ホ౯ሗ࿌᭩

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

ᖹᡂ 30 ᖺ㸦2018 ᖺ㸧12 ᭶ 27 ᪥

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

28

␎ㄒ
CAS: Chemical Abstracts Services
EIT: Eye Irritation Test
ESAC: EURL ECVAM Scientific Advisory Committee
GHS: Globally Harmonized System of Classification and Labeling of Chemicals
JaCVAM: Japanese Center for the Validation of Alternative Methods
OD: Optical Density
OECD: Organization for Economic Co-operation and Development
RhCE: Reconstructed human Cornea-like Epithelium
SDS: Sodium Dodecyl Sulphate
TG: Test Guideline
UN: United Nations
VRM: Validated Reference Method
WST: Water Soluble Tetrazolium Salt39

せ᪨෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝ㸦Reconstructed human Cornea-like Epithelium: RhCE㸧ἲࡣࠊ໬Ꮫ≀㉁
ࡢ RhCE ⤌⧊࡟ᑐࡍࡿ⣽⬊ẘᛶࢆᣦᶆ࡟⏝࠸ࠊࡑࡢ≀㉁ࡢ║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛࠶ࡾࠊOECDTG492 ࡜ࡋ࡚࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡛ UN GHS ༊ศእ≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚᥇ᢥࡉࢀ࡚࠸ࡿࠋ
LabCyte CORNEA-MODEL24 ║่⃭ᛶヨ㦂㸦LabCyte CORNEA-MODEL24 EIT㸧ࡣ RhCE ἲࡢ୍ࡘ࡛ࠊ2018 ᖺ࡟ᨵゞ TG492 ࡟㏣ຍࡉࢀࡓヨ㦂ἲ࡛࠶ࡿࠋᮏሗ࿌࡛ࡣࠊLabCyte CORNEA-MODEL24 EIT ࡢࣂࣜࢹ࣮ࢩࣙࣥ◊✲ሗ࿌᭩ࠊ➨୕⪅ホ౯ሗ࿌᭩ࠊ㛵㐃ㄽᩥ࡞࡝ࢆࡶ࡜࡟ヨ㦂ἲࡢᴫせࢆㄝ᫂ࡋJaCVAM ║่⃭ᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍ࡢពぢࢆࡲ࡜ࡵࡓࠋ
 LabCyte CORNEA-MODEL24 EIT ࡢಙ㢗ᛶ࣭ṇ☜ᛶࢆ☜ㄆࡍࡿࡓࡵࠊOECD ࡢᛶ⬟ᶆ‽㸦PerformanceStandard㸧࡛ᣦᐃࡉࢀ࡚࠸ࡿ 30 ࡢཧ↷≀㉁㸦Reference Chemicals㸧ࢆ⏝࠸࡚ 3 ᪋タ࡛㏣㉮ⓗࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀ⾜ࢃࢀࡓࠋྛ᪋タ࡛ 1 ≀㉁࠶ࡓࡾ 3 ᅇヨ㦂ࢆ⾜ࡗࡓࠋࡇࡢ㏣㉮ⓗࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡟࠾࠸࡚ LabCyte CORNEA-MODL24 EIT ࡢ᪋タෆ෌⌧ᛶࡣࡑࢀࡒࢀࡢ᪋タ࡛ 93%ࠊ97%ࠊ100%ࠊࡲࡓࠊ᪋タ㛫෌⌧ᛶࡣ 87%࡛࠶ࡗࡓࠋṇ☜ᛶࡣࠊឤᗘ 97.8%ࠊ≉␗ᗘ 68.9%ࠊṇ☜ᗘ 83.5%࡛࠶ࡗࡓࠋࡇࢀ
ࡽ෌⌧ᛶࠊṇ☜ᛶࡢ್ࡣࠊᛶ⬟ᶆ‽ࡀᐃࡵࡓᇶ‽ࢆ‶ࡓࡋ࡚࠸ࡓࠋ ௨ୖࡼࡾࠊᮏጤဨ఍ࡣࠊLabCyte CORNEA-MODEL24 EIT ࡣ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡛ UN GHS ༊ศእ≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚⏝࠸ࡿࡇ࡜ࡀ࡛ࡁࡿ࡜⤖ㄽࡋࡓࠋ410

