新規試験法提案書

眼刺激性試験代替法 再構築ヒト角膜様上皮モデル法（LabCyte CORNEA-MODEL24 Eye Irritation Test）

平成31年 2 月

国立医薬品食品衛生研究所

᪂つヨ㦂ἲᥦ᱌᭩             ᖹᡂ 31 ᖺ 2 ᭶ 19 ᪥                                 No. 2018-04║่⃭ᛶヨ㦂௦᭰ἲ ෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝἲ
㸦LabCyte CORNEA-MODEL24 Eye Irritation Test㸧࡟㛵ࡍࡿᥦ᱌ ᖹᡂ 31 ᖺ 2 ᭶ 19 ᪥࡟ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ࡟࡚㛤ദࡉࢀࡓ᪂つヨ㦂ἲホ౯఍㆟㸦㏻⛠㸸
JaCVAM ホ౯఍㆟㸧࡟࠾࠸࡚௨ୗࡢᥦ᱌ࡀ࡞ࡉࢀࡓࠋ  ᥦ᱌ෆᐜ㸸LabCyte CORNEA-MODEL24 Eye Irritation Testࡣ໬Ꮫ≀㉁࡟ࡼࡿ║่⃭ᛶࢆホ౯࡛ࡁࠊ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾࠸࡚UN GHS༊ศእ≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚ࠊ⾜ᨻⓗ฼⏝ࡀྍ⬟࡛࠶ࡿ࡜⪃࠼ࡿࠋࡇࡢᥦ᱌᭩ࡣࠊOrganisation for Economic Co-operation and Development (OECD) test Guideline(TG) 492; Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals

not requiring classification and labelling for eye irritation or serious eye damage࡞࡝ࢆࡶ࡜࡟ࠊ║่⃭ᛶヨ㦂⦅⧩ጤဨ఍࡟ࡼࡾࡲ࡜ࡵࡽࢀࡓᩥ᭩ࢆ⏝࠸࡚ࠊJaCVAMホ౯఍㆟ࡀホ౯࠾ࡼࡧ᳨ウࡋࡓ⤖ᯝࠊࡑࡢ᭷⏝ᛶࡀ☜ㄆࡉࢀࡓࡇ࡜࠿ࡽసᡂࡉࢀࡓࠋ ௨ୖࡢ⌮⏤࡟ࡼࡾࠊ⾜ᨻᙜᒁࡢᏳ඲ᛶホ౯᪉ἲ࡜ࡋ࡚ LabCyte CORNEA-MODEL24 Eye Irritation
Test ἲࢆᥦ᱌ࡍࡿࡶࡢ࡛࠶ࡿࠋ
 

኱㔝Ὀ㞝
㔝Ὀ㞝                      ᖹᯘᐜᏊ
JaCVAM ホ౯఍㆟ ㆟㛗               JaCVAM 㐠Ⴀጤဨ఍ ጤဨ㛗

i

JaCVAM ホ౯఍㆟኱ 㔝 Ὀ 㞝 㸦බ┈㈈ᅋἲே ᮌཎグᛕᶓ὾⏕࿨⛉Ꮫ᣺⯆㈈ᅋ㸧㸸ᗙ㛗஬༑ᔒⰋ᫂㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧▼ ஭ 㞝 ஧㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧஭ ୖ ᬛ ᙲ 㸦᪥ᮏච␿ẘᛶᏛ఍㸧௒ ஭ ᩍ Ᏻ 㸦᪥ᮏື≀ᐇ㦂௦᭰ἲᏛ఍㸧ᒾ℩⿱⨾Ꮚ 㸦᪥ᮏ〇⸆ᕤᴗ༠఍㸧⠛ ⏣ ࿴ ಇ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧ᮡᒣ┿⌮Ꮚ 㸦᪥ᮏ໬⢝ရᕤᴗ㐃ྜ఍㸧௰ ஭ ಇ ྖ 㸦᪥ᮏ໬Ꮫᕤᴗ༠఍㸧୰ᮧࡿࡾࡇ 㸦⊂❧⾜ᨻἲே 〇ရホ౯ᢏ⾡ᇶ┙ᶵᵓ㸧す ᕝ ⛅ ె 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧἟ ⃝  ⪽ 㸦᪥ᮏẘᛶᏛ఍㸧㔝 ཱྀ ┿ ᕼ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧*᳃ ⏣  ೺ 㸦᪥ᮏ⎔ቃኚ␗ཎᏛ఍㸧ᶓ 㛵 ༤ 㞝 㸦᪥ᮏ⓶⭵ච␿࢔ࣞࣝࢠ࣮Ꮫ఍㸧௵ᮇ㸸ᖹᡂ 28 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 30 ᖺ 3 ᭶ 31 ᪥
                   *㸸ᖹᡂ 29 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 30 ᖺ 3 ᭶ 31 ᪥
኱ 㔝 Ὀ 㞝 㸦බ┈㈈ᅋἲே ᮌཎグᛕᶓ὾⏕࿨⛉Ꮫ᣺⯆㈈ᅋ㸧㸸ᗙ㛗஬༑ᔒⰋ᫂㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧▼ ஭ 㞝 ஧㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧✄ ⱝ 㑥 ᩥ㸦᪥ᮏ໬Ꮫᕤᴗ༠఍㸧஭ ୖ ᬛ ᙲ 㸦᪥ᮏච␿ẘᛶᏛ఍㸧௒ ஭ ᩍ Ᏻ 㸦᪥ᮏື≀ᐇ㦂௦᭰ἲᏛ఍㸧ᒾ℩⿱⨾Ꮚ 㸦᪥ᮏ〇⸆ᕤᴗ༠఍㸧ஂ ಖ ᩥ ᏹ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧ᮡᒣ┿⌮Ꮚ㸦᪥ᮏ໬⢝ရᕤᴗ㐃ྜ఍㸧୰ᮧࡿࡾࡇ㸦⊂❧⾜ᨻἲே 〇ရホ౯ᢏ⾡ᇶ┙ᶵᵓ㸧す ᕝ ⛅ ె㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮῭⏕఍Ᏹ㒔ᐑ⑓㝔㸧す ᮧ ḟ ᖹ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧἟ ⃝  ⪽㸦᪥ᮏẘᛶᏛ఍㸧ᖹ ᯘ ᐜ Ꮚ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧ቑ ᮧ ೺ ୍㸦᪥ᮏ⎔ቃኚ␗ཎᏛ఍㸧ᶓ 㛵 ༤ 㞝㸦᪥ᮏ⓶⭵ච␿࢔ࣞࣝࢠ࣮Ꮫ఍㸧௵ᮇ㸸ᖹᡂ 30 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 32 ᖺ 3 ᭶ 31 ᪥
ii

JaCVAM 㐠Ⴀጤဨ఍ᖹ ᯘ ᐜ Ꮚ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧㸸ጤဨ㛗▼ ஭ Ꮥ ྖ㸦ᅜ❧ឤᰁ⑕◊✲ᡤ㸧኱ ཎ  ᣅ㸦ཌ⏕ປാ┬ ་⸆࣭⏕ά⾨⏕ᒁ ་⸆ရᑂᰝ⟶⌮ㄢ㸧ᑠᕝஂ⨾Ꮚ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ⑓⌮㒊㸧ዟ ⏣ ᬕ ᏹ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧ㅋ ⏣ Ὀ ᡂ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ⸆⌮㒊㸧໭ ᔱ  ⪽㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ẘᛶ㒊㸧 ᑠụ⣫୍㑻㸦ཌ⏕ປാ┬ ་⸆࣭⏕ά⾨⏕ᒁ ་⸆ရᑂᰝ⟶⌮ㄢ ໬Ꮫ≀㉁Ᏻ඲ᑐ⟇ᐊ㸧㧗 ᮌ ⠜ ஓ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ẘᛶ㒊 ື≀⟶⌮ᐊ㸧᮰ 㔝 ṇ ᫂㸦ཌ⏕ປാ┬ ་⸆࣭⏕ά⾨⏕ᒁ ་⸆ရᑂᰝ⟶⌮ㄢ ໬Ꮫ≀㉁Ᏻ඲ᑐ⟇ᐊ㸧⻄ ⏣ ᾈ ୍㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧ᗈ ℩ ᫂ ᙪ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ Ᏻ඲ᛶணホ౯㒊㸧➜ ᮌ  ಟ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧ῲ ᒸ  Ꮫ㸦ཌ⏕ປാ┬ ་⸆࣭⏕ά⾨⏕ᒁ ་⸆ရᑂᰝ⟶⌮ㄢ ໬Ꮫ≀㉁Ᏻ඲ᑐ⟇ᐊ㸧ᮏ 㛫 ṇ ඘ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ኚ␗㑇ఏ㒊㸧ᑠ ᓥ  ⫕ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ Ᏻ඲ᛶணホ౯㒊➨஧ᐊ㸧㸸஦ົᒁ

iii

JaCVAM statement on the LabCyte CORNEA-MODEL24 Eye Irritation Test forassessing ocular irritation

At a meeting held on 19 February 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 consider the LabCyte CORNEA-MODEL24 Eye Irritation Test to be a usefulmeans of assessing the ocular irritation potential of chemicals, which when used ina bottom-up approach to identifying chemical substances that do not requireclassification and labelling under UN GHS* is suitable for use in a regulatory
context.
This statement was prepared following a review of the Organisation for Economic Cooperation and Development (OECD) Test Guideline (TG) 492 “Reconstructed human Cornealike Epithelium (RhCE) test method for identifying chemicals not requiring classification andlabelling for eye irritation or serious eye damage” and others to acknowledge that the resultsof a review and study by the JaCVAM Regulatory Acceptance Board have confirmed theusefulness of this assay.
Based on the above, we propose the LabCyte CORNEA-MODEL24 Eye Irritation Test as a
useful means for safety assessment by regulatory agencies.
*UN GHS 䠖 United Nations Globally Harmonized System of Classification and Labelling ofChemicalsYasuo Ohno
Chairperson
erson
JaCVAMM Regulatory Acceptance Board

Yoko Hirabayashi
Chairperson
JaCVAM Steering Committee

February 19, 2019v

The JaCVAM Regulatory Acceptance Board was established by the JaCVAM Steering
Committee, and is composed of nominees from the industry and academia.
This statement was endorsed by the following members of the JaCVAM Regulatory
Acceptance Board:
Mr. Yasuo Ohno (Kihara Memorial Yokohama Foundation for the Advancement ofLife Sciences): Chairperson
Mr. Yoshiaki Ikarashi (National Institute of Health Sciences: NIHS)
Mr. Noriyasu Imai (Japanese Society for Alternatives to Animal Experiments)
Mr. Tomoaki Inoue (Japanese Society of Immunotoxicology)
Mr. Yuji Ishii (Biological Safety Research Center: BSRC, NIHS)
Ms. Yumiko Iwase (Japan Pharmaceutical Manufacturers Association)
Mr. Takeshi Morita (Japanese Environmental Mutagen Society)
Mr. Shunji Nakai (Japan Chemical Industry Association)
Ms. Ruriko Nakamura (National Institute of Technology and Evaluation)
Mr. Akiyoshi Nishikawa (BSRC, NIHS)
Mr. Satoshi Numazawa (Japanese Society of Toxicology)
Ms. Maki Noguchi (Pharmaceuticals and Medical Devices Agency) *Mr. Kazutoshi Shinoda (Pharmaceuticals and Medical Devices Agency)
Ms. Mariko Sugiyama (Japan Cosmetic Industry Association)
Mr. Hiroo Yokozeki (Japanese Society for Cutaneous Immunology and Allergy)
Term: From 1st April 2016 to 31st March 2018*: From 1st April 2017 to 31st March 2018
Mr. Yasuo Ohno (Kihara Memorial Yokohama Foundation for the Advancement ofLife Sciences): Chairperson
Ms. Yoko Hirabayashi (BSRC, NIHS)
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 (BSRC, 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 (BSRC, 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
vi

This statement was endorsed by the following members of the JaCVAM steering Committee
after receiving the report from JaCVAM Regulatory Acceptance Board:
Ms. Yoko Hirabayashi (BSRC, 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, BSRC, NIHS)
Mr. Koichi Hiruta (Pharmaceuticals and Medical Devices Agency)
Mr. Masamitsu Honma (Division of Genetics and Mutagenesis, BSRC, NIHS)
Mr. Koji Ishii (National Institute of Infectious Diseases)
Mr. Yasunari Kanda (Division of Pharmacology, BSRC, NIHS)
Mr. Satoshi Kitajima (Division of Toxicology, BSRC, NIHS)
Mr. Kouichirou Koike (Ministry of Health, Labour and Welfare)
Ms. Kumiko Ogawa (Division of Pathology, BSRC, NIHS)
Mr. Haruhiro Okuda (NIHS)
Mr. Taku Oohara (Ministry of Health, Labour and Welfare)
Mr. Atsuya Takagi (Animal Management Section of the Division of Toxicology,
BSRC, NIHS)
Mr. Masaaki Tsukano (Ministry of Health, Labour and Welfare)Mr. Hajime Kojima (Division of Risk Assessment, BSRC, NIHS): Secretaryvii║่⃭ᛶヨ㦂௦᭰ἲ ෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝἲ
㸦LabCyte CORNEA-MODEL24 Eye Irritation Test㸧┠  ḟ
ホ౯఍㆟ሗ࿌᭩ ------------------------------------------------------------------------------------ 1
ホ౯ሗ࿌᭩ ------------------------------------------------------------------------------------------ 7
OECD GUIDELINE FOR THE TESTING OF CHEMICALS 492, Reconstructed humanCornea-like Epithelium (RhCE) test method for identifying chemicals not requiring
19classification and labelling for eye irritation or serious eye damage -------------------------------1ixホ౯఍㆟ሗ࿌᭩
෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝἲ㸦RhCE ἲ㸧
LabCyte CORNEA-MODEL24 ║่⃭ᛶヨ㦂
㸦LabCyte CORNEA-MODEL24 EIT㸧JaCVAM ホ౯఍㆟

ᖹᡂ 30 ᖺ㸦2018 ᖺ㸧12 ᭶ 25 ᪥

1
1

JaCVAM ホ౯఍㆟኱ 㔝 Ὀ 㞝 㸦බ┈㈈ᅋἲே ᮌཎグᛕᶓ὾⏕࿨⛉Ꮫ᣺⯆㈈ᅋ㸧㸸ᗙ㛗஬༑ᔒⰋ᫂㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧▼ ஭ 㞝 ஧㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧✄ ⱝ 㑥 ᩥ㸦᪥ᮏ໬Ꮫᕤᴗ༠఍㸧஭ ୖ ᬛ ᙲ 㸦᪥ᮏච␿ẘᛶᏛ఍㸧௒ ஭ ᩍ Ᏻ 㸦᪥ᮏື≀ᐇ㦂௦᭰ἲᏛ఍㸧ᒾ℩⿱⨾Ꮚ 㸦᪥ᮏ〇⸆ᕤᴗ༠఍㸧ஂ ಖ ᩥ ᏹ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧ᮡᒣ┿⌮Ꮚ㸦᪥ᮏ໬⢝ရᕤᴗ㐃ྜ఍㸧୰ᮧࡿࡾࡇ㸦⊂❧⾜ᨻἲே 〇ရホ౯ᢏ⾡ᇶ┙ᶵᵓ㸧す ᕝ ⛅ ె㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮῭⏕఍Ᏹ㒔ᐑ⑓㝔㸧す ᮧ ḟ ᖹ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧἟ ⃝  ⪽㸦᪥ᮏẘᛶᏛ఍㸧ᖹ ᯘ ᐜ Ꮚ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧 ቑ ᮧ ೺ ୍㸦᪥ᮏ⎔ቃኚ␗ཎᏛ఍㸧ᶓ 㛵 ༤ 㞝㸦᪥ᮏ⓶⭵ච␿࢔ࣞࣝࢠ࣮Ꮫ఍㸧௵ᮇ䠖ᖹᡂ 30 ᖺ 4 ᭶ 1 ᪥䡚ᖹᡂ 32 ᖺ 3 ᭶ 31 ᪥22෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝἲ㸦Reconstructed human Cornea-like Epithelium Test Method: RhCE ἲ㸧ࡣࠊ࢘ࢧࢠࢆ⏝࠸ࡓ Draize ║่⃭ᛶヨ㦂ࡢ௦᭰ヨ㦂ἲ࡜ࡋ࡚ࠊ⿕㦂≀㉁ࡢࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝ⤌⧊࡟ᑐࡍࡿ⣽⬊ẘᛶࢆᣦᶆ࡟⏝࠸ࠊࡑࡢ≀㉁ࡢ║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛࠶ࡿࠋOECD TG492 ࡟ࡣࠊ࣎ࢺ࣒
࢔ࢵࣉ᪉ᘧ࡛ United Nations Globally Harmonized System of Classification and Labelling of Chemicals㸦UN
GHS㸧༊ศእ≀㉁ࢆ᳨ฟ࡛ࡁࡿ EpiOcularTM ║่⃭ᛶヨ㦂㸦EpiOcularTM Eye Irritation Test: EpiOcularTM EIT㸧࠾ࡼࡧ SkinEthicTM ࣄࢺゅ⭷ୖ⓶ࣔࢹࣝ║่⃭ᛶヨ㦂㸦SkinEthicTM Human Corneal Epithelium Eye Irritation
Test: SkinEthicTM HCE EIT㸧ࡀ ESAC㸦EURL ECVAM Scientific Advisory Committee㸧ࡢ➨୕⪅ホ౯ࢆ⤒࡚RhCE ἲࡢ᳨ド῭ࡳᶆ‽ヨ㦂ἲ㸦Validated Reference Method: VRM㸧࡜ࡋ࡚཰㍕ࡉࢀ࡚࠸ࡿ 1)ࠋࡲࡓࠊOECD
ࡣ᪂つ RhCE ⤌⧊ࢆ⏝࠸ࡓヨ㦂ἲ࡟ᑐࡋ࡚ࡑࡢᵓ㐀࠾ࡼࡧᶵᗎࡀ VRM ࡜ྠ➼࡛༑ศ࡞ಙ㢗ᛶࢆഛ࠼࡚
࠸ࡿࡇ࡜ࢆホ౯ࡍࡿᛶ⬟ᶆ‽ࢆᐃࡵ࡚࠸ࡿ 2) ࠋLabCyte CORNEA-MODEL24 ࢆ⏝࠸ࡓ║่⃭ᛶヨ㦂
㸦LabCyte CORNEA-MODEL24 Eye Irritation Test: LabCyte CORNEA-MODEL24 EIT㸧ࡣ㢮ఝヨ㦂ἲ࡜ࡋ࡚ࠊOECD ࡢᛶ⬟ᶆ‽࡛ᣦᐃࡉࢀ࡚࠸ࡿ 30 ࡢཧ↷≀㉁ࢆ⏝࠸ࡓ 3 ᪋タ࡛ࡢ㏣㉮ⓗࣂࣜࢹ࣮ࢩࣙࣥ◊✲ 3)
࠾ࡼࡧ JaCVAM ࡢ➨୕⪅ホ౯ 4)ࢆ⤒࡚ࠊ2018 ᖺ࡟ TG492 ࡟཰㍕ࡉࢀࡓ 5)ࠋJaCVAM ホ౯఍㆟ࡣࠊ║่⃭ᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍࡟ࡼࡾసᡂࡉࢀࡓࠕᨵゞ OECD TG492 ෌ᵓ⠏ゅ⭷ᵝୖ⓶ࣔࢹࣝἲ LabCyte
CORNEA-MODEL24 ║่⃭ᛶヨ㦂ホ౯ሗ࿌᭩ࠖ6)ࢆ⏝࠸࡚ࠊᮏヨ㦂ἲࡢጇᙜᛶ࡟ࡘ࠸᳨࡚ウࡋࡓࠋ㸯㸬ヨ㦂ἲࡢᐃ⩏ྡ⛠㸸෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝἲ㸦Reconstructed Human Cornea-like Epithelium Test Method: RhCEἲ㸧 LabCyte CORNEA-MODEL24 ║่⃭ᛶヨ㦂௦᭰ࡍࡿᑐ㇟ẘᛶヨ㦂㸸 Draize ║่⃭ᛶヨ㦂ἲヨ㦂ἲࡢᴫ␎㸸 RhCE ἲࡢࡦ࡜ࡘ࡛࠶ࡿ LabCyte CORNEA-MODEL24 EIT ࡛ࡣࠊࣄࢺゅ⭷ୖ⓶⣽⬊ࢆ㔜ᒙᇵ㣴ࡋࡓ LabCyte CORNEA-MODEL24 ࡟⿕㦂≀㉁ࡀᾮయࡢሙྜࡣ 1 ศ㛫ࠊᅛయࡢሙྜࡣ 24 ᫬㛫⿕㦂 ≀ ㉁ ࢆ ᭚ 㟢 ࡋ ࡓ ᚋ ࠊ WST-8 㸦 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)2Htetrazolium, monosodium salt㸧ࡢ㑏ඖ㔞ࢆࡶ࡜࡟ࡋࡓ⣽⬊⏕Ꮡ⋡ࢆᐃࡋࠊ║่⃭ᛶホ౯ࡢᣦᶆ࡜ࡋ࡚

⏝࠸ࡿࠋࡇࢀࡣࠊWST-8 ࡀ࣑ࢺࢥࣥࢻࣜ࢔ࡢ⬺Ỉ⣲㓝⣲ࡢᇶ㉁࡜࡞ࡿᛶ㉁ࢆ฼⏝ࡋࠊ⣽⬊ෆ࡟ྲྀࡾ㎸
ࡲࢀࡓ WST-8 ࡀ⬺Ỉ⣲㓝⣲࡟ࡼࡾ㑏ඖࡉࢀࠊ⏕ᡂࡉࢀࡓ࣐࣍ࣝࢨࣥ㔞㸦㟷Ⰽ㸧ࡀ⏕Ꮡ⣽⬊ᩘ࡟ẚ౛ࡍࡿࡇ࡜࡟ᇶ࡙࠸࡚࠸ࡿࠋ⿕㦂≀㉁ࡀᾮయ࣭ᅛయ࡟㛵ࢃࡽࡎࠊᖹᆒ⣽⬊⏕Ꮡ⋡ࡀ 40㸣ࢆ㉸࠼ࡓሙྜࠊ
⿕㦂≀㉁ࡣ UN GHS ศ㢮࡟࠾࠸࡚༊ศእ࡛࠶ࡿ࡜ุᐃࡍࡿࠋ࡞࠾ࠊᮏヨ㦂ἲ࡛ഇ㝧ᛶࢆᅇ㑊ࡍࡿࡓࡵ࡟ࡣࠊࡲࡓࠊUN GHS ศ㢮༊ศ 1 ࡜༊ศ 2 ࢆ㆑ูࡍࡿᚲせࡀ࠶ࡿሙྜࡣࠊ௚ࡢヨ㦂ἲ࡟ࡼࡿ㏣ຍヨ㦂ࡀᚲせ࡟࡞ࡿࠋ㸰㸬ホ౯࡟⏝࠸ࡓ㈨ᩱ࠾ࡼࡧホ౯ෆᐜࡢ⛉Ꮫⓗጇᙜᛶ║࡟␗≀ࡀධࡗࡓሙྜࠊ║ࡢ่⃭ࡣࠊ⚄⤒➼ࡢ≉ᐃࡢཷᐜయ࡟స⏝ࡍࡿሙྜࢆ㝖ࡁࠊ୍⯡࡟ゅ⭷ࡸ⤖⭷ࡢ⣽⬊യᐖ࠿ࡽጞࡲࡿࠋDraize ἲ࡟࠾ࡅࡿ║่⃭ᛶࡢ⛬ᗘࡢุᐃࡣࠊ୺࡟ゅ⭷ࡢึᮇയᐖࡢ⛬ᗘ࡟኱ࡁࡃᙳ㡪ࡉࢀࠊࡑࢀࡣゅ⭷ୖ⓶⣽⬊ࡢ⣽⬊Ṛࡢ⛬ᗘ࡜┦㛵㛵ಀ࡟࠶ࡿࠋᮏヨ㦂ἲࡣࠊࣄࢺࡢゅ⭷ୖ⓶࡟

