ホ౯ሗ࿌᭩⓶⭵⭉㣗ᛶヨ㦂௦᭰ἲࣄࢺ⾲⓶ࣔࢹࣝἲ

⓶⭵⭉㣗ᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍ ᖹᡂ 29 ᖺ㸦2017 ᖺ㸧2 ᭶ 24 ᪥

17⓶⭵⭉㣗ᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍㧘ᶫ ♸ḟ

ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ ẘᛶ㒊୰ᮧࡿࡾࡇ⊂❧⾜ᨻἲே 〇ရホ౯ᢏ⾡ᇶ┙ᶵᵓ ໬Ꮫ≀㉁⟶⌮ࢭࣥࢱ࣮㡲᪉ ╩ኵ

ఫ཭໬Ꮫᰴᘧ఍♫ ࣞࢫ࣏ࣥࢩࣈࣝࢣ࢔㒊㸦໬ᏛရᏳ඲ࢢ࣮ࣝࣉ㸧㸭᪥ᮏ໬Ꮫᕤᴗ༠఍ᑠᓥ ⫕ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶண

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ホ౯㒊

せ᪨
࢘ࢧࢠࢆ⏝࠸ࡿ⓶⭵⭉㣗ᛶヨ㦂ࡢື≀ᐇ㦂௦᭰ἲ㸦௦᭰ἲ㸧࡜ࡋ࡚⤒῭༠ຊ㛤Ⓨᶵᵓ(OECD:
Organisation for Economic Co-operation and Development㸧࡛ヨ㦂࢞࢖ࢻࣛ࢖ࣥ(TG: Test Guideline)
431 ࡜ࡋ࡚ᢎㄆࡉࢀࡓࣄࢺ⾲⓶ࣔࢹࣝࢆ⏝࠸ࡿヨ㦂ἲࡢ᭷⏝ᛶࢆホ౯ࡋࡓࠋಙ㢗ᛶ࡜ጇᙜᛶ࡜࠸
࠺どⅬ࡟࠾࠸࡚ࠊࣄࢺ⾲⓶ࣔࢹࣝࢆ⏝࠸ࡓヨ㦂ࢆホ౯ࡋࡓ⤖ᯝࠊTG431 ࡟ᥖ㍕ࡉࢀ࡚࠸ࡿࡍ࡭࡚
ࡢࣔࢹࣝ EpiSkin™ࠊEpiDerm™ࠊSkinEthic™ࠊepiCS®ࡀ⭉㣗ᛶࡢ᭷↓ࢆホ౯࡛ࡁࡿࣔࢹࣝ࡜ࡋ࡚
᥎ዡ࡛ࡁࡿ࡜⪃࠼ࡽࢀࡓࠋࡓࡔࡋࠊᅜ㐃໬Ꮫရࡢศ㢮࠾ࡼࡧ⾲♧࡟㛵ࡍࡿୡ⏺ㄪ࿴ࢩࢫࢸ࣒(UN
GHS: United Nations Globally Harmonized System of Classification and Labelling of Chemicals)ศ㢮ࡢ⣽༊ศࢆ⪃៖ࡍࡿሙྜࡣ EpiSkin™ࡀ⓶⭵⭉㣗ᛶヨ㦂ࡢ௦᭰ἲ࡜ࡋ࡚ࡶࡗ࡜ࡶ᭷⏝࡛࠶ࡿ࡜⤖ㄽ
ࡋࡓࠋ