1. ࡲ࠼࠾ࡁ෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝ㸦Reconstructed human Cornea-like Epithelium: RhCE㸧ἲࡣࠊRhCE ⤌⧊࡟ᑐࡍࡿ⿕㦂≀㉁ࡢ⣽⬊ẘᛶࢆᣦᶆ࡟║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛ࠊ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟ࡼࡾ UNGHS ༊ศእ≀㉁ࢆ᳨ฟࡍࡿࠋ
EpiOcularTM ║่⃭ᛶヨ㦂㸦EpiOcularTM Eye Irritation Test: EpiOcularTM EIT㸧࠾ࡼࡧ SkinEthicTM ࣄࢺゅ⭷ୖ⓶ࣔࢹࣝ║่⃭ᛶヨ㦂㸦SkinEthicTM Human Corneal Epithelium Eye Irritation Test: SkinEthicTM
HCE EIT㸧ࡣ ESAC ࡢ➨୕⪅ホ౯ࢆ⤒࡚ RhCE ἲࡢ᳨ド῭ࡳᶆ‽ヨ㦂ἲ㸦Validated Reference Method:VRM㸧࡜ࡋ࡚ OECD TG492 ࡟཰㍕ࡉࢀ࡚࠸ࡿ 1)ࠋࡲࡓࠊOECD ࡣ᪂つ RhCE ⤌⧊ࢆ⏝࠸ࡓヨ㦂ἲ㸦me-tooヨ㦂ἲ㸧࡟ᑐࡋ࡚ࡑࡢᵓ㐀࣭ᶵᗎࡀ VRM ࡜ྠ➼࡛࠶ࡾ༑ศ࡞ಙ㢗ᛶ࡜㛵㐃ᛶࢆഛ࠼࡚࠸ࡿࡇ࡜ࢆホ౯ࡍࡿᛶ⬟ᶆ‽㸦Performance Standard㸧ࢆᐃࡵࡓ 2) ࠋLabCyte CORNEA-MODEL24 ║่⃭ᛶヨ㦂
㸦LabCyte CORNEA-MODEL24 Eye Irritation Test: LabCyte CORNEA-MODEL24 EIT㸧ࡣ me-too ヨ㦂ἲ࡜ࡋ࡚ࠊOECD ࡢᛶ⬟ᶆ‽࡛ᣦᐃࡉࢀ࡚࠸ࡿ 30 ࡢཧ↷≀㉁㸦Reference Chemicals㸧ࢆ⏝࠸ࡓ 3 ᪋タ࡛㏣㉮ⓗࣂࣜࢹ࣮ࢩࣙࣥ◊✲ 3)ࠊJaCVAM ࡢ➨୕⪅ホ౯ 4)ࢆ⤒࡚ 2018 ᖺ࡟ TG492 ࡟཰㍕ࡉࢀࡓ 1)ࠋ