33㢮ఝࡋࡓᵓ㐀ࢆ᭷ࡍࡿ RhCE㸦LabCyte CORNEA-MODEL24㸧ࢆ⏝࠸࡚ࠊ⿕㦂≀㉁ࡢ⣽⬊ẘᛶࢆᣦᶆ࡜ࡋ
࡚║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛࠶ࡿࠋࡇࢀࡽࡢࡇ࡜࠿ࡽࠊᮏヨ㦂ἲࡣ࢘ࢧࢠࢆ⏝࠸ࡿ║่⃭ᛶヨ㦂ࡢ௦᭰ἲ࡜ࡋ࡚⛉Ꮫⓗጇᙜᛶࡀ࠶ࡿࠋ
LabCyte CORNEA-MODEL24 EIT ࡟ࡘ࠸࡚ࡣࠊ࣮ࣜࢻࣛ࣎࡜ࡣ␗࡞ࡿ 3 ᪋タ࡟࠾࠸࡚ OECD ࡢᛶ⬟ᶆ‽࡛ᣦᐃࡉࢀ࡚࠸ࡿ 30 ࡢཧ↷≀㉁ࢆ⏝࠸ࡓ㏣㉮ⓗࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀ⾜ࢃࢀࡓ 3)ࠋࡑࡢ⤖ᯝࠊLabCyte
CORNEA-MODEL24 EIT ࡣࠊUN GHS ༊ศእ≀㉁ண

ᛶࡢ᪋タෆ෌⌧ᛶࠊ᪋タ㛫෌⌧ᛶ࠾ࡼࡧṇ☜ᛶ࡟ࡘ࠸࡚ OECD ࡢᛶ⬟ᶆ‽ࡀᐃࡵࡓᇶ‽ࢆ‶ࡓࡋࡓࠋࡉࡽ࡟ JaCVAM ࡢ➨୕⪅ホ౯ 4)ࢆ⤒࡚ࠊUN GHS ༊ศእ≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚ 2018 ᖺ࡟ᨵゞ OECD TG492 ࡟㏣グࡉࢀࡓࠋJaCVAM ║่⃭ᛶヨ㦂㈨ᩱ
⦅⧩ጤဨ఍ࡣࠊࡇࢀࡽࡢ㈨ᩱࢆ⏝࠸࡚ᮏヨ㦂ἲࢆホ౯ࡋ࡚࠾ࡾࠊ⛉Ꮫⓗ࡟ጇᙜ࡛࠶ࡿ࡜⪃࠼ࡿࠋ㸱㸬ᮏヨ㦂ἲࡢ᭷⏝ᛶ࡜㐺⏝㝈⏺
RhCE ἲ࡟⏝࠸ࡿ LabCyte CORNEA-MODEL24 ࡣᕷ㈍ࡉࢀ࡚࠾ࡾࠊࡇࢀ௨እࡣ≉Ṧ࡞ᶵᮦࡸヨ⸆ࢆᚲせ࡜ࡏࡎࠊᡭᢏࡶ」㞧࡛ࡣ࡞࠸ࡇ࡜࠿ࡽᢏ⾡⛣㌿ᛶࡣ㧗࠸࡜ุ᩿࡛ࡁࡿࠋణࡋࠊධᡭࡋࡓ LabCyte
CORNEA-MODEL24 ࡀရ㉁ᇶ‽ࡢチᐜ⠊ᅖ࡟࠶ࡾࠊ࠿ࡘᐇ᪋ࡍࡿヨ㦂᪋タࡢᢏ⾡⩦ᚓࡀ࢞࢖ࢻࣛ࢖ࣥࡢ
⩦⇍ᗘ☜ㄆ≀㉁࡛☜࠿ࡵࡽࢀ࡚࠸ࡿᚲせࡀ࠶ࡿࠋUN GHS ༊ศእ≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚ࡢಙ㢗ᛶࢆㄪ࡭ࡿࡓࡵࠊ᪋タෆ෌⌧ᛶ࠾ࡼࡧ᪋タ㛫෌⌧ᛶࢆ᳨ウࡍࡿࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀ⾜ࢃࢀ࡚࠸ࡿࠋࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡟࠾࠸࡚ࠊUN GHS ศ㢮ุᐃࡢ᪋タࡈ࡜ࡢ᪋タෆ෌⌧ᛶࡣ 93-100㸣࡛࠶ࡾࠊOECD ࡢᛶ⬟ᶆ‽ࡀᐃࡵࡓᇶ‽㸦90㸣௨ୖ㸧ࢆ‶ࡓࡋ࡚࠸ࡓ 6)ࠋ
ࡲࡓࠊ᪋タ㛫෌⌧ᛶࡣࠊ87㸣࡛ࠊOECD ࡢᛶ⬟ᶆ‽ࡀᐃࡵࡓᇶ‽㸦85㸣௨ୖ㸧ࢆ‶ࡓࡋ࡚࠸ࡓ 6)ࠋࡉࡽ
࡟ࠊࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡛ᚓࡽࢀࡓࢹ࣮ࢱ࡟ࡼࡿᛶ⬟ホ౯࡛ࡣࠊឤᗘ 97.8㸣ࠊ≉␗ᗘ 68.9㸣࠾ࡼࡧṇ☜ᗘ 83.5㸣࡜࡞ࡾ OECD ࡢᛶ⬟ᶆ‽ࡀᐃࡵࡓᇶ‽㸦ឤᗘ 90%௨ୖࠊ≉␗ᗘ 60%௨ୖࠊṇ☜ᗘ 75%௨ୖ㸧ࢆ‶ࡓࡋ࡚࠸ࡓ 6)ࠋ
ణࡋࠊ⣽⬊⏕Ꮡ⋡ࡢ⟬ฟ࡟㝿ࡋࠊ⿕㦂≀㉁ࡀ WST-8 ࢆ㑏ඖࡍࡿ≀㉁ࡢሙྜࠊ࠶ࡿ࠸ࡣ࣐࣍ࣝࢨࣥ࡜ྠ
ࡌࡼ࠺࡞Ἴ㛗㸦450 nm ㏆㎶㸧࡟྾཰ࢆᣢࡘ╔Ⰽ≀㉁ࡢሙྜ࡟ࡣࠊ྾ගᗘ⿵ṇࢆ⾜࠺ᚲせࡀ࠶ࡿࠋࡑࡢᡭ
㡰࡟ࡘ࠸࡚ࡣࠊᨵゞ TG492 ࡢᮏᩥࡢㄝ᫂࠾ࡼࡧ ANNEX V ࡢࣇ࣮ࣟࢳ࣮ࣕࢺࢆཧ↷ࡍࡿᚲせࡀ࠶ࡿࠋࡲࡓࠊᮏヨ㦂ἲࢆ㐺⏝ࡍࡿ࡟ࡣࠊヨ㦂ἲࡢᛶ⬟࡜ṇ☜ᛶࡢ☜ಖࢆ⪃៖ࡋ࡚௨ୗࡢไ㝈ࡀタࡅࡽࢀࡿࠋ
1) ࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡟࠾࠸࡚⿕㦂≀㉁࡟ྵࡵࡽࢀ࡞࠿ࡗࡓẼయ㸦࢞ࢫ㸧࠾ࡼࡧ࢚࢔ࣟࢰࣝ≧≀㉁ࡣ㐺⏝࠿ࡽ㝖እࡉࢀࡿࠋ
2) UN GHS ༊ศࡢ༊ศ 1 ≀㉁࡜༊ศ 2㸦2A/2B㸧≀㉁ࡢ㆑ู࡟ࡣ⏝࠸ࡿࡇ࡜ࡣ࡛ࡁ࡞࠸ࠋ
௨ୖࡢⅬ࠿ࡽࠊTG492 ࡟‽ᣐࡋ࡚ᐇ᪋ࡋࡓሙྜࠊ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾࠸࡚ UN GHS ༊ศእ≀㉁ࢆ
᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚᭷⏝࡛࠶ࡿ࡜⪃࠼ࡿࠋ44㸲㸬┠ⓗ࡜ࡍࡿ≀㉁ཪࡣ〇ရࡢẘᛶࢆホ౯ࡍࡿヨ㦂ἲ࡜ࡋ࡚ࡢࠊ♫఍ⓗཷࡅධࢀᛶ࠾ࡼࡧ⾜ᨻୖࡢ฼⏝ࡢྍ⬟ᛶ ♫఍ⓗཷࡅධࢀᛶ㸸ᮏヨ㦂ἲࡣ RhCE ࡟ᑐࡍࡿ໬Ꮫ≀㉁ࡢ⣽⬊ẘᛶࢆᣦᶆ࡟⏝࠸࡚║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛࠶ࡾࠊ⏕ࡁࡓື≀ࢆ⏝࠸࡞࠸࡜࠸࠺Ⅼ࡛ࠊ3Rs ࡢ⢭⚄࡟ྜ⮴ࡋ࡚࠸ࡿࠋࡲࡓࠊLabCyte CORNEA-MODEL24 ࡣẚ
㍑ⓗᏳ౯࡛࠶ࡾࠊࡑࡢධᡭࡣᐜ࡛᫆ࠊ▷᫬㛫࡛ᐇ᪋࡛ࡁࡿࠋࡲࡓࠊ≉Ṧ࡞ᶵᮦࡸヨ⸆ࢆᚲせ࡜ࡏࡎࠊᚲ
せ࡞ᡭᢏࡶ」㞧࡞ࡶࡢ࡛࡞࠸ࠋࡋࡓࡀࡗ࡚ࠊධᡭࡋࡓ LabCyte CORNEA-MODEL24 ࡀရ㉁ᇶ‽ࡢチᐜ⠊ᅖ࡟࠶ࡾࠊ࠿ࡘᐇ᪋ࡍࡿヨ㦂᪋タࡢᢏ⾡⩦ᚓࡀ࢞࢖ࢻࣛ࢖ࣥࡢ⩦⇍ᗘ☜ㄆ≀㉁࡛☜࠿ࡵࡽࢀ࡚࠸ࢀࡤࠊ
ᇶᮏⓗ࡞⣽⬊ᇵ㣴ࡢᢏ⾡࡜タഛࢆ᭷ࡍࡿ᪋タ࡛ᐇ᪋ྍ⬟࡛࠶ࡾࠊᢏ⾡⛣㌿ᛶࡣ㧗࠸ࠋ௨ୖࡼࡾࠊᮏヨ㦂ἲࡢ♫఍ⓗཷࡅධࢀᛶࡣ㧗࠸ࠋ ⾜ᨻୖࡢ฼⏝ᛶ㸸ᮏヨ㦂ἲࡣࠊ໬Ꮫ≀㉁࡟ࡼࡿ║่⃭ᛶࢆホ౯࡛ࡁࠊ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾࠸࡚ UN GHS ༊ศእ≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚ࠊ⾜ᨻⓗ฼⏝ࡀྍ⬟࡛࠶ࡿ࡜⪃࠼ࡿࠋཧ⪃ᩥ⊩1) OECD (2017) Guideline for the testing of chemicals. 492, Reconstructed human Cornea-like Epithelium(RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation orserious eye damage.
2) OECD (2016) Performance standards for the assessment of proposed similar or modified in vitro reconstructed
human cornea-like epithelium (RhCE) test methods for identifying chemicals not requiring classification andlabelling for eye irritation or serious eye damage, based on the validated reference methods EpiocularTM EIT
and SkinethicTM HCE EIT described in TG492.
3) LabCyte Validation Management Team (2017) Me-too Validation Report: Validation Study for LabCyte
CORNEA-MODEL24 Eye Irritation Test (Version. 3.0).
4) Peer Review Panel (2017) Validation status of the LabCyte CORNEA-MODEL24 EYE IRRITTION TEST,
Report of the Peer Review Panel on a JaCVAM co-ordinated study programme addressing the validation statusof LabCyte CORNEA-MODEL24 EYE IRRITATION TEST for discriminating eye irritant from non-eye
irritant substances.
5) OECD (2018) Test Guideline 492. Reconstructed human Cornea-like Epithelium (RhCE) test method for
identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage.
6) JaCVAM ║่⃭ᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍㸸ホ౯ሗ࿌᭩ ෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝἲ (RhCEἲ)
LabCyte CORNEA-MODEL24║่⃭ᛶヨ㦂㸦LabCyte CORNEA-MODEL24 EIT㸧㸦2018ᖺ10᭶1᪥㸧
5
5



ホ౯ሗ࿌᭩


෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝἲ㸦RhCE ἲ㸧
LabCyte CORNEA-MODEL24 ║่⃭ᛶヨ㦂
㸦LabCyte CORNEA-MODEL24 EIT㸧║่⃭ᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍

ᖹᡂ 30 ᖺ㸦2018 ᖺ㸧12 ᭶ 27 ᪥

1
7



║่⃭ᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍ᒣᮏ┤ ᶞ 㸦⸨⏣་⛉኱Ꮫ㸧బ ࠎ ᮌ ṇ ἞ 㸦࢔ࢵࣦ࢕ྜྠ఍♫㸧➉ෆᑠ ⱑ 㸦P&G ࢖ࣀ࣮࣋ࢩࣙࣥྜྠ఍♫㸧Ἴ ከ 㔝 ᾈ ኴ 㸦࣮࣮࣍ࣘᰴᘧ఍♫㸧᳃ᮧᑠ ᓥᬛ ⨾ 㸦୍⯡㈈ᅋἲே㣗ရ⸆ရᏳ඲ࢭࣥࢱ࣮㸧⫕ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧

2
8



␎ㄒ
CAS: Chemical Abstracts Services
EIT: Eye Irritation Test
ESAC: EURL ECVAM Scientific Advisory Committee
GHS: Globally Harmonized System of Classification and Labeling of Chemicals
JaCVAM: Japanese Center for the Validation of Alternative Methods
OD: Optical Density
OECD: Organization for Economic Co-operation and Development
RhCE: Reconstructed human Cornea-like Epithelium
SDS: Sodium Dodecyl Sulphate
TG: Test Guideline
UN: United Nations
VRM: Validated Reference Method
WST: Water Soluble Tetrazolium Salt

3
9



せ᪨
෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝ㸦Reconstructed human Cornea-like Epithelium: RhCE㸧ἲࡣࠊ໬Ꮫ≀㉁
ࡢ RhCE ⤌⧊࡟ᑐࡍࡿ⣽⬊ẘᛶࢆᣦᶆ࡟⏝࠸ࠊࡑࡢ≀㉁ࡢ║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛࠶ࡾࠊOECD
TG492 ࡜ࡋ࡚࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡛ UN GHS ༊ศእ≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚᥇ᢥࡉࢀ࡚࠸ࡿࠋ
LabCyte CORNEA-MODEL24 ║่⃭ᛶヨ㦂㸦LabCyte CORNEA-MODEL24 EIT㸧ࡣ RhCE ἲࡢ୍ࡘ࡛ࠊ
2018 ᖺ࡟ᨵゞ TG492 ࡟㏣ຍࡉࢀࡓヨ㦂ἲ࡛࠶ࡿࠋᮏሗ࿌࡛ࡣࠊLabCyte CORNEA-MODEL24 EIT ࡢࣂࣜࢹ࣮ࢩࣙࣥ◊✲ሗ࿌᭩ࠊ➨୕⪅ホ౯ሗ࿌᭩ࠊ㛵㐃ㄽᩥ࡞࡝ࢆࡶ࡜࡟ヨ㦂ἲࡢᴫせࢆㄝ᫂ࡋJaCVAM ║่⃭ᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍ࡢពぢࢆࡲ࡜ࡵࡓࠋ
 LabCyte CORNEA-MODEL24 EIT ࡢಙ㢗ᛶ࣭ṇ☜ᛶࢆ☜ㄆࡍࡿࡓࡵࠊOECD ࡢᛶ⬟ᶆ‽㸦PerformanceStandard㸧࡛ᣦᐃࡉࢀ࡚࠸ࡿ 30 ࡢཧ↷≀㉁㸦Reference Chemicals㸧ࢆ⏝࠸࡚ 3 ᪋タ࡛㏣㉮ⓗࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀ⾜ࢃࢀࡓࠋྛ᪋タ࡛ 1 ≀㉁࠶ࡓࡾ 3 ᅇヨ㦂ࢆ⾜ࡗࡓࠋࡇࡢ㏣㉮ⓗࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡟࠾࠸࡚ LabCyte CORNEA-MODL24 EIT ࡢ᪋タෆ෌⌧ᛶࡣࡑࢀࡒࢀࡢ᪋タ࡛ 93%ࠊ97%ࠊ100%ࠊࡲࡓࠊ᪋タ㛫෌⌧ᛶࡣ 87%࡛࠶ࡗࡓࠋṇ☜ᛶࡣࠊឤᗘ 97.8%ࠊ≉␗ᗘ 68.9%ࠊṇ☜ᗘ 83.5%࡛࠶ࡗࡓࠋࡇࢀ
ࡽ෌⌧ᛶࠊṇ☜ᛶࡢ್ࡣࠊᛶ⬟ᶆ‽ࡀᐃࡵࡓᇶ‽ࢆ‶ࡓࡋ࡚࠸ࡓࠋ ௨ୖࡼࡾࠊᮏጤဨ఍ࡣࠊLabCyte CORNEA-MODEL24 EIT ࡣ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡛ UN GHS ༊ศእ≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚⏝࠸ࡿࡇ࡜ࡀ࡛ࡁࡿ࡜⤖ㄽࡋࡓࠋ

