║่⃭ᛶヨ㦂௦᭰ἲࡢホ౯఍㆟ሗ࿌᭩

In vitro ▷᫬㛫᭚㟢ἲ(The Short Time Exposure In Vitro Test Method: STE ἲ)

JaCVAM ホ౯఍㆟

ᖹᡂ 27 ᖺ㸦2015 ᖺ㸧12 ᭶ 18 ᪥11JaCVAM ホ౯఍㆟኱㔝Ὀ㞝㸦㐠Ⴀጤဨ఍᥎⸀㸧㸸ᗙ㛗㣤ሯᑦᩥ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 ⑓⌮㒊㸧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 ᪥22

In vitro ▷᫬㛫᭚㟢ἲ㸦The Short Time Exposure In Vitro Test Method㸸STE ἲ㸧ࡣࠊ࢘ࢧࢠࢆ⏝࠸ࡓDraize ║่⃭ᛶヨ㦂ἲࡢ௦᭰ヨ㦂ἲ࡛࠶ࡾࠊ࢘ࢧࢠゅ⭷⏤᮶ࡢୖ⓶⣽⬊ᰴ㸦SIRC ⣽⬊㸧࡟ᑐࡍࡿ໬Ꮫ≀㉁ࡢ⣽⬊ẘᛶࢆᣦᶆ࡟⏝࠸࡚║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛࠶ࡿࠋࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡟࠾࠸࡚㔜⠜࡞║ࡢᦆയࢆ㉳ࡇࡍ≀㉁㸦United Nations Globally Harmonized System of Classification and Labeling of Chemicals:
UN GHS ༊ศ 1㸧ࢆ᳨ฟࡍࡿ᪉ἲࠊ࠾ࡼࡧ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾࠸࡚║่⃭ᛶ≀㉁࡜ࡣศ㢮ࡉࢀ࡞࠸≀㉁㸦UN GHS ༊ศእ㸧ࢆ᳨ฟࡍࡿ᪉ἲ 1) ࡜ࡋ࡚ࣂࣜࢹ࣮ࢩࣙࣥࡀ⾜ࢃࢀࠊ2015 ᖺ࡟ OECD TG491 ࡜ࡋ࡚᥇ᢥࡉࢀࡓ 2㸧ࠋJaCVAM ホ౯఍㆟ࡣࠊࡇࡢ TG491 ࡢጇᙜᛶ࡟ࡘ࠸᳨࡚ウࡋࡓࠋ㸯㸬ヨ㦂ἲࡢᐃ⩏ྡ⛠㸸 In vitro ▷᫬㛫᭚㟢ἲ㸦The Short Time Exposure In Vitro Test Method㸸STE ἲ㸧௦᭰ࡍࡿᑐ㇟ẘᛶヨ㦂㸸 Draize ║่⃭ᛶヨ㦂ヨ㦂ἲࡢᴫ␎㸸ࢥࣥࣇ࢚ࣝࣥࢺ࡟࡞ࡿࡼ࠺࡟༢ᒙᇵ㣴ࡋࡓ SIRC ⣽⬊࡟⿕㦂≀㉁ᕼ㔘ᾮࢆ 5 ศ㛫᭚㟢ࡋࡓᚋࠊ⣽⬊ࢆὙίࡋࠊࡑࡢᚋࠊMTT㸦3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide㸧ࡢྲྀࡾ㎸ࡳ㔞࡛ᐃࡋࡓ⣽⬊⏕Ꮡ⋡ࢆᣦᶆ࡟⏝࠸ࡿࠋࡇࢀࡣ MTT ࡀ࣑ࢺࢥࣥࢻࣜ࢔ෆࡢ⬺Ỉ⣲㓝⣲ࡢᇶ㉁࡜࡞ࡿᛶ㉁ࢆ฼