ᮏሗ࿌᭩ࡣ LabCyte CORNEA-MODEL24 EIT ࡢࣂࣜࢹ࣮ࢩࣙࣥ◊✲ሗ࿌᭩ࠊ➨୕⪅ホ౯ሗ࿌᭩ࠊࡑࡢ௚㛵㐃ㄽᩥ࡞࡝ࢆࡶ࡜࡟ᮏヨ㦂ἲࡢᴫせࢆㄝ᫂ࡋࠊᮏጤဨ఍ࡢពぢࢆࡲ࡜ࡵࡓࡶࡢ࡛࠶ࡿࠋ2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࡜࠿ࡽጞࡲࡿࠋࡑࡢᶵᗎࡣᵝࠎ࡛࠶ࡿࡀࠊ⣽⬊ẘᛶࡀ㔜せ࡞ᙺ๭ࢆᢸࡗ࡚࠸ࡿࠋࡲࡓࠊ≀㉁ࡢ║่⃭ᛶࡣ୺࡟ゅ⭷ࡢึᮇᦆയࡢ῝ᗘ࡟ࡼࡾỴᐃࡉࢀࠊࡑࢀࡣ⣽⬊Ṛࡢ⛬ᗘ࡜┦㛵㛵ಀ࡟࠶ࡿࠋRhCE ἲࡣࠊ෌ᵓ⠏ࣄࢺゅ⭷ᵝୖ⓶ࣔࢹࣝࢆ⏝࠸࡚ࠊ⿕㦂≀㉁ࡢ⣽⬊ẘᛶࢆᣦᶆ࡜ࡋ࡚║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛࠶ࡿࠋ
LabCyte CORNEA-MODEL24 EIT ࡣࠊࣄࢺゅ⭷ୖ⓶⣽⬊ࢆ㔜ᒙᇵ㣴ࡋࡓᕷ㈍ࡢ RhCE ⤌⧊㸦LabCyteCORNEA-MODEL24㸧ࢆ⏝࠸ࡿ 5)ࠋ⿕㦂≀㉁ࡀᾮయࡢሙྜࡣ⣙ 1 ศ㛫ࠊᅛయࡢሙྜࡣ⣙ 24 ᫬㛫ࠊ⿕㦂≀㉁ࢆ LabCyte CORNEA-MODEL24 ࡟᭚㟢ࡋࡓᚋࠊWST-8 ἲࡼࡾ⟬ฟࡋࡓ⣽⬊⏕Ꮡ⋡ࢆ࢚ࣥࢻ࣏࢖ࣥࢺ࡟⏝࠸ࡿࠋࡇࢀࡣࠊ⣽⬊ෆࡢ⬺Ỉ⣲㓝⣲ࡢാࡁ࡟ࡼࡾ㟁Ꮚ࣓ࢹ࢕࢚࣮ࢱ࣮ࡢᏑᅾୗ࡛ WST-8
㸦2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt, CAS
No.193149-74-5㸧ࡀ㑏ඖࡉࢀ࡚⏕ᡂࡉࢀࡿᶳⰍ࣐࣍ࣝࢨࣥ㔞ࡀ⏕Ꮡ⣽⬊ᩘ࡟ẚ౛ࡍࡿࡇ࡜ࢆᇶᮏཎ⌮࡜ࡋ࡚࠸ࡿ 6)ࠋ⣽⬊⏕Ꮡ⋡ࡀ㝜ᛶᑐ↷࡜ẚ㍑ࡋ࡚ 40%ࢆ㉸࠼ࡓሙྜࠊ⿕㦂≀㉁ࡣ UN GHS ศ㢮࡟࠾࠸࡚༊ศእ࡛࠶ࡿ࡜ุᐃࡍࡿࠋ4㸬ヨ㦂ᡭ㡰 LabCyte CORNEA-MODEL24 EIT ࡢᡭ㡰ࢆ௨ୗ࡟♧ࡍࠋヲ⣽ࡣࠊᨵゞ TG4921)࠾ࡼࡧࣉࣟࢺࢥࣝ 7)ࢆཧ↷ࡍࡿࠋ౑⏝ࡍࡿ RhCE ⤌⧊ LabCyte CORNEA-MODEL24 ࡣᰴᘧ఍♫ࢪࣕࣃ࣭ࣥࢸ࢕ࢵࢩ࣭࢚ࣗࣥࢪࢽ࢔ࣜࣥࢢ㸦J-TECࠊ᪥ᮏ㸧ࡼࡾ㉎ධ࡛ࡁࡿࠋ