4
10



1. ࡲ࠼࠾ࡁ
෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝ㸦Reconstructed human Cornea-like Epithelium: RhCE㸧ἲࡣࠊRhCE ⤌⧊࡟ᑐࡍࡿ⿕㦂≀㉁ࡢ⣽⬊ẘᛶࢆᣦᶆ࡟║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛ࠊ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟ࡼࡾ UN
GHS ༊ศእ≀㉁ࢆ᳨ฟࡍࡿࠋ
EpiOcularTM ║่⃭ᛶヨ㦂㸦EpiOcularTM Eye Irritation Test: EpiOcularTM EIT㸧࠾ࡼࡧ SkinEthicTM ࣄࢺゅ⭷ୖ⓶ࣔࢹࣝ║่⃭ᛶヨ㦂㸦SkinEthicTM Human Corneal Epithelium Eye Irritation Test: SkinEthicTM
HCE EIT㸧ࡣ ESAC ࡢ➨୕⪅ホ౯ࢆ⤒࡚ RhCE ἲࡢ᳨ド῭ࡳᶆ‽ヨ㦂ἲ㸦Validated Reference Method:
VRM㸧࡜ࡋ࡚ OECD TG492 ࡟཰㍕ࡉࢀ࡚࠸ࡿ 1)ࠋࡲࡓࠊOECD ࡣ᪂つ RhCE ⤌⧊ࢆ⏝࠸ࡓヨ㦂ἲ㸦me-too
ヨ㦂ἲ㸧࡟ᑐࡋ࡚ࡑࡢᵓ㐀࣭ᶵᗎࡀ VRM ࡜ྠ➼࡛࠶ࡾ༑ศ࡞ಙ㢗ᛶ࡜㛵㐃ᛶࢆഛ࠼࡚࠸ࡿࡇ࡜ࢆホ౯ࡍࡿᛶ⬟ᶆ‽㸦Performance Standard㸧ࢆᐃࡵࡓ 2) ࠋLabCyte CORNEA-MODEL24 ║่⃭ᛶヨ㦂
㸦LabCyte CORNEA-MODEL24 Eye Irritation Test: LabCyte CORNEA-MODEL24 EIT㸧ࡣ me-too ヨ㦂ἲ࡜ࡋ࡚ࠊOECD ࡢᛶ⬟ᶆ‽࡛ᣦᐃࡉࢀ࡚࠸ࡿ 30 ࡢཧ↷≀㉁㸦Reference Chemicals㸧ࢆ⏝࠸ࡓ 3 ᪋タ࡛㏣㉮ⓗࣂࣜࢹ࣮ࢩࣙࣥ◊✲ 3)ࠊJaCVAM ࡢ➨୕⪅ホ౯ 4)ࢆ⤒࡚ 2018 ᖺ࡟ TG492 ࡟཰㍕ࡉࢀࡓ 1)ࠋ
ᮏሗ࿌᭩ࡣ LabCyte CORNEA-MODEL24 EIT ࡢࣂࣜࢹ࣮ࢩࣙࣥ◊✲ሗ࿌᭩ࠊ➨୕⪅ホ౯ሗ࿌᭩ࠊࡑ
ࡢ௚㛵㐃ㄽᩥ࡞࡝ࢆࡶ࡜࡟ᮏヨ㦂ἲࡢᴫせࢆㄝ᫂ࡋࠊᮏጤဨ఍ࡢពぢࢆࡲ࡜ࡵࡓࡶࡢ࡛࠶ࡿࠋ
2㸬ヨ㦂ἲࡢ఩⨨࡙ࡅ
RhCE ἲࡣࠊUN GHS ༊ศእ≀㉁㸦༢୍≀㉁࠾ࡼࡧΰྜ≀㸧ࢆ᳨ฟࡍࡿࡓࡵ࡟⏝࠸ࡿヨ㦂ἲ࡛࠶ࡿࠋ
3㸬ヨ㦂ἲࡢཎ⌮║่⃭ᛶࡣࠊ≀㉁ࡀゅ⭷ࢆྵࡴ║⾲㠃࡟᥋ゐࡋ⣽⬊യᐖࢆᘬࡁ㉳ࡇࡍࡇ࡜࠿ࡽጞࡲࡿࠋࡑࡢᶵᗎࡣ
ᵝࠎ࡛࠶ࡿࡀࠊ⣽⬊ẘᛶࡀ㔜せ࡞ᙺ๭ࢆᢸࡗ࡚࠸ࡿࠋࡲࡓࠊ≀㉁ࡢ║่⃭ᛶࡣ୺࡟ゅ⭷ࡢึᮇᦆയࡢ
῝ᗘ࡟ࡼࡾỴᐃࡉࢀࠊࡑࢀࡣ⣽⬊Ṛࡢ⛬ᗘ࡜┦㛵㛵ಀ࡟࠶ࡿࠋRhCE ἲࡣࠊ෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝࢆ⏝࠸࡚ࠊ⿕㦂≀㉁ࡢ⣽⬊ẘᛶࢆᣦᶆ࡜ࡋ࡚║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛࠶ࡿࠋ
LabCyte CORNEA-MODEL24 EIT ࡣࠊࣄࢺゅ⭷ୖ⓶⣽⬊ࢆ㔜ᒙᇵ㣴ࡋࡓᕷ㈍ࡢ RhCE ⤌⧊㸦LabCyte
CORNEA-MODEL24㸧ࢆ⏝࠸ࡿ 5)ࠋ⿕㦂≀㉁ࡀᾮయࡢሙྜࡣ⣙ 1 ศ㛫ࠊᅛయࡢሙྜࡣ⣙ 24 ᫬㛫ࠊ⿕㦂≀㉁ࢆ LabCyte CORNEA-MODEL24 ࡟᭚㟢ࡋࡓᚋࠊWST-8 ἲࡼࡾ⟬ฟࡋࡓ⣽⬊⏕Ꮡ⋡ࢆ࢚ࣥࢻ࣏࢖ࣥ
ࢺ࡟⏝࠸ࡿࠋࡇࢀࡣࠊ⣽⬊ෆࡢ⬺Ỉ⣲㓝⣲ࡢാࡁ࡟ࡼࡾ㟁Ꮚ࣓ࢹ࢕࢚࣮ࢱ࣮ࡢᏑᅾୗ࡛ WST-8
㸦2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt, CAS
No.193149-74-5㸧ࡀ㑏ඖࡉࢀ࡚⏕ᡂࡉࢀࡿᶳⰍ࣐࣍ࣝࢨࣥ㔞ࡀ⏕Ꮡ⣽⬊ᩘ࡟ẚ౛ࡍࡿࡇ࡜ࢆᇶᮏཎ⌮࡜ࡋ࡚࠸ࡿ 6)ࠋ⣽⬊⏕Ꮡ⋡ࡀ㝜ᛶᑐ↷࡜ẚ㍑ࡋ࡚ 40%ࢆ㉸࠼ࡓሙྜࠊ⿕㦂≀㉁ࡣ UN GHS ศ㢮࡟࠾࠸࡚༊ศእ࡛࠶ࡿ࡜ุᐃࡍࡿࠋ
4㸬ヨ㦂ᡭ㡰
 LabCyte CORNEA-MODEL24 EIT ࡢᡭ㡰ࢆ௨ୗ࡟♧ࡍࠋヲ⣽ࡣࠊᨵゞ TG4921)࠾ࡼࡧࣉࣟࢺࢥࣝ 7)ࢆཧ↷ࡍࡿࠋ౑⏝ࡍࡿ RhCE ⤌⧊ LabCyte CORNEA-MODEL24 ࡣᰴᘧ఍♫ࢪࣕࣃ࣭ࣥࢸ࢕ࢵࢩ࣭࢚ࣗ
ࣥࢪࢽ࢔ࣜࣥࢢ㸦J-TECࠊ᪥ᮏ㸧ࡼࡾ㉎ධ࡛ࡁࡿࠋ

5
11



4-1. RhCE ⤌⧊ࡢᶵ⬟ⓗ᮲௳ LabCyte CORNEA-MODEL24 ࡢᶵ⬟ⓗ᮲௳ࡣࠊ௨ୗࡢ࡜࠾ࡾ࡛࠶ࡿࠋ
⏕Ꮡ⋡㸸㝜ᛶᑐ↷࡟࠾࠸࡚ 0.5  OD  1.6㸦౑⏝᫬ࡢᇶ‽࡛࠶ࡾࠊ〇㐀⪅ࡢฟⲴᇶ‽ࡣ␗࡞ࡿሙྜࡶ࠶ࡿ㸧ࣂࣜ࢔ᶵ⬟㸸SDS㸦25ȝL㸧ࡢ 60 ศ㛫᭚㟢࡟࠾࠸࡚ 1.0  IC50 (mg/mL)  4.0㸦IC50㸸⣽⬊⏕Ꮡ⋡ࢆ 50%పୗࡉࡏࡿࡢ࡟ᚲせ࡞⃰ᗘ㸧 ᙧែ㸸ᑡ࡞ࡃ࡜ࡶ 3 ᒙࡢୖ⓶⣽⬊ࡀ࠶ࡾࠊࡑࡢ⾲㠃ࡣゅ໬ࡋ࡚࠸࡞࠸ ෌⌧ᛶ㸸㝧ᛶ࠾ࡼࡧ㝜ᛶᑐ↷ࡢ⤖ᯝࡣ⫼ᬒࢹ࣮ࢱࢆࡶ࡜࡟タᐃࡋࡓチᐜ್ෆ࡟࠶ࡿ
ࡇࢀࡽ㸲㡯┠ࡣ LabCyte CORNEA-MODEL24 ࡢ〇㐀⪅ࡢฟⲴᇶ‽࡜ࡋ࡚᥇⏝ࡉࢀࡿࠋ୍᪉ࠊLabCyte
CORNEA-MODEL24 ࡢ౑⏝⪅ࡣ⏕Ꮡ⋡࡜෌⌧ᛶࡢ 2 㡯┠ࢆ☜ㄆࡍࡿᚲせࡀ࠶ࡿࠋ
4-2. ⿕㦂≀㉁ࡢ㐺⏝ ≀㉁࠶ࡓࡾ 3 RhCE ⤌⧊ࢆ⏝࠸ࡿࠋ⿕㦂≀㉁ࡣㄪ〇ࡏࡎࠊࡑࡢࡶࡢࢆ⏝࠸ࡿࠋ37ÛC ௨ୗ࡛ࣆ࣌ࢵࢺ
࡛ᢅ࠼ࡿࡶࡢࡣᾮయ࡜ࡋ࡚ࠊࡑࢀ௨እࡣᅛయ࡜ࡋ࡚ヨ㦂ࢆ⾜࠺ࠋᾮయ⿕㦂≀㉁ࡢሙྜࠊLabCyte
CORNEA-MODEL24 ࡟ 50 μL ࢆ㐺⏝ࡋࠊᐊ

࡛ 1 ศ㛫㸦s5 ⛊㸧ᇵ㣴ࡍࡿࠋࡑࡢᚋࠊCa2+/Mg2+୙ྵ

Dulbecco ࣜࣥ㓟⦆⾪⏕⌮㣗ሷỈ㸦DPBS㸧ࡢỈὶ࡛ 10 ᅇ࠶ࡿ࠸ࡣࡑࢀ௨ୖὙίࡋ࡚⿕㦂≀㉁ࢆ㝖ཤࡋࠊ24s1᫬㛫ᶆ‽ᇵ㣴᮲௳ ࡛ᇵ㣴ࡍࡿࠋᅛయ⿕㦂≀㉁ࡢሙྜࠊ10 mg ࢆᆒ୍࡟㐺⏝ࡋࠊᶆ‽ᇵ㣴᮲௳࡛
24s1 ᫬㛫ᇵ㣴ࡍࡿࠋࡑࡢᚋࠊCa2+/Mg2+୙ྵ DPBS ࡢỈὶ࡛ 10 ᅇ࠶ࡿ࠸ࡣࡑࢀ௨ୖὙίࡋ࡚⿕㦂≀㉁ࢆ㝖ཤࡍࡿࠋὙίᚋࡢᇵ㣴ࡣ⾜ࢃ࡞࠸ࠋ ྠ᫬㝧ᛶᑐ↷࡟ࡣᾮయ⿕㦂≀㉁ࡢሙྜࡣ࢚ࢱࣀ࣮ࣝ㸦CAS No. 64-17-5㸧ࠊᅛయ⿕㦂≀㉁ࡢሙྜࡣࣛ
࢘ࣜࣥ㓟㸦CAS No. 143-07-7㸧ࡀ᥎ዡࡉࢀࡿࠋྠ᫬㝜ᛶᑐ↷࡟ࡣᾮయ⿕㦂≀㉁ࡢሙྜࡣ Ca2+/Mg2+୙ྵ
DPBS ࡀ᥎ዡࡉࢀࠊᅛయ⿕㦂≀㉁ࡢሙྜࡣ↓ฎ⌮ࢆ⏝࠸ࡿࠋᑐ↷≀㉁ࡢ㐺⏝࣭ᚋฎ⌮ࡣᑐ↷࡜࡞ࡿ⿕㦂≀㉁㸦ᾮయࡲࡓࡣᅛయ㸧ࡢ᮲௳࡟‽ࡌࡿࠋ
4-3. ⣽⬊⏕Ꮡ⋡ࡢ⟬ฟ⣽⬊⏕Ꮡ⋡⟬ฟ࡟ࡣ WST-8 ἲࢆ⏝࠸ࡿࠋὙί࣭ᚋᇵ㣴㸦ᾮయࡢࡳ㸧ᚋࠊRhCE ⤌⧊ࢆ WST-8 ⁐ᾮ㸦Cell Count Kit-8 ⁐ᾮ:࢔࣮ࣝᖹ⾮ሷ⁐ᾮ= 1:10㸧0.3 mL ୰࡛ 240s15 ศ㛫ᶆ‽ᇵ㣴᮲௳ୗ࡛཯ᛂࡉࡏ
ࡿࠋWST-8 ⁐ᾮ୰ࡢ࣐࣍ࣝࢨࣥࡢᐃ㔞ࡣ OD

ᐃ㸦OD450nm㸫OD650nm㸧ࡲࡓࡣ HPLC/UPLC ࡛⾜࠺ࠋ⿕㦂≀㉁ࡀ WST-8 㑏ඖ≀㉁ࡢሙྜࠊ࠶ࡿ࠸ࡣ࣐࣍ࣝࢨࣥ࡜ྠࡌࡼ࠺࡞Ἴ㛗㸦450 nm ㏆㎶㸧࡟྾཰ࢆᣢࡘ╔Ⰽ≀㉁ࡢሙྜࠊ⣽⬊⏕Ꮡ⋡ࡢ⿵ṇࢆ⾜࠺ᚲせࡀ࠶ࡿࠋ⿕㦂≀㉁ࡀ WST-8 ἲࢆᖸ΅ࡍࡿ≀㉁࡛࠶ࡿ࠿࡝࠺࠿ࡢ஦๓᳨ウ࡜ࡑࡢሙྜࡢ⣽⬊⏕Ꮡ⋡ࡢ⿵ṇࡢᡭ㡰࡟ࡘ࠸࡚ࡣ TG ᮏᩥࡢㄝ᫂࠾ࡼࡧ
ANNEX V ࡢࣇ࣮ࣟࢳ࣮ࣕࢺࢆཧ↷ࡍࡿࠋ
4-4. ヨ㦂ᡂ❧ࡢᢎㄆᇶ‽௨ୗࡢ᮲௳ࢆࡍ࡭࡚‶ࡓࡋࡓሙྜࠊヨ㦂ࡢᡂ❧ࢆᢎㄆࡍࡿࠋ
1㸧㝜ᛶᑐ↷ࡢᖹᆒ OD ࡀ 0.5  OD  1.6 ࡛࠶ࡿࡇ࡜ࠋ
2㸧㝧ᛶᑐ↷ࡢᖹᆒ⣽⬊⏕Ꮡ⋡ࡀ 40%௨ୗ࡛࠶ࡿࡇ࡜ࠋ
37s2ÛCࠊ5s1% CO2ࠊ95% ‵ᗘ

6
12



3㸧⿕㦂≀㉁࠾ࡼࡧ㝜ᛶ࣭㝧ᛶᑐ↷ࡢࡑࢀࡒࢀ࡟࠾࠸࡚⣽⬊⏕Ꮡ⋡ࡢᶆ‽೫ᕪࡀ 18%௨ୗ࡛࠶ࡿࡇ
࡜ࠋ࡞࠾ࠊ」ᩘࡢ⿕㦂≀㉁ࢆྠ᫬࡟ヨ㦂ࡋࡓሙྜࡣࠊࡇࡢ᮲௳ࢆ‶ࡓࡉ࡞࠸⿕㦂≀㉁ࡢࡳ୙ᡂ
❧࡜ࡍࡿࠋ
4-5. ุᐃ
⿕㦂≀㉁ࡢᖹᆒ⣽⬊⏕Ꮡ⋡ࡀ 40%ࢆ㉸࠼ࡓሙྜࠊ⿕㦂≀㉁ࡣ UN GHS ༊ศእ࡜ุ᩿ࡉࢀࡿࠋᖹᆒ⣽⬊⏕Ꮡ⋡ࡀ 40%௨ୗࡢሙྜࡣࠊࡑࡢ⤖ᯝࡢࡳ࡛ࡣ⿕㦂≀㉁ࡢ║่⃭ᛶࡢணࡣ࡛ࡁ࡞࠸ࠋᮏヨ㦂ἲ࡛ࡣഇ㝧ᛶࡢྍ⬟ᛶࡀ࠶ࡾࠊࡲࡓ UN GHS ༊ศ 1 ࡜༊ศ 2 ࢆ༊ู࡛ࡁ࡞࠸ࠋࡑࡢሙྜࡣࠊ⿕㦂≀㉁ࡢ║่⃭ᛶࢆศ㢮ࡍࡿ࡟ࡣ㏣ຍࡢ᝟ሗࡀᚲせ࡜࡞ࡿࠋྛ⤌⧊ࡢᐃ್࡛ࡢุᐃࡀ୍⮴ࡋ࡞࠸ࠊᖹᆒ⣽⬊⏕Ꮡ⋡ࡀ 40s5%ࡢ⠊ᅖ࡟࠶ࡿ➼ࠊᚓࡽࢀࡓ⤖ᯝࡀ࣮࣎ࢲ࣮ࣛ࢖ࣥࡢሙྜࡣ 2 ᅇ┠ࡢヨ㦂ࢆ᳨ウࡍࡿࠋ1 ᅇ┠࡜ 2 ᅇ┠ࡢヨ㦂࡛⤖ᯝࡀ୍⮴ࡋ࡞࠸ሙྜ
ࡣ 3 ᅇ┠ࡢヨ㦂ࢆ᳨ウࡍࡿࠋ⾲ 1. VRM ࡜ LabCyte CORNEA-MODEL24 EIT ࡢ୺࡞㡯┠ࡢẚ㍑
EpiOcularTM IET

SkinEthicTM HCE EITLabCyte CORNEA-MODEL24(VRM1)(VRM2)EIT

RhCE ⤌⧊ࡢ⏤᮶ࣄࢺ⾲⓶ゅ໬⣽⬊ࣄࢺ୙Ṛ໬ゅ⭷ୖ⓶⣽⬊

ࣄࢺゅ⭷ୖ⓶⣽⬊

᭚㟢㔞50 ȝL ࡲࡓࡣ50 mg (0.6 cm2 )30 ȝL ࡲࡓࡣ 30 mg (0.5 cm2 )50 ȝL ࡲࡓࡣ 10 mg (0.3 cm2 )᭚㟢᫬㛫

30 ศ㸦ᾮయ㸧ࠊ6 ᫬㛫㸦ᅛయ㸧 30 ศ㸦ᾮయ㸧ࠊ4 ᫬㛫㸦ᅛయ㸧 1 ศ㸦ᾮయ㸧ࠊ24 ᫬㛫㸦ᅛయ㸧㝧ᛶ㸸࢚ࢱࣀ࣮ࣝ㸦ᾮయ㸧ࠊࣛ࢘ࣜࣥ㓟㸦ᅛయ㸧㝧ᛶ㸸㓑㓟࣓ࢳࣝ

㝧ᛶ㸸㓑㓟࣓ࢳࣝ

㝜ᛶ㸸㉸⣧Ỉ

㝜ᛶ㸸Ca2+/Mg2+୙ྵ DPBS㝧ᛶ࣭㝜ᛶᑐ↷㝜ᛶ㸸Ca2+/Mg2+୙ྵ DPBS㸦ᾮయ㸧ࠊ↓ฎ⌮㸦ᅛయ㸧⏕Ꮡ⋡ᐃUN GHS ༊ศእุᐃᇶ

MTT ἲ

MTT ἲ

WST-8 ἲ>60%

>60%㸦ᾮయ㸧ࠊ>50%㸦ᅛయ㸧 >40%‽㸦ᖹᆒ⣽⬊⏕Ꮡ⋡㸧

5㸬㏣㉮ⓗࣂࣜࢹ࣮ࢩࣙࣥ◊✲ 㛤Ⓨ⪅ࡢ J-TEC㸦࣮ࣜࢻࣛ࣎㸧࡜ࡣ␗࡞ࡿ 3 ᪋タ㸦ᰴᘧ఍♫⸆≀Ᏻ඲ᛶヨ㦂ࢭࣥࢱ࣮ࠊ᪥ᮏࢥ࣐ࣝ
࣮ᰴᘧ఍♫ࠊᐩኈࣇ࢕࣒ࣝᰴᘧ఍♫㸧࡛ LabCyte CORNEA-MODEL24 EIT ࡢ㏣㉮ⓗࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀ⾜ࢃࢀࡓ 3)ࠋࡇࢀࡽ 3 ᪋タࡣ㏣㉮ⓗࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡢ๓࡟ᢏ⾡⛣㌿ᛶࡢ☜ㄆࢆ⾜ࡗࡓ⥲ᙜࡾ
ヨ㦂࡟ཧຍࡋࡓ᪋タ࡛࠶ࡿ 8)ࠋ⿕㦂≀㉁࡟ࡣ OECD ࡢᛶ⬟ᶆ‽࡛ᣦᐃࡉࢀ࡚࠸ࡿ 30 ࡢཧ↷≀㉁ࢆ⏝࠸ࡓ㸦Appendix 1㸧ࠋヨ㦂ࡣࡍ࡭࡚ࡢ⿕㦂≀㉁࡟ࡘ࠸࡚ 3 ᪋タ࡛ࡑࢀࡒࢀ 3 ᅇᐇ᪋ࡉࢀࡓࠋ
5-1㸬ヨ㦂ἲࡢಙ㢗ᛶ
5.1.1. ᢏ⾡⛣㌿ᛶ ᢏ⾡⛣㌿ᛶ࡟ࡘ࠸࡚ࡣࠊ㏣㉮ⓗࣂࣜࢹ࣮ࢩࣙࣥࡢᐇ᪋๓࡟⾜ࢃࢀࡓ 24 ᪋タࡀཧຍࡋࡓ⥲ᙜࡾヨ㦂

7
13



᳨࡛ウࡉࢀࠊⰋዲ࡞⤖ᯝࡀᚓࡽࢀ࡚࠸ࡿ 8)ࠋ

5-1-2. ᪋タෆ෌⌧ᛶUN GHS ศ㢮ุᐃࡢ᪋タࡈ࡜ࡢ᪋タෆ෌⌧ᛶࡣ 93%ࠊ97%ࠊ100%࡛࠶ࡗࡓࠋࡇࢀࡽࡢ್ࡣ OECDࡢᛶ⬟ᶆ‽ࡀᐃࡵࡓᇶ‽㸦90%௨ୖ㸧ࢆ‶ࡓࡋࡓ 3)ࠋ
࡞࠾ࠊ࣮ࣜࢻ࡛ࣛ࣎࠶ࡿ J-TEC ࡢ♫ෆࢹ࣮ࢱ㸦139 ≀㉁㸧࡛ࡣࠊ᪋タෆ෌⌧ᛶࡣ 97%࡛࠶ࡗࡓ 8)ࠋ
5-1-3. ᪋タ㛫෌⌧ᛶUN GHS ศ㢮ุᐃࡢ᪋タ㛫෌⌧ᛶࡣ 87%࡛࠶ࡗࡓࠋࡇࡢ್ࡣ OECD ࡢᛶ⬟ᶆ‽ࡀᐃࡵࡓᇶ‽㸦85%௨ୖ㸧ࢆ‶ࡓࡋࡓ 3)ࠋ
5-2㸬ヨ㦂ἲࡢṇ☜ᛶṇ☜ᛶࡢホ౯࡟ࡣࠊ3 ᪋タ࡛⾜ࢃࢀࡓ඲ヨ㦂ࡢࢹ࣮ࢱࢆ⏝࠸ࡓࠋࡑࡢ⤖ᯝࠊឤᗘ 97. 8%ࠊ≉␗ᗘ 68.9%ࠊ
ṇ☜ᗘ 83.5%࡛࠶ࡗࡓࠋࡇࢀࡽࡢ್ࡣ OECD ࡢᛶ⬟ᶆ‽ࡀᐃࡵࡓᇶ‽㸦ឤᗘ 90%௨ୖࠊ≉␗ᗘ 60%௨ୖࠊṇ☜ᗘ 75%௨ୖ㸧ࢆ‶ࡓࡋࡓ 3)ࠋ
࡞࠾ࠊ࣮ࣜࢻ࡛ࣛ࣎࠶ࡿ J-TEC ࡢ♫ෆࢹ࣮ࢱ㸦139 ≀㉁㸧࡛ࡣࠊឤᗘ 100.0%ࠊ≉␗ᗘ 73.0%ࠊṇ☜ᗘ 87.8%࡛࠶ࡗࡓ 8)ࠋ
6㸬ヨ㦂ἲࡢ㐺⏝⠊ᅖ
TG492 ࡣࠊヨ㦂ἲࡢ㐺ṇ࡜ṇ☜ᛶࡢ☜ಖࢆ⪃៖ࡋ࡚ RhCE ἲࡢ㐺⏝࡟௨ୗࡢไ㝈ࢆタࡅ࡚࠸ࡿࠋ
1) ࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡟࠾࠸࡚⿕㦂≀㉁࡟ྵࡲࢀ࡞࠿ࡗࡓẼయ㸦࢞ࢫ㸧࠾ࡼࡧ࢚࢔ࢰ࣮ࣝࡣ㐺⏝≀㉁࠿ࡽ㝖እࡉࢀࡿࠋ
2) UN GHS ༊ศ 1ࠊ༊ศ 2㸦2A࣭2B㸧≀㉁ࡢ᳨ฟ࡟ࡣ⏝࠸ࡿࡇ࡜ࡣ࡛ࡁ࡞࠸ࠋ
7. ᮏጤဨ఍ࡢ⤖ㄽ
LabCyte CORNEA-MODEL24 EIT ࡢ㏣㉮ⓗࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡢ⤖ᯝࠊᮏヨ㦂ἲࡣ෌⌧ᛶ࠾ࡼࡧṇ☜ᛶ࡟ࡘ࠸࡚ OECD ࡢᛶ⬟ᶆ‽ࡀᐃࡵࡓᇶ‽ࢆ‶ࡓࡋ࡚࠸ࡓࠋ
LabCyte CORNEA-MODEL24 EIT ࡟⏝࠸ࡿ RhCE ⤌⧊ࡣᕷ㈍ࡉࢀ࡚࠾ࡾࠊࡲࡓࠊRhCE ⤌⧊௨እࡣࠊ
≉Ṧ࡞ᶵᮦࡸヨ⸆ࢆᚲせ࡜ࡏࡎࠊᡭᢏࡶ」㞧࡞ࡶࡢ࡛࡞࠸ࠋධᡭࡋࡓ RhCE ⤌⧊ࡀᶵ⬟ⓗ᮲௳ࡢチᐜ⠊ᅖ࡟࠶ࡾࠊ࠿ࡘᐇ᪋ࡍࡿヨ㦂᪋タ࡜ࡋ࡚ᢏ⾡⩦ᚓࡀ࢞࢖ࢻࣛ࢖ࣥࡢ⩦⇍ᗘ☜ㄆ≀㉁ Appendix2 ࡛☜࠿ࡵࡽࢀ࡚࠸ࢀࡤࠊLabCyte CORNEA-MODEL24 EIT ࡣ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡛ UN GHS ༊ศእ≀㉁ࢆ
᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚⏝࠸ࡿࡇ࡜ࡀ࡛ࡁࡿࠊ࡜ᮏጤဨ఍ࡣ⪃࠼ࡿࠋ

