新規試験法提案書

再構築ヒト角膜様上皮モデル法（RhCE法）
SkinEthic HCE EIT
TM

平成30年 3 月

国立医薬品食品衛生研究所

᪂つヨ㦂ἲᥦ᱌᭩             ᖹᡂ 30 ᖺ 3 ᭶ 23 ᪥                                 No. 2017-04෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝἲ RhCEἲSkinEthic™ HCE EIT࡟㛵ࡍࡿᥦ᱌ ᖹᡂ 30 ᖺ 2 ᭶ 21 ᪥䛻ᕝᓮࠊᅜ❧་⸆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 ෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝἲ㸦Reconstructed human Cornea-like Epithelium Test
Method: RhCE ἲ㸧SkinEthic™ Human Corneal Epithelium (HCE) Eye Irritation Test (EIT)ࡣࠊ໬Ꮫ≀㉁࡟ࡼࡿ║่⃭ᛶࢆホ౯࡛ࡁࠊ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾࠸࡚ UN GHS ༊ศእࢆ᳨ฟࡍࡿ᪉ἲ࡜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 damage͇࡜ RhCE ἲ SkinEthic™ HCEEIT ࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩➼ࢆࡶ࡜࡟ࠊ║่⃭ᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍࡟ࡼࡾసᡂࡉࢀࡓࠕ෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝἲ Skin EthicTM HCE/S ࢆ⏝࠸ࡓ║่⃭ᛶヨ㦂ホ౯ሗ࿌᭩ࠖࢆ⏝࠸࡚ࠊ
JaCVAM ホ౯఍㆟ࡀホ౯࠾ࡼࡧ᳨ウࡋࡓ⤖ᯝࠊࡑࡢ᭷⏝ᛶࡀ☜ㄆࡉࢀࡓࡇ࡜࠿ࡽసᡂࡉࢀࡓࠋ ௨ୖࡢ⌮⏤࡟ࡼࡾࠊ⾜ᨻᙜᒁࡢᏳ඲ᛶホ౯᪉ἲ࡜ࡋ࡚ RhCE ἲ SkinEthic™ HCE EIT ࡢ౑⏝ࢆᥦ᱌ࡍࡿࡶࡢ࡛࠶ࡿࠋ
኱㔝Ὀ㞝                      すᕝ⛅ె
JaCVAM ホ౯఍㆟ ㆟㛗               JaCVAM 㐠Ⴀጤဨ఍ ጤဨ㛗

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JaCVAM ホ౯఍㆟኱ 㔝 Ὀ 㞝 㸦බ┈㈈ᅋἲே ᮌཎグᛕᶓ὾⏕࿨⛉Ꮫ᣺⯆㈈ᅋ㸧㸸ᗙ㛗㣤 ሯ ᑦ ᩥ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧*஬༑ᔒⰋ᫂ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ ⏕ά⾨⏕໬Ꮫ㒊㸧▼ ஭ 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 28 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 29 ᖺ 3 ᭶ 31 ᪥**㸸ᖹᡂ 29 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 30 ᖺ 3 ᭶ 31 ᪥

                 
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JaCVAM 㐠Ⴀጤဨ఍す ᕝ ⛅ ె㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧㸸ጤဨ㛗ᕝ す  ᚭ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲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ຍ ⸨  ⠜㸦ᅜ❧ឤᰁ⑕◊✲ᡤ㸧ㅋ ⏣ Ὀ ᡂ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ⸆⌮㒊㸧ᑠụ⣫୍㑻㸦ཌ⏕ປാ┬ ་⸆࣭⏕ά⾨⏕ᒁ ་⸆ရᑂᰝ⟶⌮ㄢ ໬Ꮫ≀㉁Ᏻ඲ᑐ⟇ᐊ㸧⠛ ⏣ ࿴ ಇ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ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ᖹ ᯘ ᐜ Ꮚ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ẘᛶ㒊㸧ᗈ ℩ ᫂ ᙪ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ Ᏻ඲ᛶண ホ౯㒊㸧ᘅ ⏣ ග ᜨ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Ώ 㑔 ఙ ୍㸦ཌ⏕ປാ┬ ་⸆࣭⏕ά⾨⏕ᒁ ་⸆ရᑂᰝ⟶⌮ㄢ㸧ᑠ ᓥ  ⫕ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ Ᏻ඲ᛶணホ౯㒊➨஧ᐊ㸧㸸஦ົᒁ

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JaCVAM statement on the RhCE test method, SkinEthic™ HCE EIT
At a meeting held on 21 February 2018 at the National Institute of Health Sciences (NIHS) in
Kawasaki, Japan, the Japanese Center for the Validation of Alternative Methods (JaCVAM)
Regulatory Acceptance Board unanimously endorsed the following statement:
Proposal: The Reconstructed human Cornea-like Epithelium Eye Irritation (RhCE) test method,
SkinEthic™ Human Corneal Epithelium (HCE) Eye Irritation Test (EIT) is a suitable
method for assessing ocular irritation potential in a regulatory context as part of abottom-up approach for identifying chemicals not requiring classification and labellingfor eye irritation or serious eye damage (No Category) under the United Nations
Globally Harmonized System of Classification and Labeling of Chemicals (UN GHS).
This statement was prepared following a review of the Organisation for Economic Co-operation
and Development (OECD) Test Guideline 492 “Reconstructed human Cornea-like Epithelium
test method for identifying chemicals not requiring classification and labelling for eye irritationTMor serious eye damage” as well as a validation report on the RhCE test method, SkinEthic HCE
EIT prepared by the Ocular Irritation Testing JaCVAM Editorial Committee to acknowledge thatthe results of a review and study by the JaCVAM Regulatory Acceptance Board have confirmed
the usefulness of this assay.
Based on the above, we propose the RhCE test method, SkinEthic™ HCE EIT as a useful means
for safety assessment by regulatory agencies.

Yasuo Ohno

Akiyoshi Nishikawa

Chairperson

Chairperson

JaCVAM Regulatory Acceptance BoardJaCVAM Steering Committee

23 March 2018v

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 of Life
Sciences) : Chairperson
Mr. Naofumi Iizuka (Pharmaceuticals and Medical Devices Agency)*
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)
Ms. Maki Noguchi (Pharmaceuticals and Medical Devices Agency)**
Mr. Satoshi Numazawa (Japanese Society of Toxicology)
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 2016 to 31st March 2017**: From 1st April 2017 to 31st March 2018vi

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

Mr. Akiyoshi Nishikawa (BSRC, NIHS): Chairperson
Mr. Toru Kawanishi (NIHS)
Mr. Manabu Fuchioka (Ministry of Health, Labour and Welfare)
Ms. Yoko Hirabayashi (Division of Toxicology, BSRC, NIHS)
Mr. Akihiko Hirose (Division of Risk Assessment, BSRC, NIHS)
Ms. Mitsue Hirota (Pharmaceutical & Medical Devices Agency)
Mr. Masamitsu Honma (Division of Genetics and Mutagenesis, BSRC, NIHS)
Mr. Yasunari Kanda (Division of Pharmacology, BSRC, NIHS)
Mr. Atsushi Kato (National Institute of Infectious Diseases)
Mr. Kouichirou Koike (Ministry of Health, Labour and Welfare)
Ms. Kumiko Ogawa (Division of Pathology, BSRC, NIHS)
Mr. Taku Oohara (Ministry of Health, Labour and Welfare)
Mr. Kazutoshi Shinoda (Pharmaceuticals and Medical Devices Agency)
Mr. Atsuya Takagi (Animal Management Section of the Division of Toxicology, BSRC,
NIHS)
Mr. Masaaki Tsukano (Ministry of Health, Labour and Welfare)
Mr. Shinichi Watanabe (Ministry of Health, Labour and Welfare)Mr. Hajime Kojima (Division of Risk Assessment, BSRC, NIHS): Secretary

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෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝἲRhCEἲ

SkinEthic™ HCE EIT┠  ḟ
ホ౯఍㆟ሗ࿌᭩ ------------------------------------------------------------------------------------ 1
ホ౯ሗ࿌᭩ ------------------------------------------------------------------------------------------ 7
OECD 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 or serious eye damage------------19ix

xホ౯఍㆟ሗ࿌᭩
෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝἲ RhCE ἲ 
SkinEthicTM HCE/S ࢆ⏝࠸ࡓ║่⃭ᛶヨ㦂 SkinEthicTM HCE EIT 

JaCVAM ホ౯఍㆟

ᖹᡂ 30 ᖺ㸦2018 ᖺ㸧2 ᭶ 21 ᪥

1

1

JaCVAM ホ౯఍㆟኱ 㔝 Ὀ 㞝 㸦බ┈㈈ᅋἲே ᮌཎグᛕᶓ὾⏕࿨⛉Ꮫ᣺⯆㈈ᅋ㸧㸸ᗙ㛗㣤 ሯ ᑦ ᩥ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧*஬༑ᔒⰋ᫂ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ ⏕ά⾨⏕໬Ꮫ㒊㸧▼ ஭ 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 28 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 29 ᖺ 3 ᭶ 31 ᪥**㸸ᖹᡂ 29 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 30 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 ࡜ࡋ࡚࣎ࢺ࣒
࢔ࢵࣉ᪉ᘧ࡛ United Nations Globally Harmonized System of Classification and Labelling of Chemicals㸦UN
GHS㸧༊ศእ≀㉁ࢆ᳨ฟ࡛ࡁࡿ RhCE ἲࡢ୍ࡘ࡜ࡋ࡚ࠊEpiOcularTM ࡀ᥇ᢥࡉࢀ࡚࠸ࡿࠋ
ࡇࡢᗘࠊSkinEthicTM
ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝ㸦SkinEthicTM HCE/S㸧ࢆ⏝࠸ࡓ║่⃭ᛶヨ㦂(SkinEthicTM HCE EIT)ࡀ RhCE ἲࡢ୍ࡘ࡜ࡋ࡚ࠊᨵゞ TG 4921)࡟᳨ド῭ࡳᶆ‽ヨ㦂ἲ࡜ࡋ࡚㏣ຍࡉࢀࡓࠋSkinEthicTM HCE/S ࢆ⏝࠸ࡿ RhCE
ἲࡣࠊEURL ECVAM(European Union Reference Laboratory for Alternatives to Animal Testing)࡜
CE(Cosmetics Europe)ࡢඹྠࣂࣜࢹ࣮ࢩࣙࣥ◊✲ 2)ࡀ⾜ࢃࢀࠊࣉࣟࢺࢥࣝࡢ᭱㐺໬ࠊ3 ᪋タࣂࣜࢹ࣮ࢩࣙ
ࣥ◊✲࠾ࡼࡧ ESAC㸦EURL ECVAM Scientific Advisory Committee㸧ࡢ➨୕⪅ホ౯ࢆ⤒࡚ࠊ2017 ᖺ 10 ᭶࡟ᨵゞ TG492 ࡟㏣グࡉࢀࡓࠋJaCVAM ホ౯఍㆟ࡣࠊ║่⃭ᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍࡟ࡼࡾసᡂࡉࢀࡓࠕ෌
ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝἲ(RhCE ἲ)Skin EthicTM HCE/S ࢆ⏝࠸ࡓ║่⃭ᛶヨ㦂(Skin EthicTM HCE EIT)
ホ౯ሗ࿌᭩ࠖ3)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: RhCE
ἲ SkinEthicTM ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝࢆ⏝࠸ࡓ║่⃭ᛶヨ㦂㸧௦᭰ࡍࡿᑐ㇟ẘᛶヨ㦂㸸Draize ║่⃭ᛶヨ㦂ἲヨ㦂ἲࡢᴫ␎㸸 RhCE ἲࡣࠊ෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝࢆ⏝࠸ࡓ║่⃭ᛶヨ㦂࡛࠶ࡿࠋRhCE ἲࡢࡦ࡜ࡘ࡛࠶ࡿ SkinEthicTM HCE EIT ࡛ࡣࠊ⿕㦂≀㉁ࡀᾮయࡢሙྜࡣ⣙ 30 ศ㛫ࠊᅛయࡢሙྜࡣ⣙ 4᫬㛫⿕㦂≀㉁ࢆ SkinEthicTM HCE/S ࡟᭚㟢ࡋࡓᚋࠊMTT ࡢ㑏ඖ㔞ࢆࡶ࡜࡟ࡋࡓ⣽⬊⏕Ꮡ⋡ࢆᐃࡋࠊ║่⃭ᛶホ౯ࡢᣦᶆ࡜ࡋ࡚⏝࠸ࡿࠋࡇࢀࡣࠊMTT ࡀ࣑ࢺࢥࣥࢻࣜ࢔ࡢ⬺Ỉ⣲㓝⣲ࡢᇶ㉁࡜࡞ࡿᛶ㉁ࢆ฼⏝ࡋࠊ⣽⬊ෆ࡟ྲྀࡾ㎸ࡲࢀࡓ MTT ࡀ⬺Ỉ⣲㓝⣲࡟ࡼࡾ㑏ඖࡉࢀࠊ⏕ᡂࡉࢀࡓ࣐࣍ࣝࢨࣥ㔞㸦㟷Ⰽ㸧ࡀ⏕Ꮡ⣽⬊ᩘ࡟ẚ౛ࡍࡿࡇ࡜࡟ᇶ࡙࠸࡚࠸ࡿ㸦MTT 㑏ඖἲ㸧ࠋ⿕㦂≀㉁ࡀᾮయࡢሙྜࠊ⣽⬊⏕Ꮡ⋡ࡀ㝜ᛶᑐ↷࡜ẚ㍑ࡋ࡚ 60㸣ࢆ㉸࠼ࡿ࡜ࠊࡲࡓࠊ⿕㦂≀㉁ࡀᅛయ
ࡢሙྜࡣ⣽⬊⏕Ꮡ⋡ࡀ 50㸣ࢆ㉸࠼ࡿ࡜ UN GHS ศ㢮࡟࠾࠸࡚༊ศእ࡛࠶ࡿ࡜ุᐃࡍࡿࠋࡇࢀ
ࡽ࢝ࢵࢺ࢜ࣇ್௨ୗࡢሙྜࡣࠊᮏヨ㦂ἲ࡛ࡣഇ㝧ᛶࡀ⏕ࡌࡿࡇ࡜ࠊ༊ศ 1 ࡜༊ศ 2 ࢆ㆑ู࡛ࡁ
࡞࠸ࡇ࡜࠿ࡽࠊ௚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࡟㢮ఝࡋࡓᵓ㐀ࢆ᭷ࡍࡿ RhCE㸦SkinEthicTM HCE/S㸧ࢆ⏝࠸࡚ࠊ⿕㦂≀㉁ࡢ⣽⬊ẘᛶࢆᣦᶆ࡜ࡋ࡚║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛࠶ࡿࠋࡇࢀࡽࡢࡇ࡜࠿ࡽࠊᮏヨ㦂ἲࡣ࢘ࢧࢠࢆ⏝࠸ࡿ║่⃭ᛶヨ㦂ࡢ௦᭰ἲ࡜ࡋ
࡚⛉Ꮫⓗጇᙜᛶࡀ࠶ࡿࠋ