511

4-1. RhCE ⤌⧊ࡢᶵ⬟ⓗ᮲௳ LabCyte CORNEA-MODEL24 ࡢᶵ⬟ⓗ᮲௳ࡣࠊ௨ୗࡢ࡜࠾ࡾ࡛࠶ࡿࠋ⏕Ꮡ⋡㸸㝜ᛶᑐ↷࡟࠾࠸࡚ 0.5  OD  1.6㸦౑⏝᫬ࡢᇶ‽࡛࠶ࡾࠊ〇㐀⪅ࡢฟⲴᇶ‽ࡣ␗࡞ࡿሙྜࡶ࠶ࡿ㸧ࣂࣜ࢔ᶵ⬟㸸SDS㸦25ȝL㸧ࡢ 60 ศ㛫᭚㟢࡟࠾࠸࡚ 1.0  IC50 (mg/mL)  4.0㸦IC50㸸⣽⬊⏕Ꮡ⋡ࢆ 50%పୗࡉࡏࡿࡢ࡟ᚲせ࡞⃰ᗘ㸧 ᙧែ㸸ᑡ࡞ࡃ࡜ࡶ 3 ᒙࡢୖ⓶⣽⬊ࡀ࠶ࡾࠊࡑࡢ⾲㠃ࡣゅ໬ࡋ࡚࠸࡞࠸ ෌⌧ᛶ㸸㝧ᛶ࠾ࡼࡧ㝜ᛶᑐ↷ࡢ⤖ᯝࡣ⫼ᬒࢹ࣮ࢱࢆࡶ࡜࡟タᐃࡋࡓチᐜ್ෆ࡟࠶ࡿ
ࡇࢀࡽ㸲㡯┠ࡣ LabCyte CORNEA-MODEL24 ࡢ〇㐀⪅ࡢฟⲴᇶ‽࡜ࡋ࡚᥇⏝ࡉࢀࡿࠋ୍᪉ࠊLabCyte
CORNEA-MODEL24 ࡢ౑⏝⪅ࡣ⏕Ꮡ⋡࡜෌⌧ᛶࡢ 2 㡯┠ࢆ☜ㄆࡍࡿᚲせࡀ࠶ࡿࠋ4-2. ⿕㦂≀㉁ࡢ㐺⏝ ≀㉁࠶ࡓࡾ 3 RhCE ⤌⧊ࢆ⏝࠸ࡿࠋ⿕㦂≀㉁ࡣㄪ〇ࡏࡎࠊࡑࡢࡶࡢࢆ⏝࠸ࡿࠋ37ÛC ௨ୗ࡛ࣆ࣌ࢵࢺ࡛ᢅ࠼ࡿࡶࡢࡣᾮయ࡜ࡋ࡚ࠊࡑࢀ௨እࡣᅛయ࡜ࡋ࡚ヨ㦂ࢆ⾜࠺ࠋᾮయ⿕㦂≀㉁ࡢሙྜࠊLabCyteCORNEA-MODEL24 ࡟ 50 μL ࢆ㐺⏝ࡋࠊᐊ

