᪂つヨ㦂ἲᥦ᱌᭩             ᖹᡂ 29 ᖺ 1 ᭶ 6 ᪥                                 No. 2016-02║่⃭ᛶヨ㦂௦᭰ἲ ෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝἲ(Reconstructed Human Cornea-like Epithelium Test Method: RhCEἲ)࡟㛵ࡍࡿᥦ᱌ ᖹᡂ 28 ᖺ 7 ᭶ 5 ᪥䛻ᮾிࠊᅜ❧་⸆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 damage͇ࢆࡶ࡜࡟ࠊ║่⃭ᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍࡟ࡼࡾసᡂࡉࢀࡓࠕ෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝἲホ౯ሗ࿌᭩ࠖࢆ⏝࠸࡚ࠊJaCVAM
ホ౯఍㆟ࡀホ౯࠾ࡼࡧ᳨ウࡋࡓ⤖ᯝࠊࡑࡢ᭷⏝ᛶࡀ☜ㄆࡉࢀࡓࡇ࡜࠿ࡽసᡂࡉࢀࡓࠋ ௨ୖࡢ⌮⏤࡟ࡼࡾࠊ⾜ᨻᙜᒁࡢᏳ඲ᛶホ౯᪉ἲ࡜ࡋ࡚෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝἲࡢ౑⏝ࢆᥦ᱌ࡍࡿࡶࡢ࡛࠶ࡿࠋ
኱㔝Ὀ㞝኱㔝Ὀ㞝                      すᕝ⛅ె
JaCVAM ホ౯఍㆟ ㆟㛗               JaCVAM 㐠Ⴀጤဨ఍ ጤဨ㛗

i

JaCVAM ホ౯఍㆟኱ 㔝 Ὀ 㞝 㸦㐠Ⴀጤဨ఍᥎⸀㸧㸸ᗙ㛗㣤 ሯ ᑦ ᩥ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧஬༑ᔒⰋ᫂㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧▼ ஭ 㞝 ஧ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧ᒾ℩⿱⨾Ꮚ㸦᪥ᮏ〇⸆ᕤᴗ༠఍㸧㔠Ꮚ࿴ᘯ㸦᪥ᮏ໬Ꮫᕤᴗ༠఍㸧⠛ ⏣ ࿴ ಇ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧ᮡᒣ┿⌮Ꮚ㸦᪥ᮏ໬⢝ရᕤᴗ㐃ྜ఍㸧㇂ᕝᾈᏊ㸦᪥ᮏື≀ᐇ㦂௦᭰ἲᏛ఍㸧すᕝ⛅ె㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧∾ ᰤ஧㸦᪥ᮏච␿ẘᛶᏛ఍㸧᳃⏣ ೺㸦᪥ᮏ⎔ቃኚ␗ཎᏛ఍㸧ᒣ⏣㝯ᚿ㸦⊂❧⾜ᨻἲே 〇ရホ౯ᢏ⾡ᇶ┙ᶵᵓ㸧ᶓ㛵༤㞝㸦᪥ᮏ⓶⭵࢔ࣞࣝࢠ࣮࣭᥋ゐ⓶⭵⅖Ꮫ఍㸧ྜྷ⏣Ṋ⨾㸦᪥ᮏẘᛶᏛ఍㸧ྜྷᮧ ຌ㸦ᗙ㛗᥎⸀㸧                  ௵ᮇ㸸ᖹᡂ 26 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 28 ᖺ 3 ᭶ 31 ᪥                 JaCVAM ホ౯఍㆟኱ 㔝 Ὀ 㞝 㸦㐠Ⴀጤဨ఍᥎⸀㸧㸸ᗙ㛗㣤 ሯ ᑦ ᩥ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧஬༑ᔒⰋ᫂ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧▼ ஭ 㞝 ஧ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧஭ ୖ ᬛ ᙲ 㸦᪥ᮏච␿ẘᛶᏛ఍㸧௒ ஭ ᩍ Ᏻ 㸦᪥ᮏື≀ᐇ㦂௦᭰ἲᏛ఍㸧ᒾ℩⿱⨾Ꮚ 㸦᪥ᮏ〇⸆ᕤᴗ༠఍㸧⠛ ⏣ ࿴ ಇ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧ᮡᒣ┿⌮Ꮚ 㸦᪥ᮏ໬⢝ရᕤᴗ㐃ྜ఍㸧௰ ஭ ಇ ྖ 㸦᪥ᮏ໬Ꮫᕤᴗ༠఍㸧୰ᮧࡿࡾࡇ 㸦⊂❧⾜ᨻἲே 〇ရホ౯ᢏ⾡ᇶ┙ᶵᵓ㸧す ᕝ ⛅ ె 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧἟ ⃝  ⪽ 㸦᪥ᮏẘᛶᏛ఍㸧᳃ ⏣  ೺ 㸦᪥ᮏ⎔ቃኚ␗ཎᏛ఍㸧ᶓ 㛵 ༤ 㞝 㸦᪥ᮏ⓶⭵࢔ࣞࣝࢠ࣮࣭᥋ゐ⓶⭵⅖Ꮫ఍㸧                ௵ᮇ㸸ᖹᡂ 28 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 30 ᖺ 3 ᭶ 31 ᪥ii


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ᑠᕝஂ⨾Ꮚ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ⑓⌮㒊㸧ຍ ⸨  ⠜ 㸦ᅜ❧ឤᰁ⑕◊✲ᡤ㸧᪥ୗ㒊ဴஓ 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⠛ ⏣ ࿴ ಇ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧㛵 㔝 ♸ Ꮚ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ⸆⌮㒊㸧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୰ ᮧ 㧗 ᩄ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧᪥ ⏣  ඘ 㸦ཌ⏕ປാ┬ ་⸆࣭⏕ά⾨⏕ᒁ ་⸆ရᑂᰝ⟶⌮ㄢ ໬Ꮫ≀㉁Ᏻ඲ᑐ⟇ᐊ㸧ᗈ ℩ ᫂ ᙪ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ 
Ᏻ඲ᛶண

ホ౯㒊㸧

ᮏ 㛫 ṇ ඘ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ኚ␗㑇ఏ㒊㸧୕ ⃝  㤾 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 Reconstructed human Cornea-like Epithelium Eye Irritation Test Method
At a meeting held on 5 July 2016 at the National Institute of Health Sciences (NIHS) in Tokyo,
Japan, the Japanese Center for the Validation of Alternative Methods (JaCVAM) Regulatory
Acceptance Board unanimously endorsed the following statement:
Proposal: The Reconstructed human Cornea-like Epithelium (RhCE) test method 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 andlabelling for 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 irritationor serious eye damage” as well as a validation report on the RhCE test method prepared by the
Ocular Irritation Testing JaCVAM Editorial Committee to acknowledge that the results of areview and study by the JaCVAM Regulatory Acceptance Board have confirmed the usefulnessof this assay.
Based on the above, we propose the RhCE test method as a useful means for safety assessment
by regulatory agencies.

Chairperson

Akiyoshi Nishikawa
Chairperson

JaCVAM Regulatory Acceptance BoardJaCVAM Steering Committee

Yasuo Ohno

6 January 2017v

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 (nominee by JaCVAM Steering Committee) : Chairperson
Mr. Naofumi Iizuka (Pharmaceuticals and Medical Devices Agency)
Mr. Yoshiaki Ikarashi (National Institute of Health Sciences: NIHS)
Mr. Yuji Ishii (Biological Safety Research Center: BSRC, NIHS)
Ms. Yumiko Iwase (Japan Pharmaceutical Manufacturers Association)
Mr. Kazuhiro Kaneko (Japan Chemical Industry Association)
Mr. Eiji Maki (Japanese Society of Immunotoxicology)
Mr. Takeshi Morita (Japanese Environmental Mutagen Society)
Mr. Akiyoshi Nishikawa (BSRC,NIHS)
Mr. Kazutoshi Shinoda (Pharmaceuticals and Medical Devices Agency)
Ms. Mariko Sugiyama (Japan Cosmetic Industry Association)
Ms. Koko Tanigawa (Japanese Society for Alternatives to Animal Experiments)
Mr. Takashi Yamada (National Institute of Technology and Evaluation)
Mr. Hiroo Yokozeki (Japanese Society for Dermatoallergology and Contact Dermatitis)
Mr. Takemi Yoshida (Japanese Society of Toxicology)
Mr. Isao Yoshimura (nominee by Chairperson)
Term: From 1st April 2014 to 31st March 2016

Mr. Yasuo Ohno (nominee by JaCVAM Steering Committee) : 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 (BSRC, NIHS)
Ms. Yumiko Iwase (Japan Pharmaceutical Manufacturers Association)
Mr. Takeshi Morita (Japanese Environmental Mutagen Society)
Mr. Shunji Nakai (Japan Chemical Industry Association)
Ms. Ruriko Nakamura (National Institute of Technology and Evaluation)
Mr. Akiyoshi Nishikawa (BSRC, NIHS)
Mr. Satoshi Numazawa (Japanese Society of Toxicology)
Mr. Kazutoshi Shinoda (Pharmaceuticals and Medical Devices Agency)
Ms. Mariko Sugiyama (Japan Cosmetic Industry Association)
Mr. Hiroo Yokozeki (Japanese Society for Dermatoallergology and Contact Dermatitis)
Term: From 1st April 2016 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. Mitsuru Hida (Ministry of Health, Labour and Welfare)
Ms. Yoko Hirabayashi (Division of Toxicology, BSRC, NIHS)
Mr. Akihiko Hirose (Division of Risk Assessment, BSRC, NIHS)
Mr. Masamitsu Honma (Division of Genetics and Mutagenesis, BSRC, NIHS)
Mr. Atsushi Kato (National Institute of Infectious Diseases)
Mr. Tetsuya Kusakabe (Ministry of Health, Labour and Welfare)
Mr. Kaoru Misawa (Ministry of Health, Labour and Welfare)
Mr. Takatoshi Nakamura (Pharmaceutical & Medical Devices Agency)
Ms. Kumiko Ogawa (Division of Pathology, BSRC, NIHS)
Ms. Yuko Sekino (Division of Pharmacology, BSRC, NIHS)
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. Hajime Kojima (Division of Risk Assessment, BSRC, NIHS): Secretary

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║่⃭ᛶヨ㦂௦᭰ἲ ෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝἲ(Reconstructed Human Cornea-like Epithelium Test Method: RhCEἲ)┠  ḟ
ホ౯఍㆟ሗ࿌᭩ ------------------------------------------------------------------------------- 1
ホ౯ሗ࿌᭩ ------------------------------------------------------------------------------------- 7
OECD GUIDELINE FOR THE TESTING OF CHEMICALS 492-------------19ixx║่⃭ᛶヨ㦂௦᭰ἲࡢホ౯఍㆟ሗ࿌᭩෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝἲ(Reconstructed Human Cornea-like Epithelium Test Method: RhCE ἲ)