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1. ヨ㦂ἲࡢ⛉Ꮫⓗ࠾ࡼࡧつไ㠃࠿ࡽࡢጇᙜᛶ
⓶⭵⭉㣗ᛶヨ㦂ࡣ⓶⭵่⃭ᛶヨ㦂ࡢ୍⎔࡜ࡋ࡚⾜ࢃࢀࠊ✀ࠎࡢ࢞࢖ࢻࣛ࢖࡛ࣥࡣ Draize ࡽ࡟ࡼࡾᥦၐࡉࢀࡓ࢘ࢧࢠࢆ⏝࠸ࡿ᪉ἲࡀ᥎ዡࡉࢀ࡚ࡁࡓ 1)ࠋࡇࡢ᪉ἲࡣ⿕㦂≀㉁ࡢ่⃭ᛶࡸ⭉㣗ᛶࢆ᳨ฟࡍࡿヨ㦂࡜ࡋ࡚㛗ࡃ౑⏝ࡉࢀ࡚ࡁࡓࡶࡢࡢࠊุᐃࢆ⫗║࡛⾜࠺ࡓࡵᐈほᛶ࡟ஈࡋࡃᐇ㦂㛫ࡸ᪋タ㛫࡛ࡢ෌⌧ᛶࡀஈࡋ࠸ࠋ᭦࡟ື≀࡟⃭ࡋ࠸③ࡳ࡜ࢫࢺࣞࢫࢆ୚࠼ࡿࡇ࡜ࡀ♫఍ⓗ࡟ၥ㢟࡜࡞ࡾࠊ௨๓ࡼࡾື≀ࢆ౑⏝ࡋ࡞࠸ື≀ᐇ㦂௦᭰ἲ㸦௨ୗࠊ௦᭰ἲ࡜グࡍ㸧ࡢ㛤Ⓨࡀษᮃࡉࢀ࡚࠸ࡓࠋ
ࡇࡢ௦᭰ἲ࡜ࡋ࡚ࠊ⤒῭༠ຊ㛤Ⓨᶵᵓ(OECD: Organisation for Economic Co-operation andDevelopment㸧࡛ヨ㦂࢞࢖ࢻࣛ࢖ࣥ(TG: Test Guideline)431 ࡟ࡣࠊ⓶⭵⭉㣗ᛶヨ㦂࡜ࡋ࡚ゅ㉁ᒙࢆ
᭷ࡋ 3 ḟඖⓗ࡟෌ᵓ⠏ࡉࢀࡓࣄࢺ⾲⓶ࣔࢹࣝࢆ౑⏝ࡋࡓホ౯᪉ἲࡀグ㍕ࡉࢀ࡚࠸ࡿ 2)ࠋࡇࡢヨ㦂ἲࡣࠊ⭉㣗ᛶ≀㉁ࡀゅ㉁ᒙࡢയᐖࡲࡓࡣゅ㉁ᒙ࡟྾཰ࡉࢀࡓᚋᣑᩓࡍࡿࡇ࡜࡟ࡼࡾࠊୗᒙࡢ⣽⬊࡟฿㐩ࡋ࡚⣽⬊ẘᛶࢆ♧ࡍ࡜࠸࠺௬ㄝ࡟ᇶ࡙ࡁࠊ⿕㦂≀㉁᭚㟢ᚋࡢ⣽⬊⏕Ꮡ⋡ࢆᣦᶆ࡟⓶⭵⭉㣗ᛶࢆホ౯ࡋ࡚࠸ࡿࠋEpiSkin™ࡸ EpiDerm™➼ࡢࣄࢺ⾲⓶ࣔࢹࣝࡣḢ⡿࡛ࡣ᪤࡟ࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀᐇ᪋ࡉࢀࠊḢᕞ࡛ࡣ໬Ꮫ≀㉁ࡢ⓶⭵⭉㣗ᛶホ౯ࢆ┠ⓗ࡜ࡋ࡚ᢎㄆࡉࢀࠊ໬Ꮫ≀㉁ࡢࣜࢫࢡ⾲♧㆑ู➼࡟฼⏝ࡉࢀ࡚࠸ࡿࠋ≉࡟᫖௒࡛ࡣᅜ㐃໬Ꮫရࡢศ㢮࠾ࡼࡧ⾲♧࡟㛵ࡍࡿୡ⏺ㄪ࿴ࢩࢫࢸ࣒(UN GHS: United Nations Globally Harmonized System of Classification and Labelling of Chemicals)ศ㢮࡟ᚑࡗ࡚ホ౯ࡉࢀࡿࢣ࣮ࢫࡀቑ࠼࡚࠸ࡿࠋࡇࡢ TG431 ࡣ᫖௒ࠊẖᖺ✀ࠎࡢⅬ࡛ᨵゞࡉࢀ࡚࠾ࡾࠊ⌧ᅾࡣ 2016 ᖺ∧࡜࡞ࡗ࡚࠸ࡿ 3)ࠋ
ᡃࡀᅜ࡛᪤Ꮡࡢ໬Ꮫ≀㉁ࢆホ౯ࡍࡿሙྜࠊOECD ࡛ᢎㄆࡉࢀࡓヨ㦂᪉ἲ࡟ࡼࡿ⤖ᯝࡣࠊ୍⯡ⓗ࡟⾜ᨻⓗ࡟ཷࡅධࢀࡽࢀࡿࡀࠊ⌧ᅾࡲ࡛௦᭰ἲ࡛ࡢ⤖ᯝࢆࡶ࡜࡟⾜ᨻⓗ࡟ホ౯ࡉࢀࡓ౛ࡣከࡃ࡞
࠸ࠋᏳ඲ᛶホ౯࡟࠾ࡅࡿ௦᭰ἲࡢᬑཬࡀษᮃࡉࢀ࡚࠸ࡿ⌧≧࡟࠾࠸࡚ࠊᡃࡀᅜ࡛ࡶ⛉Ꮫⓗ࡟ጇᙜ࡞ࡶࡢࡣ✚ᴟⓗ࡟ཷࡅධࢀࡿࡇ࡜ࡀᚲせ࡜࡞ࡗ࡚࠸ࡿࠋ࡞࠾ࠊᅜෆ௻ᴗ࠿ࡽࡶᅜ㝿ⓗ࡞ホ౯࡟⪏࠼࠺ࡿࣄࢺ⾲⓶ࣔࢹࣝࡀ㛤Ⓨࡉࢀ࡚࠸ࡿࠋᮏホ౯᭩࡛ࡣࠊOECD TG431 ࡟ᥖ㍕ࡉࢀࡓࣄࢺ⾲⓶ࣔࢹࣝ EpiSkin ™ࠊEpiDerm™ࠊSkinEthic™
࠾ࡼࡧ epiCS®ࢆ⏝࠸ࡿ⭉㣗ᛶヨ㦂ἲࡢ᭷⏝ᛶࢆホ౯ࡋࡓ 3)ࠋ
2. ヨ㦂ࣉࣟࢺࢥࣝᵓᡂࡢጇᙜᛶ 3)⿕ 㦂 ≀ ㉁ ࡀ ゅ ㉁ ᒙ ࢆ ㏻ 㐣 ࡋ ࡚ ⾲ ⓶ ⣽ ⬊ ࡟ ᭚ 㟢 ࡉ ࢀ ࠊ MTT ࠝ 3(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromideࠞࡢ㑏ඖ㔞࠿ࡽồࡵࡓ⣽⬊⏕Ꮡ⋡ࡢ๭ྜ࠿ࡽ