࡛ 1 ศ㛫㸦s5 ⛊㸧ᇵ㣴ࡍࡿࠋࡑࡢᚋࠊCa2+/Mg2+୙ྵ

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

ᐃ㸦OD450nm㸫OD650nm㸧ࡲࡓࡣ HPLC/UPLC ࡛⾜࠺ࠋ⿕㦂≀㉁ࡀ WST-8 㑏ඖ≀㉁ࡢሙྜࠊ࠶ࡿ࠸ࡣ࣐࣍ࣝࢨࣥ࡜ྠࡌࡼ࠺࡞Ἴ㛗㸦450 nm ㏆㎶㸧࡟྾཰ࢆᣢࡘ╔Ⰽ≀㉁ࡢሙྜࠊ⣽⬊⏕Ꮡ⋡ࡢ⿵ṇࢆ⾜࠺ᚲせࡀ࠶ࡿࠋ⿕㦂≀㉁ࡀ WST-8 ἲࢆᖸ΅ࡍࡿ≀㉁࡛࠶ࡿ࠿࡝࠺࠿ࡢ஦๓᳨ウ࡜ࡑࡢሙྜࡢ⣽⬊⏕Ꮡ⋡ࡢ⿵ṇࡢᡭ㡰࡟ࡘ࠸࡚ࡣ TG ᮏᩥࡢㄝ᫂࠾ࡼࡧ
ANNEX V ࡢࣇ࣮ࣟࢳ࣮ࣕࢺࢆཧ↷ࡍࡿࠋ
4-4. ヨ㦂ᡂ❧ࡢᢎㄆᇶ‽௨ୗࡢ᮲௳ࢆࡍ࡭࡚‶ࡓࡋࡓሙྜࠊヨ㦂ࡢᡂ❧ࢆᢎㄆࡍࡿࠋ
1㸧㝜ᛶᑐ↷ࡢᖹᆒ OD ࡀ 0.5  OD  1.6 ࡛࠶ࡿࡇ࡜ࠋ2㸧㝧ᛶᑐ↷ࡢᖹᆒ⣽⬊⏕Ꮡ⋡ࡀ 40%௨ୗ࡛࠶ࡿࡇ࡜ࠋ
37s2ÛCࠊ5s1% CO2ࠊ95% ‵ᗘ6123㸧⿕㦂≀㉁࠾ࡼࡧ㝜ᛶ࣭㝧ᛶᑐ↷ࡢࡑࢀࡒࢀ࡟࠾࠸࡚⣽⬊⏕Ꮡ⋡ࡢᶆ‽೫ᕪࡀ 18%௨ୗ࡛࠶ࡿࡇ࡜ࠋ࡞࠾ࠊ」ᩘࡢ⿕㦂≀㉁ࢆྠ᫬࡟ヨ㦂ࡋࡓሙྜࡣࠊࡇࡢ᮲௳ࢆ‶ࡓࡉ࡞࠸⿕㦂≀㉁ࡢࡳ୙ᡂ
❧࡜ࡍࡿࠋ
4-5. ุᐃ⿕㦂≀㉁ࡢᖹᆒ⣽⬊⏕Ꮡ⋡ࡀ 40%ࢆ㉸࠼ࡓሙྜࠊ⿕㦂≀㉁ࡣ UN GHS ༊ศእ࡜ุ᩿ࡉࢀࡿࠋᖹᆒ⣽⬊⏕Ꮡ⋡ࡀ 40%௨ୗࡢሙྜࡣࠊࡑࡢ⤖ᯝࡢࡳ࡛ࡣ⿕㦂≀㉁ࡢ║่⃭ᛶࡢணࡣ࡛ࡁ࡞࠸ࠋᮏヨ㦂ἲ࡛ࡣഇ㝧ᛶࡢྍ⬟ᛶࡀ࠶ࡾࠊࡲࡓ UN GHS ༊ศ 1 ࡜༊ศ 2 ࢆ༊ู࡛ࡁ࡞࠸ࠋࡑࡢሙྜࡣࠊ⿕㦂≀㉁ࡢ║่⃭ᛶࢆศ㢮ࡍࡿ࡟ࡣ㏣ຍࡢ᝟ሗࡀᚲせ࡜࡞ࡿࠋྛ⤌⧊ࡢᐃ್࡛ࡢุᐃࡀ୍⮴ࡋ࡞࠸ࠊᖹᆒ⣽⬊⏕Ꮡ⋡ࡀ 40s5%ࡢ⠊ᅖ࡟࠶ࡿ➼ࠊᚓࡽࢀࡓ⤖ᯝࡀ࣮࣎ࢲ࣮ࣛ࢖ࣥࡢሙྜࡣ 2 ᅇ┠ࡢヨ㦂ࢆ᳨ウࡍࡿࠋ1 ᅇ┠࡜ 2 ᅇ┠ࡢヨ㦂࡛⤖ᯝࡀ୍⮴ࡋ࡞࠸ሙྜ
ࡣ 3 ᅇ┠ࡢヨ㦂ࢆ᳨ウࡍࡿࠋ⾲ 1. VRM ࡜ LabCyte CORNEA-MODEL24 EIT ࡢ୺࡞㡯┠ࡢẚ㍑
EpiOcularTM IET

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

RhCE ⤌⧊ࡢ⏤᮶ࣄࢺ⾲⓶ゅ໬⣽⬊ࣄࢺ୙Ṛ໬ゅ⭷ୖ⓶⣽⬊ࣄࢺゅ⭷ୖ⓶⣽⬊᭚㟢㔞50 ȝL ࡲࡓࡣ50 mg (0.6 cm2 )30 ȝL ࡲࡓࡣ 30 mg (0.5 cm2 )50 ȝL ࡲࡓࡣ 10 mg (0.3 cm2 )᭚㟢᫬㛫

30 ศ㸦ᾮయ㸧ࠊ6 ᫬㛫㸦ᅛయ㸧 30 ศ㸦ᾮయ㸧ࠊ4 ᫬㛫㸦ᅛయ㸧 1 ศ㸦ᾮయ㸧ࠊ24 ᫬㛫㸦ᅛయ㸧㝧ᛶ㸸࢚ࢱࣀ࣮ࣝ㸦ᾮయ㸧ࠊࣛ࢘ࣜࣥ㓟㸦ᅛయ㸧㝧ᛶ㸸㓑㓟࣓ࢳࣝ㝧ᛶ㸸㓑㓟࣓ࢳࣝ㝜ᛶ㸸㉸⣧Ỉ㝜ᛶ㸸Ca2+/Mg2+୙ྵ DPBS㝧ᛶ࣭㝜ᛶᑐ↷㝜ᛶ㸸Ca2+/Mg2+୙ྵ DPBS㸦ᾮయ㸧ࠊ↓ฎ⌮㸦ᅛయ㸧⏕Ꮡ⋡ᐃUN GHS ༊ศእุᐃᇶMTT ἲ