JaCVAM ホ౯఍㆟

ᖹᡂ 28 ᖺ㸦2016 ᖺ㸧7 ᭶ 5 ᪥

11

JaCVAM ホ౯఍㆟኱ 㔝 Ὀ 㞝 㸦㐠Ⴀጤဨ఍᥎⸀㸧㸸ᗙ㛗㣤 ሯ ᑦ ᩥ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧஬༑ᔒⰋ᫂㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧▼ ஭ 㞝 ஧ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧ᒾ℩⿱⨾Ꮚ 㸦᪥ᮏ〇⸆ᕤᴗ༠఍㸧㔠Ꮚ࿴ᘯ㸦᪥ᮏ໬Ꮫᕤᴗ༠఍㸧⠛ ⏣ ࿴ ಇ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧ᮡᒣ┿⌮Ꮚ 㸦᪥ᮏ໬⢝ရᕤᴗ㐃ྜ఍㸧㇂ᕝᾈᏊ㸦᪥ᮏື≀ᐇ㦂௦᭰ἲᏛ఍㸧すᕝ⛅ె㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧∾ ᰤ஧㸦᪥ᮏච␿ẘᛶᏛ఍㸧᳃⏣ ೺㸦᪥ᮏ⎔ቃኚ␗ཎᏛ఍㸧ᒣ⏣㝯ᚿ㸦⊂❧⾜ᨻἲே 〇ရホ౯ᢏ⾡ᇶ┙ᶵᵓ㸧ᶓ㛵༤㞝㸦᪥ᮏ⓶⭵࢔ࣞࣝࢠ࣮࣭᥋ゐ⓶⭵⅖Ꮫ఍㸧ྜྷ⏣Ṋ⨾㸦᪥ᮏẘᛶᏛ఍㸧ྜྷᮧ ຌ㸦ᗙ㛗᥎⸀㸧                  ௵ᮇ㸸ᖹᡂ 26 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 28 ᖺ 3 ᭶ 31 ᪥                 JaCVAM ホ౯఍㆟኱ 㔝 Ὀ 㞝 㸦㐠Ⴀጤဨ఍᥎⸀㸧㸸ᗙ㛗㣤 ሯ ᑦ ᩥ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧஬༑ᔒⰋ᫂ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧▼ ஭ 㞝 ஧ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧஭ ୖ ᬛ ᙲ 㸦᪥ᮏච␿ẘᛶᏛ఍㸧௒ ஭ ᩍ Ᏻ 㸦᪥ᮏື≀ᐇ㦂௦᭰ἲᏛ఍㸧ᒾ℩⿱⨾Ꮚ 㸦᪥ᮏ〇⸆ᕤᴗ༠఍㸧⠛ ⏣ ࿴ ಇ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧ᮡᒣ┿⌮Ꮚ 㸦᪥ᮏ໬⢝ရᕤᴗ㐃ྜ఍㸧௰ ஭ ಇ ྖ 㸦᪥ᮏ໬Ꮫᕤᴗ༠఍㸧୰ᮧࡿࡾࡇ 㸦⊂❧⾜ᨻἲே 〇ရホ౯ᢏ⾡ᇶ┙ᶵᵓ㸧す ᕝ ⛅ ె 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧἟ ⃝  ⪽ 㸦᪥ᮏẘᛶᏛ఍㸧᳃ ⏣  ೺ 㸦᪥ᮏ⎔ቃኚ␗ཎᏛ఍㸧ᶓ 㛵 ༤ 㞝 㸦᪥ᮏ⓶⭵࢔ࣞࣝࢠ࣮࣭᥋ゐ⓶⭵⅖Ꮫ఍㸧                ௵ᮇ㸸ᖹᡂ 28 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ἲࡢ௦᭰ἲ࡜ࡋ࡚ࠊ⿕㦂≀㉁ࡢࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝ⤌⧊࡟ᑐࡍࡿ⣽⬊ẘᛶࢆᣦᶆ࡟⏝࠸ࠊࡑࡢ≀㉁ࡢ║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛࠶ࡿࠋ
࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾࠸࡚ United Nations Globally Harmonized System of Classification and Labelling ofChemicals㸦UN GHS㸧༊ศእ≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚ࣂࣜࢹ࣮ࢩࣙࣥࡀ⾜ࢃࢀ 1)ࠊ2015 ᖺ࡟ OECD
TG492 ࡜ࡋ࡚᥇ᢥࡉࢀࡓ 2㸧ࠋJaCVAM ホ౯఍㆟ࡣࠊ║่⃭ᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍࡟ࡼࡾసᡂࡉࢀࡓࠕ෌
ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝἲ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㸧௦᭰ࡍࡿᑐ㇟ẘᛶヨ㦂㸸 Draize ║่⃭ᛶヨ㦂ἲ
ヨ 㦂 ἲ ࡢ ᴫ ␎ 㸸  RhCE ἲ ࡣ ࠊ ෌ ᵓ ⠏ ࣄ ࢺ ゅ ⭷ ᵝ ୖ ⓶ ࣔ ࢹ ࣝ 㸦 Reconstructed human Cornea-like
Epithelium:RhCE㸧⤌⧊ࢆ⏝࠸ࡓ║่⃭ᛶヨ㦂࡛࠶ࡿࠋRhCE ἲࡢ୍ࡘ࡛࠶ࡿ EpiOcularTM ࢆ⏝࠸ࡿ║่⃭ᛶヨ㦂ἲ㸦EpiOcularTM EIT㸧࡛ࡣࠊ⿕㦂≀㉁ࡀᾮయࡢሙྜࡣ⣙ 30 ศ㛫ࠊᅛయࡢሙྜࡣ⣙ 6 ᫬㛫ࠊࣄࢺゅ⭷ᵝୖ⓶ᵓ㐀ࢆ᭷ࡍࡿ⤌⧊࡟᭚㟢ࡋࡓᚋࠊMTT ࡢྲྀࡾ㎸ࡳ㔞ࢆࡶ࡜࡟ࡋࡓ⣽⬊⏕Ꮡ⋡ࢆᐃࡋࠊ║่⃭ᛶホ౯ࡢᣦᶆ࡜ࡋ࡚⏝࠸ࡿࠋࡇࢀࡣࠊMTT ࡀ࣑ࢺࢥࣥࢻࣜ࢔ࡢ⬺Ỉ⣲㓝⣲ࡢᇶ㉁࡜࡞ࡿ
ᛶ㉁ࢆ฼⏝ࡋࠊ⣽⬊ෆ࡟ྲྀࡾ㎸ࡲࢀࡓ MTT ࡀ⬺Ỉ⣲㓝⣲࡟ࡼࡾ㑏ඖࡉࢀࠊ⏕ᡂࡉࢀࡓ࣐࣍ࣝࢨࣥ㔞㸦㟷Ⰽ㸧ࡣ⏕Ꮡ⣽⬊ᩘ࡟ẚ౛ࡍࡿࡇ࡜࡟ᇶ࡙࠸࡚࠸ࡿ㸦MTT 㑏ඖἲ㸧ࠋ 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⭷ୖ⓶ᵝᵓ㐀ࢆ᭷ࡍࡿ RhCE ⤌⧊㸦EpiOcularTM㸧ࢆ⏝࠸࡚ࠊ⿕㦂≀㉁ࡢ⣽⬊ẘᛶࢆᣦᶆ࡜ࡋ࡚║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛࠶ࡿࠋࡇࢀࡽࡢࡇ࡜࠿ࡽࠊᮏヨ㦂ἲࡣ࢘ࢧࢠࢆ⏝࠸ࡿ║่⃭ᛶヨ㦂ࡢ௦᭰ἲ࡜ࡋ࡚⛉Ꮫⓗጇᙜᛶࡀ࠶ࡿࠋ
EpiOcularTM EIT ࡟ࡘ࠸࡚ࡣࠊEURL ECVAM(European Union Reference Laboratory for Alternatives toAnimal Testing)࡜ CE(Cosmetics Europe)ࡢඹྠࣂࣜࢹ࣮ࢩࣙࣥヨ㦂 1)ࡀ⾜ࢃࢀࡓࠋࡑࡢヨ㦂࡛ࡣࠊṇ☜ᛶࡢホ౯ࡢࡓࡵ࡟ᚲせ࡞ື≀ヨ㦂ࡢࢹ࣮ࢱࢆᣢࡗ࡚࠸ࡿࡇ࡜ࢆ๓ᥦ᮲௳࡟ࠊUN GHS ༊ศ≀㉁࣭༊ศእ≀㉁ࡸᅛయ࣭ᾮయ➼ࡢࣂࣛࣥࢫࢆ⪃៖ࡋ࡚⿕㦂≀㉁ࡢ㑅ᢥࡀ⾜ࢃࢀࡓࠋࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡣࠊࢥ࣮ࢻ໬
ࡉࢀࡓ 107 ≀㉁࡟ࡘ࠸࡚⾜ࢃࢀࡓࠋࡑࡢᚋࣉࣟࢺࢥࣝࡢ᭱㐺໬ࢆ⾜ࡗ࡚ࠊ᪂ࡓ࡞ᅛయ 8 ≀㉁ࢆ㏣ຍࡋࠊ
115 ≀㉁࡛ホ౯ࡉࢀࡓࠋ

3
3

EpiOcularTM EIT ࡣࠊ෌⌧ᛶࡶṇ☜ᛶࡶ EURL ECVAM ࣂࣜࢹ࣮ࢩࣙࣥ㐠Ⴀጤဨ఍ࡢᐃࡵࡓᇶ‽ࢆ‶ࡓࡋ࡚࠾ࡾࠊࡉࡽ࡟ ESAC ࡢ➨୕⪅ホ౯ 4)ࢆ⤒࡚ࠊUN GHS ༊ศእ≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚ 2015 ᖺ 7 ᭶
࡟ OECD ࡟ࡼࡾヨ㦂࢞࢖ࢻࣛ࢖ࣥ࡟᥇ᢥࡉࢀࡓ㸦TG492㸧2)ࠋJaCVAM ║่⃭ᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍ࡣࠊ
ࡇࢀࡽࡢ㈨ᩱࢆ⏝࠸࡚ᮏヨ㦂ἲࢆホ౯ࡋ࡚࠾ࡾࠊ⛉Ꮫⓗ࡟ጇᙜ࡛࠶ࡿ࡜⪃࠼ࡿࠋ㸱㸬ᮏヨ㦂ἲࡢ᭷⏝ᛶ࡜㐺⏝㝈⏺
EpiOcularTM ࡣᕷ㈍ࡉࢀ࡚࠾ࡾࠊ⡿ᅜࡢ〇㐀ඖࡼࡾධᡭ࡛ࡁࡿࠋRhCE ⤌⧊௨እࡣࠊ≉Ṧ࡞ᶵᮦࡸヨ⸆ࢆᚲせ࡜ࡏࡎࠊ≉ู࡞ᡭᢏࡶᚲせ࡞࠸ࡇ࡜࠿ࡽᢏ⾡⛣㌿ᛶࡣ㧗࠸࡜ุ᩿࡛ࡁࡿࠋࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟࠾࠸࡚ࡶࠊཧຍ᪋タࡢᢏ⾡⪅࡟ᑐࡋ࡚ᢏ⾡⩦ᚓࡢࡓࡵࡢࢺ࣮ࣞࢽࣥࢢࡀ⾜ࢃࢀࡓ⤖ᯝࠊᢏ⾡⛣㌿ᛶ࡟ၥ㢟ࡀ࡞࠿ࡗࡓࡇ࡜ࡀ☜ㄆࡉࢀ࡚࠸ࡿࠋణࡋࠊEpiOcularTM EIT ࢆᐇ᪋ࡍࡿヨ㦂᪋タࡢᢏ⾡⩦ᚓࢆ☜࠿ࡵࡿࡓࡵࡢ⇍㐩ᗘ☜ㄆ≀㉁ࡢ୍ぴࡀ TG492 ࡟ᐃࡵࡽࢀ࡚࠾ࡾࠊࡑࢀࡽࢆ⏝࠸࡚ᢏ⾡⛣㌿ࡀ㐩ᡂ࡛ࡁࡓࡇ࡜
ࢆ☜࠿ࡵࡿᚲせࡀ࠶ࡿࠋUN GHS ༊ศእ≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚ࡢጇᙜᛶࢆㄪ࡭ࡿࡓࡵࡢࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟࠾࠸࡚᪋タෆ෌⌧ᛶࡣࠊUN GHS ศ㢮ุᐃࡢ᪋タࡈ࡜ࡢ᪋タෆ෌⌧ᛶࡣ EURL ECVAM-CE ࣂࣜࢹ࣮ࢩࣙࣥ㐠Ⴀጤဨ఍ࡀᐃࡵࡓᇶ‽㸦85%௨ୖ㸧ࢆ‶ࡓࡋ࡚࠸ࡓ 1)ࠋࡲࡓࠊ᪋タ㛫෌⌧ᛶ࡟㛵ࡋ࡚ࡣࠊᮏヨ㦂࡛ᚓࡽࢀࡓ⣽⬊⏕Ꮡ⋡㸦」ᩘᅇヨ㦂㸧ࡢᖹᆒ್ࢆࡶ࡜࡟ UN GHS ศ㢮ุᐃࢆ᪋タࡈ࡜࡟⾜࠸ࠊࡑࡢ୍⮴ᗘࢆ᳨ウࡋࡓ㸦ᅛయ≀㉁ࡢࢹ࣮ࢱࢆྵࡴ㸧ࠋࡑࡢ⤖ᯝࠊࣂࣜࢹ࣮ࢩࣙࣥ㐠Ⴀጤဨ఍ࡀᐃࡵࡓᇶ‽㸦80%௨ୖ㸧ࢆ‶ࡓࡋ࡚࠸ࡓ 1)ࠋ
UN GHS ༊ศእ≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚ヨ㦂ἲࡢṇ☜ᛶ࡟㛵ࡋ࡚ࡣࠊ≀㉁⮬యࡢⰍࡀ࣐࣍ࣝࢨࣥᐃ
㔞࡛ࡢ྾ගᗘ

ᐃࢆጉࡆࡿ 3 ≀㉁࡜ࠊᙉ࠸㑏ඖᛶ≀㉁ 1 ≀㉁ࢆ㝖ࡃ 111 ≀㉁ࡢ⤖ᯝࢆ⏝࠸࡚ホ౯ࡀ⾜ࢃࢀࡓࠋࡑࡢ⤖ᯝࠊୗグࡢࡼ࠺࡟ࠊᾮయ≀㉁ࡢࡳࡢሙྜࠊᅛయ≀㉁ࡢࡳࡢሙྜ࠾ࡼࡧᾮయ≀㉁࣭ᅛయ≀㉁ࢆྵࡵࡓሙྜ࡟ศࡅ࡚ゎᯒࡋࡓሙྜࡢ࠸ࡎࢀ࡟࠾࠸࡚ࡶࠊ୍⮴ᗘࠊഇ㝜ᛶ⋡ࠊഇ㝧ᛶ⋡ࡣࣂࣜࢹ࣮ࢩࣙ
ࣥ㐠Ⴀጤဨ఍ࡀᐃࡵࡓཷࡅධࢀᇶ‽ 1)ࢆ‶ࡓࡋ࡚࠸ࡓࠋ

EpiOcularTM EIT ࡢṇ☜ᛶ≀㉁ᩘ୍⮴ᗘ(%)

ഇ㝜ᛶ⋡(%)

ഇ㝧ᛶ⋡(%)

ᾮయ

52

81.9

1.7

33.3

ᅛయ

59

78.0

6.5

39.3

ᾮయ࣭ᅛయ

111

79.7

4.3

37.0

௦᭰ἲ࡜ࡋ࡚ࡢ
ཷࡅධࢀᇶ‽ 1)

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 75

 10

 40

║่⃭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࠸࡚ࡣࠊTG492 ࡢᮏᩥࡢㄝ᫂࠾ࡼࡧ ANNEX II ࡢࣇ࣮ࣟࢳࣕ
࣮ࢺࢆཧ↷ࡍࡿᚲせࡀ࠶ࡿࠋ44ࡲࡓࠊᮏヨ㦂ἲࢆ㐺⏝ࡍࡿ࡟ࡣࠊヨ㦂ἲࡢᛶ㉁࡜ṇ☜ᛶࡢ☜ಖࢆ⪃៖ࡋ࡚௨ୗࡢไ㝈ࡀタࡅࡽࢀࡿࠋ
1) ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟࠾࠸࡚⿕㦂≀㉁࡟ྵࡵࡽࢀ࡞࠿ࡗࡓẼయ㸦࢞ࢫ㸧࠾ࡼࡧ࢚࢔ࢰ࣮ࣝࡣ㐺⏝≀㉁࠿ࡽ㝖እࡉࢀࡿࠋ
2) UN GHS ༊ศ 1ࠊ༊ศ 2㸦2A࣭2B㸧≀㉁ࡢ᳨ฟ࡟ࡣ⏝࠸ࡿࡇ࡜ࡣ࡛ࡁ࡞࠸ࠋ
ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟⏝࠸ࡽࢀࡓΰྜ≀ࡢᩘࡣ㝈ࡽࢀ࡚࠸ࡓࡀࠊࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡜ࡣู࡟ࠊື≀ヨ㦂ࡢࢹ࣮ࢱࡀ࠶ࡿ㎰⸆ฎ᪉㸦97 ฎ᪉ࠊ࠺ࡕ༊ศእࡣ 43 ฎ᪉㸧ࢆ⏝࠸࡚ホ౯ࡀ⾜ࢃࢀࡓ 5)ࠋ⤖ᯝࡣ୍⮴ᗘ 82%ࠊഇ㝜ᛶ⋡ 9%ࠊഇ㝧ᛶ⋡ 28%࡛ࠊࡇࢀࡽࡣࣂࣜࢹ࣮ࢩࣙࣥ㐠Ⴀጤဨ఍ࡀᐃࡵࡓཷࡅධࢀᇶ‽ࢆ‶ࡓࡋ࡚࠸ࡿࡇ࡜࠿ࡽࠊΰྜ≀࡟ࡶ㐺⏝࡛ࡁࡿྍ⬟ᛶࡀ࠶ࡿࠋ
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♫఍ⓗཷࡅධ࠾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࠸ࡿࠋࡲࡓࠊEpiOcularTM ࡢධᡭࡣᐜ࡛᫆ࠊ▷᫬㛫࡛Ᏻ౯࡟ᐇ᪋࡛ࡁࠊ≉Ṧ࡞ᶵᮦࡸヨ⸆ࢆᚲせ࡜ࡏࡎࠊᚲせ࡞ᡭᢏࡶ」㞧࡞ࡶࡢ࡛࡞࠸ࠋࡋࡓࡀࡗ࡚ࠊ
ධᡭࡋࡓ EpiOcularTM 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 ⾜ᨻୖࡢ฼⏝ᛶ㸸ᮏヨ㦂ἲࡣࠊ໬Ꮫ≀㉁࡟ࡼࡿ║่⃭ᛶࢆホ౯࡛ࡁࠊ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾࠸࡚ UN GHS ༊ศእࢆ᳨ฟ
ࡍࡿ᪉ἲ࡜ࡋ࡚ࠊ⾜ᨻⓗ฼⏝ࡀྍ⬟࡛࠶ࡿ࡜⪃࠼ࡿࠋ࡞࠾ࠊEpiOcularTM EIT ௨እࡢ RhCE ἲࢆ⏝࠸ࡿሙྜ࡟ࡣࠊOECD TG492 ࡢᛶ⬟ᶆ‽࡟グ㍕ࡉࢀࡓ≀㉁ࢆ⏝࠸࡚ 6)ࠊࡑࡢጇᙜᛶࢆ☜ㄆࡋ࡚࠾ࡃᚲせࡀ࠶ࡿࠋ