⏝ࡋࠊ⣽⬊ෆ࡟ྲྀࡾ㎸ࡲࢀࡓ MTT ࡀ㑏ඖࡉࢀࠊ⏕ᡂࡍࡿ࣐࣍ࣝࢨࣥ㔞ࡀ⏕Ꮡ⣽⬊ᩘ࡟ẚ౛ࡍࡿࡇ࡜ࢆ
฼⏝ࡋ࡚࠸ࡿ㸦MTT 㑏ඖἲ㸧ࠋ      㸰㸬ホ౯࡟⏝࠸ࡓ㈨ᩱ࠾ࡼࡧホ౯ෆᐜࡢ⛉Ꮫⓗጇᙜᛶ║࡟␗≀ࡀධࡗࡓሙྜࠊ║ࡢ่⃭ᛶࡣゅ⭷ࡸ⤖⭷ࡢ⣽⬊യᐖ࠿ࡽጞࡲࡿࠋᮏヨ㦂ἲࡣࡇࢀ࡟ὀ┠ࡋ࡚ࠊゅ⭷ୖ⓶⣽⬊࡟ᑐࡍࡿ⣽⬊ẘᛶࢆᣦᶆ࡜ࡋ࡚║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛࠶ࡿࠋ║࡟⁐ᾮࡀධࡗࡓሙྜࠊࣄࢺ࡛ࡣࡑࡢ኱㒊ศࡀ 1㹼2 ศ࡛║ෆ࠿ࡽ᤼ฟࡉࢀࠊ࢘ࢧࢠ࡛ࡣ 3㹼4 ศ࡛ 80%ࡀ᤼ฟࡉࢀࡿ࡜ሗ࿌ࡉࢀ
࡚࠸ࡿ 3㸧ࠋࡇࡢࡼ࠺࡞ᐇ㝿ࡢ᭚㟢≧ἣࢆ⪃៖ࡋ࡚ࠊᮏヨ㦂ἲࡣ㏻ᖖࡢ⣽⬊ẘᛶヨ㦂࡜ẚ࡭࡚▷᫬㛫᭚㟢ࡢヨ㦂࡜ࡋ࡚タィࡉࢀ࡚࠸ࡿࠋࡇࢀࡽࡢࡇ࡜࠿ࡽࠊᮏヨ㦂ἲࡣ࢘ࢧࢠࢆ⏝࠸ࡿ║่⃭ᛶヨ㦂ࡢ௦᭰ἲ࡜
ࡋ࡚⛉Ꮫⓗጇᙜᛶࡀ࠶ࡿࠋᮏヨ㦂ἲࡣ᪥ᮏື≀ᐇ㦂௦᭰ἲᏛ఍ 4㸧࡜ JaCVAM5㸧ࡢࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࢆ⤒࡚ࠊࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡟࠾࠸࡚ UN GHS ༊ศ㸯≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࠾ࡼࡧ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾࠸࡚ UN GHS ༊ศእ≀㉁ࢆ᳨
ฟࡍࡿ᪉ἲ࡜ࡋ࡚ ICCVAM(Interagency Coordinating Committee on the Validation of Alternative
Methods)/NICEATM(National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative
Toxicological Methods)࡟ࡼࡾ➨୕⪅ホ౯ࢆཷࡅࠊࡑࡢሗ࿌᭩ࡀ 2013 ᖺ 6 ᭶࡟බ⾲ࡉࢀࡓ 6㸧ࠋࡑࡢᚋࠊ2015ᖺ 7 ᭶࡟ OECD ࡟ࡼࡾࢺࢵࣉࢲ࣭࢘ࣥ࣎ࢺ࣒࢔ࢵࣉ୧᪉ᘧ࡛฼⏝࡛ࡁࡿ║่⃭ᛶヨ㦂௦᭰ἲ࡜ࡋ࡚
TG491 ࡀ᥇ᢥࡉࢀࡓ 2㸧ࠋ