33

SkinEthicTM HCE EIT ࡟ࡘ࠸࡚ࡣࠊEURL ECVAM ࡜ CE ࡢඹྠࣂࣜࢹ࣮ࢩࣙࣥ◊✲ 2)ࡀ⾜ࢃࢀࡓࠋࡑ
ࡢᚋࣉࣟࢺࢥࣝࡢ᭱㐺໬ࢆ⾜ࡗ࡚ࠊUN GHS ༊ศ≀㉁࣭༊ศእ≀㉁ࡸᅛయ࣭ᾮయࡢࣂࣛࣥࢫࢆ⪃៖ࡋ࡚
120 ≀㉁࡟ࡘ࠸࡚ 3 ᪋タ࡛ࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀ⾜ࢃࢀࡓࠋ⿕㦂≀㉁࡟ࡣࠊMTT 㑏ඖ≀㉁ࡸ࣐࣍ࣝࢨࣥ
࡜ྠࡌࡼ࠺࡞Ἴ㛗ࢆࡶࡘ╔Ⰽ≀㉁ࡶྵࡵࡽࢀࡓࠋࡲࡓࠊࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡜ࡣู࡟ࠊ࣮ࣜࢻࣛ࣎࡟࠾
࠸࡚ 80 ≀㉁࡟ࡘ࠸࡚㏣ຍヨ㦂ࡀ⾜ࢃࢀࡓ 4,5)ࠋ
᪋タෆ෌⌧ᛶࠊ᪋タ㛫෌⌧ᛶ࠾ࡼࡧヨ㦂ἲࡢṇ☜ᛶ࡟ࡘ࠸࡚ࡶ EURL ECVAM
SkinEthicTM HCE EIT ࡣࠊࣂࣜࢹ࣮ࢩࣙࣥ㐠Ⴀጤဨ఍ࡢᐃࡵࡓᇶ‽ࢆ‶ࡓࡋࡓࠋࡉࡽ࡟ ESAC ࡢ➨୕⪅ホ౯ 6)ࢆ⤒࡚ࠊUN GHS ༊ศእ≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚ 2017 ᖺ 10 ᭶࡟ᨵゞ TG492 ࡟㏣グࡉࢀࡓࠋJaCVAM ║่⃭ᛶヨ㦂㈨ᩱ
⦅⧩ጤဨ఍ࡣࠊࡇࢀࡽࡢ㈨ᩱࢆ⏝࠸࡚ᮏヨ㦂ἲࢆホ౯ࡋ࡚࠾ࡾࠊ⛉Ꮫⓗ࡟ጇᙜ࡞ホ౯࡛࠶ࡿ࡜⪃࠼ࡿࠋ㸱㸬ᮏヨ㦂ἲࡢ᭷⏝ᛶ࡜㐺⏝㝈⏺
RhCE ἲ࡟⏝࠸ࡿ SkinEthicTM HCE/S ࡣᕷ㈍ࡉࢀ࡚࠾ࡾࠊࡇࢀ௨እࡣ≉Ṧ࡞ᶵᮦࡸヨ⸆ࢆᚲせ࡜ࡏࡎࠊᡭᢏࡶ」㞧࡛ࡣ࡞࠸ࡇ࡜࠿ࡽᢏ⾡⛣㌿ᛶࡣ㧗࠸࡜ุ᩿࡛ࡁࡿࠋణࡋࠊධᡭࡋࡓ SkinEthicTM HCE/S ࡀရ㉁ᇶ‽ࡢチᐜ⠊ᅖ࡟࠶ࡾࠊ࠿ࡘᐇ᪋ࡍࡿヨ㦂᪋タࡢᢏ⾡⩦ᚓࡀ࢞࢖ࢻࣛ࢖ࣥࡢ⇍㐩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◊✲࡟࠾࠸࡚ࠊྛ᪋タࡢ᪋タෆ෌⌧ᛶࡣࠊᾮయ⏝ࣉࣟࢺࢥ࡛ࣝ 88.3-95.0㸣ࠊᅛయ⏝ࣉࣟࢺࢥ࡛ࣝ 95.0-96.7㸣࡛࠶ࡾࠊEURL ECVAM ࣂࣜࢹ࣮ࢩࣙࣥ㐠Ⴀጤဨ఍ࡀᐃࡵࡓᇶ‽㸦85㸣௨ୖ㸧ࢆ‶ࡓࡋ࡚࠸ࡓ㸰)ࠋࡲࡓࠊ᪋タ㛫෌⌧ᛶ࡟㛵ࡋ࡚ࡣࠊᮏヨ㦂࡛ᚓࡽࢀࡓ⣽⬊⏕Ꮡ⋡㸦」ᩘᅇヨ㦂㸧ࡢᖹᆒ್ࢆࡶ࡜࡟ UN GHS ศ㢮ุᐃࢆ᪋タࡈ࡜࡟⾜࠸ࠊᾮయ⏝ࣉࣟࢺࢥ࡛ࣝ 93.3㸣ࠊᅛయ⏝ࣉࣟࢺࢥ࡛ࣝ 96.7㸣ࠊ඲య࡛ࡣ 95.0㸣࡜࡞ࡾࠊࣂࣜࢹ࣮ࢩࣙࣥ㐠Ⴀጤဨ఍ࡀᐃࡵࡓᇶ‽㸦80㸣௨ୖ㸧ࢆ‶ࡓࡋ࡚࠸ࡓ 3)ࠋࡉࡽ࡟ࠊࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡛ᚓࡽࢀࡓࢹ࣮ࢱ࡟ࡼࡿṇ☜ᛶࡢホ౯࡛ࡣࠊᾮయࣉࣟࢺࢥ࡛ࣝឤᗘ 98.3㸣ࠊ≉␗ᗘ 69.4㸣࠾ࡼࡧṇ☜ᗘ 84.8㸣ࠊᅛయ⏝ࣉࣟࢺࢥࣝ
࡛ឤᗘ 92.2㸣ࠊ≉␗ᗘ 76.6㸣࠾ࡼࡧṇ☜ᗘ 84.4㸣࡜࡞ࡾࣂࣜࢹ࣮ࢩࣙࣥ㐠Ⴀጤဨ఍ࡀᐃࡵࡓᇶ‽㸦ឤᗘ
90㸣௨ୖࠊ≉␗ᗘ 60㸣௨ୖࠊṇ☜ᗘ 75㸣௨ୖ㸧ࢆ‶ࡓࡋ࡚࠸ࡓ 3)ࠋ

4

4

⾲㸬SkinEthicTM HCE EIT ࡢṇ☜ᛶ

≀㉁ᩘ

ឤᗘ(%)

≉␗ᗘ(%)

ṇ☜ᗘ(%)

ᾮయ

60

98.3

69.4

84.8

ᅛయ

60

92.2

76.6

84.4

௦᭰ἲ࡜ࡋ࡚ࡢ
ཷࡅධࢀᇶ‽ 1)

-

 90%

60%

75%

║่⃭ᛶ௦᭰ἲ㈨ᩱ⦅⧩ጤဨ఍ࡢሗ࿌᭩㸦⾲㸯㸧ࡼࡾᘬ⏝ 3)ణࡋࠊ⣽⬊⏕Ꮡ⋡ࡢ⟬ฟ࡟ MTT 㑏ඖἲࢆ⏝࠸ࡿ௚ࡢ௦᭰ἲ࡜ྠᵝ࡟ࠊ⿕㦂≀㉁ࡀ MTT ࢆ㑏ඖࡍࡿ≀㉁ࡢሙྜࠊ࠶ࡿ࠸ࡣ࣐࣍ࣝࢨࣥ࡜ྠࡌࡼ࠺࡞Ἴ㛗㸦570 nm ㏆㎶㸧࡟྾཰ࢆᣢࡘ╔Ⰽ≀㉁ࡢሙྜ࡟ࡣࠊ྾
ගᗘ⿵ṇࢆ⾜࠺ᚲせࡀ࠶ࡿࠋࡑࡢᡭ㡰࡟ࡘ࠸࡚ࡣࠊᨵゞ TG492 ࡢᮏᩥࡢㄝ᫂࠾ࡼࡧ ANNEX IV ࡢヨ㦂ἲࢆཧ↷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࢔ࢰ࣮ࣝࡣ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≀㉁ࢆ᳨
ฟࡍࡿ᪉ἲ࡜ࡋ࡚᭷⏝࡛࠶ࡿ࡜⪃࠼ࡿࠋ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࠸ࡿࠋࡲࡓࠊSkinEthicTM HCE/S ࡢධᡭࡣᐜ࡛᫆ࠊ
▷᫬㛫࡛Ᏻ౯࡟ᐇ᪋࡛ࡁࠊ≉Ṧ࡞ᶵᮦࡸヨ⸆ࢆᚲせ࡜ࡏࡎࠊᚲせ࡞ᡭᢏࡶ」㞧࡞ࡶࡢ࡛࡞࠸ࠋࡋࡓࡀࡗ
࡚ࠊධᡭࡋࡓ SkinEthicTM HCE/S ࡀရ㉁ᇶ‽ࡢチᐜ⠊ᅖ࡟࠶ࡾࠊ࠿ࡘᐇ᪋ࡍࡿヨ㦂᪋タࡢᢏ⾡⩦ᚓࡀ࢞࢖ࢻࣛ࢖ࣥࡢ⇍㐩ᗘ☜ㄆ≀㉁࡛☜࠿ࡵࡽࢀ࡚࠸ࢀࡤࠊᇶᮏⓗ࡞⣽⬊ᇵ㣴ࡢᢏ⾡࡜タഛࢆ᭷ࡍࡿ᪋タ࡛࠶ࢀࡤᐇ᪋ྍ⬟࡛࠶ࡾࠊᢏ⾡⛣㌿ᛶࡣ㧗࠸ࠋ௨ୖࡼࡾࠊᮏヨ㦂ἲࡢ♫఍ⓗཷࡅධࢀᛶࡣ㧗࠸ࠋ55⾜ᨻୖࡢ฼⏝ᛶ㸸ᮏヨ㦂ἲࡣࠊ໬Ꮫ≀㉁࡟ࡼࡿ║่⃭ᛶࢆホ౯࡛ࡁࠊ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾࠸࡚ UN GHS ༊ศእ≀㉁ࢆ
᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚ࠊ⾜ᨻⓗ฼⏝ࡀྍ⬟࡛࠶ࡿ࡜⪃࠼ࡿࠋ
࡞࠾ࠊRhCE ἲ࡟ EpiOcularTM ࡲࡓࡣ SkinEthicTM HCE/S ௨እࢆ⏝࠸ࡿሙྜ࡟ࡣࠊOECD TG492 ࡢᛶ⬟ᶆ‽࡟グ㍕ࡉࢀࡓ≀㉁ࢆ⏝࠸࡚ࠊࡑࡢጇᙜᛶࢆ☜ㄆࡋ࡚࠾ࡃᚲせࡀ࠶ࡿ 1)ࠋ

ཧ⪃ᩥ⊩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) EURL ECVAM-CE (2014) Prospective validation study of Reconstructed Human Tissue-based test methods
for identifying chemical not requiring classification for serious eye damage/eye irritation – Validation Study
Report.
3) JaCVAM ║่⃭ᛶ௦᭰ἲ㈨ᩱ⦅⧩ጤဨ఍㸸ホ౯ሗ࿌᭩ ෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝἲ(RhCEἲ)SkinEthicTM HCE/Sࢆ⏝࠸ࡓ║่⃭ᛶヨ㦂 (SkinEthicTM HCE EIT)㸦ᖹᡂ29ᖺ 2017ᖺ 12᭶4᪥㸧4) Alépée N., et al. (2016) Multi-laboratory validation of SkinEthic HCE test method for testing serious eye
damage/eye irritation using liquid chemicals. Toxicol In Vitro 31, 43-53.
5) Alépée N., et al. (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 solidand liquid chemicals testing. Toxicol In Vitro 34, 55-70.
6) ESAC (2016) ESAC Opinion on the SkinEthicTM Human Corneal Epithelium (HCE) Eye Irritation Test (EIT).
ESAC Opinion No. 2016-02 of 24 June 2016.

66

ホ౯ሗ࿌᭩
෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝἲ RhCE ἲ 
SkinEthicTM HCE/S ࢆ⏝࠸ࡓ║่⃭ᛶヨ㦂 SkinEthicTM HCE EIT ║่⃭ᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍

ᖹᡂ 29 ᖺ㸦2017 ᖺ㸧12 ᭶ 4 ᪥17║่⃭ᛶ௦᭰ἲ㈨ᩱ⦅⧩ጤဨ఍ᒣᮏ ┤ᶞ

㸦⸨⏣ಖ೺⾨⏕኱Ꮫ㸧బࠎᮌ ṇ἞㸦࢔ࢵࣦ࢕ྜྠ఍♫㸧➉ෆ ᑠⱑ

㸦P&G ࢖ࣀ࣮࣋ࢩࣙࣥྜྠ఍♫㸧Ἴከ㔝 ᾈኴ

㸦࣮࣮࣍ࣘᰴᘧ఍♫㸧᳃ᮧ ᬛ⨾㸦୍⯡㈈ᅋἲே㣗ရ⸆ရᏳ඲ࢭࣥࢱ࣮㸧ᑠᓥ ⫕㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧

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␎ㄒ
CAS: Chemical Abstracts Services
CE: Cosmetics Europe
EIT: Eye Irritation Test
EURL ECVAM: the European Union Reference Laboratory for Alternatives to Animal Testing
ESAC: ECVAM Scientific Advisory Committee
GHS: Globally Harmonized System of Classification and Labeling of Chemicals
HCE: Human Corneal Epithelium
JaCVAM: Japanese Center for the Validation of Alternative Methods
MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
OD: optical density
OECD: Organization for Economic Co-operation and Development
SDS: sodium dodecyl sulphate
SOP: Standard Operating Procedures
TG: Test Guideline
UN: United Nations
VRM: Validated Reference Method

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せ᪨
෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝἲ㸦Reconstructed human Cornea-like Epithelium Test Method㸸RhCEἲ㸧ࡣࠊ໬Ꮫ≀㉁ࡢ RhCE ⤌⧊࡟ᑐࡍࡿ⣽⬊ẘᛶࢆᣦᶆ࡟⏝࠸ࠊࡑࡢ≀㉁ࡢ║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛࠶ࡾࠊOECD TG 492 ࡜ࡋ࡚࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡛ UN GHS ༊ศእ≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚᥇
ᢥࡉࢀ࡚࠸ࡿࠋSkinEthicTM ࣄࢺゅ⭷ୖ⓶ࣔࢹࣝࢆ⏝࠸ࡓ║่⃭ᛶヨ㦂㸦SkinEthicTM Human Corneal
Eithelium Eye Irritation Test: SkinEthicTM HCE EIT㸧ࡣ RhCE ἲࡢ୍ࡘ࡛ࠊᨵゞ TG 492 ࡟㏣ຍࡉࢀࡓ᳨ド῭ࡳᶆ‽ヨ㦂ἲ࡛࠶ࡿࠋᮏሗ࿌࡛ࡣࠊSkinEthicTM HCE/S EIT ࡢࣂࣜࢹ࣮ࢩࣙࣥ◊✲ㄽᩥࠊ➨୕⪅ホ౯ሗ࿌᭩ࠊ㛵㐃ㄽᩥ࡞࡝ࢆࡶ࡜࡟ヨ㦂ἲࡢᴫせࢆㄝ᫂ࡋ JaCVAM ║่⃭ᛶ௦᭰ἲ㈨ᩱ⦅⧩ጤဨ఍ࡢពぢࢆࡲ࡜ࡵࡓࠋ
 SkinEthicTM HCE EIT ࡢಙ㢗ᛶ࣭ṇ☜ᛶࢆ☜ㄆࡍࡿࡓࡵࠊᾮయ≀㉁࠾ࡼࡧᅛయ≀㉁ࡑࢀࡒࢀ 60 ≀㉁ࢆ⏝࠸ࡓ 3 ᪋タ࡟ࡼࡿࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀ⾜ࢃࢀࡓࠋྛ᪋タ࡛ 1 ≀㉁࠶ࡓࡾ 3 ᅇࡢヨ㦂ࢆ⾜ࡗࡓࠋࡇࡢ 3 ᪋タࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡟࠾ࡅࡿ SkinEthicTM HCE/S EIT ࡢṇ☜ᛶࡣࠊᾮయ≀㉁࠾ࡼࡧᅛ
య≀㉁࡟࠾࠸࡚ࠊࡑࢀࡒࢀឤᗘ 98.3%࠾ࡼࡧ 92.2%ࠊ≉␗ᗘ 69.4%࠾ࡼࡧ 76.6%ࠊṇ☜ᗘ 84.8%࠾ࡼ
ࡧ 84.4%࡛࠶ࡗࡓࠋᢏ⾡⛣㌿ᛶ࡟㛵ࡋ࡚ᠱᛕࡉࢀࡿ⤖ᯝࡣᚓࡽࢀ࡚࠾ࡽࡎࠊ᪋タෆ෌⌧ᛶ࠾ࡼࡧ᪋タ㛫෌⌧ᛶࡣ࡝ࡕࡽࡶ 85%௨ୖ࡛࠶ࡗࡓࠋࡇࢀࡽࡢṇ☜ᛶ࠾ࡼࡧ෌⌧ᛶࡢ್ࡣࠊࣂࣜࢹ࣮ࢩࣙࣥ㐠Ⴀጤဨ఍ࡀᐃࡵࡓᇶ‽ࢆ‶ࡓࡋ࡚࠸ࡓࠋ ᮏጤဨ఍ࡣࠊSkinEthicTM HCE/S EIT ࡣ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡛ UN GHS ༊ศእ≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜
ࡋ࡚⏝࠸ࡿࡇ࡜ࡀ࡛ࡁࡿ࡜⤖ㄽࡋࡓࠋ