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ཧ⪃ᩥ⊩
1) 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.
2) OECD (2015) Test Guideline 492. Reconstructed human Cornea-like Epithelium (RhCE) test method for
identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage.
3) JaCVAM ║่⃭ᛶ௦᭰ἲ㈨ᩱ⦅⧩ጤဨ఍㸸OECD TG492 ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝἲ (Reconstructed
Human Cornea-like Epithelium Test Method: RhCEἲ)ホ౯ሗ࿌᭩㸦2016ᖺ1᭶25᪥㸧4) ESAC (2014) ESAC Opinion based on the ESAC Scientific Peer Review of the ECVAM Eye Irritation
Validation Study (EIVS) and a related Cosmetics Europe study on HPLC/UPLC-photometry as an alternative
endpoint detection system for formazan reaction product.
5) Kolle SN et al. (2015) The EpiOcularTM eye irritation test is the method of choice for the in vitro eye irritation
testing of agrochemical formulations: Correlation analysis of EpiOcular Eye Irritation Test and BCOP test dataaccording to UN GHS, US EPA and Brazil ANVISA classification schemes. ATLA. 43, 181-198.
6) OECD (2015) Series on Testing & Assessment 216. Performance standards for the assessment of proposed
similar or modified in vitro Reconstructed human Cornea-like Epithelium (RhCE) test method for eye hazard

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2016 ᖺ 1 ᭶ 25 ᪥║่⃭ᛶヨ㦂௦᭰ἲࡢホ౯ሗ࿌᭩෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝἲ(Reconstructed Human Cornea-like Epithelium Test Method: RhCE ἲ)║่⃭ᛶ௦᭰ἲ㈨ᩱ⦅⧩ጤဨ఍

ᖹᡂ 28 ᖺ㸦2016 ᖺ㸧1 ᭶ 25 ᪥

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2016 ᖺ 1 ᭶ 25 ᪥║่⃭ᛶ௦᭰ἲ㈨ᩱ⦅⧩ጤဨ఍ᑠᆏ ᛅྖ  㸦୍⯡㈈ᅋἲே ṧ␃㎰⸆◊✲ᡤ㸧బࠎᮌ ṇ἞ 㸦࢔ࢵࣦ࢕ྜྠ఍♫㸧➉ෆ ᑠⱑ  㸦P&G ࢖ࣀ࣮࣋ࢩࣙࣥྜྠ఍♫㸧᳃ᮧ ᬛ⨾ 㸦୍⯡㈈ᅋἲே 㣗ရ⸆ရᏳ඲ࢭࣥࢱ࣮ ⛙㔝◊✲ᡤ㸧ྜྷᮧ ຌ  㸦ᮾி⌮⛉኱Ꮫྡ㄃ᩍᤵ㸧

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2016 ᖺ 1 ᭶ 25 ᪥␎ㄒ
CAS: Chemical Abstracts Services
CE: Cosmetics Europe
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
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
SOP: Standard Operating Procedures
TG: Test Guideline
UN: United Nations

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2016 ᖺ 1 ᭶ 25 ᪥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࠶ࡿࠋUN GHS ༊ศእ≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚ EURL ECVAM ࡜ CE ࡢඹྠࣂࣜࢹ࣮ࢩࣙࣥ
ࢆ⤒࡚ 2015 ᖺ࡟ OECD ࡟ࡼࡾࢸࢫࢺ࢞࢖ࢻࣛ࢖ࣥ TG 492 ࡜ࡋ࡚᥇ᢥࡉࢀࡓࠋJaCVAM ║่⃭ᛶ௦᭰ἲ㈨ᩱ⦅⧩ጤဨ఍ࡣࠊTG 492 ࡛♧ࡉࢀࡓ RhCE ἲࡢ 1 ࡘ࡛࠶ࡿ EpiOcularTM ࢆ⏝࠸ࡓ║่⃭ᛶヨ㦂࡟ࡘ࠸࡚ࠊࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩ࠊ➨୕⪅ホ౯ሗ࿌᭩ࠊ㛵㐃ㄽᩥ࡞࡝ࢆࡶ࡜࡟ヨ㦂ἲࡢᴫせ࠾ࡼࡧጤဨ఍ࡢពぢࢆࡲ࡜ࡵࡓࠋ ᚑ᮶ࡢ࢘ࢧࢠࢆ⏝࠸ࡓ Draize ἲࡢ⤖ᯝࢆᇶ‽࡜ࡋ࡚ṇ☜ࢆホ౯ࡋࡓ࡜ࡁࠊ୍⮴ᗘࡣ 79.7%ࠊഇ㝧ᛶ⋡ࡣ 37.0%ࠊഇ㝜ᛶ⋡ࡣ 4.3%࡛࠶ࡗࡓࠋᢏ⾡⛣㌿ᛶ࡟㛵ࡋ࡚ᠱᛕࡉࢀࡿ⤖ᯝࡣᚓࡽࢀ࡚࠾ࡽࡎࠊ᪋タෆ෌⌧ᛶ࠾ࡼࡧ᪋タ㛫෌⌧ᛶࡣ࡝ࡕࡽࡶ 90%௨ୖ࡛࠶ࡗࡓࠋࡇࢀࡽࡢ್ࡣࠊࣂࣜࢹ࣮ࢩࣙࣥ㐠Ⴀጤဨ఍ࡀᐃࡵࡓᇶ‽ࢆ‶ࡓࡋ࡚࠸ࡓࠋ ᮏጤဨ఍ࡣࠊRhCE ἲࡣ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡛ UN GHS ༊ศእ≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚⏝࠸ࡿࡇ࡜ࡀ࡛ࡁࡿ࡜⤖ㄽࡋࡓࠋ

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2016 ᖺ 1 ᭶ 25 ᪥

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ࠊ࠾ࡼࡧࣇࣝ࢜ࣞࢭ࢖ࣥ₃ฟヨ㦂ἲ㸦FL ἲࠊTG 460㸧ࡢ 3 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ἲ࡛ࠊࢺࢵࣉࢲ࢘ࣥ
᪉ᘧ࡟࠾࠸࡚㔜⠜࡞║ࡢയᐖࢆ㉳ࡇࡍ㸦ࡍ࡞ࢃࡕࠊUN GHS ༊ศ 1㸧≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚ࠊࡲ
ࡓ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾࠸࡚║࡟ᑐࡍࡿ㔜⠜࡞ᦆയᛶࢆ᭷ࡍࡿ≀㉁࠾ࡼࡧ║่⃭ᛶ≀㉁࡜ࡣศ㢮ࡉࢀ࡞࠸㸦ࡍ࡞ࢃࡕࠊUN GHS ༊ศእ㸧≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚⏝࠸ࡽࢀࡿࠋFL ἲࡣᒀ⣽⟶ୖ⓶⣽⬊ࡢ༢ᒙᇵ㣴ࢆ⏝࠸࡚໬Ꮫ≀㉁ࡢ᭚㟢࡟ࡼࡾ⏕ࡌࡿ⣽⬊㛫⤖ྜࡢയᐖࢆᣦᶆ࡟║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛ࠊࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡛ࡢࡳ⏝࠸ࡽࢀࡿࠋ
෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝἲ㸦Reconstructed Human Cornea-like Epithelium Test Methodࠊ௨ୗ RhCEἲ㸧ࡣࠊ⿕㦂≀㉁ࡢࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝ㸦RhCE㸧⤌⧊࡟ᑐࡍࡿ⣽⬊ẘᛶࢆᣦᶆ࡟⏝࠸ࠊࡑࡢ≀㉁ࡢ
║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛࠶ࡿࠋRhCE ⤌⧊࡟⿕㦂≀㉁ࢆ᭚㟢ࡋࡓᚋࠊ㝜ᛶᑐ↷࡜ࡢẚ㍑࡟ࡼࡾ⣽⬊⏕Ꮡ⋡ࢆ⟬ฟࡋ⿕㦂≀㉁ࡢ║่⃭ᛶࢆホ౯ࡍࡿࠋ
RhCE ἲࡣ EURL ECVAM ࡜ CE ࡢඹྠࣂࣜࢹ࣮ࢩࣙࣥ◊✲㸦EURL ECVAM-CE, 2014㸧࠾ࡼࡧ ESACࡢ➨୕⪅ホ౯㸦ESAC, 2014㸧ࢆ⤒࡚ࠊUN GHS ༊ศእ≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚ 2015 ᖺ 7 ᭶࡟ OECD࡟ࡼࡾࢸࢫࢺ࢞࢖ࢻࣛ࢖ࣥ࡟᥇ᢥࡉࢀࡓ㸦TG 492㸧ࠋᮏሗ࿌᭩ࡣࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩ࠊ➨୕⪅ホ౯ሗ࿌᭩ࠊ㛵㐃ㄽᩥ࡞࡝ࢆࡶ࡜࡟ OECD TG 492 RhCEἲࡢᴫせࢆㄝ᫂ࡋࠊᮏጤဨ఍ࡢពぢࢆࡲ࡜ࡵࡓࡶࡢ࡛࠶ࡿࠋ
2㸬ヨ㦂ἲࡢ఩⨨࡙ࡅ
RhCE ἲࡣࠊUN GHS ༊ศእ≀㉁㸦༢୍≀㉁࠾ࡼࡧΰྜ≀㸧ࢆ᳨ฟࡍࡿࡓࡵ࡟⏝࠸ࡿヨ㦂ἲ࡛࠶ࡿࠋ
3㸬ヨ㦂ἲࡢཎ⌮║่⃭ᛶࡣࠊ≀㉁ࡀゅ⭷ࢆྵࡴ║⾲㠃࡟᥋ゐࡋ⣽⬊യᐖࢆᘬࡁ㉳ࡇࡍࡇ࡜࠿ࡽጞࡲࡿࠋࡑࡢᶵᗎࡣ
ᵝࠎ࡛࠶ࡿࡀࠊ⣽⬊ẘᛶࡀ㔜せ࡞ᙺ๭ࢆᢸࡗ࡚࠸ࡿࠋࡲࡓࠊ≀㉁ࡢ║่⃭ᛶࡣ୺࡟ゅ⭷ࡢึᮇയᐖࡢ
῝ᗘ࡟ࡼࡾỴᐃࡉࢀࠊࡑࢀࡣ⣽⬊Ṛࡢ⛬ᗘ࡜┦㛵㛵ಀ࡟࠶ࡿࠋRhCE ἲࡣࠊ෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹ࡛ࣝ࠶ࡿ RhCE ⤌⧊ࢆ⏝࠸࡚ࠊ⿕㦂≀㉁ࡢ⣽⬊ẘᛶࢆᣦᶆ࡜ࡋ࡚║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛࠶ࡿࠋ
TG 492 ࡛ㄆࡵࡽࢀ࡚࠸ࡿ RhCE ἲࡣࠊࣄࢺ᪂⏕ඣໟ⓶⣽⬊⏤᮶ࡢᕷ㈍ࡢ RhCE ⤌⧊࡛࠶ࡿࠋ⿕㦂≀㉁ࡀᾮయ
EpiOcularTM ࢆ⏝࠸ࡓ║่⃭ᛶヨ㦂㸦EpiOcularTM EIT㸧࡛࠶ࡿ㸦Kaluzhny et al, 2011㸧ࡢሙྜࡣ⣙ 30 ศ㛫ࠊᅛయࡢሙྜࡣ⣙ 6 ᫬㛫ࠊRhCE ⤌⧊࡟᭚㟢ࡋࡓᚋ࡛ࡢ MTT ࡢྲྀࡾ㎸ࡳ㔞ࢆࡶ࡜࡟ࡋࡓ⣽⬊⏕Ꮡ⋡ࢆ࢚ࣥࢻ࣏࢖ࣥࢺ࡟⏝࠸ࡿࠋࡇࢀࡣࠊMTT ࡀ⬺Ỉ⣲㓝⣲ࡢᇶ㉁࡜࡞ࡿᛶ㉁ࢆ฼⏝ࡋࠊ⣽⬊ෆ࡟ྲྀࡾ㎸ࡲࢀࡓ MTT ࡀ࣑ࢺࢥࣥࢻࣜ࢔ෆ⬺Ỉ⣲㓝⣲࡟ࡼࡾ㑏ඖࡉࢀࠊ⏕ᡂࡉࢀࡓ࣐࣍ࣝ
511