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ICCVAM/NICEATM ࡟ࡼࡿࢺࢵࣉࢲ࢘ࣥ᪉ᘧࡢṇ☜ᛶࡢ᳨ウ࡟ࡣ 125 ≀㉁ࡀࠊ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧࡢṇ☜ᛶࡢ᳨ウ࡟ࡣ 130 ≀㉁ࡀ⏝࠸ࡽࢀࠊ༑ศ࡞ᩘࡀ ICCVAM/NICEATM ࡟ࡼࡾ➨୕⪅ホ౯ጤဨ఍࡟ࡼࡾホ౯ࡉࢀ࡚࠸ࡿࠋJaCVAM ║่⃭ᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍ࡣࠊࡑࢀࡽࡢ㈨ᩱࢆ⏝࠸࡚ᮏヨ㦂ἲࢆホ౯ࡋ࡚࠾ࡾࠊጇᙜ࡛࠶ࡿࠋ㸱㸬ᮏヨ㦂ἲࡢ᭷⏝ᛶ࡜㐺⏝㝈⏺
ᮏヨ㦂ἲࡢࣂࣜࢹ࣮ࢩࣙࣥࡢ⤖ᯝࠊࢺࢵࣉࢲ࢘ࣥ᪉ᘧࡢഇ㝧ᛶ⋡ࡣ 1.2%ࠊ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧࡢഇ㝜ᛶ⋡ࡣࠊ㐺⏝㝈⏺ࢆ⪃៖ࡋ࡞࠸ሙྜ 12.3% ⪃៖ࡍࡿሙྜ 1.9%࡜࠸࠺ඃࢀࡓ್࡛࠶ࡿࠋ
ᮏヨ㦂ἲࡣࠊ▷᫬㛫࡛Ᏻ౯࡟ᐇ᪋࡛ࡁࠊ౑⏝⣽⬊ࡣබⓗ࡞⣽⬊ࣂࣥࢡ࠿ࡽධᡭྍ⬟࡛࠶ࡾࠊ≉Ṧ࡞ᶵᮦࡸヨ⸆ࢆᚲせ࡜ࡋ࡞࠸ࡇ࡜࠿ࡽࠊᇶᮏⓗ࡞⣽⬊ᇵ㣴ࡢᢏ⾡࡜タഛࢆᣢࡗ࡚࠸ࡿ᪋タ࡛࠶ࢀࡤᐇ᪋ྍ⬟࡛࠶ࡾࠊᢏ⾡⛣㌿ᛶࡣ㧗࠸ࠋ2ࡘࡢࣉࣞࣂࣜࢹ࣮ࢩࣙࣥヨ㦂7,8㸧࡟࠾࠸࡚ࡶᢏ⾡⛣㌿ᛶ࡟ࡘ࠸࡚ࡢၥ㢟ࡣᣦ᦬ࡉࢀ࡚࠸࡞࠸ࡀࠊOECDࡢ࢞࢖ࢻࣛ࢖ࣥ࡟♧ࡉࢀࡓ⇍㐩ᗘ☜ㄆ≀㉁2, 9)ࢆ⏝࠸࡚ࠊᐇ᪋ࡍࡿヨ㦂᪋タ࡬㐺ษ࡞ᢏ⾡⛣㌿ࡀ㐩ᡂ࡛ࡁࡓࡇ࡜ࢆ☜࠿ࡵࡿᚲせࡀ࠶ࡿࠋᮏヨ㦂ἲ࡟ࡣヨ㦂ࡢᛶ㉁࡜ṇ☜ᛶࡢほⅬ࠿ࡽ௨ୗࡢไ㝈ࡀタࡅࡽࢀ࡚࠸ࡿࠋ1) ⏕⌮㣗ሷỈࠊ5% (w/w) DMSOࢆྵࡴ⏕⌮㣗ሷỈࠊ࠾ࡼࡧ࣑ࢿࣛࣝ࢜࢖ࣝࡢ࠸ࡎࢀ࡟ࡶ⁐ゎࡋ࡞࠸ࠊ࠶ࡿ࠸ࡣࡑࢀࡽ⁐፹୰࡛ᆒ୍ศᩓࡋ࡚࠸ࡿ≧ែࡀ5ศ௨ୖಖࡓࢀ࡞࠸≀㉁ࡣ㐺⏝୙ྍ⬟࡛࠶ࡿࠋ2) ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡛༢୍≀㉁࡟㐺⏝ࡍࡿሙྜࠊ⵨Ẽᅽࡀ6 kPaࢆ㉸࠼ࡿ≀㉁࠾ࡼࡧ⏺㠃άᛶ๣࡛ࡣ࡞࠸ᅛయࡣࠊ㧗࠸ഇ㝜ᛶ⋡ࢆ♧ࡍࡇ࡜࠿ࡽࠊ㐺⏝ྍ⬟≀㉁࠿ࡽ㝖እࡉࢀࡿࠋᅛయΰྜ≀࡟㐺⏝ࡍࡿሙྜࡣࠊ⏺㠃άᛶ๣ࡢࡳ࠿ࡽᵓᡂࡉࢀࡿ≀㉁ࡣᑐ㇟࡟ࡍࡿࡀࠊࡑࢀ௨እࡢ≀㉁ࡣ㝖እࡍࡿࠋࡲࡓࠊ⵨Ẽᅽࡀ6 