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1. ࡲ࠼࠾ࡁ
໬Ꮫ≀㉁ࡢ║่⃭ᛶࢆホ౯ࡍࡿ᪉ἲ࡜ࡋ࡚ᚑ᮶౑⏝ࡉࢀ࡚ࡁࡓࡢࡣࠊDraize ἲ࡜࠸࠺࢘ࢧࢠࢆ⏝࠸ࡓື≀ヨ㦂࡛࠶ࡿࠋࡋ࠿ࡋࠊ㏆ᖺࡢື≀⚟♴࡟ᑐࡍࡿ㛵ᚰࡢ㧗ࡲࡾࡸḢᕞ࡟࠾ࡅࡿἲつไᨵṇࡣࠊࡑࡢ௦᭰ἲࡢ㛤Ⓨ࣭ࣂࣜࢹ࣮ࢩࣙࣥࢆಁ㐍ࡋࡓࠋOECD ࡀࢸࢫࢺ࢞࢖ࢻࣛ࢖ࣥ࡜ࡋ࡚ࡍ࡛࡟᥇ᢥࡋࡓ௦
᭰ἲࡣࠊ࢘ࢩ᦬ฟゅ⭷ࡢΰ⃮࠾ࡼࡧ㏱㐣ᛶヨ㦂ἲ㸦BCOP ἲࠊTG 437㸧ࠊࢽ࣡ࢺࣜ᦬ฟ║⌫ࢆ⏝࠸ࡓ║่⃭ᛶヨ㦂ἲ㸦ICE ἲࠊTG 438㸧ࠊࣇࣝ࢜ࣞࢭ࢖ࣥ₃ฟヨ㦂ἲ㸦FL ἲࠊTG 460㸧ࠊin vitro ▷᫬㛫᭚㟢ἲ㸦STE ἲࠊTG 491㸧ࠊࡑࡋ࡚ࠊ෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝἲ㸦RhCE ἲࠊTG 492㸧ࡢ 5 ヨ㦂ἲ࡛࠶ࡿࠋBCOP ἲ࡜ ICE ἲࡣ㣗⏝࡞࡝ࡢ┠ⓗ࡛ฎศࡉࢀࡓື≀ࡼࡾ᦬ฟࡋࡓჾᐁ࣭⤌⧊ࢆ⏝࠸࡚໬Ꮫ≀㉁ࡢ᭚㟢࡟ࡼࡾ⏕ࡌࡿゅ⭷ࡢ≀⌮ⓗ≉ᛶࡢኚ໬ࢆᣦᶆ࡟║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛ࠊࢺࢵࣉࢲ࢘ࣥ᪉
ᘧ࡟࠾࠸࡚ UN GHS ༊ศ 1㸦㔜⠜࡞║ࡢയᐖࢆ㉳ࡇࡍ㸧≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚ࠊࡲࡓ࣎ࢺ࣒࢔ࢵ
ࣉ᪉ᘧ࡟࠾࠸࡚ UN GHS ༊ศእ㸦║࡟ᑐࡍࡿ㔜⠜࡞ᦆയᛶ࠾ࡼࡧ║่⃭ᛶࢆ᭷ࡍࡿ≀㉁࡜ࡣศ㢮ࡉࢀ࡞࠸㸧≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚⏝࠸ࡽࢀࡿࠋFL ἲࡣ༢ᒙᇵ㣴ࡋࡓᒀ⣽⟶ୖ⓶⣽⬊ࢆ⏝࠸࡚໬Ꮫ≀㉁ࡢ᭚㟢࡟ࡼࡾ⏕ࡌࡿ⣽⬊㛫⤖ྜࡢയᐖࢆᣦᶆ࡟║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛ࠊࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡟
࠾࠸࡚ࡢࡳ⏝࠸ࡽࢀࡿࠋSTE ἲࡣ༢ᒙᇵ㣴ࡋࡓゅ⭷⣽⬊ࢆ⏝࠸࡚≀㉁ࡢ᭚㟢࡟ࡼࡿ⣽⬊ẘᛶ㸦⣽⬊⏕Ꮡ⋡㸧ࢆᣦᶆ࡟║่⃭ᛶࢆホ౯ࡍࡿ᪉ἲ࡛ࠊࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࠾ࡼࡧ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾࠸࡚⏝࠸
ࡽࢀࡿࠋ
RhCE ἲࡣࠊ෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝ㸦RhCE㸧⤌⧊࡟ᑐࡍࡿ⿕㦂≀㉁ࡢ⣽⬊ẘᛶࢆᣦᶆ࡜ࡋࠊ
࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟ࡼࡾ UN GHS ༊ศእ≀㉁ࢆ᳨ฟࡍࡿࡇ࡜࡛ࡑࡢ≀㉁ࡢ║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛࠶ࡿࠋRhCE ἲ࡛࠶ࡿ EpiOcularTM ║่⃭ᛶヨ㦂㸦EpiOcularTM Eye Irritation Test: EpiOcularTM EIT㸧࠾ࡼࡧ SkinEthicTM ࣄࢺゅ⭷ୖ⓶ࣔࢹࣝ║่⃭ᛶヨ㦂㸦SkinEthicTM HCE EIT㸧ࡢ 2 ヨ㦂ἲ࡟ࡘ࠸࡚ EURLECVAM ࡜ CE ࡢඹྠࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀ⾜ࢃࢀࡓࠋEpiOcularTM EIT ࡣࠊࣉࣟࢺࢥࣝࡢ᭱㐺໬ࠊ㏣
ຍヨ㦂ࠊESAC ࡢ➨୕⪅ホ౯ࢆ⤒࡚ࠊRhCE ἲࡢ᳨ド῭ࡳᶆ‽ヨ㦂ἲ㸦Validated Reference Method: VRM㸧࡜ࡋ࡚ 2015 ᖺ 7 ᭶࡟ OECD ࡟ࡼࡾࢸࢫࢺ࢞࢖ࢻࣛ࢖ࣥ࡟᥇ᢥࡉࢀࡓ㸦TG 492㸧ࠋࡑࡢᚋࠊSkinEthicTM
HCE EIT ࡶࣉࣟࢺࢥࣝࡢ᭱㐺໬ࠊ3 ᪋タࣂࣜࢹ࣮ࢩࣙࣥ◊✲࠾ࡼࡧ ESAC ࡢ➨୕⪅ホ౯ࢆ⤒࡚ࠊ2 ࡘ┠ࡢ VRM㸦VMR2㸧࡜ࡋ࡚ 2017 ᖺ 10 ᭶࡟ᨵゞ TG 492 ࡟㏣ຍࡉࢀࡓࠋ
ᮏሗ࿌᭩ࡣᨵゞ TG 492 ࡢ VRM2 ࡛࠶ࡿ SkinEthicTM HCE EIT ࡢࣂࣜࢹ࣮ࢩࣙࣥ◊✲ㄽᩥࠊ➨୕⪅ホ౯ሗ࿌᭩ࠊࡑࡢ௚㛵㐃ㄽᩥ࡞࡝ࢆࡶ࡜࡟ᮏヨ㦂ἲࡢᴫせࢆㄝ᫂ࡋࠊᮏጤဨ఍ࡢពぢࢆࡲ࡜ࡵࡓࡶࡢ
࡛࠶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⭷ࡢึᮇയᐖࡢ
῝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
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ᨵゞ TG 492 ࡛㏣ຍࡉࢀࡓ VRM ࡣࠊᕷ㈍ࡢࣄࢺ୙Ṛ໬ゅ⭷ୖ⓶⣽⬊⏤᮶ࡢ RhCE ⤌⧊࡛࠶ࡿ
SkinEthicTM HCE/S ࢆ⏝࠸ࡓ║่⃭ᛶヨ㦂㸦SkinEthicTM HCE EIT㸧࡛࠶ࡿࠋ⿕㦂≀㉁ࡀᾮయࡢሙྜࡣ⣙30 ศ㛫ࠊᅛయࡢሙྜࡣ⣙ 4 ᫬㛫ࠊSkinEthicTM HCE/S ࡟᭚㟢ࡋࡓᚋࠊMTT ヨ㦂ࡼࡾ⟬ฟࡋࡓ⣽⬊⏕Ꮡ⋡ࢆ࢚ࣥࢻ࣏࢖ࣥࢺ࡟⏝࠸ࡿࠋࡇࢀࡣࠊ⣽⬊ෆ࡟ྲྀࡾ㎸ࡲࢀࡓ MTT ࡢ㑏ඖ࡟ࡼࡾ⏕ᡂࡉࢀࡓ࣐࣍ࣝ
ࢨࣥ㔞㸦㟷Ⰽ㸧ࡀ⏕Ꮡ⣽⬊ᩘ࡟ẚ౛ࡍࡿࡇ࡜ࢆᇶᮏཎ⌮࡜ࡋ࡚࠸ࡿࠋ⿕㦂≀㉁ࡀᾮయࡢሙྜࠊ⣽⬊⏕Ꮡ⋡ࡀ㝜ᛶᑐ↷࡜ẚ㍑ࡋ࡚ 60%ࢆ㉸࠼ࡿ࡜ࠊ⿕㦂≀㉁ࡣ UN GHS ศ㢮࡟࠾࠸࡚༊ศእ࡛࠶ࡿ࡜ุᐃ
ࡍࡿࠋ⿕㦂≀㉁ࡀᅛయࡢሙྜࡣ⣽⬊⏕Ꮡ⋡ࡀ 50%ࢆ㉸࠼ࡿ࡜ UN GHS ศ㢮࡟࠾࠸࡚༊ศእ࡛࠶ࡿ࡜
ุᐃࡍࡿࠋ
4㸬ヨ㦂ᡭ㡰
 SkinEthicTM HCE EIT ࡢᡭ㡰ࢆ௨ୗ࡟♧ࡍࠋヲ⣽ࡣࠊᨵゞ TG 492 ࢆཧ↷ࡍࡿࠋSkinEthicTM HCE/S ࡣ
EPISKIN ♫㸦ࣇࣛࣥࢫ㸧ࡼࡾ㉎ධ࡛ࡁࡿࠋ
4-1. RhCE ⤌⧊ࡢᶵ⬟ⓗ᮲௳ RhCE ⤌⧊ࡢᶵ⬟ⓗ᮲௳ࡣࠊ௨ୗࡢ࡜࠾ࡾ࡛࠶ࡿࠋ
⏕Ꮡ⋡㸸㝜ᛶᑐ↷࡟࠾࠸࡚ 1.0 < OD  2.5㸦౑⏝᫬ࡢᇶ‽࡛࠶ࡾࠊ〇㐀⪅ࡢฟⲴᇶ‽ࡣ␗࡞ࡿሙྜࡶ࠶ࡿ㸧ࣂࣜ࢔ᶵ⬟㸸SDS ࡢ 30 ศ㛫᭚㟢࡟࠾࠸࡚ 1.0  IC50 (mg/mL)  3.2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⤖ᯝࢆ⫼ᬒࢹ࣮ࢱࢆࡶ࡜࡟タᐃࡋࡓチᐜ್ෆ࡟࠶ࡿ
ࡇࢀࡽࡣ SkinEthicTM HCE/S ࡢ〇㐀⪅ࡢฟⲴᇶ‽࡜ࡋ࡚᥇⏝ࡉࢀࡿࠋ୍᪉ࠊSkinEthicTM HCE/S ࡢ౑⏝⪅ࡣ⏕Ꮡ⋡࡜෌⌧ᛶࢆ☜ㄆࡍࡿᚲせࡀ࠶ࡿࠋ
4-2. ⿕㦂≀㉁ࡢ㐺⏝ ≀㉁࠶ࡓࡾᑡ࡞ࡃ࡜ࡶ 2 RhCE ⤌⧊ࢆ⏝࠸ࡿࠋ⿕㦂≀㉁ࡣホ౯ᑐ㇟≀㉁ࡑࡢࡶࡢࢆ⏝࠸ࡿࠋ37ÛC ௨ୗ࡛ࣆ࣌ࢵࢺ࡛ᢅ࠼ࡿࡶࡢࡣᾮయ࡜ࡋ࡚ࠊࡑࢀ௨እࡣᅛయ࡜ࡋ࡚ヨ㦂ࢆ⾜࠺ࠋᾮయ⿕㦂≀㉁ࡢሙྜࠊ
SkinEthicTM HCE/S ࡟ 30s2 μL ࢆ㐺⏝ࡋࠊᶆ‽ᇵ㣴᮲௳1࡛ 30s2 ศ㛫ᇵ㣴ࡍࡿࠋࡑࡢᚋࠊࣜࣥ㓟⦆⾪⏕⌮㣗ሷỈ 20 mL ࡛Ὑίࡋ࡚⿕㦂≀㉁ࢆ㝖ཤࡋࠊ30s2 ศ㛫ᶆ‽ᇵ㣴᮲௳࡛ᇵᆅ࡟ᾐₕࡍࡿࠋᅛయ⿕㦂≀㉁ࡢሙྜࠊ30s2 mg ࢆᆒ୍࡟㐺⏝ࡋࠊᶆ‽ᇵ㣴᮲௳࡛ 4s0.1 ᫬㛫ᇵ㣴ࡍࡿࠋࡑࡢᚋࠊࣜࣥ㓟⦆⾪⏕⌮㣗ሷỈ 25 mL ࡛Ὑίࡋ࡚⿕㦂≀㉁ࢆ㝖ཤࡋࠊ30s2 ศ㛫ᐊ࡛ᇵᆅ࡟ᾐₕᚋࠊ᪂ࡓ࡞ᇵᆅࢆ⏝࠸࡚ᶆ‽ᇵ㣴᮲௳࡛ 18s0.5 ᫬㛫ᇵ㣴ࡍࡿࠋ ྠ᫬㝧ᛶᑐ↷࡟ࡣ㓑㓟࣓ࢳࣝ㸦CAS No. 79-20-9㸧ࠊྠ᫬㝜ᛶᑐ↷࡟ࡣࣜࣥ㓟⦆⾪⏕⌮㣗ሷỈࡀ᥎ዡࡉࢀࡿࠋᑐ↷≀㉁ࡢ㐺⏝࣭ᚋฎ⌮ࡣᑐ↷࡜࡞ࡿ⿕㦂≀㉁㸦ᾮయࡲࡓࡣᅛయ㸧ࡢ᮲௳࡟‽ࡌࡿࠋ
4-3. ⣽⬊⏕Ꮡ⋡ࡢ⟬ฟ⣽⬊⏕Ꮡ⋡⟬ฟ࡟ࡣ MTT 㑏ඖἲࢆ⏝࠸ࡿࠋᇵᆅࢆ㝖ཤࡋࠊ1 mg/mL MTT ⁐ᾮ 0.3 mL ୰࡛ 180s151