8
14



8. ᩥ⊩
1)

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

2)

OECD (2015) Performance Standards for the Assessment of Proposed Similar or Modified In Vitro
Reconstructed Human Cornea-like Epithelium (RhCE) Test Methods for Eye Hazards, Series on Testing &Assessment No. 216

3)

LabCyte Validation Management Team (2017) Me-too Validation Report: Validation Study for LabCyte
CORNEA-MODEL24 Eye Irritation Test (Version. 3.0), JaCVAM

4)Peer Review Panel (2017) Validation status of the LabCyte CORNEA-MODEL24 EYE IRRITTIONTEST, Report of the Peer Review Panel on a JaCVAM co-ordinated study programme addressing thevalidation status of LabCyte CORNEA-MODEL24 EYE IRRITION TEST for discriminating eye irritantfrom non eye irritant substances

5)

Katoh, M et al (2013) Establishment of a new in vitro test method for evaluation of eye irritation using areconstructed human corneal epithelial model, LabCyte CORNEA-MODEL, Toxicol. in Vitro 27, 2184

6)

Ishiyama, M. et al (1997) A highly water-soluble disulfonated tetrazolium salt as a chromogenic indicatorfor NADH as well as cell viability, Talana 44, 1299

7)

Japan Tissue Engineering Co., Ltd. (2017) In Vitro Eye Irritation Test Using Human Corneal Tissue
Model: LabCyte CORNEA-MODEL24 (Version. 2.5.6), JaCVAM

8)

Katoh, M et al (2017) Background Review Document: New Established In Vitro Eye Irritation Test
Method using Reconstructed Human Corneal Epithelium, LabCyte CORNEA-MODEL24 (Version 2.2),
JaCVAM

915



Appendix 1㏣㉮ⓗࣂࣜࢹ࣮ࢩࣙࣥ࡟⏝࠸ࡽࢀࡓཧ↷≀㉁CAS ␒ྕ

ᛶ≧UN GHS ศ㢮(Ethylenediaminepropyl)-trimethoxysilane

1760-24-3

ᾮయ

༊ศ 1

Methylthioglycolate

2365-48-2

ᾮయ

༊ศ 1

Tetraethylene glycol diacrylate

17831-71-9

ᾮయ

༊ศ 1

1,2-Benzisothiazol-3(2H)-one

2634-33-5

ᅛయ

༊ศ 1

2,5-Dimethyl-2,5-hexanediol

110-03-2

ᅛయ༊ศ 1

Disodium 2,2'-([1,1'-biphenyl]-4,4'-diyldivinylene)bis- (benzenesulphonate)27344-41-8

ᅛయ

༊ศ 1

62-76-0ᅛయ༊ศ 118472-51-0

ᾮయ

༊ศ 2A

gamma-Butyrolactone

96-48-0

ᾮయ༊ศ 2A

1,5-Naphthalenediol

83-56-7

ᅛయ༊ศ 2A

Sodium benzoate

532-32-1

ᅛయ

༊ศ 2A

2-Methyl-1-pentanol

105-30-6

ᾮయ

༊ศ 2B

Diethyl toluamide

134-62-3

ᾮయ

༊ศ 2B

1,4-Dibutoxy benzene

104-36-9

ᅛయ

༊ศ 2B

2,2-Dimethyl-3-methylenebicyclo [2.2.1] heptane

79-92-5

ᅛయ༊ศ 2B

1-Ethyl-3-methylimidazolium ethylsulphate342573-75-5

ᾮయ

༊ศእ

2-Ethoxyethyl methacrylate

2370-63-0ᾮయ༊ศእ

3-Phenoxybenzyl alcohol

13826-35-2

ᾮయ

༊ศእ

4-(Methylthio)-benzaldehyde

3446-89-7

ᾮయ༊ศእ

Dipropyl disulphide629-19-6ᾮయ༊ศእEthyl thioglycolate

623-51-8ᾮయ༊ศእPiperonyl butoxide51-03-6ᾮయ༊ศእPolyethylene glycol (PEG-40) hydrogenated castor oil

61788-85-0

⢓ᛶ

༊ศእ

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

101-20-2ᅛయ༊ศእ

2,2'-[[3-Methyl-4-[(4-nitrophenyl)azo]-phenyl]imino]bis-ethanol

3179-89-3ᅛయ༊ศእ

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

103597-45-1

ᅛయ

༊ศእ

4,4'-Methylene bis-(2,6-di-tert-butylphenol)

118-82-1

ᅛయ༊ศእ

Cellulose, 2-(2-hydroxy -3-(trimethylammonium) propoxy) ethyl ether chloride (91%)

68610-92-4

ᅛయ

༊ศእ

Potassium tetrafluoroborate

14075-53-7

ᅛయ

༊ศእ

Trisodium mono-(5-(1,2-dihydroxyethyl)-4-oxido-2-oxo-2,5-dihydro-furan-3-yl)
phosphate

66170-10-3

ᅛయ

༊ศእ

≀㉁ྡ

Sodium oxalate
2,4,11,13-Tetraazatetradecane-diimidamide,N,N''-bis(4-chlorophenyl)-3,12-diimino-,
di-D-gluconate (20%, aqueous)

10

16



Appendix 2
RhCE ἲࡢ⩦⇍ᗘ☜ㄆ≀㉁CAS ␒ྕ

ᛶ≧UN GHS ศ㢮

Methylthioglycolate

2365-48-2

ᾮయ

༊ศ 1

Hydroxyethyl acrylate

818-61-1

ᾮయ༊ศ 1

2,5-Dimethyl-2,5-hexanediol

110-03-2

ᅛయ༊ศ 1

Sodium oxalate

62-76-0ᅛయ༊ศ 1

2,4,11,13-Tetraazatetradecane-diimidamide, N,N’’-bis(4-chlorophenyl)3,12-diimino-,di-D-gluconate (20%, aqueous)

18472-51-0

ᾮయ

༊ศ 2A

Sodium benzoate

532-32-1

ᅛయ

༊ศ 2A

Diethyl toluamide

134-62-3

ᾮయ

༊ศ 2B

2,2-Dimethyl-3-methylenebicyclo [2.2.1] heptane

79-92-5

ᅛయ༊ศ 2B

342573-75-7

ᾮయ

༊ศእ

Dicaprylyl ether

629-82-3ᾮయ༊ศእPiperonyl butoxide51-03-6ᾮయ༊ศእPolyethylene glycol (PEG-40) hydrogenated castor oil

61788-85-0

⢓ᛶ≀

༊ศእ

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

101-20-2ᅛయ༊ศእ

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

103597-45-1

ᅛయ

༊ศእ

Potassium tetrafluoroborate

14075-53-7

ᅛయ

༊ศእ

≀㉁ྡ

1-Ethyl-3-methylimidazolium ethylsulphate

1117OECD/OCDE

492
Adopted:25 June 2018

OECD GUIDELINE FOR THE TESTING OF CHEMICALS
Reconstructed human Cornea-like Epithelium (RhCE) test method for
identifying chemicals not requiring classification and labelling for eye
irritation or serious eye damage
INTRODUCTION
1.
Serious eye damage refers to the production of tissue damage in the eye, or serious
physical decay of vision, following application of a test chemical to the anterior surface ofthe eye, which is not fully reversible within 21 days of application, as defined by the United
Nations Globally Harmonized System of Classification and Labelling of Chemicals(UN GHS) (1). Also according to UN GHS, eye irritation refers to the production ofchanges in the eye following the application of a test chemical to the anterior surface of theeye, which are fully reversible within 21 days of application. Test chemicals inducing
serious eye damage are classified as UN GHS Category 1, while those inducing eye
irritation are classified as UN GHS Category 2. Test chemicals not classified for eye
irritation or serious eye damage are defined as those that do not meet the requirements forclassification as UN GHS Category 1 or 2 (2A or 2B) i.e., they are referred to as UN GHS
No Category.
2.
The assessment of serious eye damage/eye irritation has typically involved the useof laboratory animals (OECD Test Guideline (TG) 405; adopted in 1981 and revised in
1987, 2002, 2012 and 2017) (2). The choice of the most appropriate test method and the useof this Test Guideline should be seen in the context of the OECD Guidance Document on
an Integrated Approaches on Testing and Assessment (IATA) for Serious Eye Damage andEye irritation (3).
3.
This Test Guideline describes an in vitro procedure allowing the identification ofchemicals (substances and mixtures) not requiring classification and labelling for eye
irritation or serious eye damage in accordance with UN GHS. It makes use of reconstructed
human cornea-like epithelium (RhCE) which closely mimics the histological,
morphological, biochemical and physiological properties of the human corneal epithelium.
Four other in vitro test methods have been validated, considered scientifically valid and© 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 Meeting of the
Chemicals Committee and the Working Party on Chemicals, Pesticides and Biotechnology by written procedure on 25 June 2018.guideline wasadopted by the OECD Council by written procedure on 25 June 2018.

19

2│

492

OECD/OCDE

adopted as OECD Test Guidelines (TGs) 437 (4), 438 (5), 460 (6) and 491 (7) to address
the human health endpoint serious eye damage or no classification.
4.
Three validated test methods using commercially available RhCE models areincluded in this Test Guideline. Validation studies for assessing eye irritation/serious eye
damage have been conducted (8)(9)(10)(11)(12)(13)(14)(15) (16) using the EpiOcular™
Eye Irritation Test (EIT), the SkinEthic™ Human Corneal Epithelium (HCE) EIT and the
LabCyte CORNEA-MODEL24 EIT. Each of these methods makes use of commerciallyavailable RhCE tissue constructs as test system, two of them are referred to in the following
text as the Validated Reference Methods – EpiOcular™ EIT (VRM1) and SkinEthic™
HCE EIT (VRM2), respectively. From these validation studies and their independent peer
review (10)(13) it was concluded that EpiOcular™ EIT, SkinEthic™ HCE EIT and
LabCyte CORNEA-MODEL24 EIT are able to correctly identify chemicals (bothsubstances and mixtures) not requiring classification and labelling for eye irritation orserious eye damage according to UN GHS (1), and the test methods were recommended as
scientifically valid for that purpose (14). Annexes II-V provide a synopsis of the importantelements of the test methods, as well as flowcharts providing guidance for specificsituations.
5.
It is currently generally accepted that, in the foreseeable future, no single in vitro
test method will be able to fully replace the in vivo Draize eye test (2)(16) to predict acrossthe full range of serious eye damage/eye irritation responses for different chemical classes.
However, strategic combinations of several alternative test methods within (tiered) testing
strategies such as the Bottom-Up/Top-Down approach may be able to fully replace the
Draize eye test (17). The Bottom-Up approach is designed to be used when, based on
existing information, a chemical is expected not to cause sufficient eye irritation to requirea classification, while the Top-Down approach is designed to be used when, based on
existing information, a chemical is expected to cause serious eye damage. The EpiOcular™
EIT, SkinEthic™ HCE EIT and LabCyte CORNEA-MODEL24 EIT are recommended to
identify chemicals that do not require classification for eye irritation or serious eye damage
according to UN GHS (UN GHS No Category) (1) without further testing, within a testing
strategy such as the Bottom-Up/Top-Down approach suggested by Scott et al. e.g., as an
initial step in a Bottom-Up approach or as one of the last steps in a Top-Down approach.
However, the EpiOcular™ EIT, SkinEthic™ HCE EIT and LabCyte CORNEA-MODEL24
EIT are not intended to differentiate between UN GHS Category 1 (serious eye damage)and UN GHS Category 2 (eye irritation). This differentiation will need to be addressed byanother tier of a test strategy (3). A test chemical that is identified as requiring classificationfor eye irritation/serious eye damage with EpiOcular™ EIT, SkinEthic™ HCE EIT or
LabCyte CORNEA-MODEL24 EIT will thus require additional testing (in vitro and/or invivo) to reach a definitive conclusion (UN GHS No Category, Category 2 or Category 1),
using e.g., TG 437, 438, 460, 491, or if necessary TG 405.
6.
The purpose of this Test Guideline is to describe the procedure used to evaluate theeye hazard potential of a test chemical based on its ability to induce cytotoxicity in a RhCE
tissue construct, as measured by the tetrazolium dye {TD; e.g., MTT [3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium
bromide; CAS RN 298-93-1] for VRM1 and VRM2, or WST-8 [2-(2-methoxy-4nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt;
CAS RN 193149-74-5] for LabCyte CORNEA-MODEL24 EIT } assay (18)(19) (seeparagraph 22). The viability of the RhCE tissue following exposure to a test chemical is© OECD, (2018)20

OECD/OCDE

492 │ 3determined in comparison to tissues treated with the negative control substance(%viability), and is then used to predict the eye hazard potential of the test chemical.
7.
Performance Standards (20) are available to facilitate the validation of new or
modified in vitro RhCE-based test methods similar to EpiOcular™ EIT, SkinEthic™ HCE
EIT and LabCyte CORNEA-MODEL24 EIT, in accordance with the principles of
Guidance Document No. 34 (21), and allow for timely amendment of this Test Guideline
for their inclusion. Mutual Acceptance of Data (MAD) will only be guaranteed for test
methods validated according to the Performance Standards, if these test methods have beenreviewed and included in this Test Guideline by the OECD.
8.

Definitions are provided in Annex I.

INITIAL CONSIDERATIONS AND LIMITATIONS
9.
This Test Guideline is based on commercial three-dimensional RhCE tissue
constructs that are produced using either primary human epidermal keratinocytes (i.e.,
EpiOcular™ OCL-200), human immortalized corneal epithelial cells (i.e., SkinEthic™
HCE/S), or primary human corneal epithelial cells (i.e., LabCyte CORNEA-MODEL24).
The EpiOcular™ OCL-200, SkinEthic™ HCE/S, and LabCyte CORNEA-MODEL24
RhCE tissue constructs are similar to the in vivo corneal epithelium three-dimensional
structure and are produced using cells from the species of interest (22)(23)(24). Moreover,
the test methods directly measure cytotoxicity resulting from penetration of the chemical
through the cornea and production of cell and tissue damage following chemical exposure,which determines the overall in vivo serious eye damage/eye irritation response. Celldamage can occur by several modes of action (see paragraph 21), but cytotoxicity plays animportant, if not the primary, mechanistic role in determining the overall serious eye
damage/eye irritation response of a chemical, manifested in vivo mainly by corneal opacity,
iritis, conjunctival redness and/or conjunctival chemosis, regardless of the physicochemical
processes underlying tissue damage.
10.
A wide range of chemicals, covering a variety of chemical types, chemical classes,
molecular weights, LogPs, chemical structures, etc., have been tested in the validation study
underlying this Test Guideline. The EpiOcular™ EIT validation database contained 113
chemicals in total, covering 95 different organic functional groups according to an OECDQSAR toolbox analysis (9). The majority of these chemicals represented mono-constituent
substances, but several multi-constituent substances (including 3 homopolymers, 5copolymers and 10 quasi polymers) were also included in the study. In terms of physicalstate and UN GHS Categories, the 113 tested chemicals were distributed as follows: 13
Category 1 liquids, 15 Category 1 solids, 6 Category 2A liquids, 10 Category 2A solids, 7Category 2B liquids, 7 Category 2B solids, 27 No Category liquids and 28 No Categorysolids (8). The SkinEthic™ HCE EIT validation database contained 200 chemicals in total,
covering 165 different organic functional groups (9)(11)(12). The majority of thesechemicals represented mono-constituent substances, but several multi-constituent
substances (including 10 polymers) were also included in the study. In terms of physicalstate and UN GHS Categories, the 200 tested chemicals were distributed as follows: 27
Category 1 liquids, 24 Category 1 solids, 19 Category 2A liquids, 10 Category 2A solids, 9Category 2B liquids, 8 Category 2B solids, 50 No Category liquids and 53 No Categorysolids (11)(12). The LabCyte CORNEA-MODEL24 EIT catch-up validation database© OECD, (2018)

21

4│

492

OECD/OCDE

contained 30 reference chemicals listed in the performance standards (20) of this Test
Guideline 492.
11.
This Test Guideline is applicable to substances and mixtures, and to solids, liquids,
semi-solids and waxes. The liquids may be aqueous or non-aqueous; solids may be soluble
or insoluble in water. Whenever possible, solids should be ground to a fine powder before
application; no other pre-treatment of the sample is required. Gases and aerosols have notbeen assessed in a validation study. While it is conceivable that these can be tested using
RhCE technology, the current Test Guideline does not allow testing of gases and aerosols.
12.
Test chemicals absorbing light in the same range as formazan dye (FD, naturally orafter treatment) and test chemicals able to directly reduce the vital dye TD (to FD) may
interfere with the tissue viability measurements and need the use of adapted controls forcorrections. The type of adapted controls that may be required will vary depending on thetype of interference produced by the test chemical and the procedure used to quantify each
FD (see paragraphs 37-43).
13.
Results generated in pre-validation (25)(26)(27) and validation (9)(11)(12)(15)
studies have demonstrated that EpiOcular™ EIT, SkinEthic™ HCE EIT and LabCyte
CORNEA-MODEL24 EIT are transferable to laboratories considered to be naïve in the
conduct of the assays and also to be reproducible within- and between laboratories. Basedon these studies, the level of reproducibility in terms of concordance of predictions that canbe expected from EpiOcular™ EIT from data on 113 chemicals is in the order of 95%
within laboratories and 93% between laboratories. The level of reproducibility in terms ofconcordance of predictions that can be expected from SkinEthic™ HCE EIT from data on
120 chemicals is in the order of 92% within laboratories and 95% between laboratories. The
level of reproducibility in terms of concordance of predictions that can be expected from
LabCyte CORNEA-MODEL24 EIT from data on 30 reference chemicals listed in theperformance standards (21) is in the order of 96% within laboratories and 87% between
laboratories.
14.
The EpiOcular™ EIT can be used to identify chemicals that do not requireclassification for eye irritation or serious eye damage according to the UN GHS
classification system (1). Considering the data obtained in the validation study (9), theEpiOcular™ EIT has an overall accuracy of 80% (based on 112 chemicals), sensitivity of
96% (based on 57 chemicals), false negative rate of 4% (based on 57 chemicals), specificity
of 63% (based on 55 chemicals) and false positive rate of 37% (based on 55 chemicals),
when compared to reference in vivo rabbit eye test data (OECD TG 405) (2)(16) classified
according to the UN GHS classification system (1). A study where 97 liquid agrochemical
formulations were tested with EpiOcular™ EIT demonstrated a similar performance of thetest method for this type of mixtures as obtained in the validation study (28). The
97 formulations were distributed as follows: 21 Category 1, 19 Category 2A, 14 Category
2B and 43 No Category, classified according to the UN GHS classification system (1)based on reference in vivo rabbit eye test data (OECD TG 405) (2)(16). An overall accuracyof 82% (based on 97 formulations), sensitivity of 91% (based on 54 formulations), false
negative rate of 9% (based on 54 formulations), specificity of 72% (based on 43
formulations) and false positive rate of 28% (based on 43 formulations) were obtained (28).
15.
The SkinEthic™ HCE EIT can be used to identify chemicals that do not requireclassification for eye irritation or serious eye damage according to the UN GHS
classification system (1). Considering the data obtained in the validation study (11)(12), theSkinEthic™ HCE EIT has an overall accuracy of 84% (based on 200 chemicals), sensitivity© OECD, (2018)22

OECD/OCDE

492 │ 5of 95% (based on 97 chemicals), false negative rate of 5% (based on 97 chemicals),
specificity of 72% (based on 103 chemicals) and false positive rate of 28% (based on 103
chemicals), when compared to reference in vivo rabbit eye test data (OECD TG 405)
(2)(16) classified according to the UN GHS classification system (1).
16.
The LabCyte CORNEA-MODEL EIT can be used to identify chemicals that do not
require classification for eye irritation or serious eye damage according to the UN GHS
classification system (1). Considering the data obtained in the catch up validation study(15), the LabCyte CORNEA-MODEL EIT meets the criteria for reproducibility andreproductive capacity, as required by the Performance Standards of this Gguideline (20).
Additionally, a database consisting of 139 chemicals were tested by the tissue constructdeveloper (unpublished data).
17.
The false negative rates obtained with these RhCE test methods with either
substances or mixtures are within the in vivo Draize eye test within-test variability of 12 %(29). The false positive rates obtained with any RhCE test methods with either substancesor mixtures are not critical in the context of this Test Guideline since all test chemicals that
produce a tissue viability equal or lower than the established cut-offs (see paragraph 45)
will require further testing with (an)other in vitro test method(s), or as a last option in
rabbits, depending on regulatory requirements, according to the OECD GuidanceDocument on an Integrated Approaches on Testing and Assessment for Serious Eye
Damage and Eye irritation (3). These test methods can be used for all types of chemicals,
whereby a negative result should be accepted for not classifying a chemical for eye
irritation and serious eye damage (UN GHS No Category). The appropriate regulatory
authorities should be consulted before using the EpiOcular™ EIT, SkinEthic™ HCE EIT
and LabCyte CORNEA-MODEL24 EIT under classification schemes other than UN GHS.
18.
A limitation of this Test Guideline is that it does not allow discrimination between
eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible
effects on the eye (Category 1), nor between eye irritants (optional Category 2A) and mild
eye irritants (optional Category 2B), as defined by UN GHS (1). For these purposes, further
testing with other in vitro test guidelines is required (3).
19.
The term "test chemical" is used in this Test Guideline to refer to what is being
tested1 and is not related to the applicability of the RhCE test method to the testing of
substances and/or mixtures.