kPaࢆ㉸࠼ࡿ≀㉁ࢆྵࡴΰྜ≀࡟㐺⏝ࡍࡿሙྜࡣࠊ║่⃭ᛶࡀ㐣ᑠホ౯ࡉࢀࡿྍ⬟ᛶࡀ࠶ࡿࡢ࡛ὀពࡍࡿᚲせࡀ࠶ࡿࠋ3) ࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡛༢୍≀㉁࡟㐺⏝ࡍࡿࡇ࡜࡟ࡣไ㝈ࡀ࡞ࡃࠊࣂࣜࢹ࣮ࢩࣙࣥࢹ࣮ࢱࡣ㝈ࡽࢀ࡚ࡣ࠸ࡿࡶࡢࡢࠊΰྜ≀㉁࡬ࡢ㐺⏝࡟ࡶไ㝈ࡀ࡞࠸ࠋ௨ୖࡢⅬ࠿ࡽࠊTG491 ࡟‽ᣐࡋ࡚ᐇ᪋ࡋࡓሙྜࠊࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡟࠾࠸࡚㔜⠜࡞║ࡢᦆയࢆ㉳ࡇࡍ㸦ࡍ࡞ࢃࡕࠊUN GHS ༊ศ 1㸧≀㉁ࢆ᳨ฟࡍࡿ᪉ἲࠊ࠾ࡼࡧ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾࠸࡚║่⃭ᛶ≀㉁࡜
ࡣศ㢮ࡉࢀ࡞࠸㸦ࡍ࡞ࢃࡕࠊUN GHS ༊ศእ㸧≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚᭷⏝࡛࠶ࡿࠋ㸲㸬┠ⓗ࡜ࡍࡿ≀㉁ཪࡣ〇ရࡢẘᛶࢆホ౯ࡍࡿヨ㦂ἲ࡜ࡋ࡚ࡢࠊ♫఍ⓗཷࡅධ࠾ࡼࡧ⾜ᨻୖࡢ฼⏝ࡢྍ⬟ᛶ ♫఍ⓗཷࡅධࢀᛶ㸸ᮏヨ㦂ἲࡣ࢘ࢧࢠゅ⭷⏤᮶ࡢୖ⓶⣽⬊ᰴ㸦SIRC ⣽⬊㸧࡟ᑐࡍࡿ໬Ꮫ≀㉁ࡢ⣽⬊ẘᛶࢆᣦᶆ࡟⏝࠸࡚║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛࠶ࡾࠊ⏕ࡁࡓື≀ࢆ⏝࠸࡞࠸࡜࠸࠺Ⅼ࡛ࠊ3Rs ࡢ⢭⚄࡟ྜ⮴ࡋ࡚࠸ࡿࠋࡲࡓࠊ▷᫬㛫࡛Ᏻ౯࡟ᐇ᪋࡛ࡁࠊ౑⏝⣽⬊ࡢධᡭࡣᐜ࡛᫆࠶ࡾࠊ≉Ṧ࡞ᶵᮦࡸヨ⸆ࢆᚲせ࡜ࡋ࡞࠸ࡇ࡜࠿44ࡽࠊᇶᮏⓗ࡞⣽⬊ᇵ㣴ࡢᢏ⾡࡜タഛࢆᣢࡗ࡚࠸ࡿ᪋タ࡛࠶ࢀࡤᐇ᪋ྍ⬟࡛࠶ࡾࠊᢏ⾡⛣㌿ᛶࡣ㧗࠸ࠋ௨ୖࡼࡾࠊᮏヨ㦂ἲࡣ♫఍ⓗཷࡅධࢀᛶࡀ㧗࠸ࠋ ⾜ᨻୖࡢ฼⏝ᛶ㸸ᮏヨ㦂ἲࡣࠊ໬Ꮫ≀㉁࡟ࡼࡿ║่⃭ᛶࢆホ౯࡛ࡁࠊࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡟࠾࠸࡚㔜⠜࡞║ࡢയᐖࢆ㉳ࡇࡍ≀㉁㸦UN GHS ༊ศ 1㸧≀㉁ࢆ᳨ฟࡍࡿ᪉ἲࠊ࡞ࡽࡧ࡟࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾࠸࡚║่⃭ᛶ≀㉁࡜ࡣ
ศ㢮ࡉࢀ࡞࠸≀㉁㸦UN GHS ༊ศእ㸧ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚ࠊ⾜ᨻⓗ฼⏝ࡀྍ⬟࡛࠶ࡿࠋཧ⪃ᩥ⊩1)