37s2ÛCࠊ5s1% CO2ࠊ95% ‵ᗘ

612ศ㛫ᶆ‽ᇵ㣴᮲௳࡛཯ᛂࡉࡏࠊࡑࡢᚋ 1.5 mL ࢖ࢯࣉࣟࣃࣀ࣮ࣝ㸦ࡲࡓࡣྠᵝ࡞⁐፹㸧࡛㟷Ⰽࡢ࣐࣍ࣝࢨࣥࢆᢳฟࡍࡿࠋᾮయ⿕㦂≀㉁ࡢሙྜࡣࠊRhCE ⤌⧊ࡢୖ㒊࣭ᗏ㒊୧᪉࠿ࡽᢳฟࡍࡿࠋᅛయ⿕㦂≀㉁࠾ࡼࡧ↓Ⰽ㏱࡛᫂࡞࠸ᾮయ⿕㦂≀㉁ࡢሙྜࡣࠊ⤌⧊࡟ṧᏑࡍࡿ⿕㦂≀㉁ࡢΰධࢆ᭱ᑠ㝈࡟ᢚ࠼ࡿࡓ
ࡵࠊ࣐࣍ࣝࢨࣥࡢᢳฟࡣ RhCE ⤌⧊ࡢᗏ㒊ࡢࡳ࠿ࡽ⾜࠺ࠋᾮయ⿕㦂≀㉁࡛ࡶࠊὙίࡀᅔ㞴࡞ሙྜ࡟ࡣࠊ
ᗏ㒊ࡢࡳ࠿ࡽᢳฟࢆ⾜࠺ࠋྠ᫬ᑐ↷≀㉁࡟ᑐࡋ࡚ࡢᢳฟ᪉ἲࡣ⿕㦂≀㉁࡜ྠᵝ࡟⾜࠺ࠋᢳฟࡋࡓ࣍ࣝ
࣐ࢨࣥࡢᐃ㔞ࡣ OD570nm

ᐃࡲࡓࡣ HPLC/UPLC ࡛⾜࠺ࠋ⿕㦂≀㉁ࡀ MTT 㑏ඖ≀㉁ࡢሙྜࠊ࠶ࡿ࠸ࡣ࣐࣍ࣝࢨࣥ࡜ྠࡌࡼ࠺࡞Ἴ㛗㸦570 nm ㏆㎶㸧࡟྾཰ࢆᣢࡘ╔Ⰽ≀㉁ࡢሙྜࠊ⣽⬊⏕Ꮡ⋡ࡢ⿵ṇࢆ⾜࠺ᚲせࡀ࠶ࡿࠋࡑࡢᡭ㡰࡟ࡘ࠸࡚ࡣ TG ᮏᩥࡢㄝ᫂࠾ࡼ
ࡧ ANNEX IV ࡢࣇ࣮ࣟࢳ࣮ࣕࢺࢆཧ↷ࡍࡿࠋ
4-4. ヨ㦂ᡂ❧ࡢᢎㄆᇶ‽௨ୗࡢ᮲௳ࢆࡍ࡭࡚‶ࡓࡋࡓሙྜࠊヨ㦂ࡢᡂ❧ࢆᢎㄆࡍࡿࠋ
1㸧㝜ᛶᑐ↷ࡢᖹᆒ OD ࡀ 1.0 < OD  2.5 ࡛࠶ࡿࡇ࡜ࠋ
2㸧㝧ᛶᑐ↷ࡢᖹᆒ⣽⬊⏕Ꮡ⋡ࡀᾮయ⿕㦂≀㉁࡛ࡣ 30%௨ୗࠊᅛయ⿕㦂≀㉁࡛ࡣ 20%௨ୗ࡛࠶ࡿࡇ
࡜ࠋ
3㸧⿕㦂≀㉁࠾ࡼࡧ㝜ᛶ࣭㝧ᛶᑐ↷ࡢࡑࢀࡒࢀ࡟࠾࠸࡚ 2 RhCE ⤌⧊ࡢ⣽⬊⏕Ꮡ⋡ࡢᕪࡀ 20%ᮍ‶࡛࠶ࡿࡇ࡜ࠋ3 RhCE ⤌⧊௨ୖࢆ⏝࠸ࡓሙྜࡣ⣽⬊⏕Ꮡ⋡ࡢᶆ‽೫ᕪࡀ 18%௨ୗ࡛࠶ࡿࡇ࡜ࠋ
࡞࠾ࠊ」ᩘࡢ⿕㦂≀㉁ࢆྠ᫬࡟ヨ㦂ࡋࡓሙྜࡣࠊࡇࡢ᮲௳ࢆ‶ࡓࡉ࡞࠸⿕㦂≀㉁ࡢࡳ୙ᡂ❧࡜ࡍࡿࠋ
4-5. ่⃭ᛶࡢุᐃᾮయ⿕㦂≀㉁࡛ࡣᖹᆒ⣽⬊⏕Ꮡ⋡ࡀ 60%ࢆ㉸࠼ࡓሙྜࠊ⿕㦂≀㉁ࡣ GHS ༊ศእ࡜ุ᩿ࡉࢀࡿࠋᅛ
య⿕㦂≀㉁࡛ࡣᖹᆒ⣽⬊⏕Ꮡ⋡ࡀ 50%ࢆ㉸࠼ࡓሙྜࠊ⿕㦂≀㉁ࡣ GHS ༊ศእ࡜ุ᩿ࡉࢀࡿࠋࡇࢀࡽ
ࡢ࢝ࢵࢺ࢜ࣇ್௨ୗࡢሙྜࡣࠊᮏヨ㦂ἲ࡛ࡣഇ㝧ᛶࡀ⏕ࡌ࠼ࡿࡋࠊࡲࡓ༊ศ 1 ࡜༊ศ 2 ≀㉁ࢆ㆑ู࡛
ࡁ࡞࠸ࡇ࡜࠿ࡽࠊ௚ࡢヨ㦂ἲ࡟ࡼࡿ㏣ຍヨ㦂ࡀᚲせ࡜࡞ࡿࠋᖹᆒ⣽⬊⏕Ꮡ⋡ࡀ࢝ࢵࢺ࢜ࣇ್㏆㎶ࡢሙྜ㸦s5%㸧ࡣ 2 ᅇ┠ࡢヨ㦂ࢆᐇ᪋ࡍࡿࠋ1 ᅇ┠࡜ 2 ᅇ┠ࡢヨ㦂࡛⤖ᯝࡀ୍⮴ࡋ࡞࠸ሙྜࡣ 3 ᅇ┠ࡢヨ㦂ࢆᐇ᪋ࡍࡿࠋ

5㸬ࣂࣜࢹ࣮ࢩࣙࣥ◊✲ 㛤Ⓨ⪅ࡢ L'Oréal ♫ࢆ࣮ࣜࢻࣛ࣎࡜ࡋ࡚ࠊࡉࡽ࡟ 2 ᪋タ㸦Charles River LaboratoriesࠊFlemish Institutefor Technological Research㸧ࢆຍ࠼ࠊྜィ 3 ᪋タ࡛ SkinEthicTM HCE EIT ࡢࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀ⾜ࢃࢀࡓࠋṇ☜ᛶࡢホ౯ࡢࡓࡵ࡟ᚲせ࡞ື≀ヨ㦂ࡢࢹ࣮ࢱࢆᣢࡗ࡚࠸ࡿࡇ࡜ࢆ๓ᥦ᮲௳࡟ࠊUN GHS ༊ศ≀㉁࣭༊ศእ≀㉁ࡸᅛయ࣭ᾮయ➼ࡢࣂࣛࣥࢫࢆ⪃៖ࡋ࡚⿕㦂≀㉁ࡢ㑅ᢥࡀ⾜ࢃࢀࡓ㸦Appendix 1㸧ࠋMTT㑏ඖ≀㉁ࡸ࣐࣍ࣝࢨࣥ࡜ྠࡌࡼ࠺࡞Ἴ㛗࡟྾཰ࢆᣢࡘ╔Ⰽ≀㉁ࡶྵࡵࡽࢀࡓࠋ ࢥ࣮ࢻ໬ࡉࢀࡓ 120 ≀㉁ࡍ࡭࡚ࢆ 3 ᪋タ࡛ヨ㦂ࡋࡓࠋヨ㦂ࡣࡍ࡭࡚ࡢ⿕㦂≀㉁࡟ࡘ࠸࡚ 3 ᅇᐇ᪋ࡉࢀࡓࠋ ࡲࡓࠊࡇࡢ 3 ᪋タࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡜ࡣู࡟ࠊ࣮ࣜࢻࣛ࣎࡟࠾࠸࡚ࠊ80 ≀㉁࡟ࡘ࠸࡚㏣ຍヨ㦂ࡀ⾜ࢃࢀࡓ㸦Appendix 1㸧ࠋヨ㦂ࡣࡍ࡭࡚ࡢ⿕㦂≀㉁࡟ࡘ࠸࡚ 3 ᅇᐇ᪋ࡉࢀࡓࠋ
713

6㸬ヨ㦂ἲࡢಙ㢗ᛶ
6-1. ᢏ⾡⛣㌿ᛶ ࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࢆ㛤ጞࡍࡿ๓࡟ࠊ࣮ࣜࢻࣛ࣎࡟ࡼࡾࠊཧຍ 2 ᪋タࡢᢏ⾡⪅࡟ᑐࡋ࡚ SkinEthicTM
HCE EIT ࡢᾮయ࣭ᅛయ⏝ࣉࣟࢺ࣮ࣝࢆ⏝࠸࡚ࢺ࣮ࣞࢽࣥࢢࡀ⾜ࢃࢀࡓࠋࡑࡢᚋࠊᾮయ≀㉁࠾ࡼࡧᅛయ≀㉁ࡑࢀࡒࢀ 9 ≀㉁㸦ࢥ࣮ࢻ໬㸧ࢆ⏝࠸࡚ᢏ⾡⛣㌿ࡢ☜ㄆࢆ⾜ࡗࡓࠋ1 ≀㉁࠶ࡓࡾ 3 ᅇヨ㦂ࢆᐇ᪋ࡋࡓ࡜ࡇࢁࠊⰋዲ࡞⤖ᯝࡀᚓࡽࢀࡓࠋ
࡞࠾ࠊRhCE ἲࢆᐇ᪋ࡍࡿヨ㦂᪋タࡢᢏ⾡⩦ᚓࢆ☜࠿ࡵࡿࡓࡵࡢ⇍㐩ᗘ☜ㄆ≀㉁ࡢ୍ぴࡣᨵゞ TG
492 ࡟ᥦ♧ࡉࢀ࡚࠸ࡿ㸦Appendix 2㸧ࠋ
6-2. ᪋タෆ෌⌧ᛶࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡛ࡣ᪋タࡈ࡜࡟ 1 ⿕㦂≀㉁࡟ࡘࡁ」ᩘᅇࡢヨ㦂ࢆ⾜ࡗ࡚࠸ࡿࠋGHS ศ㢮ุᐃࡢ᪋タࡈ࡜ࡢ᪋タෆ෌⌧ᛶࡣࠊᾮయ⏝ࣉࣟࢺࢥ࡛ࣝࡣ 88.3 - 95.0%ࠊᅛయ⏝ࣉࣟࢺࢥ࡛ࣝࡣ 95.0 - 96.7%
࡛࠶ࡗࡓࠋ࣮ࣜࢻ࡛ࣛ࣎⾜ࢃࢀࡓ㏣ຍヨ㦂࡛ࡢ᪋タෆ෌⌧ᛶࡣᾮయ⏝ࣉࣟࢺࢥ࡛ࣝࡣ 91.1%ࠊᅛయ⏝ࣉࣟࢺࢥ࡛ࣝࡣ 97.1%࡛࠶ࡗࡓࠋࡇࢀࡽࡢ್ࡣࣂࣜࢹ࣮ࢩࣙࣥ㐠Ⴀጤဨ఍ࡀᐃࡵࡓᇶ‽㸦85%௨ୖ㸧ࢆ‶ࡓࡋࡓࠋ
6-3. ᪋タ㛫෌⌧ᛶ᪋タࡈ࡜࡟」ᩘᅇࡢヨ㦂ࡢ⣽⬊⏕Ꮡ⋡ࢆᖹᆒࡋ࡚ GHS ศ㢮ุᐃࢆ⾜࠸ࠊ᪋タ㛫෌⌧ᛶࢆ᳨ウࡋࡓࠋࡑࡢ⤖ᯝࠊ3 ᪋タ㛫ࡢ෌⌧ᛶࡣᾮయ⏝ࣉࣟࢺࢥ࡛ࣝࡣ 93.3%ࠊᅛయ⏝ࣉࣟࢺࢥ࡛ࣝࡣ 96.7%࡛࠶ࡾࠊ
඲య࡛ࡣ 95.0%࡛࠶ࡗࡓࠋࡇࡢ್ࡣࣂࣜࢹ࣮ࢩࣙࣥ㐠Ⴀጤဨ఍ࡀᐃࡵࡓᇶ‽㸦80%௨ୖ㸧ࢆ‶ࡓࡋࡓࠋ7㸬ヨ㦂ἲࡢṇ☜ᛶṇ☜ᛶࡢホ౯࡟ࡣࠊࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡜ࡋ࡚ 3 ᪋タ࡛⾜ࢃࢀࡓ඲ヨ㦂ࡢࢹ࣮ࢱࢆ⏝࠸ࡓࠋ⤖ᯝࡣ
⾲ 1 ࡢ㏻ࡾ࡛࠶ࡿࠋ
⾲ 1. SkinEthicTM HCE EIT ࡢṇ☜ᛶ㸦3 ᪋タࣂࣜࢹ࣮ࢩࣙࣥ㸧
ホ౯㡯┠

ᾮయ

ᅛయ

ឤᗘ㸦90%㸧

98.3%

92.2%

≉␗ᗘ㸦60%㸧

69.4%

76.6%

ṇ☜ᗘ㸦75%㸧

84.8%

84.4%

㸦ཷࡅධࢀᇶ‽㸧

SkinEthicTM HCE EIT ࡢṇ☜ᛶࡣࣂࣜࢹ࣮ࢩࣙࣥ㐠Ⴀጤဨ఍ࡀᐃࡵࡓᇶ‽㸦ឤᗘ 90%௨ୖࠊ≉␗ᗘ
60%௨ୖࠊṇ☜ᗘ 75%௨ୖ㸧ࢆ‶ࡓࡋࡓࠋ࡞࠾ࠊࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡟࠾࠸࡚࣮ࣜࢻ࡛ࣛ࣎ᚓࡽࢀࡓࢹ࣮ࢱ࡜࣮ࣜࢻࣛ࣎࡟࠾࠸࡚ᐇ᪋ࡉࢀࡓ
㏣ຍヨ㦂ࡢ 80 ≀㉁㸦45 ᾮయ≀㉁ࠊ35 ᅛయ≀㉁㸧ࡢࢹ࣮ࢱࢆྜࢃࡏ࡚࣮ࣜࢻࣛ࣎࡟࠾ࡅࡿṇ☜ᛶࢆホ౯ࡋࡓሙྜࠊᾮయ≀㉁࡛ࡣឤᗘ 100%ࠊ≉␗ᗘ 65.3%ࠊṇ☜ᗘ 83.5%ࠊᅛయ≀㉁࡛ࡣឤᗘ 89.7%ࠊ≉␗ᗘ 73.6%ࠊṇ☜ᗘ 80.7%࡛࠶ࡗࡓࠋࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡢṇ☜ᛶ࡜࡯ࡰྠᵝ࡞⤖ᯝࡀᚓࡽࢀࡓࠋ