MTT ἲ

WST-8 ἲ>60%>60%㸦ᾮయ㸧ࠊ>50%㸦ᅛయ㸧 >40%‽㸦ᖹᆒ⣽⬊⏕Ꮡ⋡㸧

5㸬㏣㉮ⓗࣂࣜࢹ࣮ࢩࣙࣥ◊✲ 㛤Ⓨ⪅ࡢ J-TEC㸦࣮ࣜࢻࣛ࣎㸧࡜ࡣ␗࡞ࡿ 3 ᪋タ㸦ᰴᘧ఍♫⸆≀Ᏻ඲ᛶヨ㦂ࢭࣥࢱ࣮ࠊ᪥ᮏࢥ࣐ࣝ
࣮ᰴᘧ఍♫ࠊᐩኈࣇ࢕࣒ࣝᰴᘧ఍♫㸧࡛ LabCyte CORNEA-MODEL24 EIT ࡢ㏣㉮ⓗࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀ⾜ࢃࢀࡓ 3)ࠋࡇࢀࡽ 3 ᪋タࡣ㏣㉮ⓗࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡢ๓࡟ᢏ⾡⛣㌿ᛶࡢ☜ㄆࢆ⾜ࡗࡓ⥲ᙜࡾヨ㦂࡟ཧຍࡋࡓ᪋タ࡛࠶ࡿ 8)ࠋ⿕㦂≀㉁࡟ࡣ OECD ࡢᛶ⬟ᶆ‽࡛ᣦᐃࡉࢀ࡚࠸ࡿ 30 ࡢཧ↷≀㉁ࢆ⏝࠸ࡓ㸦Appendix 1㸧ࠋヨ㦂ࡣࡍ࡭࡚ࡢ⿕㦂≀㉁࡟ࡘ࠸࡚ 3 ᪋タ࡛ࡑࢀࡒࢀ 3 ᅇᐇ᪋ࡉࢀࡓࠋ5-1㸬ヨ㦂ἲࡢಙ㢗ᛶ
5.1.1. ᢏ⾡⛣㌿ᛶ ᢏ⾡⛣㌿ᛶ࡟ࡘ࠸࡚ࡣࠊ㏣㉮ⓗࣂࣜࢹ࣮ࢩࣙࣥࡢᐇ᪋๓࡟⾜ࢃࢀࡓ 24 ᪋タࡀཧຍࡋࡓ⥲ᙜࡾヨ㦂

