acceptable
for the proposed purpose.
Weight-of-evidence: The process of considering the strengths and weaknesses ofvarious pieces of information in reaching and supporting a conclusion concerning
the hazard potential of a chemical.

© OECD 2018
© OECD 2018

41

24
2│

438

OECD/OCDE
OECD/OCDE

replace the Draize eye test (7)(11). The Top-Down approach is designed to be

ANNEXused
2: PROFICIENCYCHEMICALS FOR THE ICE TEST METHOD
when, based on existing information, a chemical is expected to have highirritancy potential, while the Bottom-Up approach is designed to be used when,
Prior to routine use of a test method that adheres to this Test Guideline,
based on existing information, a chemical is expected not to cause sufficient eyelaboratories should demonstrate technical proficiency by correctly identifying theirritation to require a classification (7)(11). The ICE test method is an in vitro test
eye hazard classification of the 13 chemicals recommended in Table 10. The ICE
method that can be used, under certain circumstances and with specific limitations
outcomes provided represent examples of the range of responses observed during
as described in paragraphs 7 to 11 for eye hazard classification and labelling of
the evaluation studies and that may be expected (5)(18).These chemicals werechemicals. While it is not considered valid as a stand-alone replacement for the inselected to represent the range of responses for eye hazards based on results fromvivo rabbit eye test, the ICE test method is recommended as an initial step within
the in vivo rabbit eye test (TG 405) and the UN GHS classification system (i.e.,a testing strategy such as the Top-Down approach recommended within the
UN GHS Categories 1, 2A, 2B, or No Category) (4)(26). Other selection criteria
OECD GD 263 (7) to identify chemicals inducing serious eye damage, i.e.,
were, to the extent possible that these chemicals produced reproducible results inchemicals to be classified as UN GHS Category 1 without further testing (4). The
the ICE test method, are commercially available and have high quality in vivo
ICE test method is also recommended to identify chemicals that do not requirereference data available. Reference data are available in the SSD (5). In situationsclassification for eye irritation or serious eye damage as defined by the UN GHS
where a listed chemical is unavailable or cannot be used for other justified(No Category) (4), and may therefore be used as an initial step within a Bottomreasons, another chemical fulfilling the criteria described above, e.g. from theUp testing strategy approach (OECD GD 263 (7). However, a chemical that is notchemicals used in the evaluation and validation of the ICE test method could be
predicted as causing serious eye damage or as not classified for eye
used (5) (18). Such deviations should however be justified.
irritation/serious eye damage with the ICE test method would require additional
information to establish a definitive classification. Choice of the most appropriate
Table 10. Recommended chemicals for demonstrating technical proficiency
test method(s) and use of this Test Guideline should be seen in the context of thewith ICEOECD Guidance Document on an Integrated Approach on Testing andAssessment for Serious Eye Damage and Eye irritation (7). Furthermore, theChemical
CASRN
ChemicalPhysical
In Vivo UN GHS
ICE
appropriate regulatory authorities 1 should be consulted before using
the ICE test
Class
Form
Classification2
UN GHS
method in a Bottom-Up approach for classification schemes other Classification
than the UN3,4
GHS.

Benzalkonium
8001-54-5
Onium
LiquidCategory 1
Category 1
chloride (10%)
compound3.
The purpose of this Test Guideline is to describe the procedures used to
Chlorhexidine
55-56-1
Amine,
Solid
Category 1
Category 1
evaluate the eye hazard potentialof a test chemical as measured by its ability toamidineinduce or not induce
toxicityAlkaliin the enucleatedof chicken.
ToxicCategory
effects 1to
Sodium1310-73-2
Liquid eyes Category1
the cornea
hydroxide(10%) are measured by (i) a qualitative assessment of opacity, (ii) a
qualitative assessment
damage to Solidepithelium Category
based 1on application
Imidazole288-32-4 ofHeterocyclic
Category 1offluorescein to theeye (fluorescein
measurement
Trichloroacetic
76-03-9
Carboxylicretention),
Liquid (iii) a quantitative
Category 1
Category 1of
thickness (swelling),and (iv) a qualitative evaluation of macroscopic
acidincreased
(30%)
acid
2,6- morphological4659-45-4
2A corneal
No predictions
damage to Acylthe halidesurface ofLiquid
the treatedCategory
eyes. The
opacity,4
Dichlorobenzcanbemadeswelling, and damage assessments following exposure to a test chemicalareoyl chloride
assessed individually and then combined to derive an Eye Irritancy
Classification.
Ammonium6484-52-2
Inorganic
Solidmay also
Category
2A5 as an
Noadditional
predictions
Furthermore, histopathologicalobservations
be usednitratesalt
can be made 4
endpoint to potentially improve the prediction of UN GHS Category 1 nonSodium
Alkali
LiquidCategory 2B
extreme pH (2 1310-73-2< pH < 11.5) detergents
andsurfactants(see paragraphsNo8 predictions
and 56).
hydroxide (1%)
can be made 4
Dimethyl
67-68-5
Organic
LiquidNo Category
No Category
4.
Definitions are provided
in Annex 1.
sulfoxide
sulphurcompoundINITIALCONSIDERATIONSAND
Ethyl trimethyl3938-95-2
Ester LIMITATIONS
LiquidNo Category
No Category
acetate5.
This Test
Guideline
is based onLiquid
the protocolsuggested in No
theCategory
OECD
Methylcyclo96-37-7
Hydrocarbon
No Category
Guidance Document 160 (12),which was originally adopted in 2011 and further
pentane(cyclic)
n-Hexane
110-54-3
Hydrocarbon
Noinformation
Category
No Category
updated in 2017and 2018.
The protocolLiquid
is based on
obtainedfrompublished protocols (13) (14)(acyclic)(15) (16) (17).
Triacetin102-76-1
Lipid
LiquidNo Category
No Category©©OECD
OECD2018
2018