814

8㸬ヨ㦂ἲࡢ㐺⏝⠊ᅖ
TG 492 ࡣࠊヨ㦂ἲࡢᛶ㉁࡜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) GHS ༊ศ 1ࠊ༊ศ 2㸦2A࣭2B㸧≀㉁ࡢ᳨ฟ࡟ࡣ⏝࠸ࡿࡇ࡜ࡣ࡛ࡁ࡞࠸ࠋ
9. ᮏጤဨ఍ࡢ⤖ㄽ
SkinEthicTM HCE EIT ࡢ 3 ᪋タ࡟ࡼࡿࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡢ⤖ᯝࠊᮏヨ㦂ἲࡣ෌⌧ᛶ࠾ࡼࡧṇ☜ᛶ࡟ࡘ࠸࡚ࣂࣜࢹ࣮ࢩࣙࣥ㐠Ⴀጤဨ఍ࡢᐃࡵࡓᇶ‽ࢆ‶ࡓࡋ࡚࠸ࡓࠋᮏጤဨ఍ࡣࣂࣜࢹ࣮ࢩࣙࣥ㐠Ⴀጤဨ఍ࡢᐃࡵࡓᇶ‽ࡣጇᙜ࡛࠶ࡿ࡜⪃࠼ࡿࠋ
SkinEthicTM HCE EIT ࡟⏝࠸ࡿ RhCE ⤌⧊ࡣᕷ㈍ࡉࢀ࡚࠾ࡾࠊࡲࡓࠊRhCE ⤌⧊௨እࡣࠊ≉Ṧ࡞ᶵᮦࡸヨ⸆ࢆᚲせ࡜ࡏࡎࠊᡭᢏࡶ」㞧࡞ࡶࡢ࡛࡞࠸ࠋ
ධᡭࡋࡓ RhCE ⤌⧊ࡀᶵ⬟ⓗ᮲௳ࡢチᐜ⠊ᅖ࡟࠶ࡾࠊ
࠿ࡘᐇ᪋ࡍࡿヨ㦂᪋タ࡜ࡋ࡚ࡢᢏ⾡⩦ᚓࡀ࢞࢖ࢻࣛ࢖ࣥࡢ⇍㐩ᗘ☜ㄆ≀㉁࡛☜࠿ࡵࡽࢀ࡚࠸ࢀࡤࠊ
SkinEthicTM HCE EIT ࡣ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡛ UN GHS ༊ศእ≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚⏝࠸ࡿࡇ࡜ࡀ
࡛ࡁࡿࠊ࡜ᮏጤဨ఍ࡣ⪃࠼ࡿࠋ
10. ᩥ⊩
1)

Alépée N et al (2016) Multi-laboratory validation of SkinEthic HCE test method for testing serious eye
damage/eye irritation using liquid chemicals. Toxicol In Vitro 31, 43-53.

2)

Alépée N et al (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 tosolid and liquid chemicals testing. Toxicol In Vitro 34, 55-70.

3)

ESAC (2016) ESAC Opinion on the SkinEthic™ Human Corneal Epithelium (HCE) Eye Irritation Test
(EIT). ESAC Opinion No. 2016-02 of 24 June 2016.

4)

OECD (2017) 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.

915

Appendix 1ࣂࣜࢹ࣮ࢩࣙࣥ࡟⏝࠸ࡽࢀࡓ≀㉁
3 ᪋タ

࣮ࣜࢻࣛ࣎

ࣂࣜࢹ࣮ࢩࣙࣥ

㏣ຍࢹ࣮ࢱ

120

80

༊ศ 1

32

19

༊ศ 2A

17

12

༊ศ 2B

13

4

༊ศእ

58

45

ᾮయ

60

45

ᅛయ

60

35

Aromatic

21

20

Alcohol

19

17

Ester

17

12

Heterocyclic

13

12

Halogenated9

14

Carboxylic acid

11

7

Amine

3

10

Phenol121

Acrylate

6

2

Ether

0

8

Salt

6

2

Polyether

6

1

Nitrile2

4

Onium Compound0

6

Silicium42

Ketone3

2

Pyrimidine

1

4

Thioether

4

1



඲య
║่⃭ᛶ GHS ศ㢮≀㉁ࡢ≉ᛶ 

Functional Group Classὀ㸧ࣂࣜࢹ࣮ࢩࣙࣥ◊✲ㄽᩥ㸦Alépée et al., 2016a, 2016b㸧ࢆࡶ࡜࡟సᡂࠋFunctional Group Class ࡣ≀㉁ᩘࡢከ࠸ࡶࡢࢆᢳฟࠋ≀㉁࡟ࡼࡗ࡚ࡣ」ᩘࡢ Functional Group Class ࡟ࡲࡓࡀࡗ࡚࠸ࡿሙྜࡶ࠶
ࡿࠋ

10
16

Appendix 2
RhCE ἲࡢ⇍㐩ᗘ☜ㄆ≀㉁≀㉁ྡ

CAS ␒ྕ

ᛶ≧

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 118472-51-0

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

1-Ethyl-3-methylimidazolium ethylsulphate342573-75-7

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

101-20-2

ᅛయ༊ศእ103597-45-1

ᅛయ

༊ศእ

14075-53-7

ᅛయ

༊ศእ

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

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

11
17

18

OECD/OCDE

492
Adopted:
9 October 2017

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 physicaldecay 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, as defined by the United Nations Globally Harmonized
System of Classification and Labelling of Chemicals (UN GHS) (1). Also according to UN GHS, eyeirritation refers to the production of changes in the eye following the application of a test chemical to theanterior surface of the eye, 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 areclassified 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 for classification 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 use of 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 use of this Test Guideline should be seen in thecontext of the OECD Guidance Document on an Integrated Approaches on Testing and Assessment for
Serious Eye Damage and Eye irritation (39).
3.
This Test Guideline describes an in vitro procedure allowing the identification of chemicals(substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damagein accordance with UN GHS. It makes use of reconstructed human cornea-like epithelium (RhCE) whichclosely mimics the histological, morphological, biochemical and physiological properties of the humancorneal epithelium. Four other in vitro test methods have been validated, considered scientifically valid andadopted as OECD Test Guidelines (TGs) 437 (3), 438 (4), 460 (5) and 491 (6) to address the human healthendpoint serious eye damage/eye irritation.

1© OECD, (2017)
You are free to use this material
http://www.oecd.org/termsandconditions/.

subject

to

the

terms

and

conditions

availableatThis Guideline was adopted by the OECD Council by written procedure on 9 October 2017 [C(2017)97].

19

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OECD/OCDE

4.
Two validated test methods using commercially available RhCE models are included in this Test
Guideline. Validation studies for assessing eye irritation/serious eye damage have been conducted(7)(8)(9)(10)(11)(12)(13) using the EpiOcular™ Eye Irritation Test (EIT) and the SkinEthic™ Human
Corneal Epithelium (HCE) Eye Irritation Test (EIT). Each of these methods makes use of commerciallyavailable RhCE tissue constructs as test system, which are referred to in the following text as the Validated
Reference Methods – VRM 1 and VRM2, respectively. From these validation studies and their
independent peer review (9)(12) it was concluded that the EpiOcular™ EIT and SkinEthic™ HCE EIT areable to correctly identify chemicals (both substances and mixtures) not requiring classification andlabelling for eye irritation or serious eye damage according to UN GHS (1), and the test methods wererecommended as scientifically valid for that purpose (13).
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)(14) to predict across the 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 (15). The Bottom-Up approach (15) 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 (15) is designed to be used when, based on existing
information, a chemical is expected to cause serious eye damage. The EpiOcular™ EIT and SkinEthic™
HCE EIT are recommended to identify chemicals that do not require classification for eye irritation orserious eye damage according to UN GHS (UN GHS No Category) (1) without further testing, within atesting strategy such as the Bottom-Up/Top-Down approach suggested by Scott et al. e.g., as an initial stepin a Bottom-Up approach or as one of the last steps in a Top-Down approach (15). However, theEpiOcular™ EIT and SkinEthic™ HCE 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 beaddressed by another tier of a test strategy (15). A test chemical that is identified as requiring classificationfor eye irritation/serious eye damage with EpiOcular™ EIT or SkinEthic™ HCE EIT will thus require
additional testing (in vitro and/or in vivo) to reach a definitive conclusion (UN GHS No Category,Category 2 or Category 1), using e.g., TG 437, 438, 460 or 491.
6.
The purpose of this Test Guideline is to describe the procedure used to evaluate the eye hazard
potential of a test chemical based on its ability to induce cytotoxicity in a RhCE tissue construct, asmeasured by the MTT assay (16) (see paragraph 21). The viability of the RhCE tissue following exposure
to a test chemical is determined 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 (17) are available to facilitate the validation of new or modified in vitro
RhCE-based test methods similar to EpiOcular™ EIT and SkinEthic™ HCE EIT, in accordance with theprinciples of Guidance Document No. 34 (18), 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 been reviewed and included in this Test
Guideline by the OECD.
DEFINITIONS
8.

Definitions are provided in Annex I.

2© OECD, (2017)20

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492

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) or humanimmortalized corneal epithelial cells (i.e., SkinEthic™ HCE/S). The EpiOcular™ OCL-200 and
SkinEthic™ HCE/S RhCE tissue constructs are similar to the in vivo corneal epithelium three-dimensional
structure and are produced using cells from the species of interest (19)(20). Moreover, the test methods
directly measure cytotoxicity resulting from penetration of the chemical through the cornea and productionof cell and tissue damage; the cytotoxic response then determines the overall in vivo serious eye
damage/eye irritation outcome. Cell damage can occur by several modes of action (see paragraph 20), but
cytotoxicity plays an important, if not the primary, mechanistic role in determining the overall serious eye
damage/eye irritation response of a chemical, manifested in vivo mainly by corneal opacity, iritis,
conjunctival redness and/or conjunctival chemosis, regardless of the physicochemical processes underlying
tissue damage.
10.
A wide range of chemicals, covering a large 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 OECD QSAR toolbox analysis (8). The majority ofthese chemicals represented mono-constituent substances, but several multi-constituent substances(including 3 homopolymers, 5 copolymers and 10 quasi polymers) were also included in the study. In
terms of physical state 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, 7 Category 2B
liquids, 7 Category 2B solids, 27 No Category liquids and 28 No Category solids (8). The SkinEthic™
HCE EIT validation database contained 200 chemicals in total, covering 165 different organic functionalgroups (8)(10)(11). The majority of these chemicals 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, 9 Category 2B liquids, 8 Category
2B solids, 50 No Category liquids and 53 No Category solids (10)(11).
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 not been assessed in a validation study. While it isconceivable 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 MTT formazan (naturally or after treatment)
and test chemicals able to directly reduce the vital dye MTT (to MTT formazan) may interfere with thetissue viability measurements and need the use of adapted controls for corrections. The type of adapted
controls that may be required will vary depending on the type of interference produced by the test chemical
and the procedure used to quantify MTT formazan (see paragraphs 36-42).
13.
Results generated in pre-validation (21)(22) and full validation (8)(10)(11) studies havedemonstrated that both EpiOcular™ EIT and SkinEthic™ HCE EIT are transferable to laboratories
considered to be naïve in the conduct of the assays and also to be reproducible within- and between
laboratories. Based on these studies, the level of reproducibility in terms of concordance of predictions thatcan be expected from EpiOcular™ EIT from data on 113 chemicals is in the order of 95% within
3© OECD, (2017)

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OECD/OCDE

laboratories and 93% between laboratories. The level of reproducibility in terms of concordance ofpredictions 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.
14.
The EpiOcular™ 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 dataobtained in the validation study (8), the EpiOcular™ 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), whencompared to reference in vivo rabbit eye test data (OECD TG 405) (2)(14) 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 the test method for this type of mixtures as
obtained in the validation study (23). 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)(14). An overall accuracy of
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 (23).
15.
The SkinEthic™ HCE EIT can be used to identify chemicals that do not require classification foreye irritation or serious eye damage according to the UN GHS classification system (1). Considering thedata obtained in the validation study (10)(11), the SkinEthic™ HCE EIT has an overall accuracy of 84%(based on 200 chemicals), sensitivity of 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)(14) classified
according to the UN GHS classification system (1).
16.
The false negative rates obtained with both RhCE test methods, with either substances or
mixtures, fall within the 12% overall probability that chemicals are identified as either UN GHS Category
2 or UN GHS No Category by the in vivo Draize eye test, in repeated tests; this is due to the method's
inherent within-test variability (24). The false positive rates obtained with both RhCE test methods witheither substances or 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 44) will require
further testing with other in vitro test methods, or as a last option in rabbits, depending on regulatory
requirements, using a sequential testing strategy in a weight-of-evidence approach. These test methods canbe used for all types of chemicals, whereby a negative result should be accepted for not classifying achemical for eye irritation and serious eye damage (UN GHS No Category). The appropriate regulatory
authorities should be consulted before using the EpiOcular™ EIT and SkinEthic™ HCE EIT underclassification schemes other than UN GHS.
17.
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 methods isrequired.