713᳨࡛ウࡉࢀࠊⰋዲ࡞⤖ᯝࡀᚓࡽࢀ࡚࠸ࡿ 8)ࠋ

5-1-2. ᪋タෆ෌⌧ᛶUN GHS ศ㢮ุᐃࡢ᪋タࡈ࡜ࡢ᪋タෆ෌⌧ᛶࡣ 93%ࠊ97%ࠊ100%࡛࠶ࡗࡓࠋࡇࢀࡽࡢ್ࡣ OECDࡢᛶ⬟ᶆ‽ࡀᐃࡵࡓᇶ‽㸦90%௨ୖ㸧ࢆ‶ࡓࡋࡓ 3)ࠋ
࡞࠾ࠊ࣮ࣜࢻ࡛ࣛ࣎࠶ࡿ J-TEC ࡢ♫ෆࢹ࣮ࢱ㸦139 ≀㉁㸧࡛ࡣࠊ᪋タෆ෌⌧ᛶࡣ 97%࡛࠶ࡗࡓ 8)ࠋ
5-1-3. ᪋タ㛫෌⌧ᛶUN GHS ศ㢮ุᐃࡢ᪋タ㛫෌⌧ᛶࡣ 87%࡛࠶ࡗࡓࠋࡇࡢ್ࡣ OECD ࡢᛶ⬟ᶆ‽ࡀᐃࡵࡓᇶ‽㸦85%௨ୖ㸧ࢆ‶ࡓࡋࡓ 3)ࠋ
5-2㸬ヨ㦂ἲࡢṇ☜ᛶṇ☜ᛶࡢホ౯࡟ࡣࠊ3 ᪋タ࡛⾜ࢃࢀࡓ඲ヨ㦂ࡢࢹ࣮ࢱࢆ⏝࠸ࡓࠋࡑࡢ⤖ᯝࠊឤᗘ 97. 8%ࠊ≉␗ᗘ 68.9%ࠊ
ṇ☜ᗘ 83.5%࡛࠶ࡗࡓࠋࡇࢀࡽࡢ್ࡣ OECD ࡢᛶ⬟ᶆ‽ࡀᐃࡵࡓᇶ‽㸦ឤᗘ 90%௨ୖࠊ≉␗ᗘ 60%௨ୖࠊṇ☜ᗘ 75%௨ୖ㸧ࢆ‶ࡓࡋࡓ 3)ࠋ
࡞࠾ࠊ࣮ࣜࢻ࡛ࣛ࣎࠶ࡿ J-TEC ࡢ♫ෆࢹ࣮ࢱ㸦139 ≀㉁㸧࡛ࡣࠊឤᗘ 100.0%ࠊ≉␗ᗘ 73.0%ࠊṇ☜ᗘ 87.8%࡛࠶ࡗࡓ 8)ࠋ
6㸬ヨ㦂ἲࡢ㐺⏝⠊ᅖ
TG492 ࡣࠊヨ㦂ἲࡢ㐺ṇ࡜ṇ☜ᛶࡢ☜ಖࢆ⪃៖ࡋ࡚ RhCE ἲࡢ㐺⏝࡟௨ୗࡢไ㝈ࢆタࡅ࡚࠸ࡿࠋ1) ࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡟࠾࠸࡚⿕㦂≀㉁࡟ྵࡲࢀ࡞࠿ࡗࡓẼయ㸦࢞ࢫ㸧࠾ࡼࡧ࢚࢔ࢰ࣮ࣝࡣ㐺⏝≀㉁࠿ࡽ㝖እࡉࢀࡿࠋ2) UN GHS ༊ศ 1ࠊ༊ศ 2㸦2A࣭2B㸧≀㉁ࡢ᳨ฟ࡟ࡣ⏝࠸ࡿࡇ࡜ࡣ࡛ࡁ࡞࠸ࠋ
7. ᮏጤဨ఍ࡢ⤖ㄽ
LabCyte CORNEA-MODEL24 EIT ࡢ㏣㉮ⓗࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡢ⤖ᯝࠊᮏヨ㦂ἲࡣ෌⌧ᛶ࠾ࡼࡧṇ☜ᛶ࡟ࡘ࠸࡚ OECD ࡢᛶ⬟ᶆ‽ࡀᐃࡵࡓᇶ‽ࢆ‶ࡓࡋ࡚࠸ࡓࠋ
LabCyte CORNEA-MODEL24 EIT ࡟⏝࠸ࡿ RhCE ⤌⧊ࡣᕷ㈍ࡉࢀ࡚࠾ࡾࠊࡲࡓࠊRhCE ⤌⧊௨እࡣࠊ
≉Ṧ࡞ᶵᮦࡸヨ⸆ࢆᚲせ࡜ࡏࡎࠊᡭᢏࡶ」㞧࡞ࡶࡢ࡛࡞࠸ࠋධᡭࡋࡓ RhCE ⤌⧊ࡀᶵ⬟ⓗ᮲௳ࡢチᐜ⠊ᅖ࡟࠶ࡾࠊ࠿ࡘᐇ᪋ࡍࡿヨ㦂᪋タ࡜ࡋ࡚ᢏ⾡⩦ᚓࡀ࢞࢖ࢻࣛ࢖ࣥࡢ⩦⇍ᗘ☜ㄆ≀㉁ Appendix2 ࡛☜࠿ࡵࡽࢀ࡚࠸ࢀࡤࠊLabCyte CORNEA-MODEL24 EIT ࡣ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡛ UN GHS ༊ศእ≀㉁ࢆ
᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚⏝࠸ࡿࡇ࡜ࡀ࡛ࡁࡿࠊ࡜ᮏጤဨ఍ࡣ⪃࠼ࡿࠋ8148. ᩥ⊩1)

OECD (2018) 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.