PRINCIPLE OF THE TEST20.
The test chemical is applied topically to a minimum of two three-dimensional
RhCE tissue constructs and tissue viability is measured following exposure and a posttreatment incubation period. The RhCE tissues are reconstructed from primary humanepidermal keratinocytes, human immortalized corneal epithelial cells, or primary culture
human corneal epithelial cells, which have been cultured for several days to form a
stratified, highly differentiated squamous epithelium morphologically similar to that found1

In June 2013, the Joint Meeting agreed that where possible, a more consistent use of the term “testchemical” describing what is being tested should now be applied in new and updated Test Guidelines.

© OECD, (2018)

23

6│

492

OECD/OCDE

in the human cornea. The EpiOcular™ and LabCyte CORNEA-MODEL24 RhCE tissue
construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing
a corneal-like structure analogous to that found in vivo (24). The SkinEthic™ HCE RhCE
tissue construct consists of at least 4 viable layers of cells including columnar basal cells,
transitional wing cells and superficial squamous cells similar to that of the normal humancorneal epithelium (23)(30).
21.
Chemical-induced serious eye damage/eye irritation, manifested in vivo mainly bycorneal opacity, iritis, conjunctival redness and/or conjunctival chemosis, is the result of acascade of events beginning with penetration of the chemical through the cornea and/or
conjunctiva and production of damage to the cells. Cell damage can occur by several modesof action, including: cell membrane lysis (e.g., by surfactants, organic solvents);
coagulation of macromolecules (particularly proteins) (e.g., by surfactants, organic
solvents, alkalis and acids); saponification of lipids (e.g., by alkalis); and alkylation or othercovalent interactions with macromolecules (e.g., by bleaches, peroxides and alkylators)(17)(31)(32). However, it has been shown that cytotoxicity plays an important, if not the
primary, mechanistic role in determining the overall serious eye damage/eye irritation
response of a chemical regardless of the physicochemical processes underlying tissue
damage (33)(34). Moreover, the serious eye damage/eye irritation potential of a chemical isprincipally determined by the extent of initial injury (31), which correlates with the extent
of cell death (33) and with the extent of the subsequent responses and eventual outcomes(36). Thus, slight irritants generally only affect the superficial corneal epithelium, the mildand moderate irritants damage principally the epithelium and superficial stroma and thesevere irritants damage the epithelium, deep stroma and at times the corneal endothelium(34)(37). The measurement of viability of the RhCE tissue construct after topical exposure
to a test chemical to identify chemicals not requiring classification for serious eye
damage/eye irritancy (UN GHS No Category) is based on the assumption that all chemicals
inducing serious eye damage or eye irritation will induce cytotoxicity in the corneal
epithelium and/or conjunctiva.
22.
RhCE tissue viability is classically measured by enzymatic conversion of TD (MTTfor VRM1 and VRM2, or WST-8 for LabCyte CORNEA-MODEL24 EIT) by the viable
cells of the tissue into coloured FD (blue MTT formazan or yellow WST-8 formazan). BlueMTT FD is quantitatively measured after extraction from tissues (18), whereas yellow
WST-8 FD does not require extraction because of its water solubility and is quantitatively
measured directly from the WST-8 solution on which the tissues are incubated during the
WST-8 assay (19). Chemicals not requiring classification and labelling according to UN
GHS (No Category) are identified as those that do not decrease tissue viability below adefined threshold (i.e., tissue viability > 60% for EpiOcular™ EIT and SkinEthic™ HCE
EITL2; > 50% for SkinEthic™ HCE EITS3; or > 40% for LabCyte CORNEA-MODEL24
EIT) (see paragraph 45).

2

EITL: EIT for liquids in the case of SkinEthic™ HCE

3

EITS: EIT for solids in the case of SkinEthic™ HCE© OECD, (2018)24

OECD/OCDE

492 │ 7DEMONSTRATION OF PROFICIENCY
23.
Prior to routine use of RhCE test methods for regulatory purposes, laboratories
should demonstrate technical proficiency by correctly predicting the fifteen proficiency
chemicals listed in Table 1. These chemicals were selected from the chemicals used in thevalidation studies of the VRM1 and VRM2 (9)(11)(12). The selection includes, to theextent possible, chemicals that: (i) cover different physical states; (ii) cover the full range ofin vivo serious eye damage/eye irritation responses based on high quality results obtained in
the reference in vivo rabbit eye test (OECD TG 405) (2)(16) and the UN GHS classification
system (i.e., Categories 1, 2A, 2B, or No Category) (1); (iii) cover the various in vivo
drivers of classification (29)(38); (iv) are representative of the chemical classes used in thevalidation study (9)(11)(12); (v) cover a good and wide representation of organic functionalgroups (9)(11)(12); (vi) have chemical structures that are well-defined (9)(11)(12); (vii) arecoloured and/or direct TD reducers; (viii) produced reproducible results in RhCE test
methods during their validations; (ix) were correctly predicted by RhCE test methods
during their validation studies; (x) cover the full range of in vitro responses based on highquality RhCE test methods data (0 to 100% viability); (xi) are commercially available; and(xii) are not associated with prohibitive acquisition and/or disposal costs. In situations
where a listed chemical is unavailable or cannot be used for other justified reasons, anotherchemical fulfilling the criteria described above, e.g. from the chemicals used in thevalidation of the VRMs, could be used. Such deviations should however be justified.
Table 1: List of proficiency chemicals
Chemical Name

CAS
RN

Organic Functional
Group1

Physical state

VRM1
viability
(%)2

VRM2
viability(%)3

LabCyteviability(%)3-1

VRM
Prediction

TS Reducer

Colourinterf.

No
prediction
can be madeNo
prediction
can be madeNo
prediction
can be madeNo
prediction
can be madeY
(strong)

N

N

N

N

N

N

N

In Vivo Category 14
Methylthioglycolate

236548-2

Carboxylic acid ester;ThioalcoholL

10.9±6.4

5.5±7.4

1.7±1.2

Hydroxyethyl acrylate

818-611

Acrylate;

L

7.5±4.755

1.6±1.0

7.5±4.75

2,5-Dimethyl-2,5hexanediol

110-032

AlcoholS

2.3±0.2

0.2±0.1

2.8±2.6

Sodium oxalate

62-76-0Oxocarboxylic acidS

29.0±1.2

5.3±4.1

3.7±1.5

2,4,11,13Tetraazatetradecanediimidamide,
N,N''bis(4-chlorophenyl)3,12-diimino-,
di-Dgluconate(20%, aqueous) 6
Sodium benzoate

1847251-0

Aromatic heterocyclic
halide; Aryl halide;
Dihydroxyl group;
GuanidineL

In Vivo Category 2A4
4.0±1.1
1.3±0.6

0.4±0.4

No
prediction
can be madeNY(weak)

532-321Aryl; Carboxylic acidS

0.6±0.1

2.9±2.6

No
prediction
can be madeN

N

Diethyl toluamide

134-623

Benzamide

L

In Vivo Category 2B4
15.6±6.3
2.8±0.932.4±9.3

NN

2,2-Dimethyl-3methylenebicyclo [2.2.1]
heptane79-92-5

Alkane, branched withtertiary carbon;
Alkene;Bicycloheptane;
Bridged-ringSNo
prediction
can be madeNo
prediction
can be madeN

N

Alcohol

3.5±2.6

4.7±1.515.8±1.1

2.2±2.6© OECD, (2018)

25

8│

492

OECD/OCDE
carbocycles;
Cycloalkane
In Vivo No Category4

1-Ethyl-3methylimidazolium
ethylsulphate
Dicaprylyl ether
Piperonyl butoxide
Polyethylene
glycol(PEG-40) hydrogenatedcastor oil
1-(4-Chlorophenyl)-3(3,4-dichlorophenyl)urea
2,2'-Methylene-bis-(6(2H-benzotriazol-2- yl)4-(1,1,3,3tetramethylbutyl)phenol)
Potassium
tetrafluoroborate

342573-75-5
629-823
51-03-6
6178885-0
101-202103597-45-11407553-7

Alkoxy; Ammonium
salt; Aryl; Imidazole;
Sulphate
Alkoxy;Ether
Alkoxy; Benzodioxole;
Benzyl; Ether
Acylal; Alcohol; Allyl;
Ether
Aromatic heterocyclic
halide; Aryl halide;
Urea derivatives
Alkane branched withquaternary carbon;
Fused carbocyclicaromatic; Fused
saturated heterocycles;
Precursors quinoid
compounds; tert-Butyl
Inorganic Salt

L

79.9±6.4

79.4±6.2

48.0±8.9

No Cat

N

N

L

97.8±4.3

95.2±3.0

92.7±5.0

No CatN

N

L

104.2±4.2

96.5±3.595.6±14.0

No CatN

N

Viscous

77.6±5.489.1±2.962.6±11.5

No CatN

N

S

106.7±5.3

101.9±6.
677.8±9.0

No CatN

N

S

102.7±13.
497.7±5.690.2±5.8

No CatN

N

S

88.6±3.3

92.9±5.1

66.6±0.2

No CatN

N

Abbreviations: CASRN = Chemical Abstracts Service Registry Number; UN GHS = United Nations
Globally Harmonized System of Classification and Labelling of Chemicals (1); VRM1 = Validated
Reference Method, EpiOcular™ EIT; VRM2 = Validated Reference Method, SkinEthic™ HCE EIT;
Colour interf. = colour interference with the standard absorbance (Optical Density (OD)) measurement ofFD.
1
Organic functional group assigned according to an OECD Toolbox 3.1 nested analysis (9).
2Based on results obtained with EpiOcular™ EIT in the EURL ECVAM/Cosmetics Europe Eye Irritation
Validation Study (EIVS) (9).
3Based on results obtained with SkinEthic™ HCE EIT in the validation study (11)(12).
3-1Based on results obtained with LabCyte CORNEA-MODEL24 EIT in the validation study (15).
4Based on results from the in vivo rabbit eye test (OECD TG 405) (2)(15) and using the UN GHS (1).
5
Based on results obtained in the CEFIC Consortium for in vitro Eye Irritation testing strategy (CON4EI)
Study.
6
Classification 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
corneal opacity in one animal recovered to a score of zero by day 7 or earlier. The one animal that did not
fully recover by day 7 had a corneal opacity score of 1 (at day 7) that fully recovered at day 9.
24.
As part of the proficiency testing, it is recommended that users verify the barrier
properties of the tissues after receipt as specified by the RhCE tissue construct producer(see paragraphs 26, 28 and 31). This is particularly important if tissues are shipped overlong distance / time periods. Once a test method has been successfully established andproficiency in its use has been acquired and demonstrated, such verification will not benecessary on a routine basis. However, when using a test method routinely, it isrecommended to continue to assess the barrier properties at regular intervals.

© OECD, (2018)26

OECD/OCDE

492 │ 9PROCEDURE
25.
The test methods currently covered by this Test Guideline are the scientificallyvalid EpiOcular™ EIT, SkinEthic™ HCE EIT and LabCyte CORNEA-MODEL24
EIT(10)(13)(14), the first two being referred as the Validated Reference Methods(VRM1 and VRM2, respectively). The Standard Operating Procedures (SOP) for the RhCE
test methods are available and should be employed when implementing and using the test
methods in a laboratory (39)(40)(41). The following paragraphs and Annex II describe themain components and procedures of the RhCE test methods.

© OECD, (2018)

27

10 │

492

OECD/OCDERhCE TEST METHOD Components
GENERAL CONDITIONS
26.
Relevant human-derived cells should be used to reconstruct the cornea-likeepithelium three-dimensional tissue, which should be composed of progressively stratified
but not cornified cells. The RhCE tissue construct is prepared in inserts with a porous
synthetic membrane through which nutrients can pass to the cells. Multiple layers of viable,
non-keratinized epithelial cells should be present in the reconstructed cornea-likeepithelium. The RhCE tissue construct should have the epithelial surface in direct contact
with air so as to allow for direct topical exposure of test chemicals in a fashion similar to
how the corneal epithelium would be exposed in vivo. The RhCE tissue construct should
form a functional barrier with sufficient robustness to resist rapid penetration of cytotoxic
benchmark substances, e.g., Triton X-100 or sodium dodecyl sulphate (SDS). The barrier
function should be demonstrated and may be assessed by determination of either the
exposure time required to reduce tissue viability by 50% (ET50) upon application of abenchmark substance at a specified, fixed concentration (e.g., 100 μL of 0.3% (v/v) Triton
X-100), or the concentration at which a benchmark substance reduces the viability of thetissues by 50% (IC50) following a fixed exposure time (e.g., 30 minutes treatment with 50
μL SDS or 60 minutes treatment with 25 μL SDS) (see paragraph 31). The containment
properties of the RhCE tissue construct should prevent the passage of test chemical aroundthe edge of the viable tissue, which could lead to poor modelling of corneal exposure. The
human derived cells used to establish the RhCE tissue construct should be free ofcontamination by bacteria, viruses, mycoplasma, and fungi. The sterility of the tissue
construct should be checked by the supplier for absence of contamination by fungi andbacteria.

FUNCTIONAL CONDITIONS
Viability
27.
The assay used for quantifying tissue viability is the TD (MTT for VRM1 andVRM2, or WST-8 for LabCyte CORNEA-MODEL24 EIT) assay (18)(20). Viable cells of
the RhCE tissue construct reduce the vital dye MTT into a blue MTT formazan precipitate,
which is then extracted from the tissue using isopropanol (or a similar solvent).
Alternatively, viable cells of the RhCE tissue construct reduce the vital dye WST-8 into awater soluble yellow formazan. The extracted FD may be quantified using either a standard
absorbance (Optical Density (OD)) measurement or an HPLC/UPLC-spectrophotometry
procedure (42). The OD of the blank solution alone (which is the extraction solvent for
MTT assay, or the diluted WST-8 medium for the WST-8 assay) should be sufficiently
small, i.e., OD < 0.1. Users of the RhCE tissue construct should ensure that each batch of
the RhCE tissue construct used meets defined criteria for the negative control. Acceptability
ranges for the negative control OD values for the VRMs are given in Table 2. An
HPLC/UPLC-spectrophotometry user should use the negative control OD ranges provided© OECD, (2018)28

OECD/OCDE

492 │ 11

in Table 2 as the acceptance criterion for the negative control. It should be documented in
the test report that the tissues treated with the negative control substance are stable inculture (provide similar tissue viability measurements) for the duration of the test exposure
period. A similar procedure should be followed by the tissue producer as part of the quality
control tissue batch release, but in this case different acceptance criteria than thosespecified in Table 2 may apply. An acceptability range (upper and lower limit) for thenegative control OD values (in the QC test method conditions) should be established by the
RhCE tissue construct developer/supplier.
Table 2: Acceptability ranges for negative control OD values (for the test method users)
Test Method
EpiOcular™ EIT (OCL-200) – VRM1

Lower acceptance limit

Upper acceptance limit

1

> 0.8

< 2.5

> 1.0

≤ 2.5

≥ 0.5

≤ 1.6

(for both the liquids and the solids protocols)

SkinEthic™ HCE EIT (HCE/S) – VRM2
(for both the liquids and the solids protocols)

LabCyte CORNEA-MODEL24 EIT(for both the liquids and the solids protocols)
1

This acceptance limit considers the possibility of extended shipping/storage time (e.g. > 4 days), which has beenshown not to impact on the performance of the test method (43).

Barrier function
28.
The RhCE tissue construct should be sufficiently thick and robust to resist the rapid
penetration of cytotoxic benchmark substances, as estimated e.g. by ET50 (Triton X-100) or
by IC50 (SDS) (Table 3). The barrier function of each batch of the RhCE tissue constructused should be demonstrated by the RhCE tissue construct developer/vendor upon supplyof the tissues to the end user (see paragraph 31).

Morphology
29.
Histological examination of the RhCE tissue construct should demonstrate humancornea-like epithelium structure (including at least 3 layers of viable epithelial cells and a
non-keratinized surface). For the three test methods, appropriate morphology has been
established by the developer/supplier and therefore does not need to be demonstrated again
by a test method user for each tissue batch used.

Reproducibility
30.
The results of the positive and negative controls of the test method should
demonstrate reproducibility over time.

Quality control (QC)
31.
The RhCE tissue construct should only be used if the developer/supplierdemonstrates that each batch of the RhCE tissue construct used meets defined production
release criteria, among which those for viability (paragraph 27) and barrier function (see© OECD, (2018)

29

12 │

492

OECD/OCDE

paragraph 28) are the most relevant. An acceptability range (upper and lower limits) for thebarrier functions as measured by the ET50 or IC50 (see paragraphs 26 and 28) should beestablished by the RhCE tissue construct developer/supplier. The ET50 and IC50
acceptability range used as QC batch release criterion by the developer/supplier of the
RhCE tissue constructs used in the test methodsis given in Table 3. Data demonstratingcompliance with all production release criteria should be provided by the RhCE tissue
construct developer/supplier to the test method users so that they are able to include thisinformation in the test report. Only results produced with tissues fulfilling all of theseproduction release criteria can be accepted for reliable prediction of chemicals not requiringclassification and labelling for eye irritation or serious eye damage in accordance with UN
GHS.
Table 3: QC batch release criteria
Test Method
EpiOcular™ EIT (OCL-200) – VRM1(100 μL of 0.3% (v/v) Triton X-100)
SkinEthic™ HCE EIT (HCE/S) – VRM2
(30 minutes treatment with 50 μL SDS)
LabCyte CORNEA-MODEL24 EIT –(60 minutes treatment with 25 μL SDS)

Lower acceptance limit
ET50 = 12.2 minutes

Upper acceptance limit
ET50 = 37.5 minutes

IC50 = 1.0 mg/mL

IC50 = 3.2 mg/mL

IC50 = 1.0 mg/mL

IC50 = 4.0 mg/mL

Application of the Test Chemical and Control Substances
32.
At least two tissue replicates should be used for each test chemical and each control
substance in each run. Two different treatment protocols are used, one for liquid test
chemicals and one for solid test chemicals (39)(40)(41). For the two VRMs, the tissue
construct surface should be moistened with calcium and magnesium-free Dulbecco's
Phosphate Buffered Saline (Ca2+/Mg2+-free DPBS) before application of test chemicals, tomimic the wet conditions of the human eye. The treatment of the tissues is initiated with
exposure to the test chemical(s) and control substances. For any treatment protocols in anyof the two VRMs, a sufficient amount of test chemical or control substance should beapplied to uniformly cover the epithelial surface while avoiding an infinite dose (seeparagraphs 33 and 34) (Annex II).
33.
Test chemicals that can be pipetted at 37°C or lower temperatures (using a positive
displacement pipette, if needed) are treated as liquids in the three test methdos, otherwise
they should be treated as solids (see paragraph 34). In the test methods, liquid test chemical
are evenly spread over the tissue surface (i.e. a minimum of 60 μL/cm2 application) (seeAnnex II, (39)(40)(41). Capillary effects (surface tension effects) that may occur due to thelow volumes applied to the insert (on the tissue surface) should be avoided to the extent
possible to guarantee the correct dosing of the tissue. Tissues treated with liquid test
chemicals are incubated for 1 minute (LabCyte CORNEA-MODEL24 EIT) or 30 minutes(VRM1 and VRM2) at the standard conditions of each method. At the end of the exposure
period, the liquid test chemical and the control substances should be carefully removed
from the tissue surface by extensive rinsing with Ca2+/Mg2+-free DPBS at room
temperature. For the two VRMs, this rinsing step should be followed by a post-exposure
immersion in fresh medium at room temperature (to remove any test chemical absorbed© OECD, (2018)30

OECD/OCDE

492 │ 13

into the tissue) for a pre-defined period of time that varies depending on the VRM used. For
VMR1 and for LabCyte CORNEA-MODEL24 EIT, a post-exposure incubation in fresh
medium at standard culture conditions is applied prior to performing the TD assay (seeAnnex II, (39)(40)(41)).
34.
Test chemicals that cannot be pipetted at temperatures up to 37°C are treated assolids in the three test methods. The amount of test chemical applied should be sufficient tocover the entire surface of the tissue, i.e. a minimum of 33 mg/cm2 application should be
used (Annex II). Whenever possible, solids should be tested as a fine powder. Tissues
treated with solid test chemicals are incubated for a pre-defined period of time (dependingon the method used) at standard culture conditions (see Annex II, (39)(40)(41)). At the endof the exposure period, the solid test chemical and the control substances should be
carefully removed from the tissue surface by extensive rinsing with Ca2+/Mg2+-free DPBS
at room temperature. For the two VRMs, this rinsing step should be followed by a postexposure immersion in fresh medium at room temperature (to remove any test chemical
absorbed into the tissue) for a pre-defined period of time that varies depending on the VRM
used, followed by a post-exposure incubation in fresh medium at standard culture
conditions, prior to performing the TD assay (see Annex II, (39)(40)(41)(42)).
35.
Concurrent negative and positive controls should be included in each run todemonstrate that the viability (determined with the negative control) and the sensitivity(determined with the positive control) of the tissues are within acceptance ranges defined
based on historical data. The concurrent negative control also provides the baseline(100% tissue viability) to calculate the relative percent viability of the tissues treated withthe test chemical (%Viabilitytest). The recommended positive control substance to be usedwith the VRMs is neat methyl acetate (CAS RN. 79-20-9, commercially available frome.g., Sigma-Aldrich, Cat# 45997; liquid) for both liquids and solids protocols. The
recommended positive control substances to be used with the LabCyte CORNEAMODEL24 EIT are ethanol (CAS RN. 64-17-5) for liquids protocol and lauric acid (CASRN 143-07-7) for solids protocol. The recommended negative control substances to be usedwith the VRM1 and VRM2 are ultrapure H2O and Ca2+/Mg2+-free DPBS for both liquids
and solids protocols, respectively. The recommended negative control to be used with the
LabCyte CORNEA-MODEL24 EIT is Ca2+/Mg2+-free DPBS (CAS RN. 64-17-5) for
liquids protocol and no treatment for solids protocol. These were the control substancesused in the validation studies of the VRMs and are those for which most historical data
exist. The use of suitable alternative positive or negative control substances should bescientifically and adequately justified. Negative and positive controls should be tested withthe same protocol(s) as the one(s) used for the test chemicals included in the run (i.e. forliquids and/or solids). This application should be followed by the treatment exposure,rinsing, a post-exposure immersion, and post-exposure incubation where applicable, as
described for controls run concurrently to liquid test chemicals (see paragraph 33) or forcontrols run concurrently to solid test chemicals (see paragraph 34), prior to performing the
TD assay (see paragraph 36) (39)(40)(41). One single set of negative and positive controls
is sufficient for all test chemicals of the same physical state (liquids or solids) included in
the same run.