4© OECD, (2017)22

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492

18.
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 TEST19.
The test chemical is applied topically to a minimum of two three-dimensional RhCE tissue
constructs and tissue viability is measured following exposure and a post-treatment incubation period. The
RhCE tissues are reconstructed from primary human epidermal keratinocytes or human immortalized
corneal epithelial cells, which have been cultured for several days to form a stratified, highly differentiatedsquamous epithelium morphologically similar to that found in the human cornea. The EpiOcular™ RhCE
tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornealike structure analogous to that found in vivo. The SkinEthic™ HCE RhCE tissue construct consists of atleast 4 viable layers of cells including columnar basal cells, transitional wing cells and superficial
squamous cells similar to that of the normal human corneal epithelium (20)(26).
20.
Chemical-induced serious eye damage/eye irritation, manifested in vivo mainly by corneal
opacity, iritis, conjunctival redness and/or conjunctival chemosis, is the result of a cascade of events
beginning with penetration of the chemical through the cornea and/or conjunctiva and production ofdamage to the cells. Cell damage can occur by several modes of action, including: cell membrane lysis(e.g., by surfactants, organic solvents); coagulation of macromolecules (particularly proteins) (e.g., bysurfactants, organic solvents, alkalis and acids); saponification of lipids (e.g., by alkalis); and alkylation orother covalent interactions with macromolecules (e.g., by bleaches, peroxides and alkylators) (15)(27)(28).
However, it has been shown that cytotoxicity plays an important, if not the primary, mechanistic role indetermining the overall serious eye damage/eye irritation response of a chemical regardless of thephysicochemical processes underlying tissue damage (29)(30). Moreover, the serious eye damage/eye
irritation potential of a chemical is principally determined by the extent of initial injury (31), whichcorrelates with the extent of cell death (29) and with the extent of the subsequent responses and eventual
outcomes (32). Thus, slight irritants generally only affect the superficial corneal epithelium, the mild andmoderate irritants damage principally the epithelium and superficial stroma and the severe irritants damage
the epithelium, deep stroma and at times the corneal endothelium (30)(33). The measurement of viabilityof the RhCE tissue construct after topical exposure to a test chemical to identify chemicals not requiringclassification 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.
21.
RhCE tissue viability is classically measured by enzymatic conversion of the vital dye MTT [3(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide; CAS
number 298-93-1] by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively
measured after extraction from tissues (16). Chemicals not requiring classification and labelling according
to UN GHS (No Category) are identified as those that do not decrease tissue viability below a defined
threshold (i.e., tissue viability > 60%, in EpiOcular™ EIT and SkinEthic™ HCE EITL2, or > 50%, in
SkinEthic™ HCE EITS3) (see paragraph 44).
1

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

2

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

3

EITS: EIT for solids in the case of SkinEthic™ HCE

5© OECD, (2017)

23

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OECD/OCDE

DEMONSTRATION OF PROFICIENCY
22.
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 the validation studies of the VRMs
(8)(10)(11). The selection includes, to the extent possible, chemicals that: (i) cover different physicalstates; (ii) cover the full range of in vivo serious eye damage/eye irritation responses based on high quality
results obtained in the reference in vivo rabbit eye test (OECD TG 405) (2)(14) and the UN GHS
classification system (i.e., Categories 1, 2A, 2B, or No Category) (1); (iii) cover the various in vivo driversof classification (24)(25); (iv) are representative of the chemical classes used in the validation study(8)(10)(11); (v) cover a good and wide representation of organic functional groups (8)(10)(11); (vi) havechemical structures that are well-defined (8)(10)(11); (vii) are coloured and/or direct MTT reducers; (viii)
produced reproducible results in RhCE test methods during their validations; (ix) were correctly predictedby RhCE test methods during their validation studies; (x) cover the full range of in vitro responses basedon high quality 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 isunavailable or cannot be used for other justified reasons, another chemical fulfilling the criteria describedabove, e.g. from the chemicals used in the validation of the VRM, could be used. Such deviations should
however be justified.
Table 1: List of proficiency chemicals

Chemical Name

CASRN

Organic
Functional
Group1VRM1
viability(%)2
In Vivo Category 14

Methylthioglycolate

2365-48-2

Carboxylic acid
ester;
ThioalcoholL

10.9±6.4

5.5±7.4

Hydroxyethyl acrylate

818-61-1

Acrylate;
Alcohol

L

7.5±4.75

1.6±1.0

2,5-Dimethyl-2,5hexanediol

110-03-2

AlcoholS

2.3±0.2

0.2±0.1

Sodium oxalate

62-76-0Oxocarboxylicacid

S

29.0±1.2

5.3±4.1

Physical
State

VRM2
viability(%)3

In Vivo Category 2A42,4,11,13Tetraazatetradecanediimidamide,
N,N''bis(4-chlorophenyl)3,12-diimino-, di-Dgluconate(20%, aqueous) 6
Sodium benzoate

No
prediction
can be madeNo
prediction
can be madeNo
prediction
can be madeNo
prediction
can be made

MTT
Reducer

Colourinterf.

Y(strong)

N

N

N

N

N

N

N

18472-51-0

Aromatic
heterocyclic
halide; Aryl
halide;Dihydroxyl
group;
GuanidineL

4.0±1.1

1.3±0.6

No
prediction
can be madeNY(weak)

532-32-1

Aryl;Carboxylic acidS

3.5±2.6

0.6±0.1

No
prediction
can be madeN

N

In Vivo Category 2B4

6© OECD, (2017)24

VRM
Prediction

OECD/OCDE
Chemical Name
Diethyl toluamide

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

492

CASRN

Organic
Functional
Group1

Physical
State

VRM1
viability
(%)2

VRM2
viability(%)3

VRM
Prediction

MTT
Reducer

Colourinterf.

134-62-3

BenzamideL

15.6±6.3

2.8±0.9

No
prediction
can be madeNN

79-92-5

Alkane,branched withtertiary carbon;
Alkene;Bicycloheptane;
Bridged-ringcarbocycles;
Cycloalkane

S

4.7±1.515.8±1.1

No
prediction
can be madeN

N

In Vivo No Category4
1-Ethyl-3methylimidazolium
ethylsulphate342573-75-5

Dicaprylyl ether

629-82-3

Piperonyl butoxide51-03-6

Polyethylene(PEG-40)hydrogenatedoilglycolcastor61788-85-0

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

101-20-22,2'-Methylene-bis-(6(2H-benzotriazol-2yl)-4-(1,1,3,3tetramethylbutyl)phenol)

103597-45-1

Potassium
tetrafluoroborate

14075-53-7

Alkoxy;Ammoniumsalt; Aryl;
Imidazole;
Sulphate
Alkoxy;Ether
Alkoxy;Benzodioxole;
Benzyl; Ether
Acylal;Alcohol; Allyl;
Ether
Aromatic
heterocyclic
halide; Aryl
halide; Urea
derivatives
Alkanebranched withquaternarycarbon; Fused
carbocyclicaromatic; Fused
saturatedheterocycles;
Precursors
quinoidcompounds;
tert-Butyl
Inorganic Salt

L

79.9±6.4

79.4±6.2

No Cat

N

N

L

97.8±4.3

95.2±3.0

No CatN

N

L

104.2±4.2

96.5±3.5

No CatN

N

Viscous77.6±5.489.1±2.9

No CatN

N

S

106.7±5.3

101.9±6.6

No CatN

N

S

102.7±13.4

97.7±5.6

No CatN

N

S

88.6±3.3

92.9±5.1

No CatN

N

Abbreviations: CASRN = Chemical Abstracts Service Registry Number; UN GHS = United Nations Globally Harmonized Systemof 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 of MTT formazan.
1
Organic functional group assigned according to an OECD Toolbox 3.1 nested analysis (8).
2Based on results obtained with EpiOcular™ EIT in the EURL ECVAM/Cosmetics Europe Eye Irritation Validation Study (EIVS)(8).
3
Based on results obtained with SkinEthic™ HCE EIT in the validation study (10)(11).

7© OECD, (2017)

25

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OECD/OCDE

4

Based on results from the in vivo rabbit eye test (OECD TG 405) (2)(14) and using the UN GHS (1).
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.
523.
As part of the proficiency testing, it is recommended that users verify the barrier properties of thetissues after receipt as specified by the RhCE tissue construct producer (see paragraphs 25, 27 and 30).
This is particularly important if tissues are shipped over long distance / time periods. Once a test method
has been successfully established and proficiency in its use has been acquired and demonstrated, suchverification will not be necessary on a routine basis. However, when using a test method routinely, it isrecommended to continue to assess the barrier properties at regular intervals.
PROCEDURE
24.
The test methods currently covered by this Test Guideline are the scientifically valid EpiOcular™
EIT and SkinEthic™ HCE EIT (9)(12)(13), referred to as the Validated Reference Method (VRM1 andVRM2, 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 (34)(35). The
following paragraphs and Annex II describe the main components and procedures of the RhCE test
methods.
RHCE TEST METHOD COMPONENTS
General conditions
25.
Relevant human-derived cells should be used to reconstruct the cornea-like epithelium threedimensional tissue, which should be composed of progressively stratified but not cornified cells. The
RhCE tissue construct is prepared in inserts with a porous synthetic membrane through which nutrients canpass to the cells. Multiple layers of viable, non-keratinized epithelial cells should be present in thereconstructed cornea-like epithelium. The RhCE tissue construct should have the epithelial surface indirect 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 beassessed by determination of either the exposure time required to reduce tissue viability by 50% (ET 50)
upon application of a benchmark 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 the tissues by
50% (IC50) following a fixed exposure time (e.g., 30 minutes treatment with 50 μL SDS) (see paragraph30). The containment properties of the RhCE tissue construct should prevent the passage of test chemicalaround the edge of the viable tissue, which could lead to poor modelling of corneal exposure. The humanderived cells used to establish the RhCE tissue construct should be free of contamination by bacteria,
viruses, mycoplasma, and fungi. The sterility of the tissue construct should be checked by the supplier forabsence of contamination by fungi and bacteria.

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Functional conditions
Viability
26.
The assay used for quantifying tissue viability is the MTT assay (16). 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). The extracted MTT formazan may be quantified
using either a standard absorbance (Optical Density (OD)) measurement or an HPLC/UPLCspectrophotometry procedure (36). The OD of the extraction solvent alone should be sufficiently small, i.e.,
OD < 0.1. Users of the RhCE tissue construct should ensure that each batch of the RhCE tissue constructused 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 thenegative control OD ranges provided 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 arestable in culture (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 those specified in Table 2 may apply. An
acceptability range (upper and lower limit) for the negative 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

Lower acceptance limit

Upper acceptance limit> 0.81

< 2.5

> 1.0

≤ 2.5

EpiOcular™ EIT (OCL-200) – VRM1
(for both the liquids and the solids protocols)

SkinEthic™ HCE EIT (HCE/S) – VRM2
(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 been shown not to
impact on the performance of the test method (37).

Barrier function
27.
The RhCE tissue construct should be sufficiently thick and robust to resist the rapid penetrationof 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 construct used should be demonstrated by the RhCE
tissue construct developer/vendor upon supply of the tissues to the end user (see paragraph 30).
Morphology
28.
Histological examination of the RhCE tissue construct should demonstrate human cornea-likeepithelium structure (including at least 3 layers of viable epithelial cells and a non-keratinized surface). For
the VRMs, appropriate morphology has been established by the developer/supplier and therefore does notneed to be demonstrated again by a test method user for each tissue batch used.

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Reproducibility
29.
The results of the positive and negative controls of the test method should demonstrate
reproducibility over time.
Quality control (QC)30.
The RhCE tissue construct should only be used if the developer/supplier demonstrates that eachbatch of the RhCE tissue construct used meets defined production release criteria, among which those for
viability (paragraph 26) and barrier function (see paragraph 27) are the most relevant. An acceptability
range (upper and lower limits) for the barrier functions as measured by the ET50 or IC50 (see paragraphs 25
and 26) should be established 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 VRMs) is given in Table 3. Data demonstrating compliance 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 this information in the test report. Only results produced with tissues
fulfilling all of these production release criteria can be accepted for reliable prediction of chemicals not
requiring classification and labelling for eye irritation or serious eye damage in accordance with UN GHS.
Table 3: QC batch release criterion
Test Method

Lower acceptance limit

Upper acceptance limit

EpiOcular™ EIT (OCL-200) – VRM1(100 μL of 0.3% (v/v) Triton X-100)

ET50 = 12.2 min

ET50 = 37.5 min

SkinEthic™ HCE EIT (HCE/S) – VRM2
(30 minutes treatment with 50 μL SDS)

IC50 = 1 mg/mL

IC50 = 3.2 mg/mL

Application of the Test Chemical and Control Substances
31.
At least two tissue replicates should be used for each test chemical and each control substance ineach run. Two different treatment protocols are used, one for liquid test chemicals and one for solid test
chemicals (34)(35).For both methods and protocols, the tissue construct surface should be moistened withcalcium and magnesium-free Dulbecco's Phosphate Buffered Saline (Ca2+/Mg2+-free DPBS) before
application of test chemicals, to mimic the wet conditions of human eye. The treatment of the tissues is
initiated with exposure to the test chemical(s) and control substances. For both treatment protocols in both
VRMs, a sufficient amount of test chemical or control substance should be applied to uniformly cover theepithelial surface while avoiding an infinite dose (see paragraphs 32 and 33) (Annex II).
32.
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 VRMs, otherwise they should be treated as solids (seeparagraph 33). In the VRMs, liquid test chemical are evenly spread over the tissue surface (i.e. a minimumof 60 μL/cm2 application) (see Annex II, (33)(34)). Capillary effects (surface tension effects) that may
occur due to the low 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 30 min at standard culture conditions (37±2oC, 5±1% CO2, ≥95% RH). At the end of the10© OECD, (2017)28

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exposure period, the liquid test chemical and the control substances should be carefully removed from thetissue surface by extensive rinsing with Ca2+/Mg2+-free DPBS at room temperature. This rinsing step isfollowed by a post-exposure 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. For
VMR1 only, a post-exposure incubation in fresh medium at standard culture conditions is applied prior toperforming the MTT assay (see Annex II, (34)(35)).
33.
Test chemicals that cannot be pipetted at temperatures up to 37°C are treated as solids in the
VRMs. The amount of test chemical applied should be sufficient to cover the entire surface of the tissue,
i.e. a minimum of 60 mg/cm2 application should be used (Annex II). Whenever possible, solids should betested as a fine powder. Tissues treated with solid test chemicals are incubated for a pre-defined period oftime (depending on the VRM used) at standard culture conditions (see Annex II, (34) (35)). At the end of
the exposure period, the solid test chemical and the control substances should be carefully removed fromthe tissue surface by extensive rinsing with Ca2+/Mg2+-free DPBS at room temperature. This rinsing step isfollowed by a post-exposure 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, and a
post-exposure incubation in fresh medium at standard culture conditions, prior to performing the MTT
assay (see Annex II, (34)(35)).
34.
Concurrent negative and positive controls should be included in each run to demonstrate that theviability (determined with the negative control) and the sensitivity (determined with the positive control) of
the tissues are within acceptance ranges defined based on historical data. The concurrent negative control
also provides the baseline (100% tissue viability) to calculate the relative percent viability of the tissues
treated with the test chemical (%Viabilitytest). The recommended positive control substance to be used with
the VRMs is neat methyl acetate (CAS No. 79-20-9, commercially available from e.g., Sigma-Aldrich,Cat# 45997; liquid). The recommended negative control substances to be used with the VRM1 and VRM2
are ultrapure H2O and Ca2+/Mg2+-free DPBS, respectively. These were the control substances used in thevalidation studies of the VRMs and are those for which most historical data exist. The use of suitable
alternative positive or negative control substances should be scientifically and adequately justified.
Negative and positive controls should be tested with the same protocol(s) as the one(s) used for the test
chemicals included in the run (i.e. for liquids and/or solids). This application should be followed by thetreatment exposure, rinsing, a post-exposure immersion, and post-exposure incubation where applicable, as
described for controls run concurrently to liquid test chemicals (see paragraph 32) or for controls run
concurrently to solid test chemicals (see paragraph 33), prior to performing the MTT assay (see paragraph35) (34)(35). 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
35.
The MTT assay is a standardised quantitative method (16) that should be used to measure tissue
viability under this Test Guideline. It is compatible with use in a three-dimensional tissue construct. The
MTT assay is performed immediately following the post-exposure incubation period. In the VRMs, the
RhCE tissue construct sample is placed in 0.3 mL of MTT solution at 1 mg/mL for 180±15 min at standard
culture conditions. The vital dye MTT is reduced into a blue MTT formazan precipitate by the viable cellsof the RhCE tissue construct. The precipitated blue MTT formazan product is then extracted from thetissue using an appropriate volume of isopropanol (or a similar solvent) (34)(35). Tissues tested with liquid
test chemicals should be extracted from both the top and the bottom of the tissues, while tissues tested withsolid test chemicals and coloured liquids should be extracted from the bottom of the tissue only (to11© OECD, (2017)