2)

OECD (2015) Performance Standards for the Assessment of Proposed Similar or Modified In Vitro
Reconstructed Human Cornea-like Epithelium (RhCE) Test Methods for Eye Hazards, Series on Testing &Assessment No. 2163)

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

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

5)

Katoh, M et al (2013) Establishment of a new in vitro test method for evaluation of eye irritation using areconstructed human corneal epithelial model, LabCyte CORNEA-MODEL, Toxicol. in Vitro 27, 21846)

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

7)

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

8)

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

915

Appendix 1㏣㉮ⓗࣂࣜࢹ࣮ࢩࣙࣥ࡟⏝࠸ࡽࢀࡓཧ↷≀㉁CAS ␒ྕ

ᛶ≧UN GHS ศ㢮(Ethylenediaminepropyl)-trimethoxysilane

1760-24-3

ᾮయ

༊ศ 1

Methylthioglycolate

2365-48-2

ᾮయ

༊ศ 1

Tetraethylene glycol diacrylate

17831-71-9

ᾮయ

༊ศ 1

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

2634-33-5

ᅛయ

༊ศ 1

2,5-Dimethyl-2,5-hexanediol

110-03-2

ᅛయ༊ศ 1

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

ᅛయ༊ศ 162-76-0

ᅛయ

༊ศ 1

18472-51-0

ᾮయ

༊ศ 2A

gamma-Butyrolactone

96-48-0

ᾮయ༊ศ 2A

1,5-Naphthalenediol

83-56-7

ᅛయ༊ศ 2A

Sodium benzoate

532-32-1

ᅛయ

༊ศ 2A

2-Methyl-1-pentanol

105-30-6

ᾮయ

༊ศ 2B

Diethyl toluamide

134-62-3

ᾮయ

༊ศ 2B

1,4-Dibutoxy benzene

104-36-9

ᅛయ

༊ศ 2B

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

79-92-5

ᅛయ༊ศ 2B

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

ᾮయ

༊ศእ

2-Ethoxyethyl methacrylate

2370-63-0ᾮయ༊ศእ

3-Phenoxybenzyl alcohol

13826-35-2

ᾮయ

༊ศእ

4-(Methylthio)-benzaldehyde

3446-89-7

ᾮయ༊ศእ

Dipropyl disulphide629-19-6

ᾮయ༊ศእEthyl thioglycolate

623-51-8ᾮయ༊ศእPiperonyl butoxide51-03-6ᾮయ

༊ศእPolyethylene glycol (PEG-40) hydrogenated castor oil

61788-85-0

⢓ᛶ

༊ศእ

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

101-20-2ᅛయ༊ศእ

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

3179-89-3ᅛయ༊ศእ

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

103597-45-1ᅛయ༊ศእ

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

118-82-1ᅛయ

༊ศእ

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

68610-92-4ᅛయ

༊ศእ

Potassium tetrafluoroborate

14075-53-7

ᅛయ

༊ศእ

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

66170-10-3

ᅛయ༊ศእ≀㉁ྡ

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

1016



Appendix 2
RhCE ἲࡢ⩦⇍ᗘ☜ㄆ≀㉁CAS ␒ྕ

ᛶ≧UN GHS ศ㢮

Methylthioglycolate

2365-48-2ᾮయ

༊ศ 1Hydroxyethyl acrylate

818-61-1ᾮయ

༊ศ 1

2,5-Dimethyl-2,5-hexanediol

110-03-2ᅛయ༊ศ 1Sodium oxalate

62-76-0ᅛయ༊ศ 1

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

18472-51-0

ᾮయ

༊ศ 2A

Sodium benzoate

532-32-1

ᅛయ

༊ศ 2A

Diethyl toluamide

134-62-3

ᾮయ

༊ศ 2B

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

79-92-5

ᅛయ༊ศ 2B

342573-75-7

ᾮయ

༊ศእ

Dicaprylyl ether

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

61788-85-0⢓ᛶ≀༊ศእ

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

101-20-2ᅛయ༊ศእ

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

103597-45-1

ᅛయ

༊ศእ

Potassium tetrafluoroborate

14075-53-7

ᅛయ༊ศእ≀㉁ྡ

1-Ethyl-3-methylimidazolium ethylsulphate1117