Tissue Viability Measurements
36.
The TD assay is a standardised quantitative method (18)(19) that should be used tomeasure tissue viability under this Test Guideline. It is compatible with use in a three-© OECD, (2018)

31

14 │

492

OECD/OCDE

dimensional tissue construct. The TD assay is performed immediately following the postexposure procedures. The VRMs use the MTT assay. In the VRMs, the RhCE tissue
construct sample is placed in 0.3 mL of MTT solution at 1 mg/mL for 180±15 minutes atstandard culture conditions. The vital dye MTT is reduced into a blue MTT formazan
precipitate by the viable cells of the RhCE tissue construct. The precipitated blue MTT
formazan product is then extracted from the tissue using an appropriate volume ofisopropanol (or a similar solvent) (39)(40). Tissues tested with liquid test chemicals should
be extracted from both the top and the bottom of the tissues, while tissues tested with solid
test chemicals and coloured liquids should be extracted from the bottom of the tissue only
(to minimise any potential contamination of the isopropanol extraction solution with any
test chemical that may have remained on the tissue). Tissues tested with liquid test
chemicals that are not readily washed off may also be extracted from the bottom of thetissue only. LabCyte CORNEA-MODEL24 EIT uses the WST-8 assay. In the LabCyte
CORNEA-MODEL24 EIT, the RhCE tissue construct sample is placed in 0.3 mL of diluted
WST-8 solution, prepared according to the standard operating procedures (41), for 240
minutes at standard culture conditions and the vital dye WST-8 is reduced into a yellow
WST-8 formazan by the viable cells of the RhCE tissue construct, which is dissolved into
the diluted WST-8 solution (41). The concurrently tested negative and positive control
substances should be treated similarly to the tested chemical. In the VRM1 and VRM2, theextracted MTT formazan may be quantified either by a standard absorbance (OD)measurement at 570 nm using a filter band pass of maximum ± 30 nm or by using an
HPLC/UPLC-spectrophotometry procedure (see paragraph 43) (12)(42). In the LabCyte
CORNEA-MODEL24 EIT, the WST-8 formazan may be directly quantified (i.e. without
the need of an extraction procedure) either by a standard absorbance (OD) measurement at
450 nm using a filter band pass of maximum ±30 nm or by using an HPLC/UPLCspectrophotometry procedure (see paragraph 43) .
37.
Optical properties of the test chemical or its chemical action on TD (MTT for
VRM1 and VRM2, or WST-8 for LabCyte CORNEA-MODEL24 EIT) may interfere withthe measurement of FD leading to a false estimate of tissue viability. Test chemicals may
interfere with the measurement of FD by direct reduction of the TD into coloured FD (blue
MTT formazan or yellow WST-8 formazan) and/or by colour interference if the test
chemical absorbs, naturally or due to treatment procedures, in the same OD range as FD(i.e., MTT formazan: around 570 nm; WST-8 formazan: around 450 nm). Pre-checks
should be performed before testing to allow identification of potential direct TD reducers
and/or colour interfering chemicals and additional controls should be used to detect andcorrect for potential interference from such test chemicals (see paragraphs 38-42). This isespecially important when a specific test chemical is not completely removed from the
RhCE tissue construct by rinsing or when it penetrates the cornea-like epithelium and istherefore present in the RhCE tissue constructs when the TD assay is performed. For test
chemicals absorbing light in the same range as FD (naturally or after treatment), which are
not compatible with the standard absorbance (OD) measurement of FD due to strong
interference, i.e., strong absorption at 570±30 nm (with MTT formazan) or 450±30 nm(with WST-8 formazan), an HPLC/UPLC-spectrophotometry procedure to measure FD
may be employed (see paragraphs 42 and 43) (11)(40). A detailed description of how to
detect and correct for direct TD reduction and interferences by colouring agents is available
in the test methods’ respective SOPs (39)(40)(41). Illustrative flowcharts providing
guidance on how to identify and handle direct TD-reducers and/or colour interferingchemicals for VRM1, VRM2 and LabCyte CORNEA-MODEL24 EIT are also provided in
Annexes III, IV and V, respectively.
© OECD, (2018)32

OECD/OCDE

492 │ 1538.
To identify potential interference by test chemicals absorbing light in the samerange as FD (naturally or after treatment) and decide on the need for additional controls, thetest chemical is added to water and/or isopropanol and incubated for an appropriate time atroom temperature (see Annex II, (39)(40)(41). If the test chemical in water and/or
isopropanol absorbs sufficient light in the range of 570±20 nm for VRM1 (see Annex III),or if a coloured solution is obtained when mixing the test chemical with water for VRM2
(see Annex IV) and LabCyte CORNEA-MODEL24 EIT(see Annex V), the test chemical ispresumed to interfere with the standard absorbance (OD) measurement of FD and further
colurant controls should be performed or, alternatively, an HPLC/UPLC-spectrophotometry
procedure should be used in which case these controls are not required (see paragraphs 42
and 43 and Annexes III, IV and V)(39)(40)(41). When performing the standard absorbance(OD) measurement, each interfering test chemical should be applied on at least two viabletissue replicates, which undergo the entire testing procedure but are incubated with medium
instead of TD solution during the TD incubation step, to generate a non-specific colour inliving tissues (NSCliving) control (39)(40)(41). The NSCliving control needs to be performed
concurrently to the testing of the coloured test chemical and, in case of multiple testing, an
independent NSCliving control needs to be conducted with each test performed (in each run)due to the inherent biological variability of living tissues. True tissue viability is calculated
as: the percent tissue viability obtained with living tissues exposed to the interfering test
chemical and incubated with the MTT or WST-8 solution (%Viabilitytest) minus the percent
non-specific colour obtained with living tissues exposed to the interfering test chemical andincubated with medium without MTT or WST-8, run concurrently to the test being
corrected (%NSCliving), i.e., True tissue viability = [%Viabilitytest] - [%NSCliving].
39.
To identify direct MTT or WST-8 reducers, each test chemical should be added to
freshly prepared TD. An appropriate amount of test chemical is added to a TD solution andthe mixture is incubated for approximately 3 or 4 hours at standard culture conditions (seeAnnexes III, IV and V) (39)(40)(41). If the TD mixture containing the test chemical (orsuspension for insoluble test chemicals) turns blue/purple (for MTT solution) or
yellow/orange (for WST solution), the test chemical is presumed to directly reduce the TD
and a further functional check on non-viable RhCE tissue constructs should be performed,
independently of using the standard absorbance (OD) measurement or an HPLC/UPLCspectrophotometry procedure. This additional functional check employs killed tissues thatpossess only residual metabolic activity but absorb and retain the test chemical in a similar
way as viable tissues. Killed tissues of VRM1 are prepared by exposure to low temperature("freeze-killed"). Killed tissues of VRM2 are prepared by prolonged incubation (e.g., atleast 24±1 hours) in water followed by storage to low temperature ("water-killed"). Killed
tissues of LabCyte CORNEA-MODEL24 EIT are prepared by freezing tissues at -80°C or
below, for 30 minutes twice (“freeze-killed”). Each TD reducing test chemical is applied on
at least two killed tissue replicates, which undergo the entire testing procedure, to generatea non-specific TD reduction (NSMTT or NSWST) control (39)(40)(41). A single NSMTTor NSWST control is sufficient per test chemical regardless of the number of independent
tests/runs performed. True tissue viability is calculated as: the percent tissue viability
obtained with living tissues exposed to the TD reducer (%Viabilitytest) minus the percent
non-specific TD reduction obtained with the killed tissues exposed to the same reducer,
calculated relative to the negative control run concurrently to the test being corrected(%NSMTT or %NSWST), i.e. true tissue viability = [%Viabilitytest] - [%NSMTT
or %NSWST].

© OECD, (2018)

33

16 │

492

OECD/OCDE

40.
Test chemicals that are identified as producing both colour interference (seeparagraph 38) and direct TD reduction (see paragraph 39) will also require a third set ofcontrols when performing the standard absorbance (OD) measurement, apart from the
NSMTT or NSWST, and NSCliving controls described in the previous paragraphs. This is
usually the case with darkly coloured test chemicals absorbing light in the range of 570±30nm for MTT formazan (e.g., blue, purple, black) or with lightly colored test chemicals
absorbing light in the range of 450±30 nm for WST-8 formazan (e.g. yellow, orange)
because their intrinsic colour impedes the assessment of their capacity to directly reduce
MTT or WST-8 as described in paragraph 39. This forces the use of NSMTT or NSWST
controls, by default, together with the NSCliving controls. Test chemicals for which both
NSMTT or NSWST, and NSCliving controls are performed may be absorbed and retained byboth living and killed tissues. Therefore, in this case, the NSMTT or NSWST control may
not only correct for potential direct TD reduction by the test chemical, but also for colour
interference arising from the absorption and retention of the test chemical by killed tissues.
This could lead to double correction for colour interference since the NSCliving control
already corrects for colour interference arising from the absorption and retention of the test
chemical by living tissues. To avoid a possible double correction for colour interference, athird control for non-specific colour in killed tissues (NSCkilled) needs to be performed (seeAnnexes III and IV)(39)(40)(41). In this additional control, the test chemical is applied on
at least two killed tissue replicates, which undergo the entire testing procedure but are
incubated with medium instead of TD solution during the TD incubation step. A single
NSCkilled control is sufficient per test chemical regardless of the number of independent
tests/runs performed, but should be performed concurrently to the NSMTT or NSWST
control and with the same tissue batch. True tissue viability is calculated as: the percent
tissue viability obtained with living tissues exposed to the test chemical (%Viabilitytest)
minus %NSMTT or %NSWST minus %NSCliving plus the percent non-specific colour
obtained with killed tissues exposed to the interfering test chemical and incubated withmedium without TD, calculated relative to the negative control ran concurrently to the test
being corrected (%NSCkilled), i.e., True tissue viability = [%Viabilitytest] - [%NSMTT
or %NSWST] - [%NSCliving] + [%NSCkilled].
41.
It is important to note that non-specific TD reduction and non-specific colour
interferences may increase the OD (when performing standard absorbance measurements)of the sample above the linearity range of the spectrophotometer and that non-specific TD
reduction can also increase the FD peak area (when performing HPLC/UPLCspectrophotometry measurements) of the sample above the linearity range of thespectrophotometer. On this basis, when using RhCEs, it is important for each laboratory to
determine the OD/peak area linearity range of their spectrophotometer with MTT formazan(CAS RN# 57360-69-7) which is commercially available from e.g., Sigma-Aldrich(Cat# M2003), or WST-8 formazan (CAS RN193149-76-7), commercially available from
Dojindo Molecular Technologies.
42.
The standard absorbance (OD) measurement using a spectrophotometer isappropriate to assess direct TD-reducers and colour interfering test chemicals, when theobserved interference with the measurement of FD is not strong (i.e., the ODs of thesamples obtained with the test chemical without any correction for direct TD reduction
and/or colour interference are within the linear range of the spectrophotometer).
Nevertheless, results for test chemicals producing %NSMTT or %NSWST and/or%NSCliving ≥ 60% (VRM1, and VRM2 for liquids protocol) or 50% (VRM2 for solids
protocol) or 40% (LabCyte CORNEA-MODEL24 EIT) of the negative control should be© OECD, (2018)34

OECD/OCDE

492 │ 17taken with caution as this is the established cut-off used in the VRMs to distinguishclassified from not classified chemicals (see paragraph 45). Standard absorbance (OD) can
however not be measured when the interference with the measurement of FD is strong (i.e.,leading to uncorrected ODs of the test samples falling outside of the linear range of thespectrophotometer). Coloured test chemicals or test chemicals that become coloured incontact with water or isopropanol that interfere strongly with the standard absorbance (OD)measurement of FD may still be assessed using HPLC/UPLC-spectrophotometry (seeAnnexes III, IV and V). This is because the HPLC/UPLC system allows for the separationof the FD from the chemical before its quantification (40). For this reason, NSCliving or
NSCkilled controls are never required when using HPLC/UPLC-spectrophotometry,independently of the chemical being tested. NSMTT or NSWST controls should
nevertheless be used if the test chemical is suspected to directly reduce TD (following theprocedure described in paragraph 39). NSMTT or NSWST controls should also be usedwith test chemicals having a colour (intrinsic or appearing when in water) that impedes theassessment of their capacity to directly reduce TD as described in paragraph 39. Whenusing HPLC/UPLC-spectrophotometry to measure FD, the percent tissue viability is
calculated as percent FD peak area obtained with living tissues exposed to the test chemical
relative to the FD peak obtained with the concurrent negative control. For test chemicals
able to directly reduce TD, true tissue viability is calculated as: %Viabilitytest minus%NSMTT or %NSWST, as described in the last sentence of paragraph 39. Finally, it
should be noted that direct TD-reducers or direct TD-reducers that are also colour
interfering, which are retained in the tissues after treatment and reduce TD so strongly thatthey lead to ODs (using standard OD measurement) or peak areas (using UPLC/HPLCspectrophotometry) of the tested samples that fall outside of the linearity range of thespectrophotometer cannot be assessed with RhCE test methods, although these are expected
to occur in only very rare situations.
43.
HPLC/UPLC-spectrophotometry may be used with all types of test chemicals(coloured, non-coloured, TD-reducers and non-TD reducers) for measurement of FD(12)(42). Due to the diversity of HPLC/UPLC-spectrophotometry systems, it is not feasible
for each user to establish the exact same system conditions. As such, qualification of the
HPLC/UPLC-spectrophotometry system should be demonstrated before its use to quantify
TD from samples by meeting the acceptance criteria for a set of standard qualification
parameters based on those described in the U.S. Food and Drug Administration guidance
for industry on bioanalytical method validation (42)(44). These key parameters and theiracceptance criteria are shown in Annex IV. Once the acceptance criteria defined in Annex
VI have been met, the HPLC/UPLC-spectrophotometry system is considered qualified andready to measure FD under the experimental conditions described in this Test Guideline.

Acceptance Criteria
44.
For each run using RhCE tissue batches that met the quality control (see paragraph31), tissues treated with the negative control substance should exhibit OD reflecting thequality of the tissues that followed shipment, receipt steps and all protocol processes andshould not be outside the historically established boundaries described in Table 2 (seeparagraph 27). Similarly, tissues treated with the positive control substance, i.e., methyl
acetate (for VRM1 and VRM2), ethanol (for LabCyte CORNEA-MODEL24 EIT withliquid protocol) or lauric acid (LabCyte CORNEA-MODEL24 EIT with solid protocol),should show a mean tissue viability < 50% relative to the negative control in the VRM1
with either the liquids or the solids protocols, ≤ 30% (liquids protocol) or ≤ 20% (solids© OECD, (2018)

35

18 │

492

OECD/OCDE

protocol) relative to the negative control in the VRM2, and ≤ 40% relative to the negative
control in the LabCyte CORNEA-MODEL24 EIT, thus reflecting the ability of the tissues
to respond to an irritant test chemical under the conditions of the test method (39)(40)(41).
The variability between tissue replicates of test chemicals and control substances should
fall within the accepted limits (i.e., the difference of viability between two tissue replicatesshould be less than 20% or the standard deviation (SD) between three tissue replicatesshould not exceed 18%). If either the negative control or positive control included in a run
is outside of the accepted ranges, the run is considered "non-qualified" and should be
repeated. If the variability between tissue replicates of a test chemical is outside of theaccepted range, the test must be considered "non-qualified" and the test chemical should be
re-tested.

Interpretation of Results and Prediction Model45.
The OD values/peak areas obtained with the replicate samples for each test
chemical should be used to calculate the mean percent tissue viability (mean between tissue
replicates) normalised to the negative control, which is set at 100%. The percentage tissue
viability cut-off value for identifying test chemicals not requiring classification for eye
irritation or serious eye damage (UN GHS No Category) is given in Table 4. Results should
thus be interpreted as follows:



x

x

The test chemical is identified as not requiring classification and labelling according
to UN GHS (No Category) if the mean percent tissue viability after exposure andpost-exposure incubation is more than (>) the established percentage tissue viability
cut-off value, as shown in Table 4. In this case no further testing in other test
methods is required.
If the mean percent tissue viability after exposure and post-exposure incubation isless than or equal (≤) to the established percentage tissue viability cut-off value, noprediction can be made from this result in isolation, as shown in Table 4. In thiscase, further testing with other test methods will be required because RhCE test
methods show a certain number of false positive results (see paragraphs 14-16) andcannot resolve between UN GHS Categories 1 and 2 (see paragraph 18). The “No
prediction can be made” result of the RhCE test in these cases, would require
additional information for classification purposes [see (3) for guidance].
Table 4: Prediction Models according to UN GHS classification

Test Method

No Category
Mean tissue viability > 60%

No prediction can be madeMean tissue viability ≤ 60%

Mean tissue viability > 60%

Mean tissue viability ≤ 60%

SkinEthic™ HCE EIT(for the solids’ protocol)

Mean tissue viability > 50%

Mean tissue viability ≤ 50%

LabCyte CORNEA-MODEL24 EIT(for both protocols)

Mean tissue viability > 40%

Mean tissue viability ≤ 40%

EpiOcular™ EIT(for both protocols)
SkinEthic™ HCE EIT(for the liquids’ protocol)© OECD, (2018)36

OECD/OCDE

492 │ 19

46.
A single test composed of at least two tissue replicates should be sufficient for a test
chemical when the result is unequivocal. However, in cases of borderline results, such as
non-concordant replicate measurements and/or mean percent tissue viability equal to60±5% (VRM1, and VRM2 for liquids’ protocol), 50±5% (VRM2 for solids’ protocol) or
40±5% (LabCyte CORNEA-MODEL24 EIT), a second test should be considered, as well
as a third one in case of discordant results between the first two tests.
47.
Benchmark chemicals may be useful for evaluating the serious eye damage/eye
irritation potential of unknown test chemicals, or product class, or for evaluating therelative ocular toxicity potential of a classified chemical within a specific range of positive
responses.

DATA AND REPORTING
DATA
48.
Data from individual replicate tissues in a run (e.g., OD values/FD peak areas andcalculated percent tissue viability data for the test chemical and controls, and the final
RhCE test method prediction) should be reported in tabular form for each test chemical,
including data from repeat tests, as appropriate. In addition, mean percent tissue viability
and difference of viability between two tissue replicates (if n=2 replicate tissues) or SD (ifn≥3 replicate tissues) for each individual test chemical and control should be reported. Any
observed interferences of a test chemical with the measurement of FD through direct TD
reduction and/or coloured interference should be reported for each tested chemical.

TEST REPORT49.

The test report should include the following information:

Test chemical
Mono-constituent substancex

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

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

Storage conditions and stability to the extent available.

© OECD, (2018)

37

20 │

492

OECD/OCDE

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

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

Storage conditions and stability to the extent available.

Positive and Negative Control Substances

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

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

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

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

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

RhCE Tissue Construct and Protocol Used (providing rationale for the choices, if
applicable)
Test Method Conditions


RhCE tissue construct used, including batch number;

Wavelength and band pass (if applicable) used for quantifying FD, and linearity rangeof measuring device (e.g., spectrophotometer);Description of the method used to quantify FD;© OECD, (2018)38

OECD/OCDE

492 │ 21

Description of the HPLC/UPLC-spectrophotometry system used, if applicable;Complete supporting information for the specific RhCE tissue construct used
including its performance. This should include, but is not limited to:i.

Viability quality control (supplier)ii. Viability under test method conditions (user);iii. Barrier function quality control;
iv. Morphology, if available;v.

Reproducibility and predictive capacity;

vi. Other quality controls (QC) of the RhCE tissue construct, if available;

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

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

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

Acceptable variability between tissue replicates for the test chemical;

Test Procedure:

Details of the test procedure used;

Doses of test chemical and control substances used;

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

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

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



If applicable, results of controls used for direct TD-reducers and/or coloured test
chemicals, including OD value or FD peak area, %NSMTT or %NSWST, %NSCliving,

Results:© OECD, (2018)

39

22 │

492

OECD/OCDE%NSCkilled, Difference between tissue replicates or SD, final correct percent tissue
viability, and final prediction;

Results obtained with the test chemical(s) and control substances in relation to thedefine run and test acceptance criteria;

Description of other effects observed, e.g., coloration of the tissues by a coloured test
chemical;Discussion of the results
Conclusion© OECD, (2018)40

OECD/OCDE

492 │ 23

LITERATURE
1)

UN (2017). United Nations Globally Harmonized System of Classification and Labelling ofChemicals (GHS). ST/SG/AC.10/30/Rev.7, Seventh Revised Edition, New York and Geneva: United
Nations.
Availableat:[http://www.unece.org/fileadmin/DAM/trans/danger/publi/ghs/ghs_rev07/English/ST-SG-AC10-30Rev7e.pdf].

2)

OECD (2012). Guideline for Testing of Chemicals No. 405: Acute Eye Irritation/Corrosion.
Organisation for Economic Cooperation and Development, Paris. Available at:[http://www.oecd.org/env/testguidelines].[

3)OECD (2018). Guidance Document on an Integrated Approaches on Testing and Assessment for
Serious Eye Damage and Eye irritation. Series on Testing and Assessment No.263. ENV
Publications, Organisation for Economic Cooperation and Development, Paris.

4)OECD (2013). Guideline for Testing of Chemicals No. 437: Bovine Corneal Opacity andPermeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii)
Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage. Organisation for
Economic
Cooperation
andDevelopment,
Paris.
Availableat:[http://www.oecd.org/env/testguidelines].

5)

OECD (2013). Guideline for Testing of Chemicals No. 438: Isolated Chicken Eye Test Method for
Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring
Classification. Organisation for Economic Cooperation and Development, Paris. Available at:[http://www.oecd.org/env/testguidelines].