2016 ᖺ 1 ᭶ 25 ᪥ࢨࣥ㔞㸦㟷Ⰽ㸧ࡀ⏕Ꮡ⣽⬊ᩘ࡟ẚ౛ࡍࡿࡇ࡜ࢆᇶᮏཎ⌮࡜ࡋ࡚࠸ࡿ㸦MTT 㑏ඖἲ㸧ࠋ⣽⬊⏕Ꮡ⋡ࡀ㝜ᛶᑐ↷࡜ẚ㍑ࡋ࡚ 60%ࢆ㉸࠼ࡿሙྜࠊ⿕㦂≀㉁ࡣ UN GHS ศ㢮࡟࠾࠸࡚༊ศእ࡛࠶ࡿ࡜ุᐃࡍࡿࠋ
4㸬ヨ㦂ᡭ㡰 ௨ୗ࡟♧ࡍᡭ㡰ࡣࠊTG 492 ࡛ㄆࡵࡽࢀ࡚࠸ࡿ EpiOcularTM EIT ࡟ࡘ࠸࡚ࡢࡶࡢ࡛࠶ࡿࠋEpiOcularTMࡣ࢔࣓ࣜ࢝ࡢ〇㐀ඖࡼࡾ㉎ධ࡛ࡁࡿࠋヲ⣽ࡣࠊTG 492 ࠾ࡼࡧ SOP㸦SOP Version 9, 2015㸧ࢆཧ↷ࡍࡿࡇ࡜ࠋ
4-1. RhCE ⤌⧊ࡢᶵ⬟ⓗ᮲௳ RhCE ⤌⧊ࡢᶵ⬟ⓗ᮲௳࡟ࡣࠊRhCE ⤌⧊ࡢ⏕Ꮡ⋡࡜ࣂࣜ࢔ᶵ⬟ࡀྵࡲࢀࠊࡑࡢチᐜ⠊ᅖࡣ௨ୗࡢ
࡜࠾ࡾ࡛࠶ࡿࠋ
⏕Ꮡ⋡㸸㝜ᛶᑐ↷࡟࠾࠸࡚ 0.8 < OD < 2.5ࣂࣜ࢔ᶵ⬟㸸0.3% Triton X-100 ࡟ᑐࡋ 12.2  ET50 (min)  37.5
㸦ET50㸸⣽⬊⏕Ꮡ⋡ࢆ 50%పୗࡉࡏࡿࡢ࡟ᚲせ࡞᭚㟢᫬㛫㸧ࡇࢀࡽࡣ RhCE ⤌⧊ࡢ〇㐀⪅ࡢฟⲴᇶ‽࡜ࡋ࡚ࡶ᥇⏝ࡉࢀࡿࠋࡲࡓࠊ〇㐀⪅ࡣ⤌⧊Ꮫⓗ᳨ᰝ࡛ RhCE⤌⧊ࡀࣄࢺࡢゅ⭷ୖ⓶ᵝᵓ㐀ࢆ᭷ࡍࡿࡇ࡜ࢆಖドࡋ࡚࠾ࡃᚲせࡀ࠶ࡿࠋ୍᪉ࠊRhCE ⤌⧊ࡢ౑⏝⪅ࡣ
ヨ㦂࡛ᚓࡽࢀࡓ㝧ᛶ࠾ࡼࡧ㝜ᛶᑐ↷ࡢ⤖ᯝࢆ⫼ᬒࢹ࣮ࢱ࡜ẚ㍑ࡋ࡚ࠊ෌⌧ᛶࢆ☜ㄆࡍࡿᚲせࡀ࠶ࡿࠋ
4-2. ⿕㦂≀㉁ࡢ㐺⏝ ≀㉁࠶ࡓࡾᑡ࡞ࡃ࡜ࡶ 2 ⤌⧊ࢆ⏝࠸ࡿࠋ⿕㦂≀㉁ࡣㄪ〇ࡏࡎࠊཎయࡑࡢࡶࡢࢆ⏝࠸ࡿࠋ37ÛC ௨ୗ࡛ࣆ࣌ࢵࢺ࡛ᢅ࠼ࡿࡶࡢࡣᾮయ࡜ࡋ࡚ࠊࡑࢀ௨እࡣᅛయ࡜ࡋ࡚ヨ㦂ࢆ⾜࠺ࠋ≉࡟ࠊ⿕㦂≀㉁ࡀ⢓ᛶࡢ࠶ࡿ≀㉁ࡸᶞ⬡࣭࣡ࢵࢡࢫ࣭ࢤࣝ≧≀㉁ࡢሙྜࡣࠊ37±1ÛC ࡛ 15±1 ศ㛫Ỉᾎ࡛࢖࣮ࣥ࢟ࣗ࣋ࢺࡋ࡚ᾮయ
࠿ᅛయ࠿ࢆุ᩿ࡍࡿࠋRhCE ⤌⧊ࡣࣜࣥ㓟⦆⾪⏕⌮㣗ሷỈ㸦Mg2+/Ca2+ free㸧ࢆ⏝࠸࡚ 30±2 ศ㛫ᶆ‽ᇵ㣴᮲௳1࡛๓ฎ⌮ࡍࡿࠋᾮయ⿕㦂≀㉁ࡢሙྜࠊRhCE ⤌⧊࡟ 50 μL ࢆ㐺⏝ࡋࠊᶆ‽ᇵ㣴᮲௳࡛ 30±2 ศ㛫ᇵ㣴ࡍࡿࠋࡑࡢᚋࠊࣜࣥ㓟⦆⾪⏕⌮㣗ሷỈ࡛༑ศ࡟Ὑίࡋ࡚⿕㦂≀㉁ࢆ㝖ཤࡋࠊ12±2 ศ㛫ᐊ࡛ᇵᆅ࡟ᾐₕᚋࠊ᪂ࡓ࡞ᇵᆅࢆ⏝࠸࡚ᶆ‽ᇵ㣴᮲௳࡛ 120±15 ศ㛫ᇵ㣴ࡍࡿࠋᅛయ⿕㦂≀㉁ࡢሙྜࠊRhCE⤌⧊ࡢ⾲㠃ࢆそ࠺ࡢ࡟༑ศ࡞㔞ࠊ⣙ 50 mg ࢆᆒ୍࡟㐺⏝ࡋࠊᶆ‽ᇵ㣴᮲௳࡛ 6±0.25 ᫬㛫ᇵ㣴ࡍࡿࠋࡑࡢᚋࠊࣜࣥ㓟⦆⾪⏕⌮㣗ሷỈ࡛༑ศ࡟Ὑίࡋ࡚⿕㦂≀㉁ࢆ㝖ཤࡋࠊ25±2 ศ㛫ᐊ࡛ᇵᆅ࡟ᾐₕᚋࠊ᪂ࡓ࡞ᇵᆅࢆ⏝࠸࡚ᶆ‽ᇵ㣴᮲௳࡛ 18±0.25 ᫬㛫ᇵ㣴ࡍࡿࠋ 㝧ᛶᑐ↷࡟ࡣ㓑㓟࣓ࢳࣝ㸦CAS No. 79-20-9㸧ࠊ㝜ᛶᑐ↷࡟ࡣ㉸⣧Ỉࡀ᥎ዡࡉࢀࡿࠋᑐ↷≀㉁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 ୰࡛ 180±10ศ㛫ᶆ‽ᇵ㣴᮲௳࡛཯ᛂࡉࡏࠊࡑࡢᚋ 2 mL ࢖ࢯࣉࣟࣃࣀ࣮ࣝ㸦ࡲࡓࡣྠᵝ࡞⁐፹㸧࡛㟷Ⰽࡢ࣐࣍ࣝ
ࢨࣥࢆᢳฟࡍࡿࠋᾮయ⿕㦂≀㉁ࡢሙྜࡣࠊRhCE ⤌⧊ࡢୖ㒊࣭ᗏ㒊୧᪉࠿ࡽᢳฟࡍࡿࠋᅛయ⿕㦂≀㉁࠾ࡼࡧⰍࡢ௜࠸࡚࠸ࡿᾮయ⿕㦂≀㉁ࡢሙྜࡣࠊ⤌⧊࡟ṧᏑࡍࡿ⿕㦂≀㉁ࡢΰධࢆ᭱ᑠ㝈࡟ᢚ࠼ࡿࡓࡵࠊ
1

ᬯᡤࠊ37s1ÛCࠊ5s1% CO2ࠊ㧗‵ᗘ

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2016 ᖺ 1 ᭶ 25 ᪥࣐࣍ࣝࢨࣥࡢᢳฟࡣ RhCE ⤌⧊ࡢᗏ㒊ࡢࡳ࠿ࡽ⾜࠺ࠋᾮయ⿕㦂≀㉁࡛ࡶࠊὙίࡋ࡙ࡽ࠸ሙྜ࡟ࡣࠊᗏ
㒊ࡢࡳ࠿ࡽᢳฟࢆ⾜࠺ࠋྠ᫬ᑐ↷≀㉁࡟ᑐࡋ࡚ࡢᢳฟ᪉ἲࡣ⿕㦂≀㉁࡜ྠࡌ࡜ࡍࡿࠋᢳฟࡋࡓ࣐࣍ࣝ
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྾ගᗘ⿵ṇࢆ⾜࠺ᚲせࡀ࠶ࡿࠋࡑࡢᡭ㡰࡟ࡘ࠸࡚ࡣ TG ᮏᩥࡢㄝ᫂࠾ࡼࡧ
ANNEX II ࡢࣇ࣮ࣟࢳ࣮ࣕࢺࢆཧ↷ࡍࡿࡇ࡜ࠋ
4-4. ヨ㦂ᡂ❧ࡢᢎㄆᇶ‽௨ୗࡢ᮲௳ࢆ‶ࡓࡋࡓሙྜࠊヨ㦂ࡢᡂ❧ࢆᢎㄆࡍࡿࠋ
1㸧㝜ᛶᑐ↷ࡢ OD ࡀ 0.8 < OD < 2.5 ࡛࠶ࡿࡇ࡜ࠋ
2㸧㝧ᛶᑐ↷ࡢᖹᆒ⣽⬊⏕Ꮡ⋡ࡀ 50%ᮍ‶࡛࠶ࡿࡇ࡜ࠋ
3㸧⿕㦂≀㉁࠾ࡼࡧ㝜ᛶ࣭㝧ᛶᑐ↷ࡢࡑࢀࡒࢀ࡟࠾࠸࡚ࠊ2 ⤌⧊ࢆ⏝࠸ࡓሙྜࡣ⣽⬊⏕Ꮡ⋡ࡢᕪࡀ
20%ᮍ‶ࠊ3 ⤌⧊௨ୖࢆ⏝࠸ࡓሙྜࡣ⣽⬊⏕Ꮡ⋡ࡢᶆ‽೫ᕪࡀ 18%௨ୗ࡛࠶ࡿࡇ࡜ࠋ
4-5. ่⃭ᛶࡢุᐃ่⃭ᛶุᐃࡢ࢝ࢵࢺ࢜ࣇ್ࡣᖹᆒ⣽⬊⏕Ꮡ⋡ 60%࡛࠶ࡿࠋࡍ࡞ࢃࡕࠊᖹᆒ⣽⬊⏕Ꮡ⋡ࡀ 60%ࢆ㉸࠼ࡓሙྜࠊ⿕㦂≀㉁ࡣ GHS ༊ศእ࡜ุ᩿ࡉࢀࡿࠋᖹᆒ⣽⬊⏕Ꮡ⋡ࡀ 60%௨ୗࡢሙྜࠊᮏヨ㦂ἲ࡛ࡣഇ㝧ᛶࡀ⏕ࡌ࠼ࡿࡋࠊࡲࡓ༊ศ 1 ࡜༊ศ 2 ࢆ᳨ฟ࡛ࡁ࡞࠸ࡇ࡜࠿ࡽࠊ௚ࡢヨ㦂ἲ࡟ࡼࡿ㏣ຍヨ㦂ࡀᚲせ࡜࡞ࡿࠋ5㸬ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂 EURL ECVAM ࡜ CE ࡢ༠ྠ࡛ EpiOcularTM EIT ࡢࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡀ⾜ࢃࢀࡓࠋṇ☜ᛶࡢホ౯ࡢࡓࡵ࡟ᚲせ࡞ື≀ヨ㦂ࡢࢹ࣮ࢱࢆᣢࡗ࡚࠸ࡿࡇ࡜ࢆ๓ᥦ᮲௳࡟ࠊUN GHS ༊ศ≀㉁࣭༊ศእ≀㉁ࡸᅛ
య࣭ᾮయ➼ࡢࣂࣛࣥࢫࢆ⪃៖ࡋ࡚⿕㦂≀㉁ࡢ㑅ᢥࡀ⾜ࢃࢀࡓ㸦Appendix 1㸧ࠋ ࢥ࣮ࢻ໬ࡉࢀࡓ 107 ≀㉁࡟ࡘ࠸࡚ࡍ࡭࡚ࢆ 3 ᪋タ࡛ヨ㦂ࡋࡓ㸦ᮏヨ㦂㸧ࠋヨ㦂ࡣࡍ࡭࡚ࡢ⿕㦂≀㉁࡟ࡘ࠸࡚ 3 ᅇᐇ᪋ࡉࢀࡓࠋᅛయ≀㉁࡟ᑐࡋ࡚ࡣࠊࡑࡢᚋࣉࣟࢺࢥ࣮ࣝࡢ᭱㐺໬㸦⿕㦂≀㉁ࡢ᭚㟢᫬㛫
ࢆ 90±5 ศ࠿ࡽ 6±0.25 ᫬㛫࡟ኚ᭦㸧ࡀ⾜ࢃࢀࡓࡓࡵࠊ107 ≀㉁୰ࡢᅛయ࠾ࡼࡧ᪂ࡓ࡞ᅛయ 8 ≀㉁࡟ࡘ࠸࡚ࠊ㏣ຍヨ㦂ࡀ 1 ᪋タ࡛⾜ࢃࢀࡓࠋࡇࡢࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡛ࡣ྾ගᗘ࡛࣐࣍ࣝࢨࣥࢆᐃ㔞ࡋࠊ⣽⬊⏕Ꮡ⋡ࢆồࡵ࡚࠸ࡿࠋHPLC/UPLCࢆ฼⏝ࡋࡓ࣐࣍ࣝࢨࣥᐃ㔞࣭⣽⬊⏕Ꮡ⋡⟬ฟ࡟ࡘ࠸࡚ࡣࠊCE ࡟ࡼࡾ⓶⭵่⃭ᛶヨ㦂࡟⏝࠸ࡽࢀࡿࣄ
ࢺ⤌⧊ࣔࢹࣝ࡜ྜࢃࡏ࡚ࣂࣜࢹ࣮ࢩࣙࣥࡀ⾜ࢃࢀ࡚࠸ࡿࠋ
6㸬ヨ㦂ἲࡢಙ㢗ᛶ
6-1. ᢏ⾡⛣㌿ᛶ ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࢆ㛤ጞࡍࡿ๓࡟ࠊࣂࣜࢹ࣮ࢩࣙࣥཧຍ᪋タࡢᢏ⾡⪅㸦᪋タᙜࡓࡾ 3 ྡ㸧࡟ᑐࡋ࡚ᢏ⾡⩦ᚓࡢࡓࡵࡢࢺ࣮ࣞࢽࣥࢢࢆ⾜ࡗࡓࠋࡑࡢᚋ࡛ࠊ8 ≀㉁ࢆ⏝࠸࡚ᢏ⾡⛣㌿ࡢ☜ㄆࢆ⾜ࡗࡓ࡜ࡇࢁࠊ␗࡞ࡿุᐃࡣ 1 ᪋タࡢᢏ⾡⪅ 1 ྡࡢ 1 ᾮయ≀㉁ࡢࡳ࡛࠶ࡗࡓࠋEpiOcularTM EIT ࢆᐇ᪋ࡍࡿヨ㦂᪋タࡢᢏ⾡⩦ᚓࢆ☜࠿ࡵࡿࡓࡵࡢ⇍㐩ᗘ☜ㄆ≀㉁ࡢ୍ぴࡣࠊAppendix 2 ࡟࠶ࡿࠋ7
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2016 ᖺ 1 ᭶ 25 ᪥

