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28␎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

39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࡜⤖ㄽࡋࡓࠋ410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࡟᥇ᢥࡋࡓ௦᭰ἲࡣࠊ࢘ࢩ᦬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࡟࠾࠸࡚⏝࠸
ࡽࢀࡿࠋRhCE ἲࡣࠊ෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝ㸦RhCE㸧⤌⧊࡟ᑐࡍࡿ⿕㦂≀㉁ࡢ⣽⬊ẘᛶࢆᣦᶆ࡜ࡋࠊ࣎ࢺ࣒࢔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
511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⏕ᡂࡉࢀࡓ࣐࣍ࣝࢨࣥ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࡜ุᐃࡍࡿࠋ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࠶ࡿࠋ4-2. 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 ୰࡛ 180s15
137s2ÛCࠊ5s1% CO2ࠊ95% ‵ᗘ612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࡟ࡣࠊᗏ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࠾ࡼࡧ ANNEX IV ࡢࣇ࣮ࣟࢳ࣮ࣕࢺࢆཧ↷ࡍࡿࠋ
4-4. ヨ㦂ᡂ❧ࡢᢎㄆᇶ‽௨ୗࡢ᮲௳ࢆࡍ࡭࡚‶ࡓࡋࡓሙྜࠊヨ㦂ࡢᡂ❧ࢆᢎㄆࡍࡿࠋ1㸧㝜ᛶᑐ↷ࡢᖹᆒ OD ࡀ 1.0 < OD  2.5 ࡛࠶ࡿࡇ࡜ࠋ2㸧㝧ᛶᑐ↷ࡢᖹᆒ⣽⬊⏕Ꮡ⋡ࡀᾮయ⿕㦂≀㉁࡛ࡣ 30%௨ୗࠊᅛయ⿕㦂≀㉁࡛ࡣ 20%௨ୗ࡛࠶ࡿࡇ
࡜ࠋ3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࡜ุ᩿ࡉࢀࡿࠋᅛయ⿕㦂≀㉁࡛ࡣᖹᆒ⣽⬊⏕Ꮡ⋡ࡀ 50%ࢆ㉸࠼ࡓሙྜࠊ⿕㦂≀㉁ࡣ GHS ༊ศእ࡜ุ᩿ࡉࢀࡿࠋࡇࢀࡽࡢ࢝ࢵࢺ࢜ࣇ್௨ୗࡢሙྜࡣࠊᮏ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 TG492 ࡟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࠸ࡿࠋ1) ࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡟࠾࠸࡚⿕㦂≀㉁࡟ྵࡲࢀ࡞࠿ࡗࡓẼయ㸦࢞ࢫ㸧࠾ࡼࡧ࢚࢔ࢰ࣮ࣝࡣ㐺⏝≀㉁࠿ࡽ㝖እࡉࢀࡿࠋ2) 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 eyedamage/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 foridentifying chemicals not requiring classification and labelling for eye irritation or serious eye damage.

915

Appendix 1ࣂࣜࢹ࣮ࢩࣙࣥ࡟⏝࠸ࡽࢀࡓ≀㉁
3 ᪋タ

࣮ࣜࢻࣛ࣎

ࣂࣜࢹ࣮ࢩࣙࣥ㏣ຍࢹ࣮ࢱ

12080

༊ศ 132

19

༊ศ 2A

1712

༊ศ 2B

134

༊ศእ

5845

ᾮయ

6045

ᅛయ

60

35

Aromatic

21

20

Alcohol

19

17

Ester

1712

Heterocyclic

13

12

Halogenated914

Carboxylic acid

117Amine

310Phenol

121

Acrylate

62

Ether

08Salt62

Polyether

61

Nitrile24

Onium Compound

06

Silicium42

Ketone32

Pyrimidine

14Thioether

41඲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

1016

Appendix 2RhCE 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