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minimise any potential contamination of the isopropanol extraction solution with any test chemical that
may have remained on the tissue). Tissues tested with liquid test chemicals that are not readily washed off
may also be extracted from the bottom of the tissue only. The concurrently tested negative and positive
control substances should be treated similarly to the tested chemical. The extracted MTT formazan may bequantified 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 42) (11)(36).
36.
Optical properties of the test chemical or its chemical action on MTT may interfere with themeasurement of MTT formazan leading to a false estimate of tissue viability. Test chemicals may interferewith the measurement of MTT formazan by direct reduction of the MTT into blue MTT formazan and/or
by colour interference if the test chemical absorbs, naturally or due to treatment procedures, in the same
OD range as MTT formazan (i.e., around 570 nm). Pre-checks should be performed before testing to allow
identification of potential direct MTT reducers and/or colour interfering chemicals and additional controls
should be used to detect and correct for potential interference from such test chemicals (see paragraphs 3741). This is especially important when a specific test chemical is not completely removed from the RhCE
tissue construct by rinsing or when it penetrates the cornea-like epithelium and is therefore present in the
RhCE tissue constructs when the MTT assay is performed. For test chemicals absorbing light in the samerange as MTT formazan (naturally or after treatment), which are not compatible with the standard
absorbance (OD) measurement of MTT formazan due to too strong interference, i.e., strong absorption at
570±30 nm, an HPLC/UPLC-spectrophotometry procedure to measure MTT formazan may be employed(see paragraphs 41 and 42) (11)(36). A detailed description of how to detect and correct for direct MTT
reduction and interferences by colouring agents is available in the VRMs SOPs (34)(35). Illustrative
flowcharts providing guidance on how to identify and handle direct MTT-reducers and/or colour
interfering chemicals for VRM1 and VRM2 are also provided in Annexes III and IV, respectively.
37.
To identify potential interference by test chemicals absorbing light in the same range as MTT
formazan (naturally or after treatment) and decide on the need for additional controls, the test chemical isadded to water and/or isopropanol and incubated for an appropriate time at room temperature (see Annex
II, (34)(35)). If the test chemical in water and/or isopropanol absorbs sufficient light in the range of 570±20nm for VRM1 (see Annex III), or if a coloured solution is obtained when mixing the test chemical withwater for VRM2 (see Annex IV), the test chemical is presumed to interfere with the standard absorbance(OD) measurement of MTT formazan and further colourant controls should be performed or, alternatively,an HPLC/UPLC-spectrophotometry procedure should be used in which case these controls are not required(see paragraphs 41 and 42 and Annexes III and IV)(34)(35). When performing the standard absorbance(OD) measurement, each interfering test chemical should be applied on at least two viable tissue replicates,
which undergo the entire testing procedure but are incubated with medium instead of MTT solution during
the MTT incubation step, to generate a non-specific colour in living tissues (NSCliving) control (34)(35).
The NSCliving control needs to be performed concurrently to the testing of the coloured test chemical and, incase 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 andincubated with MTT solution (%Viabilitytest) minus the percent non-specific colour obtained with living
tissues exposed to the interfering test chemical and incubated with medium without MTT, run concurrently
to the test being corrected (%NSCliving), i.e., True tissue viability = [%Viabilitytest] - [%NSCliving].
38.
To identify direct MTT reducers, each test chemical should be added to freshly prepared MTT
solution. An appropriate amount of test chemical is added to a MTT solution and the mixture is incubatedfor approximately 3 hours at standard culture conditions (see Annexes III and IV)(34)(35). If the MTT
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mixture containing the test chemical (or suspension for insoluble test chemicals) turns blue/purple, the test
chemical is presumed to directly reduce MTT 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/UPLC-spectrophotometry procedure. This additional functional check employs killed tissues thatpossess only residual metabolic activity but absorb and retain the test chemical in a similar way as viabletissues. Killed tissues of VRM1 are prepared by exposure to low temperature ("freeze-killed"). Killed
tissues of VRM2 are prepared by prolonged incubation (e.g., at least 24±1 hours) in water followed bystorage to low temperature ("water-killed"). Each MTT reducing test chemical is applied on at least two
killed tissue replicates, which undergo the entire testing procedure, to generate a non-specific MTT
reduction (NSMTT) control (34)(35). A single NSMTT control is sufficient per test chemical regardless ofthe number of independent tests/runs performed. True tissue viability is calculated as: the percent tissue
viability obtained with living tissues exposed to the MTT reducer (%Viabilitytest) minus the percent nonspecific MTT reduction obtained with the killed tissues exposed to the same MTT reducer, calculated
relative to the negative control run concurrently to the test being corrected (%NSMTT), i.e., True tissue
viability = [%Viabilitytest] - [%NSMTT].
39.
Test chemicals that are identified as producing both colour interference (see paragraph 37) anddirect MTT reduction (see paragraph 38) will also require a third set of controls when performing thestandard absorbance (OD) measurement, apart from the NSMTT and NSCliving controls described in theprevious paragraphs. This is usually the case with darkly coloured test chemicals absorbing light in therange of 570±30 nm (e.g., blue, purple, black) because their intrinsic colour impedes the assessment of
their capacity to directly reduce MTT as described in paragraph 38. This forces the use of NSMTT
controls, by default, together with the NSCliving controls. Test chemicals for which both NSMTT and
NSCliving controls are performed may be absorbed and retained by both living and killed tissues. Therefore,
in this case, the NSMTT control may not only correct for potential direct MTT reduction by the test
chemical, but also for colour interference arising from the absorption and retention of the test chemical bykilled tissues. This could lead to double correction for colour interference since the NSC living control
already corrects for colour interference arising from the absorption and retention of the test chemical byliving tissues. To avoid a possible double correction for colour interference, a third control for non-specificcolour in killed tissues (NSCkilled) needs to be performed (see Annexes III and IV)(34)(35). In thisadditional control, the test chemical is applied on at least two killed tissue replicates, which undergo theentire testing procedure but are incubated with medium instead of MTT solution during the MTT
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 control and withthe same tissue batch. True tissue viability is calculated as: the percent tissue viability obtained with living
tissues exposed to the test chemical (%Viabilitytest) minus %NSMTT minus %NSCliving plus the percent
non-specific colour obtained with killed tissues exposed to the interfering test chemical and incubated withmedium without MTT, calculated relative to the negative control ran concurrently to the test being
corrected (%NSCkilled), i.e., True tissue viability = [%Viabilitytest] - [%NSMTT] - [%NSCliving] +[%NSCkilled].
40.
It is important to note that non-specific MTT reduction and non-specific colour interferences may
increase the OD (when performing standard absorbance measurements) of the tissue extract above thelinearity range of the spectrophotometer and that non-specific MTT reduction can also increase the MTT
formazan peak area (when performing HPLC/UPLC-spectrophotometry measurements) of the tissue
extract above the linearity range of the spectrophotometer. On this basis, it is important for each laboratory
to determine the OD/peak area linearity range of their spectrophotometer with e.g., MTT formazan (CAS #13© OECD, (2017)

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57360-69-7), commercially available from e.g., Sigma-Aldrich (Cat# M2003), before initiating the testingof test chemicals for regulatory purposes.
41.
The standard absorbance (OD) measurement using a spectrophotometer is appropriate to assess
direct MTT-reducers and colour interfering test chemicals, when the observed interference with themeasurement of MTT formazan is not too strong (i.e., the ODs of the tissue extracts obtained with the test
chemical without any correction for direct MTT reduction and/or colour interference are within the linear
range of the spectrophotometer). Nevertheless, results for test chemicals producing %NSMTT and/or%NSCliving ≥ 60% (VRM1, and VRM2 for liquids’ protocol) or 50% (VRM2 for solids’ protocol) of thenegative control should be taken with caution as this is the established cut-off used in the VRMs todistinguish classified from not classified chemicals (see paragraph 44). Standard absorbance (OD) can
however not be measured when the interference with the measurement of MTT formazan is too strong (i.e.,leading to uncorrected ODs of the test tissue extracts falling outside of the linear range of thespectrophotometer). Coloured test chemicals or test chemicals that become coloured in contact with wateror isopropanol that interfere too strongly with the standard absorbance (OD) measurement of MTT
formazan may still be assessed using HPLC/UPLC-spectrophotometry (see Annexes III and IV). This isbecause the HPLC/UPLC system allows for the separation of the MTT formazan from the chemical before
its quantification (36). For this reason, NSCliving or NSCkilled controls are never required when using
HPLC/UPLC-spectrophotometry, independently of the chemical being tested. NSMTT controls should
nevertheless be used if the test chemical is suspected to directly reduce MTT (following the proceduredescribed in paragraph 38). NSMTT controls should also be used with test chemicals having a colour(intrinsic or appearing when in water) that impedes the assessment of their capacity to directly reduce MTT
as described in paragraph 38. When using HPLC/UPLC-spectrophotometry to measure MTT formazan, thepercent tissue viability is calculated as percent MTT formazan peak area obtained with living tissues
exposed to the test chemical relative to the MTT formazan peak obtained with the concurrent negative
control. For test chemicals able to directly reduce MTT, true tissue viability is calculated as: %Viability test
minus %NSMTT, as described in the last sentence of paragraph 38. Finally, it should be noted that direct
MTT-reducers or direct MTT-reducers that are also colour interfering, which are retained in the tissues
after treatment and reduce MTT so strongly that they lead to ODs (using standard OD measurement) or
peak areas (using UPLC/HPLC-spectrophotometry) of the tested tissue extracts that fall outside of thelinearity range of the spectrophotometer cannot be assessed with RhCE test methods, although these are
expected to occur in only very rare situations.
42.
HPLC/UPLC-spectrophotometry may be used with all types of test chemicals (coloured, noncoloured, MTT-reducers and non-MTT reducers) for measurement of MTT formazan (11)(36). Due to thediversity of HPLC/UPLC-spectrophotometry systems, it is not feasible for each user to establish the exact
same system conditions. As such, qualification of the HPLC/UPLC-spectrophotometry system should bedemonstrated before its use to quantify MTT formazan from tissue extracts by meeting the acceptancecriteria 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 (36)(38). These key parameters
and their acceptance criteria are shown in Annex V. Once the acceptance criteria defined in Annex V havebeen met, the HPLC/UPLC-spectrophotometry system is considered qualified and ready to measure MTT
formazan under the experimental conditions described in this Test Guideline.
Acceptance Criteria
43.
For each run using RhCE tissue batches that met the quality control (see paragraph 30), tissues
treated with the negative control substance should exhibit OD reflecting the quality of the tissues that14
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followed shipment, receipt steps and all protocol processes and should not be outside the historicallyestablished boundaries described in Table 2 (see paragraph 26). Similarly, tissues treated with the positive
control substance, i.e., methyl acetate, should show a mean tissue viability < 50% relative to the negative
control in the VRM1 with either the liquids' or the solids' protocols, and ≤ 30% (liquids’ protocol) or ≤20% (solids’ protocol) relative to the negative control in the VRM2, thus reflecting the ability of the tissues
to respond to an irritant test chemical under the conditions of the test method (34)(35). 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 replicates should be less than 20% or the standard
deviation (SD) between three tissue replicates should not exceed 18%). If either the negative control orpositive 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 Model
44.
The OD values/peak areas obtained with the replicate tissue extracts 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

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 and post-exposure incubation ismore 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.

x

If the mean percent tissue viability after exposure and post-exposure incubation is less than orequal (≤) to the established percentage tissue viability cut-off value, no prediction can be made,as shown in Table 4. In this case, further testing with other test methods will be required because
RhCE test methods show a certain number of false positive results (see paragraphs 14-15) andcannot resolve between UN GHS Categories 1 and 2 (see paragraph 17).
Table 4: Prediction Models according to UN GHS classification
VRM
EpiOcular™ EIT(for both protocols)
SkinEthic™ HCE EIT(for the liquids’ protocol)
SkinEthic™ HCE EIT(for the solids’ protocol)

No Category

No prediction can be madeMean tissue viability > 60%

Mean tissue viability ≤ 60%

Mean tissue viability > 60%

Mean tissue viability ≤ 60%

Mean tissue viability > 50%

Mean tissue viability ≤ 50%

45.
A single test composed of at least two tissue replicates should be sufficient for a test chemical
when the result is unequivocal. However, in cases of borderline results, such as non-concordant replicate
measurements and/or mean percent tissue viability equal to 60±5% (VRM1, and VRM2 for liquids’
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protocol) or 50±5% (VRM2 for solids’ protocol), a second test should be considered, as well as a third one
in case of discordant results between the first two tests.
46.
Different percentage tissue viability cut-off values distinguishing classified from non-classified
test chemicals may be considered for specific types of mixtures, where appropriate and justifiable, in order
to increase the overall performance of the test method for those types of mixtures (see paragraph 14).
Benchmark chemicals may be useful for evaluating the serious eye damage/eye irritation potential of
unknown test chemicals or product class, or for evaluating the relative ocular toxicity potential of aclassified chemical within a specific range of positive responses.

DATA AND REPORTING
Data
47.
Data from individual replicate tissues in a run (e.g., OD values/MTT formazan 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 (if n≥3 replicate tissues) for each individual test chemical andcontrol should be reported. Any observed interferences of a test chemical with the measurement of MTT
formazan through direct MTT reduction and/or coloured interference should be reported for each tested
chemical.
Test Report
48.

The test report should include the following information:
Test ChemicalxMono-constituent substance- Chemical identification, such as IUPAC or CAS name(s), CAS registry number(s), SMILES
or InChI code, structural formula, and/or other identifiers;- Physical state, volatility, pH, LogP, molecular weight, chemical class, and additional relevant
physicochemical properties relevant to the conduct of the study, to the extent available;- Purity, chemical identity of impurities as appropriate and practically feasible, etc.;- Treatment prior to testing, if applicable (e.g., warming, grinding);- Storage conditions and stability to the extent available.

xMulti-constituent substance, UVCB and mixture- Characterisation as far as possible by e.g., chemical identity (see above), purity, quantitative
occurrence and relevant physicochemical properties (see above) of the constituents, to theextent available;
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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 ultrapure H2O or Ca 2+/Mg2+-free
DPBS, if applicable;- Justification for the use of a different positive control than neat methyl acetate, 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 MTT formazan, andlinearity range of measuring device (e.g., spectrophotometer);- Description of the method used to quantify MTT formazan;- 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;
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OECD/OCDE- 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 MTT-reducers and/or colouring test chemicals, if
applicable;- Number of tissue replicates used per test chemical and controls (positive control, negative
control, NSMTT, NSCliving and NSCkilled, if applicable);
Results- Tabulation of data from individual test chemicals and control substances for each run(including repeat experiments where applicable) and each replicate measurement,
including OD value or MTT formazan peak area, percent tissue viability, mean percent
tissue viability, Difference between tissue replicates or SD, and final prediction;- If applicable, results of controls used for direct MTT-reducers and/or coloured test chemicals,
including OD value or MTT formazan peak area, %NSMTT, %NSCliving, %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 the define 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
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492

LITERATURE

1) UN. (2015). United Nations Globally Harmonized System of Classification and Labelling of Chemicals(GHS). ST/SG/AC.10/30/Rev.6, Sixth Revised Edition, New York and Geneva: United Nations. Available
at: [http://www.unece.org/fileadmin/DAM/trans/danger/publi/ghs/ghs_rev06/English/ST-SG-AC10-30Rev6e.pdf].
2) OECD. (2012). Guideline for Testing of Chemicals No. 405: Acute Eye Irritation/Corrosion.
Organisationfor
Economic
Cooperation
andDevelopment,
Paris.
Availableat:[http://www.oecd.org/env/testguidelines].
3) OECD. (2013). Guideline for Testing of Chemicals No. 437: Bovine Corneal Opacity and Permeability
Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring
Classification for Eye Irritation or Serious Eye Damage. Organisation for Economic Cooperation andDevelopment, Paris. Available at: [http://www.oecd.org/env/testguidelines].
4) 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].
5) 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].
6) 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 andDevelopment, Paris. Available at: [http://www.oecd.org/env/testguidelines].
7) 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 of Reconstructed
Human Tissue Models for Eye Irritation Testing. ALTEX 27, Special Issue 2010, 261-266.
8) EC EURL ECVAM. (2014). The EURL ECVAM - Cosmetics Europe prospective validation study ofReconstructed 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].
9) 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
19© OECD, (2017)