42

OECD/OCDE

ch is designed to be
xpected to have highned to be used when,o cause sufficient eye
hod is an in vitro testth specific limitations
ation and labelling of
eplacement for the in
an initial step within
mmended within theus eye damage, i.e.,
urther testing (4). Thes that do not require
ined by the UN GHS
tep within a Bottoma chemical that is notclassified for eye
uld require additional
the most appropriate
in the context of thech on Testing and
7). Furthermore, there using the ICE test
es other than the UN

438 │ 25

Abbreviations: CASRN = Chemical Abstracts Service Registry Number; ICE: Isolated Chicken Eyetest; n.a.: not available; UN GHS = United Nations Globally Harmonized System of Classificationand Labelling of Chemicals (4).
1
Chemical classes were assigned to each chemical using a standard classification scheme, based on
the National Library of Medicine Medical Subject Headings (MeSH) classification system(available at http//www.nlm.nih.gov/mesh)
2
Based on results from the in vivo rabbit eye test (OECD TG 405) and using the UN GHS (4)(26).
3Based on results in ICE as described in table 7.
4
Combination of ICE scores other than the ones described in table 6 for the identification of GHS
no-category and GHS Category 1 (see table 7)
5Classification as 2A or 2B depends on the interpretation of the UN GHS criterion for
distinguishing between these two categories, i.e. 1 out of 3 vs. 2 out of 3 animals with effects at day
7 necessary to generate a Category 2A classification. The in vivo study included 3 animals. All
endpoints apart from conjunctiva redness in one animal recovered to a score of zero by day 7 orearlier. The one animal that did not fully recover by day 7 had a conjunctiva redness score of 1 (atday 7) that fully recovered at day 10.

e procedures used to
sured by its ability token. Toxic effects to
nt of opacity, (ii) a
on application of
ative measurement ofation of macroscopic
The corneal opacity,
a test chemical areitancy Classification.
used as an additional
HS Category 1 nonaragraphs 8 and 56).

gested in the OECDd in 2011 and further
mation obtained from© OECD 2018
© OECD 2018

43

44

438

OECD/OCDE

Abbreviations:

CASRN

=

Chemical

Abstracts

Service

Registry

Number;© OECD 2018

NPCM:

Prior to routine use of ICE histopathology in addition to the standard ICE test method for the limited use domain of non-extreme pH (2 < pH <11.5) detergents and surfactants, laboratories should demonstrate technical proficiency by correctly identifying the eye hazard classification of
the 6 chemicals recommended in Table 11. These chemicals were selected to represent the range of responses for eye hazards based on resultsfrom the in vivo rabbit eye test (TG 405) and the UN GHS classification system (i.e., UN GHS Categories 1, 2, or No Category) (4)(26). Other
selection criteria were, to the extent possible that these chemicals produced reproducible results in the ICE histopathology, are commerciallyavailable and have high quality in vivo reference data available. In situations where a listed chemical is unavailable or cannot be used for otherjustified reasons, another chemical fulfilling the criteria described above, e.g. from the chemicals used in the evaluation of the ICE
histopathology could be used (10). Such deviations should however be justified.