6)

OECD (2012). Guideline for Testing of Chemicals No. 460: Fluorescein Leakage Test Method for
Identifying Ocular Corrosives and Severe Irritants. Organisation for Economic Co-operation andDevelopment, Paris. Available at: [http://www.oecd.org/env/testguidelines].

7)

OECD (2015). Guideline for Testing of Chemicals No. 491: Short Time Exposure In Vitro Test
Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring
Classification for Eye Irritation or Serious Eye Damage. Organisation for Economic Co-operation
and Development, Paris. Available at: [http://www.oecd.org/env/testguidelines].

8)

Freeman, S.J., Alépée N., Barroso, J., Cole, T., Compagnoni, A., Rubingh, C., Eskes, C., Lammers,J., McNamee, P., Pfannenbecker, U., Zuang, V. (2010). Prospective Validation Study ofReconstructed Human Tissue Models for Eye Irritation Testing. ALTEX 27, Special Issue 2010, 261266.

9)

EC EURL ECVAM. (2014). The EURL ECVAM - Cosmetics Europe prospective validation studyof Reconstructed human Cornea-like Epithelium (RhCE)-based test methods for identifying
chemicals not requiring classification and labelling for serious eye damage/eye irritation: Validation
Study
Report.
EUR
28125
EN;
doi:10.2787/41680.
Availableat:[http://publications.jrc.ec.europa.eu/repository/handle/JRC100280].
© OECD, (2018)

41

24 │

492

OECD/OCDE10)EURL ECVAM Science Advisory Committee. (2014). ESAC Opinion on the EURL ECVAM Eye
Irritation Validation Study (EIVS) on EpiOcularTM EIT and SkinEthicTM HCE and a related Cosmetics
Europe study on HPLC/UPLC-spectrophotometry as an alternative endpoint detection system for
MTT-formazan. ESAC opinion No. 2014-03 of 17 November 2014; EUR 28173 EN; doi:
10.2787/043697. Available at: [http://publications.jrc.ec.europa.eu/repository/handle/JRC103702].

11)

Alépée, N., Leblanc, V., Adriaens, E., Grandidier, M.H., Lelièvre, D, Meloni, M., Nardelli, L.,
Roper, C.S, Santirocco, E., Toner, F., Van Rompay, A., Vinall, J., Cotovio, J. (2016). Multilaboratory validation of SkinEthic HCE test method for testing serious eye damage/eye irritation
using liquid chemicals. Toxicol. In Vitro 31, 43-53.

12)

Alépée, N., Adriaens, E., Grandidier, M.H., Meloni, M., Nardelli, L., Vinall, C.J., Toner, F., Roper,
C.S, Van Rompay, A.R., Leblanc, V., Cotovio, J. (2016). Multi-laboratory evaluation of SkinEthic
HCE test method for testing serious eye damage/eye irritation using solid chemicals and overall
performance of the test method with regard to solid and liquid chemicals testing. Toxicol. In Vitro
34, 55-70.

13)

EURL ECVAM Science Advisory Committee. (2016). ESAC Opinion on the SkinEthic™ Human
Corneal Epithelium (HCE) Eye Irritation Test (EIT). ESAC Opinion No. 2016-02 of 24 June
2016;EUR
28175
EN;
doi
:
10.2787/390390.
Availableat:[http://publications.jrc.ec.europa.eu/repository/handle/JRC103704].

14)

Nakahara, S., Kojima, H., Omori, T., Yamashita, A., Endo, M., Satake, M., Nishiura, H., Shinoda,
S., Hagiwara, S., Kasahara, T., Tahara, H., Yamamoto, Y., Ikeda, H., Yoshitake, Y., Lee, J. Ik., Han,
Y., Lee, S., Sugawara, K., Katoh, M. A catch-up validation study of an in vitro eye irritation test
method using reconstructed human corneal epithelial tissue, LabCyte CORNEA-MODEL24.
(Manuscript submitted).

15)

OECD (2018). Peer Review Report on Validation status of the LabCyte CORNEA-MODEL24 EYE
IRRITATION TEST, OECD Series on Testing and Assessment No.282. ENV Publications,
Organisation for Economic Cooperation and Development, Paris.

16)

Draize, J.H., Woodard, G., Calvery, H.O. (1944). Methods for the Study of Irritation and Toxicity of
Substances Applied Topically to the Skin and Mucous Membranes. Journal of Pharmacol. and Exp.
Therapeutics 82, 377-390.

17)

Scott, L., Eskes, C., Hoffmann, S., Adriaens, E., Alépée, N., Bufo, M., Clothier, R., Facchini, D.,
Faller, C., Guest, R., Harbell, J., Hartung, T., Kamp, H., Le Varlet, B., Meloni, M., McNamee, P.,
Osborne, R., Pape, W., Pfannenbecker, U., Prinsen, M., Seaman, C., Spielman, H., Stokes, W.,
Trouba, K., Van den Berghe, C., Van Goethem, F., Vassallo, M., Vinardell, P., Zuang, V. (2010). A
Proposed Eye Irritation Testing Strategy to Reduce and Replace In Vivo Studies Using Bottom-Up
and Top-Down Approaches. Toxicol. In Vitro 24, 1-9.

18)

Mosmann, T. (1983). Rapid Colorimetric Assay for Cellular Growth and Survival: Application to
Proliferation and Cytotoxicity Assays. J. Immunol. Methods 65, 55-63.

19)

Tominaga, H., Ishiyama, M., Ohseto. F., Sasamoto, K., Hamamoto, T., Suzuki, K., Watanabe, M.,(1999). A water-soluble tetrazolium salt useful for colorimetric cell viability assay. Anal. Commun.
36, 47–50.

© OECD, (2018)42

OECD/OCDE

492 │ 25

20)

OECD (2016). Series on Testing and Assessment No. 216: Performance Standards for theAssessment of Proposed Similar or Modified In Vitro Reconstructed Human Cornea-Like
Epithelium (RhCE) Test Methods for Identifying Chemicals not Requiring Classification andLabelling for Eye Irritation or Serious Eye Damage, Based on the Validated Reference Methods
EpiOcular™ EIT and SkinEthic™ HCE EIT described in TG 492. Organisation for Economic
Cooperation and Development, Paris. Available at: [http://www.oecd.org/env/testguidelines].

21)

OECD (2005). Series on Testing and Assessment No. 34: Guidance Document on the Validation andInternational Acceptance of New or Updated Test Methods for Hazard Assessment. Organisation for
Economic
Cooperation
andDevelopment,
Paris.
Availableat:[http://www.oecd.org/env/testguidelines].

22)

Kaluzhny, Y., Kandárová, H., Hayden, P., Kubilus, J., d'Argembeau-Thornton, L., Klausner, M.
(2011). Development of the EpiOcular™ Eye Irritation Test for Hazard Identification and Labellingof Eye Irritating Chemicals in Response to the Requirements of the EU Cosmetics Directive and
REACH Legislation. Altern. Lab. Anim. 39, 339-364.

23)

Nguyen, D.H., Beuerman, R.W., De Wever, B., Rosdy, M. (2003). Three-dimensional construct of
the human corneal epithelium for in vitro toxicology. In: Salem, H., Katz, S.A. (Eds), Alternative
Toxicological Methods, CRC Press, pp. 147-159.

24)

Katoh, M., Uemura, N., Hamajima, F., Ogasawar,a T., Hata, K. (2012). Morphologicalcharacterization of a reconstructed human corneal epithelial model (LabCyte CORNEA-MODEL) as
an alternative to the draize eye test for the assessment of eye irritation. AATEX. 17, 22-28.

25)

Pfannenbecker, U., Bessou-Touya, S., Faller, C., Harbell, J., Jacob, T., Raabe, H., Tailhardat, M.,
Alépée, N., De Smedt, A., De Wever, B., Jones, P., Kaluzhny, Y., Le Varlet, B., McNamee, P.,
Marrec-Fairley, M., Van Goethem, F. (2013). Cosmetics Europe multi-laboratory pre-validation of
the EpiOcular™ reconstituted Human Tissue Test Method for the Prediction of Eye Irritation.
Toxicol. In Vitro 27, 619-626.

26)

Alépée, N., Bessou-Touya, S., Cotovio, J., de Smedt, A., de Wever, B., Faller, C., Jones, P., Le
Varlet, B., Marrec-Fairley, M., Pfannenbecker, U., Tailhardat, M., van Goethem, F., McNamee, P.
(2013). Cosmetics Europe Multi-Laboratory Pre-Validation of the SkinEthic™ Reconstituted Human
Corneal Epithelium Test Method for the Prediction of Eye Irritation. Toxicol. In Vitro 27, 14761488.

27)

Katoh, M., Hamajima, F., Ogasawar,a T., Hata, K.. (2013). Establishment of a new in vitro test
method for evaluation of eye irritancy using a reconstructed human corneal epithelial model,
LabCyte CORNEA-MODEL. Toxicol. In Vitro. 27, 2184-2192.

28)

Kolle, S.N., Moreno, M.C.R., Mayer, W., van Cott, A., van Ravenzwaay, B., Landsiedel, R. (2015).
The EpiOcular™ Eye Irritation Test is the Method of Choice for In Vitro Eye Irritation Testing ofAgrochemical Formulations: Correlation Analysis of EpiOcular™ Eye Irritation Test and BCOP
Test Data to UN GHS, US EPA and Brazil ANIVSA Classifications. Altern. Lab. Anim. 43, 1-18.

29)

Adriaens, E., Barroso, J., Eskes, C., Hoffmann, S., McNamee, P., Alépée, N., Bessou-Touya, S., De
Smedt, A., De Wever, B., Pfannenbecker, U., Tailhardat, M., Zuang, V. (2014). Retrospective
Analysis of the Draize Test for Serious Eye Damage/Eye Irritation: Importance of Understanding the© OECD, (2018)

43

26 │

492

OECD/OCDEIn Vivo Endpoints Under UN GHS/EU CLP for the Development and Evaluation of In Vitro Test
Methods. Arch. Toxicol. 88, 701-723.
30)

Meloni, M., De Servi, B., Marasco, D., Del Prete, S. (2011). Molecular mechanism of ocular surface
damage: Application to an in vitro dry eye model on human corneal epithelium. Molecular Vision17, 113-126.

31)

Hackett, R.B., McDonald, T.O. (1991). Eye Irritation. In Advances in Modern Toxicology:
Dermatoxicology Marzulli F.N.and Maibach H.I. (Eds.), 4th Edition, pp. 749–815. Washington, DC,
USA: Hemisphere Publishing Corporation.

32)

Fox, D.A., Boyes, W.K. (2008). Toxic Responses of the Ocular and Visual System. In Cassaret andDoull’s Toxicology: The Basic Science of Poisons Klaassen C.D.(Ed.), 7th Edition, pp. 665–697.
Withby, ON, Canada: McGraw-Hill Ryerson.

33)

Jester, J.V., Li, H.F., Petroll, W.M., Parker, R.D., Cavanagh, H.D., Carr, G.J., Smith, B., Maurer,
J.K. (1998). Area and Depth of Surfactant Induced Corneal Injury Correlates with Cell Death. Invest.
Ophthalmol. Vis. Sci. 39, 922–936.

34)

Maurer, J.K., Parker, R.D., Jester, J.V. (2002). Extent of Corneal Injury as the Mechanistic Basis for
Ocular Irritation: Key Findings and Recommendations for the Development of Alternative Assays.
Reg. Tox. Pharmacol. 36, 106-117.

35)

Jester, J.V., Li, L., Molai, A., Maurer, J.K. (2001). Extent of Corneal Injury as a Mechanistic Basisfor Alternative Eye Irritation Tests. Toxicol. In Vitro 15, 115–130.

36)

Jester, J.V., Petroll, W.M., Bean, J., Parker, R.D., Carr, G.J., Cavanagh, H.D., Maurer, J.K. (1998).
Area and Depth of Surfactant-Induced Corneal Injury Predicts Extent of Subsequent Ocular
Responses. Invest. Ophthalmol. Vis. Sci. 39, 2610–2625.

37)

Jester, J.V. (2006). Extent of Corneal Injury as a Biomarker for Hazard Assessment and theDevelopment of Alternative Models to the Draize Rabbit Eye Test. Cutan. Ocul. Toxicol. 25, 41–54.

38)

Barroso, J., Pfannenbecker, U., Adriaens, E., Alépée, N., Cluzel, M., De Smedt, A., Hibatallah, J.,
Klaric, M., Mewes, K.R., Millet, M., Templier, M., McNamee, P. (2017). Cosmetics Europe
compilation of historical serious eye damage/eye irritation in vivo data analysed by drivers ofclassification to support the selection of chemicals for development and evaluation of alternative
methods/strategies: the Draize eye test Reference Database (DRD). Arch. Toxicol. 91, 521-547.

39)

EpiOcular™ EIT SOP, Version 8. (March 05, 2013). EpiOcular™ EIT for the Prediction of Acute
Ocular Irritation of Chemicals. Available at: [http://www.ecvam-dbalm.jrc.ec.europa.eu/].

40)

SkinEthic™ HCE EIT SOP, Version 1. (July 20, 2015). SkinEthic™ HCE Eye Irritation Test (EITL
for Liquids, EITS for Solids) for the Prediction of Acute Ocular Irritation of Chemicals. Available at:[http://www.ecvam-dbalm.jrc.ec.europa.eu/].

41) LabCyte CORNEA-MODEL24 EIT SOP, Version 2.5.6. (February, 2017). LabCyte CORNEAMODEL24
eyeirritation
test
operation[http://www.jacvam.jp/files/doc/06_11/06_11_E1.pdf].

protocol.

Availableat:© OECD, (2018)44

OECD/OCDE

492 │ 27

42)

Alépée, N., Barroso, J., De Smedt, A., De Wever, B., Hibatallah, J., Klaric, M., Mewes, K.R., Millet,
M., Pfannenbecker, U., Tailhardat, M., Templier, M., McNamee, P. (2015). Use of HPLC/UPLCSpectrophotometry for Detection of Formazan in In Vitro Reconstructed Human Tissue (RhT)-Based
Test Methods Employing the MTT-Reduction Assay to Expand their Applicability to Strongly
Coloured Test Chemicals. Toxicol. In Vitro 29, 741-761.

43)

Kaluzhny, Y., Kandárová, H., Handa, Y., DeLuca, J., Truong, T., Hunter, A., Kearney, P.,d'Argembeau-Thornton, L., Klausner, M. (2015). EpiOcular™ Eye Irritation Test (EIT) for Hazard
Identification and Labeling of Eye Irritating Chemicals: Protocol Optimization for Solid Materials
and Extended Shipment Times. Altern. Lab Anim. 43, 101-127.

© OECD, (2018)

45

28 │

492

OECD/OCDEANNEX I
DEFINITIONS

Accuracy: The closeness of agreement between test method results and accepted reference values. It is ameasure of test method performance and one aspect of “relevance.” The term is often used interchangeably
with “concordance”, to mean the proportion of correct outcomes of a test method (21).
Benchmark chemical: A chemical used as a standard for comparison to a test chemical. A benchmark
chemical should have the following properties: (i) consistent and reliable source(s) for its identification andcharacterisation; (ii) structural, functional and/or chemical or product class similarity to the chemical(s)being tested; (iii) known physicochemical characteristics; (iv) supporting data on known effects; and (v)known potency in the range of the desired response.
Bottom-Up approach: Step-wise approach used for a test chemical suspected of not requiringclassification and labelling for eye irritation or serious eye damage, which starts with the determination ofchemicals not requiring classification and labelling (negative outcome) from other chemicals (positive
outcome) (3).
Chemical: A substance or mixture.
Concordance: See "Accuracy".
Cornea: The transparent part of the front of the eyeball that covers the iris and pupil and admits light tothe interior.
CV: Coefficient of Variation.
Dev: Deviation.
EIT: Eye Irritation Test.
EURL ECVAM: European Union Reference Laboratory for Alternatives to Animal Testing.
Eye irritation: Production of changes in the eye following the application of a test chemical to the anterior
surface of the eye, which are fully reversible within 21 days of application. Interchangeable with
“Reversible effects on the eye” and with “UN GHS Category 2” (1).
ET50: Exposure time required to reduce tissue viability by 50% upon application of a benchmark chemical
at a specified, fixed concentration.
False negative rate: The proportion of all positive substances falsely identified by a test method as
negative. It is one indicator of test method performance.
False positive rate: The proportion of all negative substances that are falsely identified by a test method as
positive. It is one indicator of test method performance.
Formazan dye (FD): Chromogenic product of the reduction of MTT and WST-8.
Hazard: Inherent property of an agent or situation having the potential to cause adverse effects when an
organism, system or (sub) population is exposed to that agent.
HCE: SkinEthic™ Human Corneal Epithelium.

© OECD, (2018)46

OECD/OCDE

492 │ 29

HPLC: High Performance Liquid Chromatography.
IC50: Concentration at which a benchmark chemical reduces the viability of the tissues by 50% following afixed exposure time (e.g., 30 or 60 minutes treatment with SDS).
Infinite dose: Amount of test chemical applied to the RhCE tissue construct exceeding the amountrequired to completely and uniformly cover the epithelial surface.
Irreversible effects on the eye: See “Serious eye damage”.
LLOQ: Lower Limit of Quantification.
LogP: Logarithm of the octanol-water partitioning coefficient
Mixture: A mixture or a solution composed of two or more substances in which they do not react (1).
Mono-constituent substance: A substance, defined by its quantitative composition, in which one main
constituent is present to at least 80% (w/w).
Multi-constituent substance: A substance, defined by its quantitative composition, in which more than
one main constituent is present in a concentration ≥ 10% (w/w) and < 80% (w/w). A multi-constituent
substance is the result of a manufacturing process. The difference between mixture and multi-constituent
substance is that a mixture is obtained by blending of two or more substances without chemical reaction. A
multi-constituent substance is the result of a chemical reaction.
MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide.
(CAS RN 298-93-1)
Negative control: A sample containing all components of a test system and treated with a substanceknown not to induce a positive response in the test system. This sample is processed with test chemicaltreated samples and other control samples and is used to determine 100% tissue viability.
Not Classified: Chemicals that are not classified for Eye irritation (UN GHS Category 2, 2A, or 2B) or
Serious eye damage (UN GHS Category 1). Interchangeable with “UN GHS No Category”.
NSCkilled: Non-Specific Colour in killed tissues.
NSCliving: Non-Specific Colour in living tissues.
NSMTT: Non-Specific MTT reduction.
NSWST: Non-Specific WST-8 reduction.
OD: Optical Density.
Performance standards: Standards, based on a validated test method which was considered scientificallyvalid, that provide a basis for evaluating the comparability of a proposed test method that ismechanistically and functionally similar. Included are: (i) essential test method components; (ii) a
minimum list of Reference Chemicals selected from among the chemicals used to demonstrate theacceptable performance of the validated test method; and (iii) the comparable levels of accuracy andreliability, based on what was obtained for the validated test method, that the proposed test method should
demonstrate when evaluated using the minimum list of Reference Chemicals (21).
Positive control: A sample containing all components of a test system and treated with a substance known
to induce a positive response in the test system. This sample is processed with test chemical-treatedsamples and other control samples. To ensure that variability in the positive control response across time
can be assessed, the magnitude of the positive response should not be excessive.
Relevance: Description of relationship of the test to the effect of interest and whether it is meaningful and© OECD, (2018)

47

30 │

492

OECD/OCDE

useful for a particular purpose. It is the extent to which the test correctly measures or predicts the
biological effect of interest. Relevance incorporates consideration of the accuracy (concordance) of a test
method (19).
Reliability: Measures of the extent that a test method can be performed reproducibly within and between
laboratories over time, when performed using the same protocol. It is assessed by calculating intra- andinter-laboratory reproducibility and intra-laboratory repeatability (21).
Replacement test: A test which is designed to substitute for a test that is in routine use and accepted forhazard identification and/or risk assessment, and which has been determined to provide equivalent or
improved protection of human or animal health or the environment, as applicable, compared to theaccepted test, for all possible testing situations and chemicals (21).
Reproducibility: The agreement among results obtained from repeated testing of the same test chemicalusing the same test protocol (See "Reliability") (21).
Reversible effects on the eye: See “Eye irritation”.
RhCE: Reconstructed human Cornea-like Epithelium.
Run: A run consists of one or more test chemicals tested concurrently with a negative control and with apositive control.
SD: Standard Deviation.
Sensitivity: The proportion of all positive/active test chemicals that are correctly classified by the test. It isa measure of accuracy for a test method that produces categorical results, and is an important considerationin assessing the relevance of a test method (21).
Serious eye damage: Production of tissue damage in the eye, or serious physical decay of vision,
following application of a test chemical to the anterior surface of the eye, which is not fully reversible
within 21 days of application. Interchangeable with “Irreversible effects on the eye” and with “UN GHS
Category 1” (1).
Standard Operating Procedures (SOP): Formal, written procedures that describe in detail how specificroutine, and test-specific, laboratory operations should be performed. They are required by GLP.
Specificity: The proportion of all negative/inactive test chemicals that are correctly classified by the test. It
is a measure of accuracy for a test method that produces categorical results and is an importantconsideration in assessing the relevance of a test method (21).
Substance: Chemical elements and their compounds in the natural state or obtained by any production
process, including any additive necessary to preserve the stability of the product and any impurities
deriving from the process used, but excluding any solvent which may be separated without affecting thestability of the substance or changing its composition (1).
Test: A single test chemical concurrently tested in a minimum of two tissue replicates as defined in thecorresponding SOP.
Tetrazolium dye (TD) : Tetrazolium salts MTT and WST-8.
Tissue viability: Parameter measuring total activity of a cell population in a reconstructed tissue as theirability to reduce the vital dye MTT, which, depending on the endpoint measured and the test design used,correlates with the total number and/or vitality of living cells.
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) fromother chemicals (negative outcome) (3).
© OECD, (2018)48

OECD/OCDE

492 │ 31

Test chemical: The term "test chemical" is used to refer to what is being tested.
Tiered testing strategy: A stepwise testing strategy, which uses test methods in a sequential manner. Allexisting information on a test chemical is reviewed at each tier, using a weight-of-evidence process, todetermine if sufficient information is available for a hazard classification decision, prior to progression to
the next tier in the strategy. If the hazard potential/potency of a test chemical can be assigned based on theexisting information at a given tier, no additional testing is required (21).
ULOQ: Upper Limit of Quantification.
United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UNGHS): A system proposing the classification of chemicals (substances and mixtures) according tostandardised types and levels of physical, health and environmental hazards, and addressing corresponding
communication elements, such as pictograms, signal words, hazard statements, precautionary statements
and safety data sheets, so that to convey information on their adverse effects with a view to protect people(including employers, workers, transporters, consumers and emergency responders) and the environment(1).
UN GHS Category 1: See “Serious eye damage”.
UN GHS Category 2: See “Eye irritation”.
UN GHS No Category: Chemicals that do not meet the requirements for classification as UN GHS
Category 1 or 2 (2A or 2B). Interchangeable with “Not Classified”.
UPLC: Ultra-High Performance Liquid Chromatography.
UVCB: substances of unknown or variable composition, complex reaction products or biologicalmaterials.
Valid test method: A test method considered to have sufficient relevance and reliability for a specificpurpose and which is based on scientifically sound principles. A test method is never valid in an absolutesense, but only in relation to a defined purpose (21).
Validated test method: A test method for which validation studies have been completed to determine therelevance (including accuracy) and reliability for a specific purpose. It is important to note that a validated
test method may not have sufficient performance in terms of accuracy and reliability to be foundacceptable for the proposed purpose (21).
VRM: Validated Reference Method.
VRM1: EpiOcular™ EIT is referred as the Validated Reference Method 1.
VRM2: SkinEthic™ HCE EIT is referred to as the Validated Reference Method 2.
Weight-of-evidence: The process of considering the strengths and weaknesses of various pieces of
information in reaching and supporting a conclusion concerning the hazard potential of a test chemical.
WST: Water soluble tetrazolium salt.
WST-8: Water soluble tetrazolium salt-8. [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4disulfophenyl)-2H-tetrazolium, monosodium salt. (CAS RN 193149-74-5).