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Europe study on HPLC/UPLC-spectrophotometry as an alternative endpoint detection system for MTTformazan. 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].
10) 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). Multi-laboratory
validation of SkinEthic HCE test method for testing serious eye damage/eye irritation using liquid
chemicals. Toxicol. In Vitro 31, 43-53.
11) 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 ofthe test method with regard to solid and liquid chemicals testing. Toxicol. In Vitro 34, 55-70.
12) 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].
13) EC EURL ECVAM. (2016). Recommendation on the Use of the Reconstructed human Cornea-like
Epithelium (RhCE) Test Methods for Identifying Chemicals not Requiring Classification and Labellingfor Serious Eye Damage/Eye Irritation According to UN GHS. (Manuscript in Preparation).
14) 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.
15) 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.
16) Mosmann, T. (1983). Rapid Colorimetric Assay for Cellular Growth and Survival: Application to
Proliferation and Cytotoxicity Assays. J. Immunol. Methods 65, 55-63.
17) OECD. (2016). Series on Testing and Assessment No. 216: 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 and Labelling 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].
18) 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 and Development, Paris. Available at: [http://www.oecd.org/env/testguidelines].
20© OECD, (2017)38

OECD/OCDE

49219) 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 Labelling of Eye
Irritating Chemicals in Response to the Requirements of the EU Cosmetics Directive and REACH
Legislation. Altern. Lab. Anim. 39, 339-364.
20) Nguyen, D.H., Beuerman, R.W., De Wever, B., Rosdy, M. (2003). Three-dimensional construct of thehuman corneal epithelium for in vitro toxicology. In: Salem, H., Katz, S.A. (Eds), Alternative
Toxicological Methods, CRC Press, pp. 147-159.
21) 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., MarrecFairley, 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.
22) 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, 1476-1488.
23) 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.
24) 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 Analysisof the Draize Test for Serious Eye Damage/Eye Irritation: Importance of Understanding the In Vivo
Endpoints Under UN GHS/EU CLP for the Development and Evaluation of In Vitro Test Methods. Arch.
Toxicol. 88, 701-723.
25) 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 ofhistorical serious eye damage/eye irritation in vivo data analysed by drivers of classification to support
the selection of chemicals for development and evaluation of alternative methods/strategies: the Draize
eye test Reference Database (DRD). Arch. Toxicol. 91, 521-547.
26) Meloni, M., De Servi, B., Marasco, D., Del Prete, S. (2011). Molecular mechanism of ocular surface
damage: Application to an in vitro dry eye model on human corneal epithelium. Molecular Vision 17,
113-126.
27) 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.
28) 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.
21© OECD, (2017)

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492

OECD/OCDE

29) 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.
30) 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.
31) Jester, J.V., Li, L., Molai, A., Maurer, J.K. (2001). Extent of Corneal Injury as a Mechanistic Basis for
Alternative Eye Irritation Tests. Toxicol. In Vitro 15, 115–130.
32) 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.
33) Jester, J.V. (2006). Extent of Corneal Injury as a Biomarker for Hazard Assessment and the Development of
Alternative Models to the Draize Rabbit Eye Test. Cutan. Ocul. Toxicol. 25, 41–54.
34) 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/].
35) 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/].
36) 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.
37) Kaluzhny, Y., Kandárová, H., Handa, Y., DeLuca, J., Truong, T., Hunter, A., Kearney, P., d'ArgembeauThornton, L., Klausner, M. (2015). EpiOcular™ Eye Irritation Test (EIT) for Hazard Identification andLabeling of Eye Irritating Chemicals: Protocol Optimization for Solid Materials and Extended Shipment
Times. Altern. Lab Anim. 43, 101-127.
38) US FDA (2001). Guidance for Industry: Bioanalytical Method Validation. U.S. Department of Health and
HumanServices,
Food
and
Drug
Administration.
May
2001.
Availableat:[http://www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf].
39) OECD (2017). 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.

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ANNEX 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 (16).
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 determinationof chemicals not requiring classification and labelling (negative outcome) from other chemicals (positive
outcome).
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.
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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.
Hazard: Inherent property of an agent or situation having the potential to cause adverse effects when an
organism, system or (sub) population is exposed to that agent.
HCE: SkinEthic™ Human Corneal Epithelium.
HPLC: High Performance Liquid Chromatography.
IC50: Concentration at which a benchmark chemical reduces the viability of the tissues by 50% following afixed exposure time (e.g., 30 minutes treatment with SDS).
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 substanceis the result of a manufacturing process. The difference between mixture and multi-constituent substance isthat a mixture is obtained by blending of two or more substances without chemical reaction. A multiconstituent substance is the result of a chemical reaction.
MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide.
Negative control: A sample containing all components of a test system and treated with a substance known
not to induce a positive response in the test system. This sample is processed with test chemical-treatedsamples 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.
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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 (16).
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-treated samples
and other control samples. To ensure that variability in the positive control response across time can beassessed, the magnitude of the positive response should not be excessive.
Relevance: Description of relationship of the test to the effect of interest and whether it is meaningful anduseful for a particular purpose. It is the extent to which the test correctly measures or predicts the
biological effect of interest. Relevance incorporates consideration of the accuracy (concordance) of a test
method (16).
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 (16).
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 (16).
Reproducibility: The agreement among results obtained from repeated testing of the same test chemicalusing the same test protocol (See "Reliability") (16).
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 (16).
Serious eye damage: Production of tissue damage in the eye, or serious physical decay of vision, following
application of a test substance to the anterior surface of the eye, which is not fully reversible within 21 daysof application. Interchangeable with “Irreversible effects on the eye” and with “UN GHS Category 1” (1).
25
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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 important considerationin assessing the relevance of a test method (16).
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.
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).
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
the existing information at a given tier, no additional testing is required (16).
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”.
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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 (16).
Validated test method: A test method for which validation studies have been completed to determine therelevance (including accuracy) and reliability for a specific purpose. It is important to note that a validated
test method may not have sufficient performance in terms of accuracy and reliability to be found acceptable
for the proposed purpose (16).
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 substance.

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OECD/OCDEANNEX IIAt least 2

Number of tissue
replicates© OECD, (2017)28

At least 2

0.6 cm2

0.6 cm2

Model surface

Protocols

Solids(not pipetteable)

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

Test Method
Components

At least 2

0.5 cm2

Liquids and viscous(pipetteable)

At least 2

0.5 cm2

Solids(not pipetteable)

SkinEthic™ HCE EIT(VRM 2)

MAIN TEST METHOD COMPONENTS OF THE RhCE TEST METHODS VALIDATED FOR IDENTIFYING CHEMICALS NOTREQUIRING CLASSIFICATION AND LABELLING FOR EYE IRRITATION OR SEERIOUS EYE DAMAGE

492

47

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)Æ if the OD of the test chemical atÆ if the OD of the test chemical
570±20 nm, after subtraction of theat 570±20 nm after subtraction of
OD for isopropanol or water is >the OD for isopropanol or water
0.08 (which corresponds tois > 0.08 (which corresponds toapproximately 5% of the mean OD
approximately 5% of the meanof the negative control), living
OD of the negative control),adapted controls should be
living adapted controls should be
performed.
performed.

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)

EpiOcular™ EIT(VRM 1)© OECD, (2017)29

50 μL + 1 mL MTT 1 mg/mL
50 mg + 1 mL MTT 1 mg/mL
solution for 180±15 min
solution for 180±15 min
at 37±2ºC, 5±1% CO2, ≥95% RH at 37±2ºC, 5±1% CO2, ≥95% RH
Æ if solution turns
Æ if solution turns blue/purple,
Pre-check for direct
blue/purple,freeze-killed adapted
freeze-killed adapted controls
MTT reduction
controls should be performed
should be performed(50 μL of sterile deionized water in (50 μL of sterile deionized water
MTT solution is used as negativein MTT solution is used ascontrol)negative control)

Pre-checkfor colour
interference

Test Method
Components

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
should be performed(30 μL of sterile deionized waterin MTT solution is used as
negative control)Æ if test chemical is coloured,
living adapted controls shouldbe performed

492

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

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

SkinEthic™ HCE EIT(VRM 2)

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

OECD/OCDE

48
3 times in 100 mLof Ca2+/Mg2+-free DPBS

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

Rinsing attemperature

50 μL H2O
Tested concurrently

50 μL H2O
Tested concurrently© OECD, (2017)30

18 h (r 0.25 h) in culture medium
at 37±2ºC, 5±1% CO2, ≥95%
RH

120 min (r 15 min) in culture
medium at 37±2ºC, 5±1% CO2,≥95% RH

Post-exposure
incubation

Negative control

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

12 min (r 2 min) at RT in culture
mediumPost-exposure
immersionroom

6 hours (r 0.25 h)
in culture medium
at 37±2ºC, 5±1% CO2, ≥95% RH

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

Exposuretime andtemperature

18 h (r 0.5 h) in culture medium
at 37±2ºC, 5±1% CO2, ≥95%
RH
30 ± 2μL Ca2+/Mg2+-free DPBS
Tested concurrently

30 ± 2μL Ca2+/Mg2+-free DPBS
Tested concurrently

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

30 min (r 2 min) at 37oC, 5%
CO2, 95% RH in culture
mediumnone

25 mL Ca2+/Mg2+-free DPBS

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

20 mL Ca2+/Mg2+-free DPBS

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

50 mg (83.3 mg/cm2) using acalibrated tool (e.g., a levelled For viscous, use a nylon meshspoonful calibrated to hold 50 mg
of sodium chloride).

50 μL (83.3 μL/cm2)

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

20 μL Ca2+/Mg2+-free DPBS
20 μL Ca2+/Mg2+ -free DPBS
for 30 r 2 min at 37±2ºC, 5±1% for 30r2 min at 37±2ºC, 5±1%
CO2, ≥95% RH, protected from CO2, ≥95% RH, protected fromlight.
light.
10 μL Ca2+/Mg2+-free DPBS
+ 30 r 2 μL (60 μL/cm2)

SkinEthic™ HCE EIT(VRM 2)

EpiOcular™ EIT(VRM 1)

OECD/OCDE

Treatment doses
and application

Pre-treatment

Test Method
Components

492

49

© OECD, (2017)31

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

Quality

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

TissueControl

30 min treatment with SDS (50μL)
1.0 mg/mL ≤ IC50 ≤ 3.5 mg/mL

30 min treatment with SDS (50μL)
1.0 mg/mL ≤ IC50 ≤ 3.2 mg/mL

570 nm (540 - 600 nm)
without reference filter

570 nm (550-590 nm)
without reference filter

570 nm (550 - 590 nm)
without reference filter

570 nm (540 - 600 nm)
without reference filter

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

4 h with shaking (~120 rpm) at
RT or at least overnight
without shaking at 4-10°C

2-3 h with shaking (~120 rpm) at
RT or overnight at 4-10°C

2-3 h with shaking (~120 rpm) at
RT or overnight at 4-10°C

Extraction time andtemperature

OD reading

1.5 mL isopropanol(extraction from bottom of
insert)

1.5 mL isopropanol(extraction from top and bottom
of insert)

2 mL isopropanol
2 mL isopropanol(extraction from top and bottom of (extraction from bottom of insert
insert by piercing the tissue)
by piercing the tissue)

Extraction solvent

180 min (r 15 min) at 37±2ºC,
5±1% CO2, ≥95% RH

300 μL 1 mg/mL

30 ± 2μL Methyl acetate
Tested concurrently

180 min (r 15 min) at 37±2ºC,
5±1% CO2, ≥95% RH

300 μL 1 mg/mL

30 ± 2μL Methyl acetate
Tested concurrently

SkinEthic™ HCE EIT(VRM 2)492

180 min (r 15 min) at 37±2ºC,
5±1% CO2, ≥95% RH

180 min (r 15 min) at 37±2ºC,
5±1% CO2, ≥95% RH

300 μL 1 mg/mL

300 μL 1 mg/mL

MTT solution

MTT incubation
time andtemperature

50 μL Methyl acetate
Tested concurrently

50 μL Methyl acetate
Tested concurrently

EpiOcular™ EIT(VRM 1)

Positive control

Test Method
Components

OECD/OCDE

50
EpiOcular™ EIT(VRM 1)

OECD/OCDE© OECD, (2017)32

1. Mean OD of the tissue
replicates treated with thenegative control should be >1.0 and ≤ 2.5
2. Mean viability of the tissue
replicates exposed for 4 hours
with the positive control,
expressed as % of the negative
control, should be ≤ 20%
3. The difference of viability
between two tissue replicatesshould be less than 20%.

SkinEthic™ HCE EIT(VRM 2)

1. Mean OD of the tissue replicates 1. Mean OD of the tissue 1. Mean OD of the tissue
treated with the negative control replicates treated with the replicates treated with theshould be > 0.8 and < 2.5
negative control should be > 0.8 negative control should be >2. Mean viability of the tissue and < 2.5
1.0 and ≤ 2.5
replicates exposed for 30 min 2. Mean viability of the tissue 2. Mean viability of the tissue
with the positive control, replicates exposed for 6 hours replicates exposed for 30 min
Acceptance Criteria
expressed as % of the negative with the positive control, with the positive control,
expressed as % of the negative expressed as % of the negative
control, should be < 50%
3. The difference of viability control, should be < 50%
control, should be ≤ 30%
between two tissue replicates 3. The difference of viability 3. The difference of viability
between two tissue replicates between two tissue replicatesshould be less than 20%.
should be less than 20%.
should be less than 20%.

Test Method
Components

492OECD/OCDE

492

ANNEX IIIILLUSTRATIVE FLOWCHART PROVIDING GUIDANCE ON HOW TO IDENTIFY AND
HANDLE DIRECT MTT-REDUCERS AND/OR COLOUR INTERFERING CHEMICALS, BASEDON 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

33© OECD, (2017)

51

492

OECD/OCDEANNEX IVILLUSTRATIVE FLOWCHART PROVIDING GUIDANCE ON HOW TO IDENTIFY AND
HANDLE DIRECT MTT-REDUCERS AND/OR COLOUR INTERFERING CHEMICALS, BASEDON 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

34© OECD, (2017)52

Final %viability =%uncorrected test viability %NSCliving

No controls
are requiredOECD/OCDE

492

ANNEX VKEY PARAMETERS AND ACCEPTANCE CRITERIA FOR QUALIFICATION OF AN
HPLC/UPLC-SPECTROPHOTOMETRY SYSTEM FOR MEASUREMENT OFMTT FORMAZAN EXTRACTED FROM RhCE TISSUE CONSTRUCTS
Parameter

Selectivity

Precision

Protocol Derived from FDA Guidance (36)(38)
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)

Accuracy

Quality Controls in isopropanol (n=5)

Matrix EffectQuality Controls in living blank (n=5)

CarryoverAnalysis of isopropanol after an ULOQ2 standard

Reproducibility(intra-day)

Reproducibility(inter-day)
Short Term Stabilityof MTT Formazan in
RhCE Tissue Extract
Long Term Stabilityof MTT Formazan in
RhCE Tissue
Extract, if required

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 ofthe preparation and after 24 hours of storage at room
temperature
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%

1

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.

2

35© OECD, (2017)53

54