ANNEX 3: PROFICIENCY CHEMICALS FOR THE ICE HISTOPATHOLOGY TO BE USED IN ADDITION TO
THE STANDARD ICE TEST METHOD FOR THE LIMITED APPLICABILITY DOMAIN OF NON-EXTREME
PH (2 < PH < 11.5) DETERGENTS AND SURFACTANTS

26 │

45

Anionic

Cationic
Cationic
Anionic
Cationic

98-09-9

140-72-7
140-72-7
137-16-6
140-72-7

Benzensulphonylchloride

bromidebromideCetylpiridinium(10%)

Cetylpiridinium(1%)

N-Lauroyl sarcosine Na salt
(10%)

Cetylpiridinium(0.1%)

LiquidLiquidLiquidLiquidLiquidLiquidPhysicalForm

No Category

Category 2ACategory 2ACategory 1

Category 1

Category 1

In Vivo
Classification1© OECD 2018

3Based on ICE histopathology criteria as described in tables 8 and 9 and within the revised OECD GD 160 (12).

2Based on results in ICE as described in table 7.

1Based on results from the in vivo rabbit eye test (OECD TG 405) and using the UN GHS (4)(26).

Abbreviations: CASRN = Chemical Abstracts Service Registry Number; NPCM: No Prediction Can Be Made

bromideCationic

8001-54-5

Benzalkonium(5%)

chlorideSurfactant
type

CASRN

ChemicalNo predictions canbe madeNo predictions canbe madeNo predictions canbe madeNo predictions canbe madeCategory 1

Category 1Standard ICE UN
GHS classification2438 │ 27

No predictions can be madein 3 out of 3 laboratories

No predictions can be madein 3 out of 3 laboratories

No predictions can be madein 3 out of 3 laboratories

Category 1 (vacuolation)in 3 out of 3 laboratories

Category 1 (necrosis andvacuolation)in 3 out of 3 laboratories

Category 1 (erosion)in 3 out of 3 laboratories

ICE Histopathology UN
GHS classification 3

Table 11. Recommended chemicals for demonstrating technical proficiency with ICE histopathology

OECD/OCDE

28
2│

438

OECD/OCDE
OECD/OCDE

replace the Draize eye test (7)(11). The Top-Down approach is designed to be

ANNEXused
4 when, based on existing information, a chemical is expected to have highirritancy potential, while the Bottom-Up approach is designed to be used when,
Figure 1. Diagrams of the ice superfusion apparatus and eye clamps
based on existing information, a chemical is expected not to cause sufficient eye
irritation to require a classification (7)(11). The ICE test method is an in vitro test
method that can be used, under certain circumstances and with specific limitations
as described in paragraphs 7 to 11 for eye hazard classification and labelling ofchemicals. While it is not considered valid as a stand-alone replacement for the invivo rabbit eye test, the ICE test method is recommended as an initial step withina testing strategy such as the Top-Down approach recommended within theOECD GD 263 (7) to identify chemicals inducing serious eye damage, i.e.,chemicals to be classified as UN GHS Category 1 without further testing (4). The
ICE test method is also recommended to identify chemicals that do not requireclassification for eye irritation or serious eye damage as defined by the UN GHS(No Category) (4), and may therefore be used as an initial step within a BottomUp testing strategy approach (OECD GD 263 (7). However, a chemical that is notpredicted as causing serious eye damage or as not classified for eye
irritation/serious eye damage with the ICE test method would require additional
information to establish a definitive classification. Choice of the most appropriate
test method(s) and use of this Test Guideline should be seen in the context of theOECD Guidance Document on an Integrated Approach on Testing andAssessment for Serious Eye Damage and Eye irritation (7). Furthermore, theappropriate regulatory authorities should be consulted before using the ICE test
method in a Bottom-Up approach for classification schemes other than the UN
GHS.
3.
The purpose of this Test Guideline is to describe the procedures used toevaluate the eye hazard potential of a test chemical as measured by its ability to
induce or not induce toxicity in the enucleated eyes of chicken. Toxic effects tothe cornea are measured by (i) a qualitative assessment of opacity, (ii) a
qualitative assessment of damage to epithelium based on application offluorescein to the eye (fluorescein retention), (iii) a quantitative measurement of
increased thickness (swelling), and (iv) a qualitative evaluation of macroscopic
morphological damage to the surface of the treated eyes. The corneal opacity,swelling, and damage assessments following exposure to a test chemical areassessed individually and then combined to derive an Eye Irritancy Classification.
Furthermore, histopathological observations may also be used as an additional
endpoint to potentially improve the prediction of UN GHS Category 1 nonextreme pH (2 < pH < 11.5) detergents and surfactants (see paragraphs 8 and 56).
4.

Definitions are provided in Annex 1.

INITIAL CONSIDERATIONS AND LIMITATIONS
5. See (25)ThisforTest
Guideline
is based ontheprotocolapparatus
suggested
inclamp.
the OECD
Note:
additional
generic descriptionsof thesuperfusionand eye

Guidance Document 160 (12), which was originally adopted in 2011 and further
updated in 2017 and 2018. The protocol is based on information obtained frompublished protocols (13) (14) (15) (16) (17).

©©OECD
OECD20182018

46