Scott, L., et al. (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): 1-9.

2)

OECD (2015) Test Guideline 491. Short Time Exposure in vitro test method for identifying 1) Chemicals
inducing serious eye damage and ii) Chemicals not requiring classification for eye irritation or serious eyedamage.

3)

Mikkelson TJ, et al. (1973). Altered bioavailability of drugs in the eye due to 34 drug-protein interaction. J.
Pharm. Sci.1648-1653.

4)

Sakaguchi et al. (2011) Validation study of the Short Time Exposure (STE) test to assess the eye irritation
potential of chemicals. Toxicol. In Vitro, 25(4): 796-809.

5)

Kojima et al. (2013) Second-phase validation study of the short time exposure test for assessment of eyeirritation potency of chemicals. Toxicol. In Vitro, 27(6): 1855-1869

6)

ICCVAM (2013) Short Time Exposure (STE) test method, Summary review document.

7)

Takahashi et al. (2009) Inter-laboratory study of short time exposure (STE) test for predicting eye irritation
potential of chemicals and correspondence to globally harmonized system (GHS) classification. J Toxicol
Sci, 34(6): 611-626.

8)

Takahashi et al. (2010) An interlaboratory study of the short time exposure (STE) test using SIRC cells forpredicting eye irritation potential. J. Cutan. Ocul. Toxicol., 29(2): 77-90.

9)JaCVAM ║่⃭ᛶ௦᭰ἲ㈨ᩱ⦅⧩ጤဨ఍㸸OECD TG491 in vitro ▷᫬㛫᭚㟢ἲ㸦The Short TimeExposure In Vitro Test Method㸸STEἲ㸧ホ౯ሗ࿌᭩㸦2015ᖺ9᭶29᪥㸧

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