6-2. ᪋タෆ෌⌧ᛶࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡛ࡣ᪋タࡈ࡜࡟ 1 ⿕㦂≀㉁࡟ࡘࡁ」ᩘᅇࡢヨ㦂ࢆ⾜ࡗ࡚࠸ࡿࠋᮏヨ㦂࡛ࡣࠊ
GHS ศ㢮ุᐃࡢ᪋タࡈ࡜ࡢ᪋タෆ෌⌧ᛶࡣ 94.2–96.2%࡛࠶ࡗࡓ㸦ᅛయ≀㉁ࡢࢹ࣮ࢱࢆྵࡴ㸧ࠋ1 ᪋タ࡛⾜ࢃࢀࡓ㏣ຍヨ㦂࡛ࡢ᪋タෆ෌⌧ᛶࡣ 96.6%࡛࠶ࡗࡓࠋࡇࢀࡽࡢ್ࡣ EURL ECVAM-CE ࣂࣜࢹ࣮ࢩࣙࣥ㐠Ⴀጤဨ఍ࡀᐃࡵࡓᇶ‽㸦85%௨ୖ㸧ࢆ‶ࡓࡋࡓࠋ
6-3. ᪋タ㛫෌⌧ᛶᮏヨ㦂࡛ᚓࡽࢀࡓ⣽⬊⏕Ꮡ⋡㸦」ᩘᅇヨ㦂㸧ࡢᖹᆒ್ࢆࡶ࡜࡟ GHS ศ㢮ุᐃࢆ᪋タࡈ࡜࡟⾜࠸ࠊࡑࡢ୍⮴ᗘࢆ᳨ウࡋࡓ㸦ᅛయ≀㉁ࡢࢹ࣮ࢱࢆྵࡴ㸧ࠋࡑࡢ⤖ᯝࠊGHS ศ㢮ุᐃࡢ᪋タ㛫෌⌧ᛶࡣ 93.2%
࡛࠶ࡗࡓࠋࡇࡢ್ࡣࣂࣜࢹ࣮ࢩࣙࣥ㐠Ⴀጤဨ఍ࡀᐃࡵࡓᇶ‽㸦80%௨ୖ㸧ࢆ‶ࡓࡋࡓࠋࣉࣟࢺࢥࣝ᭱
㐺໬ᚋࡢᅛయ≀㉁࡟ᑐࡋ࡚ࡢ GHS ศ㢮ุᐃࡢ᪋タ㛫෌⌧ᛶࡢࢹ࣮ࢱࡣࠊ㏣ຍヨ㦂ࢆ 1 ᪋タࡢࡳ࡛⾜ࡗࡓࡓࡵᏑᅾࡋ࡞࠸ࠋࣂࣜࢹ࣮ࢩࣙࣥ㐠Ⴀጤဨ఍ࡣࠊ᪋タෆ෌⌧ᛶࡣᮏヨ㦂࣭㏣ຍヨ㦂࡜ࡶ㧗࠸್࡛࠶ࡾࠊࡲࡓࠊྠ᫬࡟ࣂࣜࢹ࣮ࢩࣙࣥࢆ⾜ࡗࡓูࡢ RhCE ἲ࡟࠾࠸࡚᭚㟢᫬㛫ࢆኚ᭦ࡋࡓ㝿࡟ࠊኚ᭦๓
࡜ẚ㍑ࡋ࡚ኚ᭦ᚋ࡛ࡶྠ➼ࡢ㧗࠸᪋タ㛫෌⌧ᛶࡀᚓࡽࢀ࡚࠸ࡿࡇ࡜࠿ࡽࠊࣉࣟࢺࢥࣝ᭱㐺໬ᚋࡢ᪋タ㛫෌⌧ᛶࡢ☜ㄆࡣᚲせ࡞࠸࡜ุ᩿ࡋ࡚࠸ࡿࠋESAC ࡢ➨୕⪅ホ౯࡛ࡣࠊࣉࣟࢺࢥࣝ᭱㐺໬ᚋࡢ᪋タ㛫෌⌧ᛶࡀ࡞࠸Ⅼࡀ኱ࡁ࡞ᨭ㞀࡟࡞ࡿ࡜ࡣ⪃࠼࡚࠸࡞࠸ࠋ
7㸬ヨ㦂ἲࡢṇ☜ᛶࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩࡛ࡢṇ☜ᛶࡢホ౯࡟ࡣࠊࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡛⏝࠸ࡽࢀࡓ 115 ≀㉁ࡢ࠺ࡕ≀㉁⮬యࡢⰍࡀ࣐࣍ࣝࢨࣥᐃ㔞࡛ࡢ OD

ᐃࢆጉࡆࡿ 3 ≀㉁࡜ᙉ࠸㑏ඖᛶ≀㉁ 1 ≀㉁ࢆ㝖ࡃ 111 ≀㉁ࡢ

⤖ᯝࡀ⏝࠸ࡽࢀࡓࠋᡂ❧ࡋࡓ඲ヨ㦂ࡢࢹ࣮ࢱࢆ⏝࠸࡚ồࡵࡓ⤖ᯝࡣ⾲ 1 ࡢ㏻ࡾ࡛࠶ࡿࠋEpiOcularTM
EIT ࡢṇ☜ᛶࡣࣂࣜࢹ࣮ࢩࣙࣥ㐠Ⴀጤဨ఍ࡀᐃࡵࡓཷࡅධࢀᇶ‽ࢆ‶ࡓࡋ࡚࠸ࡿࠋ

⾲ 1. EpiOcularTM EIT ࡢṇ☜ᛶ㸫඲ヨ㦂ࢹ࣮ࢱ≀㉁ᩘ

୍⮴ᗘᗘ(%)

ഇ㝜ᛶ⋡(%)

ഇ㝧ᛶ⋡(%)

ᾮయ

52

81.9

1.7

33.3

ᅛయ

59

78.0

6.5

39.3

ᾮయ࣭ᅛయ

111

79.7

4.3

37.0

௦᭰ἲ࡜ࡋ࡚ࡢ
ཷࡅධࢀᇶ‽

-

 75

 10

 40

ESAC ࡢ➨୕⪅ホ౯࡛ࡣࠊ⿕㦂≀㉁ࡈ࡜࡟ุᐃࢆ᭱㧗ࢫࢥ࢔㸦Best-caseࠊᚓࡽࢀࡓヨ㦂ࢹ࣮ࢱࡢ࠺
ࡕ୍ࡘ࡛ࡶື≀ヨ㦂ࡢࢹ࣮ࢱ࡜୍⮴ࡋࡓุᐃࡀ࠶ࢀࡤࡑࢀࢆ᥇⏝㸧࡜᭱పࢫࢥ࢔㸦Worst-caseࠊᚓࡽࢀࡓヨ㦂ࢹ࣮ࢱࡢ࠺ࡕ୍ࡘ࡛ࡶື≀ヨ㦂ࡢࢹ࣮ࢱ࡜୍⮴ࡋ࡞࠿ࡗࡓุᐃࡀ࠶ࢀࡤࡑࢀࢆ᥇⏝㸧ࡢ 2 ࣃࢱ࣮࡛ࣥ☜ᐃࡋࠊࡑࡢ⤖ᯝࢆ⏝࠸࡚ṇ☜ᛶࡢ᳨ウࢆࡋ࡚࠸ࡿࠋࡑࡢ⤖ᯝࡣ⾲ 2 ࡢ㏻ࡾ࡛࠶ࡿࠋ᭱పࢫࢥ
࢔࡛ࡣഇ㝧ᛶ⋡࡟ࡘ࠸࡚ࡣཷࡅධࢀᇶ‽ࢆ‶ࡓࡉ࡞࠿ࡗࡓࡀࠊࡑࢀ௨እࡢṇ☜ᛶࡢᩘ್ࡣࡍ࡭࡚ཷࡅ
ධࢀᇶ‽ࢆ‶ࡓࡋࡓࠋᮏヨ㦂ἲࡢண⢭ᗘࡣ᭱పࢫࢥ࢔࡜᭱㧗ࢫࢥ࢔ࡢ㛫࡟࡞ࡿࡇ࡜ࢆ⪃៖ࡍࡿ࡜ࠊ

8
14

2016 ᖺ 1 ᭶ 25 ᪥

EpiOcularTM EIT ࡢṇ☜ᛶࡣࣂࣜࢹ࣮ࢩࣙࣥ㐠Ⴀጤဨ఍ࡀᐃࡵࡓཷࡅධࢀᇶ‽ࢆ‶ࡓࡍ࡜➨୕⪅ホ౯ࡣ⤖ㄽࡋࡓࠋ࡞࠾ࠊ➨୕⪅ホ౯࡛ࡣࠊࣂࣜࢹ࣮ࢩ࣏࣮ࣙࣥࣞࢺ࡛㝖እࡉࢀࡓᙉ࠸㑏ඖᛶ≀㉁ࡶ⤌ࡳධࢀࡽࢀ࡚࠸ࡿࠋ
⾲ 2. EpiOcularTM EIT ࡢṇ☜ᛶ㸫≀㉁ࡈ࡜ࡢࢹ࣮ࢱ≀㉁ᩘ

୍⮴ᗘᗘ(%)

ഇ㝜ᛶ⋡(%)

ഇ㝧ᛶ⋡(%)

ᾮయ

53

75.5

7.7

40.7

ᅛయ

59

76.3

6.5

42.9

ᾮయ࣭ᅛయ

112

76.1

7.0

41.1

ᾮయ

53

84.9

0.0

29.6

ᅛయ

59

79.7

6.5

35.7

ᾮయ࣭ᅛయ

112

82.1

3.5

32.7

௦᭰ἲ࡜ࡋ࡚ࡢ
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-

 75

 10

 40

᭱పࢫࢥ࢔

᭱㧗ࢫࢥ࢔

ࣂࣜࢹ࣮ࢩࣙࣥ࡟⏝࠸ࡽࢀࡓΰྜ≀ࡢᩘࡣ㝈ࡽࢀ࡚࠸ࡓࠋࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡜ࡣู࡟ࠊື≀ヨ㦂ࡢࢹ࣮ࢱࡀ࠶ࡿ㎰⸆ฎ᪉㸦97 ฎ᪉ࠊ࠺ࡕ༊ศእࡣ 43 ฎ᪉㸧ࢆ⏝࠸࡚ホ౯ࢆ⾜ࡗࡓሗ࿌࡛ࡣࠊṇ☜ᗘ
82%ࠊഇ㝜ᛶ⋡ 9%ࠊഇ㝧ᛶ⋡ 28%࡛࠶ࡗࡓ㸦Kolle et al, 2015㸧ࠋ8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ヨ㦂࡟࠾࠸࡚⿕㦂≀㉁࡟ྵࡵࡽࢀ࡞࠿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
EpiOcularTM EIT ࡣ෌⌧ᛶࡶṇ☜ᛶࡶࣂࣜࢹ࣮ࢩࣙࣥ㐠Ⴀጤဨ఍ࡢᐃࡵࡓᇶ‽ࢆ‶ࡓࡋ࡚࠾ࡾࠊᮏጤ
ဨ఍ࡣࣂࣜࢹ࣮ࢩࣙࣥ㐠Ⴀጤဨ఍ࡢᐃࡵࡓᇶ‽ࡣጇᙜ࡛࠶ࡿ࡜⪃࠼ࡿࠋEpiOcularTM EIT ࡟⏝࠸ࡿ
RhCE ⤌⧊ࡣᕷ㈍ࡉࢀ࡚࠾ࡾࠊ࢔࣓ࣜ࢝ࡢ〇㐀ඖࡼࡾධᡭ࡛ࡁࡿࠋRhCE ⤌⧊௨እࡣࠊ≉Ṧ࡞ᶵᮦࡸヨ⸆ࢆᚲせ࡜ࡏࡎࠊᚲせ࡞ᡭᢏࡶ」㞧࡞ࡶࡢ࡛࡞࠸ࠋධᡭࡋࡓ RhCE ⤌⧊ࡀᶵ⬟ⓗ᮲௳ࡢチᐜ⠊ᅖ࡟࠶ࡾࠊ࠿ࡘᐇ᪋ࡍࡿヨ㦂᪋タࡢᢏ⾡⩦ᚓࡀ࢞࢖ࢻࣛ࢖ࣥࡢ⇍㐩ᗘ☜ㄆ≀㉁࡛☜࠿ࡵࡽࢀ࡚࠸ࢀࡤࠊ
EpiOcularTM EIT ࡣ UN GHS ༊ศእ≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚⏝࠸ࡿࡇ࡜ࡀ࡛ࡁࡿࠋ
EpiOcualrTM EIT ࡣ RhCE ἲࢆ௦⾲ࡍࡿࡶࡢ࡜⪃࠼ࡽࢀࡿࡢ࡛ࠊTG 492 ࡟‽ᣐࡋ࡚ᐇ᪋ࡋࡓሙྜࠊ
RhCE ἲࡣ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡛ UN GHS ༊ศእ≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚⏝࠸ࡿࡇ࡜ࡀ࡛ࡁࡿࠊ࡜ᮏ
ጤဨ఍ࡣ⪃࠼ࡿࠋ

915

2016 ᖺ 1 ᭶ 25 ᪥

10. ᩥ⊩
EpiOcularTM EIT SOP, Version 9 (April 6, 2015). EpiOcluarTM EIT for the prediction of acuteoculatr irritation of chemicals. Available at: http:// https://eurl-ecvam.jrc.ec.europa.eu/ (as of
Nov. 2015)
ESAC (2014) ESAC Opinion based on the ESAC Scientific Peer Review of the ECVAM Eye
Irritation Validation Study (EIVS) and a related Cosmetics Europe study on HPLC/UPLCphotometry as an alternative endpoint detection system for formazan reaction product.
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.
Kaluzhny Y et al (2011) Development of the EpiOcularTM 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. ATLA. 39, 339-64.
Kolle SN et al (2015). The EpiOcular™ Eye Irritation Test is the method of choice for in vitro
eye irritation testing of agrochemical formulations: Correlation analysis of EpiOcular Eye
Irritation Test and BCOP test data according to UN GHS, US EPA and Brazil ANVISA
classification schemes. ATLA. 43, 181-198.
OECD (2012) Test Guideline 437. Bovine Corneal Opacity and Permeability test method for
identifying i) chemicals inducing serious eye damage and ii) chemicals not requiringclassification for eye irritation or serious eye damage.
OECD (2012) Test Guideline 438. Isolated Chicken Eye test method for identifying i) chemicals
inducing serious eye damage and ii) chemicals not requiring classification for eye irritation orserious eye damage.
OECD (2012) Test Guideline 460. Fluorescein Leakage test method for identifying ocularcorrosives and severe irritants.
OECD (2015) Test Guideline 492. Reconstructed human Cornea-like Epithelium (RhCE) test
method for identifying chemicals not requiring classification and labelling for eye irritation orserious eye damage.
OECD (2015) Series on Testing & Assessment 216. Performance standards for the assessment of proposed
similar or modified in vitro Reconstructed human Cornea-like Epithelium (RhCE) test method for eye hazard