© OECD, (2018)

49

50

492
ANNEX II

OECD/OCDE

50 mg + 1 mL H2O for 60 min
at 37±2ºC, 5±1% CO2, ≥95%
RH
(non-coloured test chemicals)
and/or50 mg + 2 mL isopropanol
mixed for 2-3h at RT (coloredand non-colored test chemicals)

50 μL + 1 mL H2O for 60
min at 37±2ºC, 5±1% CO2,≥95% RH (non-coloured
test chemicals), or
50 μL + 2 mL isopropanol
mixed for 2-3h at RT(coloured test chemicals)

Pre-check forcolourinterferenceÆ if the OD of the test
chemical at 570±20 nm after
subtraction of the OD for
isopropanol or water is > 0.08
(which corresponds toapproximately 5% of the mean
OD of the negative control),living adapted controls shouldbe performed.

At least 2

At least 2

Number of tissue
replicatesÆ if the OD of the test
chemical at 570±20 nm,
after subtraction of the OD
for isopropanol or water is> 0.08 (which corresponds
to approximately 5% of themean OD of the negative
control), living adapted
controls should be

0.6 cm2

0.6 cm2

Model surface

ProtocolSolids(not pipetteable)

EpiOcular™ EIT(VRM 1)Liquids(pipetteable at 37±1°C or
lower temperatures for 15
min)

Test Method
ComponentÆ if test chemical iscoloured, living
adapted controls
should be performed

10 μL + 90 μL H2O
mixed for 30±2 min
at Room
Temperature (RT,18-28oC)

At least 2

0.5 cm2

Liquids and viscous(pipetteable)

Æ if test chemical iscoloured, living adapted
controls should be
performed

10 mg + 90 μL H2O mixed
for 30±2
min at RT

At least 2

0.5 cm2

Solids(not pipetteable)

SkinEthic™ HCE EIT(VRM 2)

If test chemical iscoloured, living adapted
controls should be
performed

50 μL + 0.5 mL distilledwater mixed for 15
minutes at 37±2ºC, 5±1%
CO2, ≥95% RH.

3 tissues

0.3 cm2

Liquid(pipetteable)© OECD, (2018)

If test chemical iscoloured, living adapted
controls should be
performed

10 mg + 0.5 mL distilledwater mixed for 15
minutes at 37±2ºC, 5±1%
CO2, ≥95% RH.

3 tissues

0.3 cm2

Solid(not pipetteable)

LabCyte CORNEA-MODEL24 EIT

MAIN TEST METHOD COMPONENTS OF THE RhCE TEST METHODS VALIDATED FOR IDENTIFYINGCHEMICALS NOT REQUIRING CLASSIFICATION AND LABELLING FOR EYE IRRITATION OR SEERIOUS
EYE DAMAGE

32 │

51

30 min (r 2 min)in culture medium
at 37±2ºC, 5±1% CO2, ≥95%
RH

50 μL (83.3 μL/cm2)

30 min (r 2 min)in culture medium
at 37±2ºC, 5±1% CO2,≥95% RH

For viscous, use a nylon
mesh

4 hours (r 0.1 h)in culture medium
at 37±2ºC, 5±1% CO2, ≥95%
RH

30 μL Ca2+/Mg2+-free DPBS
+ 30 r 2 mg (60 mg/cm2)

10 μL Ca2+/Mg2+-freeDPBS+ 30 r 2 μL (60 μL/cm2)

50 mg (83.3 mg/cm2) using
a calibrated tool (e.g., alevelled spoonful calibrated
to hold 50 mg of sodium
chloride).
6 hours (r 0.25 h)
in culture medium
at 37±2ºC, 5±1% CO2,≥95% RH--

20 μL Ca2+/Mg2+ -freeDPBS
for 30r2 min at 37±2ºC,
5±1% CO2, ≥95% RH,
protected from light.

Solid
30 mg + 300 μL MTT
1 mg/mL solution for 180±
15 min at 37±2ºC, 5±1%
CO2, ≥95% RH
Æ if solution turns
blue/purple, water-killed
adapted controls should be
performed(30 μL of sterile deionizedwater in MTT solution isused as negative control)

Liquid
30 μL + 300 μL MTT 1
mg/mL solution for 180±
15 min at 37±2ºC, 5±1%
CO2, ≥95% RH
Æ if solution turns
blue/purple, water-killed
adapted controls shouldbe performed(30 μL of sterile
deionized water in MTT
solution is used as
negative control)

SkinEthic™ HCE EIT(VRM 2)Solid
50 mg + 1 mL MTT 1
mg/mL solution for 180±15min
at 37±2ºC, 5±1% CO2,≥95% RHÆ if solution turns
blue/purple, freeze-killed
adapted controls should be
performed(50 μL of sterile deionizedwater in MTT solution isused as negative control)

EpiOcular™ EIT(VRM 1)Liquid
50 μL + 1 mL MTT 1
mg/mL solution for 180±15min
at 37±2ºC, 5±1% CO2, ≥95%
RH
Æ if solution turns
blue/purple,freeze-killed
adapted controls should be
performed(50 μL of sterile deionizedwater in MTT solution isused as negative control)
20 μL Ca2+/Mg2+-free DPBS
for 30 r 2 min at 37±2ºC,
5±1% CO2, ≥95% RH,
protected from light.

© OECD, (2018)

Exposure time
and temperature

Treatment doses
and application

Pre-treatment

Pre-check fordirect MTT
reduction

ProtocolTest Method
Component

performed.

OECD/OCDE

1 minutes (± 5 second)in culture medium
at RT

50 μL (167 μL/cm2)

24 hours (± 1 hr)
in culture medium
at 37±2ºC, 5±1% CO2,≥95% RH

10 mg (33 mg/cm2)-

10 mg + 300 μL diluted
WST-8 medium for240±20 minutes at
37±2ºC, 5±1% CO2,≥95% RH
If solution turns yellow,
freeze-killed adapted
controls should be
performed.

50 μL + 300 μL diluted
WST-8 medium for240±20 minutes at
37±2ºC, 5±1% CO2,≥95% RH
If solution turns yellow,
freeze-killed adapted
controls should be
performed.

-

Solid

LiquidLabCyte CORNEA-MODEL24 EIT

492 │ 33

52

Protocol180 min (r 15 min) at
37±2ºC, 5±1% CO2,≥95% RH
2 mL isopropanol(extraction from bottom
of insert by piercing thetissue)2-3 h with shaking

180 min (r 15 min) at

37±2ºC, 5±1% CO2, ≥95%
RH
2 mL isopropanol(extraction from top andbottom of insert bypiercing the tissue)

2-3 h with shaking (~120

Tetrasolium salt
incubation andtemperature

Extraction time

Extraction
solvent

300 μL 1 mg/mL

300 μL 1 mg/mL

Tetrasolium salt
solution

50 μL Methyl acetate

Tested concurrently

Tested concurrently

50 μL Methyl acetate

Tested concurrently

50 μL H2O

medium at 37±2ºC,
5±1% CO2, ≥95% RH

culture medium at
37±2ºC, 5±1% CO2, ≥95%
RH
50 μL H2O

Solid
18 h (r 0.25 h) in culture

Liquid120 min (r 15 min) in

Tested concurrently

Positive control

Negative control

Post-exposure
incubation

EpiOcular™ EIT(VRM 1)25 min (r 2 min) at RT in
culture medium

12 min (r 2 min) at RT in
culture medium

Post-exposure
immersion

Test Method
Component

3 times in 100 mLof Ca2+/Mg2+-free DPBS

3 times in 100 mLof Ca2+/Mg2+-free DPBS

492

Rinsing at room
temperature

34 │

Solid

4 h with shaking (~120(extraction from top
and bottom of insert)

37±2ºC, 5±1% CO2,≥95% RH
1.5 mL isopropanol

180 min (r 15 min) at

300 μL 1 mg/mL

Tested concurrently

At least 2 h with(extraction from bottom
of insert)

37±2ºC, 5±1% CO2,≥95% RH
1.5 mL isopropanol

180 min (r 15 min) at

300 μL 1 mg/mL

Tested concurrently

Tested concurrently
30 ± 2μL Methyl acetate

Tested concurrently
30 ± 2μL Methyl acetate

medium at 37±2ºC,
5±1% CO2, ≥95% RH

18 h (r 0.5 h) in culture

30 ± 2μL Ca2+/Mg2+-freeDPBS

Liquid30 ± 2μL Ca2+/Mg2+-free
DPBS

none

30 min (r 2 min) at RT in
culture medium

25 mL Ca2+/Mg2+-free DPBS

SkinEthic™ HCE EIT(VRM 2)30 min (r 2 min) at
37oC, 5% CO2, 95% RH
in culture medium

20 mL Ca2+/Mg2+-free
DPBS

OECD/OCDE-

10 times or over bystreamof Ca2+/Mg2+ -free DPBS

Liquid© OECD, (2018)

Not required

Not required

Not required

Not required

240 minutes (± 15
minutes) at 37±2ºC,
5±1% CO2, ≥95% RH

300 μL of diluted
WST-8 solution (10fold dilution of CCK8)

10 mg Lauric acid
Tested concurrently

No treatment
Tested concurrently

Solid

240 minutes (± 15 minutes)
at 37±2ºC, 5±1% CO2, ≥95%
RH

300 μL of diluted WST-8
solution (10 fold dilution of
CCK-8)

50 μL Ethanol
Tested concurrently

50 μL Ca2+/Mg2+ -free DPBS
Tested concurrently

none

LabCyte CORNEA-MODEL24 EIT

24 h (± 1 h) in culture
medium at 37±2ºC, 5±1%
CO2, ≥95% RH-

10 times or over bystreamof Ca2+/Mg2+ -free DPBS

53

Solid
1. Mean OD of the tissue
replicates treated with
the negative control
should be > 0.8 and <2.5
2. Mean viability of thetissue replicates exposedfor 6 hours with thepositive control,
expressed as % of thenegative control, shouldbe < 50%
3. The difference ofviability between two
tissue replicates shouldbe less than 20%.

Liquid

1. Mean OD of the tissue
replicates treated with
the negative control
should be > 0.8 and <2.5
2. Mean viability of thetissue replicates exposedfor 30 minutes with thepositive control,
expressed as % of thenegative control, shouldbe < 50%
3. The difference ofviability between two
tissue replicates shouldbe less than 20%.

EpiOcular™ EIT(VRM 1)

12.2 min ≤ ET50 ≤ 37.5
minTreatment with 100 μL of
0.3% (v/v) Triton X-100

Treatment with 100 μL of
0.3% (v/v) Triton X-100

12.2 min ≤ ET50 ≤ 37.5
min

570 nm (550-590 nm)
without reference filter

570 nm (550 - 590 nm)(~120 rpm) at RT or
overnight at 4-10°C

without reference filterrpm) at RT or overnightat 4-10°C© OECD, (2018)

AcceptanceCriteria

ProtocolTest Method
Component

Tissue Quality
Control

OD reading

and temperature

1.0 mg/mL ≤ IC50 ≤ 3.2
mg/mL

30 min treatment with SDS(50 μL)without reference filter

570 nm (540 - 600 nm)

shaking (~120 rpm) at
RT

1. Mean OD of the tissue
replicates treated with
the negative control
should be > 1.0 and ≤2.5
2. Mean viability of thetissue replicates exposedfor 30 minutes with thepositive control,
expressed as % of thenegative control, shouldbe ≤ 30%
3. The difference ofviability between two
tissue replicates shouldbe less than 20%.

Liquid
1. Mean OD of the tissue
replicates treated with
the negative control
should be > 1.0 and ≤2.5
2. Mean viability of thetissue replicates exposedfor 4 hours with thepositive control,
expressed as % of thenegative control, shouldbe ≤ 20%
3. The difference ofviability between two
tissue replicates shouldbe less than 20%.

Solid

SkinEthic™ HCE EIT(VRM 2)

1.0 mg/mL ≤ IC50 ≤ 3.5
mg/mL

30 min treatment with SDS(50 μL)without reference filterrpm) at RT or at least
overnight without
shaking at 4-10°C
570 nm (540 - 600 nm)

OECD/OCDE

60 minutes treatment
with SDS (25 μL)
1.0 mg/mL ≤ IC50 ≤ 4.0
mg/mL

450 nmwith reference filter(650 nm)

1. Mean OD of the tissue
replicates treated with
the negative control
should be ≥ 0.5 and ≤1.3.
2. Mean viability of thetissue replicates treatedwith the positive control
should be ≤ 40%
3. The standard deviation(SD) between threetissue replicates should
not exceed 18%

Liquid

1. Mean OD of the tissue
replicates treated with
the negative control
should be ≥ 0.5 and ≤1.3.
2. Mean viability of thetissue replicates treatedwith the positive control
should be ≤ 40%
3. The standard deviation(SD) between threetissue replicates should
not exceed 18%

Solid

LabCyte CORNEA-MODEL24 EIT

60 minutes treatment withSDS (25 μL)
1.0 mg/mL ≤ IC50 ≤ 4.0
mg/mL

450 nmwith reference filter (650 nm)

492 │ 35

36 │

492

OECD/OCDEANNEX IIIILLUSTRATIVE FLOWCHART PROVIDING GUIDANCE ON HOW TOIDENTIFY AND HANDLE DIRECT MTT-REDUCERS AND/OR COLOUR
INTERFERING CHEMICALS, BASED ON THE VRM1 SOP
PRE-CHECK FOR
COLOUR INTERFERENCE

Incubate 50 μL or 50 mg oftest chemical in 1 mL ofwater for 1 hour at standard
culture conditions

Incubate 50 μL or 50 mg oftest chemical in 2 mL ofisopropanol for 2-3 hours atroom temperature

Is the OD at 570±20 nm
higher than 0.08?

PRE-CHECK FOR
DIRECT MTT REDUCTION
Incubate 50 μL or 50 mg oftest chemical in 1 mL of
1 mg/mL MTT solution for
3 hours at standard culture
conditions

No

Yes

Is the colour of the chemical
too strong to allow a
conclusive pre-check fordirect MTT reduction?

Does the mixture turnblue/purple?

No

Incubate 50 μL or 50 mg oftest chemical in 1 mL of
1 mg/mL MTT solution for
3 hours at standard culture
conditionsNo

Use OD or
HPLC/UPLCspectrophotometry

Yes

Use OD or
HPLC/UPLCspectrophotometry

Does the mixture turnblue/purple?

Yes
Consider one of thetwo following options

Use HPLC/UPLCspectrophotometry

PRE-CHECKFOR
DIRECT MTTREDUCTIONNo

Use OD

Use OD

Perform living-tissue control concurrently
with every test performed, following fulltesting procedure but incubating withmedium instead of MTT (= %NSCliving)

Perform living-tissue control concurrently
with every test performed, following fulltesting procedure but incubating withmedium instead of MTT (= %NSCliving)Use HPLC/UPLCspectrophotometryAND
Perform killed-tissue control following fulltesting procedure (= %NSMTT)(one is sufficient to correct multiple tests)

Perform killed-tissue control following fulltesting procedure (= %NSMTT)(one is sufficient to correct multiple tests)
AND
Perform killed-tissue control following fulltesting procedure but incubating withmedium instead of MTT (= % NSCkilled)(one is sufficient to correct multiple tests)

No controls
are required

Final %viability =%uncorrected test viability - %NSMTT

Final %viability =%uncorrected test viability %NSCliving - %NSMTT + %NSCkilled

Final %viability =%uncorrected test viability %NSCliving

No controls
are required© OECD, (2018)

54

OECD/OCDE

492 │ 37

ANNEX IVILLUSTRATIVE FLOWCHART PROVIDING GUIDANCE ON HOW TOIDENTIFY AND HANDLE DIRECT MTT-REDUCERS AND/OR COLOUR
INTERFERING CHEMICALS, BASED ON THE VRM2 SOP
PRE-CHECK FOR
COLOUR INTERFERENCE
Incubate 10 μL or 10 mg oftest chemical in 90 μL ofwater for 30 minutes atroom temperature

Does the mixture turncoloured?

PRE-CHECK FOR
DIRECT MTT REDUCTION
Incubate 30 μL or 30 mg oftest chemical in 300 μL of
1 mg/mL MTT solution for
3 hours at standard culture
conditions

No

Yes

Is the colour of the chemical
too strong to allow a
conclusive pre-check fordirect MTT reduction?

Does the mixture turnblue/purple?

No

Incubate 30 μL or 30 mg oftest chemical in 300 μL of
1 mg/mL MTT solution for
3 hours at standard culture
conditionsNo

Use OD or
HPLC/UPLCspectrophotometry

Yes

Use OD or
HPLC/UPLCspectrophotometry

Does the mixture turnblue/purple?

Yes
Consider one of thetwo following options

Use HPLC/UPLCspectrophotometry

PRE-CHECKFOR
DIRECT MTTREDUCTIONNo

Use OD

Use OD

Perform living-tissue control concurrently
with every test performed, following fulltesting procedure but incubating withmedium instead of MTT (= %NSCliving)

Perform living-tissue control concurrently
with every test performed, following fulltesting procedure but incubating withmedium instead of MTT (= %NSCliving)Use HPLC/UPLCspectrophotometryAND
Perform killed-tissue control following fulltesting procedure (= %NSMTT)(one is sufficient to correct multiple tests)

Perform killed-tissue control following fulltesting procedure (= %NSMTT)(one is sufficient to correct multiple tests)
AND
Perform killed-tissue control following fulltesting procedure but incubating withmedium instead of MTT (= % NSCkilled)(one is sufficient to correct multiple tests)

No controls
are required

Final %viability =%uncorrected test viability - %NSMTT

Final %viability =%uncorrected test viability %NSCliving - %NSMTT + %NSCkilled

Final %viability =%uncorrected test viability %NSCliving

No controls
are required© OECD, (2018)

55

38 │

492

OECD/OCDEANNEX VILLUSTRATIVE FLOWCHART PROVIDING GUIDANCE ON HOW TOIDENTIFY AND HANDLE DIRECT WST-REDUCERS AND/OR COLOUR
INTERFERING CHEMICALS, BASED ON THE LABCYTE CORNEA-MODEL24
EIT SOP
PRE-CHECK FOR
COLOUR INTERFERENCE
Incubate 50 μL or 10 mg oftest chemical in 300 μL ofwater for 15 minutes at room
temperature
Does the mixture turncoloured?

PRE-CHECK FOR
DIRECT WST REDUCTION
Incubate 50 μL or 10 mg oftest chemical in 300 μL of
WST-8 solution for 4 hours atstandard culture conditionNo

Yes
Is the colour of the chemical
too strong to allow a
conclusive pre-check fordirect WST-8 reduction?

No
Does the mixture turn yellow?

Incubate 50 μL or 10 mg oftest chemical in 300 μL of
WST-8 solution for 4 hours atstandard culture condition

PRE-CHECKFOR DIRECT
WST
REDUCTION

Does the mixture turn yellow?
No

Yes
Consider one of the two
following options

Yes

No

Use OD or
HPLC/UPLCspectrophotometry

Use OD or
HPLC/UPLCspectrophotometry

Use
HPLC/UPLCspectrophotometry

Perform killed-tissue control following
full testing procedure (= %NSWST)(oneis sufficient to correct multiple tests)

Use OD

Use OD

Perform living-tissue control
concurrently with every test performed,
following full testing procedure but
incubating with medium instead of WST8 (= %NSCliving)

Perform living-tissue control
concurrently with every test performed,
following full testing procedure but
incubating with medium instead of WST8 (= %NSCliving)

Use
HPLC/UPLCspectrophotometry

Perform killed-tissue control following
full testing procedure (= %NSWST)(oneis sufficient to correct multiple tests)
Perform killed-tissue control following
full testing procedure but incubating withmedium instead of WST-8(= %NSCkilled)(one is sufficient tocorrect multiple tests)

No controls
are required

Final % viability =%uncorrected test viability - %NSWST

Final% viability =%uncorrected viability %NSCliving - %NSWST + %NSCkilled

Final% viability =%uncorrected viability - %NSClivind

No controls
are required© OECD, (2018)56

OECD/OCDE

492 │ 39

ANNEX VI
KEY PARAMETERS AND ACCEPTANCE CRITERIA FOR QUALIFICATIONOF AN HPLC/UPLC-SPECTROPHOTOMETRY SYSTEM FOR MEASUREMENTOF MTT FORMAZAN EXTRACTED FROM RhCE TISSUE CONSTRUCTS

Parameter
Selectivity

Accuracy

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

Matrix EffectQuality Controls in living blank (n=5)

CarryoverAnalysis of isopropanol after an ULOQ2 standard

Reproducibility(intra-day)

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

Precision

Reproducibility(inter-day)

Short Term Stabilityof MTT Formazan in
RhCE Tissue Extract
Long Term Stability of
MTT Formazan in
RhCE Tissue Extract,
if required
1
2

Quality Controls in living blank (n=3) analysed the day ofthe preparation and after several days of storage at -20°C

Acceptance Criteria
Areainterference ≤ 20% of
AreaLLOQ1

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

LLOQ: Lower Limit of Quantification, defined to cover 1-2% tissue viability, i.e., 0.8 μg/mL.
ULOQ: Upper Limit of Quantification, defined to be at least two times higher than the highest expected
MTT formazan concentration in isopropanol extracts from negative controls (~70 μg/mL in the VRM),
i.e., 200 μg/mL.

© OECD, (2018)57