1016

2016 ᖺ 1 ᭶ 25 ᪥

Appendix 1ࣂࣜࢹ࣮ࢩࣙࣥ࡟⏝࠸ࡽࢀࡓ≀㉁ࡢ࢝ࢸࢦ࣮ࣜࡈ࡜ࡢᩘ

115

඲య
║่⃭ᛶ GHS ศ㢮
༊ศ 1

30

༊ศ 2A

16

༊ศ 2B

14

༊ศእ

55

≀㉁ࡢ≉ᛶ 
ᾮయ

53

ᅛయ

62

Chemical Class
Ether

25

Alcohol

19

Aryl

18

Carboxylic acid ester

16

Carboxylic acid14

Alkoxy13

Aromatic heterocyclic halide

10

Aryl halide

10

Alkyl halide

9

Phenol

9

Allyl

6

Ammonium salt

6

Benzyl6

Aliphatic Amine, tertiary5

Alkane, branched with tertiary carbon

5

Aromatic amine

5

Dihydroxyl group5

Guanidine

5

Saturated heterocyclic fragment

5

Sulfate5ὀ㸧ࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩ࢆࡶ࡜࡟సᡂࠋChemical Class ࡣ≀㉁ᩘࡢከ࠸ࡶࡢࢆᢳฟࡋࡓࠋ≀㉁࡟ࡼ
ࡗ࡚ࡣ」ᩘࡢ Chemical Class ࡟ࡲࡓࡀࡗ࡚࠸ࡿሙྜࡶ࠶ࡿࠋ

11
17

2016 ᖺ 1 ᭶ 25 ᪥

Appendix 2
RhCE ἲࡢ⇍㐩ᗘ☜ㄆ≀㉁≀㉁ྡ

CAS ␒ྕ

ᛶ≧

GHS ศ㢮

Methylthioglycolate

2365-48-2

ᾮయ

༊ศ 1

Tetraethylene glycol diacrylate

17831-71-9

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

1,5-Naphthalenediol

83-56-7

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

Dipropyl disulphide629-19-6ᾮయ༊ศእPiperonyl butoxide51-03-6ᾮయ༊ศእ

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

ᾮయ

༊ศእ

Polyethylene glycol (PEG-40) hydrogenated castor oil

61788-85-0

⢓ᛶ≀

༊ศእ

Potassium tetrafluoroborate

14075-53-7

ᅛయ

༊ศእ

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

101-20-2

ᅛయ༊ศእ103597-45-1

ᅛయ

༊ศእ

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

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

1218

OECD/OCDE

492$GRSWHG
-XO\
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
Serious eye damageUHIHUVWRWKHSURGXFWLRQRIWLVVXHGDPDJHLQWKHH\HRUVHULRXVSK\VLFDOGHFD\RI YLVLRQ IROORZLQJ DSSOLFDWLRQ RI D WHVW FKHPLFDO WR WKH DQWHULRU VXUIDFH RI WKH H\H ZKLFK LV QRW IXOO\UHYHUVLEOHZLWKLQGD\VRIDSSOLFDWLRQDVGHILQHGE\WKH8QLWHG1DWLRQV*OREDOO\+DUPRQL]HG6\VWHPRI&ODVVLILFDWLRQDQG/DEHOOLQJRI&KHPLFDOV 81*+6   $OVRDFFRUGLQJWR81*+6eye irritationUHIHUVWRWKHSURGXFWLRQRIFKDQJHVLQWKHH\HIROORZLQJWKHDSSOLFDWLRQRIDWHVWFKHPLFDOWRWKHDQWHULRUVXUIDFHRI
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Table 1: List of proficiency chemicals
Chemical Name

CASRN

Organic
Functional
Group1

PhysicalState

VRM
viability(%)2

VRM
Prediction

MTT
Reducer

Colourinterf.

In Vivo Category 13
Methylthioglycolate
Tetraethylene glycol
diacrylate
2,5-Dimethyl-2,5hexanediol

2365-48-2

Carboxylic acid
ester;
ThioalcoholL

10.9±6.4

Cat 2 / Cat 1

Y(strong)N

17831-71-9

Acrylate; EtherL

34.9±15.3

Cat 2 / Cat 11

1

110-03-2

AlcoholS

2.3±0.2

Cat 2 / Cat 1

1

1


© OECD, (2015)

23

492

OECD/OCDE
Chemical Name
Sodium oxalate

CASRN

Organic
Functional
Group1

PhysicalState

VRM
viability(%)2

VRM
Prediction

MTT
Reducer

Colourinterf.

62-76-0Oxocarboxylicacid

S

29.0±1.2

Cat 2 / Cat 11

1

In Vivo Category 2A3
2,4,11,13Tetraazatetradecanediimidamide,
N,N''bis(4-chlorophenyl)3,12-diimino-, di-Dgluconate(20%, aqueous) 4

18472-51-0

1,5-Naphthalenediol

83-56-7

Aromatic
heterocyclic
halide; Aryl
halide;Dihydroxyl
group;
Guanidine
Fusedcarbocyclicaromatic;
Naphthalene;
PhenolL

4.0±1.1

Cat 2 / Cat 1

N

Y(weak)

S

21.0±7.4

Cat 2 / Cat 1

Y(medium)

N

In Vivo Category 2B3Diethyl toluamide

134-62-3

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

79-92-5

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

L

15.6±6.3

Cat 2 / Cat 1

N

N

S

4.7±1.5

Cat 2 / Cat 1

N

N

/

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1

1

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1

1

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1

1

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1

1

6

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1

1

6

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1R&DW

1

1

In Vivo No Category3
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24

OECD/OCDE
Chemical Name
3RWDVVLXPWHWUDIOXRURERUDWH

492

CASRN

Organic
Functional
Group1

PhysicalState

VRM
viability(%)2

VRM
Prediction

MTT
Reducer

Colourinterf.

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Abbreviations: CASRN = Chemical Abstracts Service Registry Number; UN GHS = United Nations Globally Harmonized Systemof Classification and Labelling of Chemicals (1); VRM = Validated Reference Method, i.e., EpiOcular™ 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 in the EURL ECVAM/Cosmetics Europe Eye Irritation Validation Study (EIVS) (7)(9)(10).
3Based on results from the in vivo rabbit eye test (OECD TG 405) (2)(12) and using the UN GHS (1).
4Classification 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.

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RHCE TEST METHOD COMPONENTS
General conditions
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25

492

OECD/OCDEFunctional conditions
Viability

7KHDVVD\XVHGIRUTXDQWLI\LQJWLVVXHYLDELOLW\LVWKH077DVVD\  9LDEOHFHOOVRIWKH5K&(WLVVXHFRQVWUXFWUHGXFHWKHYLWDOG\H077LQWRDEOXH077IRUPD]DQSUHFLSLWDWHZKLFKLVWKHQH[WUDFWHG
IURP WKHWLVVXH XVLQJ LVRSURSDQRO RU DVLPLODU VROYHQW  7KHH[WUDFWHG077IRUPD]DQPD\EHTXDQWLILHGXVLQJ HLWKHU D VWDQGDUG DEVRUEDQFH 2SWLFDO 'HQVLW\ 2'  PHDVXUHPHQW RU DQ +3/&83/&
VSHFWURSKRWRPHWU\SURFHGXUH  7KH2'RIWKHH[WUDFWLRQVROYHQWDORQHVKRXOGEHVXIILFLHQWO\VPDOOLH
2'7KH5K&(WLVVXHFRQVWUXFWXVHUVVKRXOGHQVXUHWKDWHDFKEDWFKRIWKH5K&(WLVVXHFRQVWUXFWXVHG
PHHWV GHILQHG FULWHULD IRU WKH QHJDWLYH FRQWURO $Q DFFHSWDELOLW\ UDQJH XSSHU DQG ORZHU OLPLW  IRU WKHQHJDWLYH FRQWURO 2' YDOXHV LQ WKH WHVW PHWKRG FRQGLWLRQV  VKRXOG EH HVWDEOLVKHG E\ WKH 5K&( WLVVXH
FRQVWUXFW GHYHORSHUVXSSOLHU $FFHSWDELOLW\ UDQJHV IRU WKH QHJDWLYH FRQWURO 2' YDOXHV IRU WKH 950 DUHJLYHQLQ7DEOH  $Q+3/&83/&VSHFWURSKRWRPHWU\XVHUVKRXOGXVHWKHQHJDWLYHFRQWURO2'UDQJHVSURYLGHGLQ7DEOHDVWKHDFFHSWDQFHFULWHULRQIRUWKHQHJDWLYHFRQWURO,WVKRXOGEHGRFXPHQWHGWKDWWKH
WLVVXHV WUHDWHG ZLWK WKH QHJDWLYH FRQWURO VXEVWDQFH DUH VWDEOH LQ FXOWXUH SURYLGH VLPLODU WLVVXH YLDELOLW\PHDVXUHPHQWV IRUWKHGXUDWLRQRIWKHWHVWH[SRVXUHSHULRG© OECD, (2015)
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26

OECD/OCDE





492

Table 2: Acceptability ranges for negative control OD values
Test Method

Lower acceptance limit

Upper acceptance limit!



(SL2FXODU(,7 2&/ ±950
IRUERWKWKHOLTXLGVDQGWKHVROLGVSURWRFROV 

7KLVDFFHSWDQFHOLPLWFRQVLGHUVWKHSRVVLELOLW\RIH[WHQGHGVKLSSLQJVWRUDJHWLPH HJ!GD\V ZKLFKKDVEHHQVKRZQQRW WR
LPSDFWRQWKHSHUIRUPDQFHRIWKHWHVWPHWKRG  Barrier function

7KH5K&(WLVVXHFRQVWUXFWVKRXOGEHVXIILFLHQWO\WKLFNDQGUREXVWWRUHVLVWWKHUDSLGSHQHWUDWLRQRIF\WRWR[LF EHQFKPDUN VXEVWDQFHV HJ 7ULWRQ ; DV HVWLPDWHG E\ HJ (7 7DEOH   7KH EDUULHUIXQFWLRQ RI HDFK EDWFK RI WKH 5K&( WLVVXH FRQVWUXFW XVHG VKRXOG EH GHPRQVWUDWHG E\ WKH 5K&( WLVVXH
FRQVWUXFWGHYHORSHUYHQGRUXSRQVXSSO\RIWKHWLVVXHVWRWKHHQGXVHU VHHSDUDJUDSK Morphology+LVWRORJLFDO H[DPLQDWLRQ RI WKH 5K&( WLVVXH FRQVWUXFW VKRXOG GHPRQVWUDWH KXPDQ FRUQHDOLNHHSLWKHOLXPVWUXFWXUH LQFOXGLQJDWOHDVWOD\HUVRIYLDEOHHSLWKHOLDOFHOOVDQGDQRQNHUDWLQL]HGVXUIDFH EXWLVQRWUHTXLUHGIRUHDFKWLVVXHEDWFKXVHG)RUWKH950DSSURSULDWHPRUSKRORJ\KDVEHHQHVWDEOLVKHGDQG
WKHUHIRUHGRHVQRWQHHGWREHGHPRQVWUDWHGDJDLQE\DWHVWPHWKRGXVHUIRUHDFKWLVVXHEDWFKXVHG
Reproducibility

7KH UHVXOWV RI WKH SRVLWLYH DQG QHJDWLYH FRQWUROV RI WKH WHVW PHWKRG VKRXOG GHPRQVWUDWH
UHSURGXFLELOLW\RYHUWLPH
Quality control (QC)
7KH5K&(WLVVXHFRQVWUXFWVKRXOGRQO\EHXVHGLIWKHGHYHORSHUVXSSOLHUGHPRQVWUDWHVWKDWHDFKEDWFKRIWKH5K&(WLVVXHFRQVWUXFWXVHGPHHWVGHILQHGSURGXFWLRQUHOHDVHFULWHULDDPRQJZKLFKWKRVHIRU
YLDELOLW\ VHHSDUDJUDSK DQGEDUULHUIXQFWLRQ VHHSDUDJUDSK DUHWKHPRVWUHOHYDQW$QDFFHSWDELOLW\UDQJH XSSHUDQGORZHUOLPLW IRUWKHEDUULHUIXQFWLRQDVPHDVXUHGE\WKH(7  VHHSDUDJUDSKVDQG VKRXOGEHHVWDEOLVKHGE\WKH5K&(WLVVXHFRQVWUXFWGHYHORSHUVXSSOLHU7KH(7 DFFHSWDELOLW\UDQJHXVHG
DV4&EDWFKUHOHDVHFULWHULRQE\WKHGHYHORSHUVXSSOLHURIWKH(SL2FXODU2&/WLVVXHFRQVWUXFW XVHG
LQWKH950 LVJLYHQLQ7DEOH'DWDGHPRQVWUDWLQJFRPSOLDQFHZLWKDOOSURGXFWLRQUHOHDVHFULWHULDVKRXOG
EHSURYLGHGE\WKH5K&(WLVVXHFRQVWUXFWGHYHORSHUVXSSOLHUWRWKHWHVWPHWKRGXVHUVVRWKDWWKH\DUHDEOH
WR LQFOXGH WKLV LQIRUPDWLRQ LQ WKH WHVW UHSRUW 2QO\ UHVXOWV SURGXFHG ZLWK WLVVXHV IXOILOOLQJ DOO RI WKHVH
SURGXFWLRQUHOHDVHFULWHULDFDQEHDFFHSWHGIRUUHOLDEOHSUHGLFWLRQRIFKHPLFDOVQRWUHTXLULQJFODVVLILFDWLRQ
DQGODEHOOLQJIRUH\HLUULWDWLRQRUVHULRXVH\HGDPDJHLQDFFRUGDQFHZLWK81*+6© OECD, (2015)

27

492

OECD/OCDE
Table 3: QC batch release criterion

Test Method
(SL2FXODU(,7 2&/ 950
/RI YY 7ULWRQ;

Lower acceptance limit

Upper acceptance limit(7 PLQ

(7 PLQ
Application of the Test Chemical and Control Substances
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HDFKUXQ%HIRUHH[SRVXUHWRWHVWFKHPLFDOVRUDFRQWUROVXEVWDQFHWKHWLVVXHVXUIDFHRIWKH950LVSUH
WUHDWHGZLWK/RIFDOFLXPDQGPDJQHVLXPIUHH'XOEHFFR V3KRVSKDWH%XIIHUHG6DOLQH &D0JIUHH
'3%6  DQG LQFXEDWHG LQ WKH GDUN DW & LQ D KXPLGLILHG DWPRVSKHUH RI  &2  LQ DLU VWDQGDUGFXOWXUHFRQGLWLRQV IRUPLQXWHVWRPLPLFWKHZHWFRQGLWLRQVRIKXPDQH\H$IWHUWKLVPLQXWHVSUHWUHDWPHQW WUHDWPHQW RI WKH WLVVXHV LV LQLWLDWHG ZLWK H[SRVXUH WR WKH WHVW FKHPLFDO V  DQG FRQWUROVXEVWDQFHV7ZRGLIIHUHQWWUHDWPHQWSURWRFROVDUHXVHGRQHIRUOLTXLGWHVWFKHPLFDOVDQGRQHIRUVROLGWHVWFKHPLFDOV  ,QHLWKHUFDVHDVXIILFLHQWDPRXQWRIWHVWFKHPLFDORUFRQWUROVXEVWDQFHVKRXOGEHDSSOLHGWRXQLIRUPO\ FRYHU WKH HSLWKHOLDO VXUIDFH ZKLOH DYRLGLQJ DQ LQILQLWH GRVH VHH SDUDJUDSKV  DQG  $FFRUGLQJ WR WKH WHVW SURFHGXUH LW LV HVVHQWLDO WKDW WKH WLVVXH YLDELOLW\ PHDVXUHPHQWV DUH QRW PDGHLPPHGLDWHO\ DIWHU H[SRVXUH WR WKH WHVW FKHPLFDO EXW UDWKHU DIWHU D VXIILFLHQWO\ ORQJ SRVWH[SRVXUH
LQFXEDWLRQ SHULRG LQ IUHVK PHGLXP  DIWHU WKH WHVW FKHPLFDO KDV EHHQ ULQVHG IURP WKH WLVVXH 7KLV SHULRG
DOORZVERWKIRUUHFRYHU\IURPZHDNF\WRWR[LFHIIHFWVDQGIRUDSSHDUDQFHRIFOHDUF\WRWR[LFHIIHFWV
7HVWFKHPLFDOVWKDWFDQEHSLSHWWHGDW&RUORZHUWHPSHUDWXUHV XVLQJDSRVLWLYHGLVSODFHPHQWSLSHWWHLIQHHGHG DUHWUHDWHGDVOLTXLGVLQWKH9509LVFRXVZD[\UHVLQRXVDQGJHOOLNHWHVWFKHPLFDOVZLWK XQFOHDU SK\VLFDO VWDWH VKRXOG EH LQFXEDWHG DW & IRU  PLQXWHV EHIRUH GHFLGLQJ ZKLFKWUHDWPHQW SURWRFRO WR XVH ,I VXFK WHVW FKHPLFDOV EHFRPH SLSHWWDEOH DIWHU WKLV LQFXEDWLRQ SHULRG XVLQJ D
SRVLWLYHGLVSODFHPHQWSLSHWWHLIQHFHVVDU\ WKH\VKRXOGEHWUHDWHGDVOLTXLGVDQGVKRXOGEHDSSOLHGWRWKH
WLVVXHVGLUHFWO\IURPWKHZDWHUEDWK DW& RWKHUZLVHWKH\VKRXOGEHWUHDWHGDVVROLGV VHHSDUDJUDSK  ,Q WKH 950 ȝ/ RI OLTXLGWHVW FKHPLFDO DUH HYHQO\ VSUHDG RYHU WKH FP RI WKH WLVVXHVXUIDFH
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HQGRIWKHH[SRVXUHSHULRGWKHWHVWFKHPLFDODQGFRQWUROVXEVWDQFHVVKRXOGEHFDUHIXOO\UHPRYHGIURPWKH
WLVVXH VXUIDFH E\ H[WHQVLYH ULQVLQJ ZLWK &D0JIUHH '3%6 DW URRP WHPSHUDWXUH 7KLV ULQVLQJ VWHS LV
IROORZHGE\DPLQXWHVSRVWH[SRVXUHLPPHUVLRQLQIUHVKPHGLXPDWURRPWHPSHUDWXUH WRUHPRYHDQ\
WHVWFKHPLFDODEVRUEHGLQWRWKHWLVVXH DQGDPLQXWHVSRVWH[SRVXUHLQFXEDWLRQLQIUHVKPHGLXPDW
VWDQGDUGFXOWXUHFRQGLWLRQVSULRUWRSHUIRUPLQJWKH077DVVD\  
7HVW FKHPLFDOV WKDW FDQQRW EH SLSHWWHG DW WHPSHUDWXUHV XS WR & DUH WUHDWHG DV VROLGV LQ WKH
950,QWKH950DSSUR[LPDWHO\PJRIVROLGWHVWFKHPLFDODUHHYHQO\DSSOLHGRYHUWKHFPRIWKH
WLVVXHVXUIDFHXVLQJDFDOLEUDWHGWRRO HJDOHYHOOHGVSRRQIXOFDOLEUDWHGWRKROGPJRIVRGLXPFKORULGH 
DSSUR[LPDWHO\PJFPDSSOLFDWLRQ 7KHDPRXQWRIWHVWFKHPLFDODSSOLHGVKRXOGEHVXIILFLHQWWRFRYHU
WKHHQWLUHVXUIDFHRIWKHWLVVXH:KHQHYHUSRVVLEOHVROLGVVKRXOGEHWHVWHGDVDILQHSRZGHU7LVVXHVWUHDWHG
ZLWK VROLG WHVW FKHPLFDOV DQG ZLWK FRQWURO VXEVWDQFHV WHVWHG FRQFXUUHQWO\ WR VROLG WHVW FKHPLFDOV DUHLQFXEDWHG IRU  KRXUV DW VWDQGDUG FXOWXUH FRQGLWLRQV $W WKH HQG RI WKH H[SRVXUH SHULRG WKH WHVWFKHPLFDODQGFRQWUROVXEVWDQFHVVKRXOGEHFDUHIXOO\UHPRYHGIURPWKHWLVVXHVXUIDFHE\H[WHQVLYHULQVLQJZLWK &D0JIUHH '3%6 DW URRP WHPSHUDWXUH 7KLV ULQVLQJ VWHS LV IROORZHG E\ D  PLQXWHV SRVWH[SRVXUHLPPHUVLRQLQIUHVKPHGLXPDWURRPWHPSHUDWXUH WRUHPRYHDQ\WHVWFKHPLFDODEVRUEHGLQWRWKH
WLVVXH DQGDQKRXUVSRVWH[SRVXUHLQFXEDWLRQLQIUHVKPHGLXPDWVWDQGDUGFXOWXUHFRQGLWLRQVSULRUWRSHUIRUPLQJWKH077DVVD\  © OECD, (2015)

28

OECD/OCDE

492

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&RQFXUUHQWQHJDWLYHDQGSRVLWLYHFRQWUROVVKRXOGEHLQFOXGHGLQHDFKUXQWRGHPRQVWUDWHWKDWWKHYLDELOLW\ GHWHUPLQHGZLWKWKHQHJDWLYHFRQWURO DQGWKHVHQVLWLYLW\ GHWHUPLQHGZLWKWKHSRVLWLYHFRQWURO RIWKHWLVVXHVDUHZLWKLQDFFHSWDQFHUDQJHVGHILQHGEDVHGRQKLVWRULFDOGDWD7KH FRQFXUUHQWQHJDWLYH FRQWURODOVR SURYLGHV WKH EDVHOLQH  WLVVXH YLDELOLW\  WR FDOFXODWH WKH UHODWLYH SHUFHQW YLDELOLW\ RI WKH WLVVXHVWUHDWHGZLWKWKHWHVWFKHPLFDO 9LDELOLW\WHVW 7KHUHFRPPHQGHGSRVLWLYHFRQWUROVXEVWDQFHWREHXVHGZLWKWKH 950 LV QHDW PHWK\O DFHWDWH &$6 1R  6LJPD$OGULFK &DW  OLTXLG  7KHUHFRPPHQGHG QHJDWLYH FRQWURO VXEVWDQFH WR EH XVHG ZLWK WKH 950 LV XOWUDSXUH + 2 7KHVH ZHUH WKHFRQWURO VXEVWDQFHV XVHGLQWKH SUHYDOLGDWLRQDQG YDOLGDWLRQ VWXGLHV RI WKH950 DQGDUH WKRVHIRU ZKLFKPRVWKLVWRULFDOGDWDH[LVW7KHXVHRIVXLWDEOHDOWHUQDWLYHSRVLWLYHRUQHJDWLYHFRQWUROVXEVWDQFHVVKRXOGEH
VFLHQWLILFDOO\ DQG DGHTXDWHO\ MXVWLILHG 6HSDUDWH QHJDWLYH DQG SRVLWLYH FRQWUROV DUH QHHGHG IRU OLTXLG DQGVROLG WHVW FKHPLFDOV )RU FRQWUROV SHUIRUPHG FRQFXUUHQWO\ WR OLTXLG WHVW FKHPLFDOV  ȝ/ RI WKH QHJDWLYHFRQWURO DQG SRVLWLYH FRQWURO VXEVWDQFHV VKRXOG EH DSSOLHG WR WKH WLVVXHV H[DFWO\ DV IRU WKH OLTXLG WHVWFKHPLFDOV IROORZHG E\ D  PLQXWHV H[SRVXUH DW VWDQGDUG FXOWXUH FRQGLWLRQV ULQVLQJ D  PLQXWHVSRVWH[SRVXUH LPPHUVLRQ LQ IUHVK PHGLXP DW URRP WHPSHUDWXUH DQG D  PLQXWHV SRVWH[SRVXUH
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Tissue Viability Measurements

7KH077DVVD\LVDVWDQGDUGLVHGTXDQWLWDWLYHPHWKRG  WKDWVKRXOGEHXVHGWRPHDVXUHWLVVXHYLDELOLW\XQGHUWKLV7HVW*XLGHOLQH,WLVFRPSDWLEOHZLWKXVHLQDWKUHHGLPHQVLRQDOWLVVXHFRQVWUXFW7KH
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VWDQGDUG FXOWXUH FRQGLWLRQV 7KH YLWDO G\H 077 LV UHGXFHG LQWR D EOXH 077 IRUPD]DQ SUHFLSLWDWH E\ WKHYLDEOHFHOOVRIWKH5K&(WLVVXHFRQVWUXFW7KHSUHFLSLWDWHGEOXH077IRUPD]DQ SURGXFWLVWKHQH[WUDFWHG
IURPWKHWLVVXHXVLQJP/RILVRSURSDQRO RUDVLPLODUVROYHQW 7LVVXHVWHVWHGZLWKOLTXLGWHVWFKHPLFDOVVKRXOG EH H[WUDFWHG IURP ERWK WKH WRS DQG WKH ERWWRP RI WKH WLVVXHV ZKLOH WLVVXHV WHVWHG ZLWK VROLG WHVWFKHPLFDOV DQG FRORXUHG OLTXLGV VKRXOG EH H[WUDFWHG IURP WKH ERWWRP RI WKH WLVVXH RQO\ WR PLQLPLVH DQ\
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29

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31

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© OECD, (2015)
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32

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492

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33

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34

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Conclusion© OECD, (2015)
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35

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Multi-constituent substance: $VXEVWDQFHGHILQHGE\LWVTXDQWLWDWLYHFRPSRVLWLRQLQZKLFKPRUHWKDQRQH
PDLQFRQVWLWXHQWLVSUHVHQWLQDFRQFHQWUDWLRQ ZZ DQG ZZ $PXOWLFRQVWLWXHQWVXEVWDQFH
LVWKHUHVXOWRIDPDQXIDFWXULQJSURFHVV7KHGLIIHUHQFHEHWZHHQPL[WXUHDQGPXOWLFRQVWLWXHQWVXEVWDQFHLVWKDW D PL[WXUH LV REWDLQHG E\ EOHQGLQJ RI WZR RU PRUH VXEVWDQFHV ZLWKRXW FKHPLFDO UHDFWLRQ $ PXOWLFRQVWLWXHQWVXEVWDQFHLVWKHUHVXOWRIDFKHPLFDOUHDFWLRQ
MTT: 'LPHWK\OWKLD]RO\O GLSKHQ\OWHWUD]ROLXPEURPLGH7KLD]RO\OEOXHWHWUD]ROLXPEURPLGH
Negative control:$VDPSOHFRQWDLQLQJDOOFRPSRQHQWVRIDWHVWV\VWHPDQGWUHDWHGZLWKDVXEVWDQFHNQRZQQRW WR LQGXFH D SRVLWLYH UHVSRQVH LQ WKH WHVW V\VWHP 7KLV VDPSOH LV SURFHVVHG ZLWK WHVW FKHPLFDOWUHDWHG
VDPSOHVDQGRWKHUFRQWUROVDPSOHVDQGLVXVHGWRGHWHUPLQHWLVVXHYLDELOLW\
Not Classified: &KHPLFDOV WKDW DUH QRW FODVVLILHG IRU (\H LUULWDWLRQ 81 *+6 &DWHJRU\  $ RU %  RU
6HULRXVH\HGDPDJH 81*+6&DWHJRU\ ,QWHUFKDQJHDEOHZLWK³81*+61R&DWHJRU\´NSCkilled: 1RQ6SHFLILF&RORXULQNLOOHGWLVVXHVNSCliving: 1RQ6SHFLILF&RORXULQOLYLQJWLVVXHVNSMTT: 1RQ6SHFLILF077UHGXFWLRQ
OD:2SWLFDO'HQVLW\
Performance standards: 6WDQGDUGVEDVHGRQDYDOLGDWHGWHVWPHWKRGZKLFKZDVFRQVLGHUHGVFLHQWLILFDOO\YDOLG WKDW SURYLGH D EDVLV IRU HYDOXDWLQJ WKH FRPSDUDELOLW\ RI D SURSRVHG WHVW PHWKRG WKDW LV
PHFKDQLVWLFDOO\ DQG IXQFWLRQDOO\ VLPLODU ,QFOXGHG DUH L  HVVHQWLDO WHVW PHWKRG FRPSRQHQWV LL  D
PLQLPXP OLVW RI 5HIHUHQFH &KHPLFDOV VHOHFWHG IURP DPRQJ WKH FKHPLFDOV XVHG WR GHPRQVWUDWH WKHDFFHSWDEOH SHUIRUPDQFH RI WKH YDOLGDWHG WHVW PHWKRG DQG LLL  WKH FRPSDUDEOH OHYHOV RI DFFXUDF\ DQG© OECD, (2015)
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40

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492

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UHOLDELOLW\EDVHGRQZKDWZDVREWDLQHGIRUWKHYDOLGDWHGWHVWPHWKRGWKDWWKHSURSRVHGWHVWPHWKRGVKRXOG
GHPRQVWUDWHZKHQHYDOXDWHGXVLQJWKHPLQLPXPOLVWRI5HIHUHQFH&KHPLFDOV  
Positive control: $VDPSOHFRQWDLQLQJDOOFRPSRQHQWVRIDWHVWV\VWHPDQGWUHDWHGZLWKDVXEVWDQFHNQRZQWRLQGXFHDSRVLWLYHUHVSRQVHLQWKHWHVWV\VWHP7KLVVDPSOHLVSURFHVVHGZLWKWHVWFKHPLFDOWUHDWHGVDPSOHV
DQG RWKHU FRQWURO VDPSOHV 7R HQVXUH WKDW YDULDELOLW\ LQ WKH SRVLWLYH FRQWURO UHVSRQVH DFURVV WLPH FDQ EH
DVVHVVHGWKHPDJQLWXGHRIWKHSRVLWLYHUHVSRQVHVKRXOGQRWEHH[FHVVLYH
Relevance: 'HVFULSWLRQRIUHODWLRQVKLSRIWKHWHVWWRWKHHIIHFWRILQWHUHVWDQGZKHWKHULWLVPHDQLQJIXODQG
XVHIXO IRU D SDUWLFXODU SXUSRVH ,W LV WKH H[WHQW WR ZKLFK WKH WHVW FRUUHFWO\ PHDVXUHV RU SUHGLFWV WKHELRORJLFDOHIIHFWRILQWHUHVW5HOHYDQFHLQFRUSRUDWHVFRQVLGHUDWLRQRIWKHDFFXUDF\ FRQFRUGDQFH RIDWHVWPHWKRG  
Reliability: 0HDVXUHVRIWKHH[WHQWWKDWDWHVWPHWKRGFDQEHSHUIRUPHGUHSURGXFLEO\ZLWKLQDQGEHWZHHQODERUDWRULHV RYHU WLPH ZKHQ SHUIRUPHG XVLQJ WKH VDPH SURWRFRO ,W LV DVVHVVHG E\ FDOFXODWLQJ LQWUD DQGLQWHUODERUDWRU\UHSURGXFLELOLW\DQGLQWUDODERUDWRU\UHSHDWDELOLW\  
Replacement test: $WHVWZKLFKLVGHVLJQHGWRVXEVWLWXWHIRUDWHVWWKDWLVLQURXWLQHXVHDQGDFFHSWHGIRU
KD]DUG LGHQWLILFDWLRQ DQGRU ULVN DVVHVVPHQW DQG ZKLFK KDV EHHQ GHWHUPLQHG WR SURYLGH HTXLYDOHQW RU
LPSURYHG SURWHFWLRQ RI KXPDQ RU DQLPDO KHDOWK RU WKH HQYLURQPHQW DV DSSOLFDEOH FRPSDUHG WR WKHDFFHSWHGWHVWIRUDOOSRVVLEOHWHVWLQJVLWXDWLRQVDQGFKHPLFDOV  
Reproducibility: 7KHDJUHHPHQWDPRQJUHVXOWVREWDLQHGIURPUHSHDWHGWHVWLQJRIWKHVDPHWHVWFKHPLFDOXVLQJWKHVDPHWHVWSURWRFRO 6HH5HOLDELOLW\   
Reversible effects on the eye: 6HH³(\HLUULWDWLRQ´
RhCE: 5HFRQVWUXFWHGKXPDQ&RUQHDOLNH(SLWKHOLXP
Run: $UXQFRQVLVWVRIRQHRUPRUHWHVWFKHPLFDOVWHVWHGFRQFXUUHQWO\ZLWKDQHJDWLYHFRQWURODQGZLWKDSRVLWLYHFRQWURO
SD: 6WDQGDUG'HYLDWLRQ
Sensitivity: 7KHSURSRUWLRQRIDOOSRVLWLYHDFWLYHWHVWFKHPLFDOVWKDWDUHFRUUHFWO\FODVVLILHGE\WKHWHVW,WLV
DPHDVXUHRIDFFXUDF\IRUDWHVWPHWKRGWKDWSURGXFHVFDWHJRULFDOUHVXOWVDQGLVDQLPSRUWDQWFRQVLGHUDWLRQ
LQDVVHVVLQJWKHUHOHYDQFHRIDWHVWPHWKRG  Serious eye damage: 3URGXFWLRQRIWLVVXHGDPDJHLQWKHH\HRUVHULRXVSK\VLFDOGHFD\RIYLVLRQIROORZLQJDSSOLFDWLRQRIDWHVWVXEVWDQFHWRWKHDQWHULRUVXUIDFHRIWKHH\HZKLFKLVQRWIXOO\UHYHUVLEOHZLWKLQGD\V
RIDSSOLFDWLRQ,QWHUFKDQJHDEOHZLWK³,UUHYHUVLEOHHIIHFWVRQWKHH\H´DQGZLWK³81*+6&DWHJRU\´  Standard Operating Procedures (SOP): )RUPDOZULWWHQSURFHGXUHVWKDWGHVFULEHLQGHWDLOKRZVSHFLILF
URXWLQHDQGWHVWVSHFLILFODERUDWRU\RSHUDWLRQVVKRXOGEHSHUIRUPHG7KH\DUHUHTXLUHGE\*/3Specificity: 7KHSURSRUWLRQRIDOOQHJDWLYHLQDFWLYHWHVWFKHPLFDOVWKDWDUHFRUUHFWO\FODVVLILHGE\WKHWHVW,W
LVDPHDVXUHRIDFFXUDF\IRUDWHVWPHWKRGWKDWSURGXFHVFDWHJRULFDOUHVXOWVDQGLVDQLPSRUWDQWFRQVLGHUDWLRQ
LQDVVHVVLQJWKHUHOHYDQFHRIDWHVWPHWKRG  © OECD, (2015)
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41

492

OECD/OCDE
Substance: &KHPLFDO HOHPHQWV DQG WKHLU FRPSRXQGV LQ WKH QDWXUDO VWDWH RU REWDLQHG E\ DQ\ SURGXFWLRQSURFHVV LQFOXGLQJ DQ\ DGGLWLYH QHFHVVDU\ WR SUHVHUYH WKH VWDELOLW\ RI WKH SURGXFW DQG DQ\ LPSXULWLHVGHULYLQJIURPWKHSURFHVVXVHGEXWH[FOXGLQJDQ\VROYHQWZKLFKPD\EHVHSDUDWHGZLWKRXWDIIHFWLQJWKH
VWDELOLW\RIWKHVXEVWDQFHRUFKDQJLQJLWVFRPSRVLWLRQ  
Test: $VLQJOHWHVWFKHPLFDOFRQFXUUHQWO\WHVWHGLQDPLQLPXPRIWZRWLVVXHUHSOLFDWHVDVGHILQHGLQWKH
FRUUHVSRQGLQJ623

Tissue viability:3DUDPHWHUPHDVXULQJWRWDODFWLYLW\RIDFHOOSRSXODWLRQLQDUHFRQVWUXFWHGWLVVXHDVWKHLU
DELOLW\WRUHGXFHWKHYLWDOG\H077ZKLFKGHSHQGLQJRQWKHHQGSRLQWPHDVXUHGDQGWKHWHVWGHVLJQXVHGFRUUHODWHVZLWKWKHWRWDOQXPEHUDQGRUYLWDOLW\RIOLYLQJFHOOVTop-Down approach: 6WHSZLVHDSSURDFKXVHGIRUDFKHPLFDOVXVSHFWHGRIFDXVLQJVHULRXVH\HGDPDJHZKLFK VWDUWV ZLWK WKH GHWHUPLQDWLRQ RI FKHPLFDOV LQGXFLQJ VHULRXV H\H GDPDJH SRVLWLYH RXWFRPH  IURPRWKHUFKHPLFDOV QHJDWLYHRXWFRPH 
Test chemical: 7KHWHUPWHVWFKHPLFDOLVXVHGWRUHIHUWRZKDWLVEHLQJWHVWHG

Tiered testing strategy: $VWHSZLVHWHVWLQJVWUDWHJ\ZKLFKXVHVWHVWPHWKRGVLQDVHTXHQWLDOPDQQHU$OO
H[LVWLQJ LQIRUPDWLRQ RQ D WHVW FKHPLFDO LV UHYLHZHG DW HDFK WLHU XVLQJ D ZHLJKWRIHYLGHQFH SURFHVV WR
GHWHUPLQHLIVXIILFLHQWLQIRUPDWLRQLVDYDLODEOHIRUDKD]DUGFODVVLILFDWLRQGHFLVLRQSULRUWRSURJUHVVLRQWR
WKH QH[W WLHULQWKHVWUDWHJ\ ,I WKH KD]DUG SRWHQWLDOSRWHQF\RI D WHVW FKHPLFDO FDQ EH DVVLJQHG EDVHG RQ
WKHH[LVWLQJLQIRUPDWLRQDWDJLYHQWLHUQRDGGLWLRQDOWHVWLQJLVUHTXLUHG  
ULOQ: 8SSHU/LPLWRI4XDQWLILFDWLRQ
United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UNGHS): $ V\VWHP SURSRVLQJ WKH FODVVLILFDWLRQ RI FKHPLFDOV VXEVWDQFHV DQG PL[WXUHV  DFFRUGLQJ WR
VWDQGDUGLVHG W\SHV DQGOHYHOVRISK\VLFDOKHDOWKDQGHQYLURQPHQWDOKD]DUGVDQGDGGUHVVLQJFRUUHVSRQGLQJFRPPXQLFDWLRQHOHPHQWV VXFK DV SLFWRJUDPV VLJQDO ZRUGV KD]DUG VWDWHPHQWV SUHFDXWLRQDU\ VWDWHPHQWV
DQG VDIHW\ GDWDVKHHWVVRWKDWWRFRQYH\LQIRUPDWLRQRQWKHLUDGYHUVHHIIHFWVZLWKDYLHZWRSURWHFWSHRSOH
LQFOXGLQJ HPSOR\HUV ZRUNHUV WUDQVSRUWHUV FRQVXPHUV DQG HPHUJHQF\ UHVSRQGHUV  DQG WKH HQYLURQPHQW 
UN GHS Category 1: 6HH³6HULRXVH\HGDPDJH´UN GHS Category 2: 6HH³(\HLUULWDWLRQ´UN GHS No Category: &KHPLFDOV WKDW GR QRW PHHW WKH UHTXLUHPHQWV IRU FODVVLILFDWLRQ DV 81 *+6
&DWHJRU\RU $RU% ,QWHUFKDQJHDEOHZLWK³1RW&ODVVLILHG´
UPLC: 8OWUD+LJK3HUIRUPDQFH/LTXLG&KURPDWRJUDSK\
UVCB: VXEVWDQFHV RI XQNQRZQ RU YDULDEOH FRPSRVLWLRQ FRPSOH[ UHDFWLRQ SURGXFWV RU ELRORJLFDOPDWHULDOVValid test method: $ WHVW PHWKRG FRQVLGHUHG WR KDYH VXIILFLHQW UHOHYDQFH DQG UHOLDELOLW\ IRU D VSHFLILF
SXUSRVHDQGZKLFKLVEDVHGRQVFLHQWLILFDOO\VRXQGSULQFLSOHV$WHVWPHWKRGLVQHYHUYDOLGLQDQDEVROXWH
VHQVHEXWRQO\LQUHODWLRQWRDGHILQHGSXUSRVH  © OECD, (2015)
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42

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492

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Validated test method: $WHVWPHWKRGIRUZKLFKYDOLGDWLRQVWXGLHVKDYHEHHQFRPSOHWHGWRGHWHUPLQHWKH
UHOHYDQFH LQFOXGLQJDFFXUDF\ DQGUHOLDELOLW\IRUDVSHFLILFSXUSRVH,WLVLPSRUWDQWWRQRWHWKDWDYDOLGDWHG
WHVWPHWKRGPD\QRWKDYHVXIILFLHQWSHUIRUPDQFHLQWHUPVRIDFFXUDF\DQGUHOLDELOLW\WREHIRXQGDFFHSWDEOH
IRUWKHSURSRVHGSXUSRVH  
VRM: 9DOLGDWHG5HIHUHQFH0HWKRG
Weight-of-evidence: 7KH SURFHVV RI FRQVLGHULQJ WKH VWUHQJWKV DQG ZHDNQHVVHV RI YDULRXV SLHFHV RI
LQIRUPDWLRQLQUHDFKLQJDQGVXSSRUWLQJDFRQFOXVLRQFRQFHUQLQJWKHKD]DUGSRWHQWLDORIDWHVWVXEVWDQFH© OECD, (2015)
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43

492

OECD/OCDEANNEX IIILLUSTRATIVE FLOWCHART PROVIDING GUIDANCE ON HOW TO IDENTIFY AND
HANDLE DIRECT MTT-REDUCERS AND/OR COLOUR INTERFERING CHEMICALS, BASEDON THE VRM SOP© OECD, (2015)
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44

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OECD/OCDE


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492

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

6HOHFWLYLW\3UHFLVLRQProtocol Derived from FDA Guidance (29)(31)$QDO\VLVRILVRSURSDQROOLYLQJEODQN LVRSURSDQROH[WUDFW
IURPOLYLQJ5K&(WLVVXHFRQVWUXFWVZLWKRXWDQ\WUHDWPHQW 
GHDGEODQN LVRSURSDQROH[WUDFWIURPNLOOHG5K&(WLVVXH
FRQVWUXFWVZLWKRXWDQ\WUHDWPHQW DQGRIDG\H HJPHWK\OHQHEOXH 
4XDOLW\&RQWUROV LH077IRUPD]DQDWJP/
JP/DQGJP/ LQLVRSURSDQRO Q  $FFXUDF\

4XDOLW\&RQWUROVLQLVRSURSDQRO Q  

0DWUL[(IIHFW

4XDOLW\&RQWUROVLQOLYLQJEODQN Q  &DUU\RYHU

$QDO\VLVRILVRSURSDQRODIWHUDQ8/24VWDQGDUG

Acceptance Criteria$UHDLQWHUIHUHQFHRI$UHD//24
&9RUIRU
WKH//24
'HYRU
IRU//24
0DWUL[(IIHFW
$UHDLQWHUIHUHQFHRI$UHD//24

LQGHSHQGHQWFDOLEUDWLRQFXUYHV EDVHGRQFRQVHFXWLYHGLOXWLRQVRI077IRUPD]DQLQLVRSURSDQROVWDUWLQJDW
8/24LHJP/ &DOLEUDWLRQ&XUYHV
4XDOLW\&RQWUROVLQLVRSURSDQRO Q  
'HYRU
IRU//24
'D\FDOLEUDWLRQFXUYHDQG4XDOLW\&RQWUROVLQ
LVRSURSDQRO Q  
4XDOLW\&RQWUROV'HY'D\FDOLEUDWLRQFXUYHDQG4XDOLW\&RQWUROVLQ
5HSURGXFLELOLW\DQG&9
LVRSURSDQRO Q  
LQWHUGD\ 'D\FDOLEUDWLRQFXUYHDQG4XDOLW\&RQWUROVLQLVRSURSDQRO Q  
6KRUW7HUP6WDELOLW\ 4XDOLW\&RQWUROVLQOLYLQJEODQN Q  DQDO\VHGWKHGD\RI
RI077)RUPD]DQLQ
WKHSUHSDUDWLRQDQGDIWHUKRXUVRIVWRUDJHDWURRP'HY
5K&(7LVVXH([WUDFWWHPSHUDWXUH
/RQJ7HUP6WDELOLW\
RI077)RUPD]DQLQ 4XDOLW\&RQWUROVLQOLYLQJEODQN Q  DQDO\VHGWKHGD\RI
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5K&(7LVVXHWKHSUHSDUDWLRQDQGDIWHUVHYHUDOGD\VRIVWRUDJHDW&([WUDFWLIUHTXLUHG
5HSURGXFLELOLW\LQWUDGD\ 

//24/RZHU/LPLWRI4XDQWLILFDWLRQGHILQHGWRFRYHUWLVVXHYLDELOLW\LHJP/
8/24 8SSHU /LPLW RI 4XDQWLILFDWLRQ GHILQHG WR EH DW OHDVW WZR WLPHV KLJKHU WKDQ WKH KLJKHVW H[SHFWHG 077 IRUPD]DQ
FRQFHQWUDWLRQLQLVRSURSDQROH[WUDFWVIURPQHJDWLYHFRQWUROV aJP/LQWKH950 LHJP/© OECD, (2015)
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