⓶⭵⭉㣗ᛶࢆุᐃࡍࡿࠋࡑࢀࡽࡢᴫせࢆ⾲㸯࡟ࡲ࡜ࡵࡓࠋᐇ㦂᧯సୖࡢ㐪࠸ࡣࠊ๓ᇵ㣴ἲ࠾ࡼࡧᰁⰍᾮࡢ⏝㔞ࠊ⤌⧊࠿ࡽࡢᢳฟἲ➼࡛࠶ࡾࠊᇶᮏⓗ࡞᭚㟢᫬㛫࡜ุᐃᇶ‽࡟ࡘ࠸࡚ࡣ⾲ 2 ࢆཧ↷ࡉࢀࡓ࠸ࠋヨ㦂ࣉࣟࢺࢥࣝ࡜ࡋ࡚ࠊEpiDerm™ࢆ౛࡟ㄝ᫂ࡍࡿࠋ6 well ࣉ࣮ࣞࢺࡢྛ well ࡟ᇵ㣴ᾮ 1 mL ࢆຍ࠼ࡓᚋ࡟ࣄࢺ⾲⓶ࣔࢹࣝࢆ⨨ࡁࠊ⿕㦂≀㉁ࡀᾮ≧ࡢሙྜࡣࣆ࣌ࢵࢱ࣮࡛ 50 μLࠊ⢊ᮎ࡞࡝ᅛᙧ
ࡢሙྜࡣ 25 mg ࢆ 25 μL ࡢỈ࡜ྜࢃࡏࠊࣔࢹࣝୖᒙ࡟㐺⏝ࡍࡿ (n=2)ࠋ⿕㦂≀㉁ࢆ 3 ศࡲࡓࡣ 60ศฎ⌮ᚋࠊࣉ࣮ࣞࢺ࠿ࡽ⿕㦂≀㉁ࢆࢹ࢝ࣥࢸ࣮ࢩࣙࣥ࡟ࡼࡾ㝖ཤࡋࡓᚋࠊ10 mL ࡢ PBS ࡛ 2㹼3
ᅇ㍍ࡃὙίࡍࡿࠋὙίᚋࠊ࣮࣌ࣃ࣮ࢱ࢜ࣝ➼࡛Ỉศࢆษࡾࠊ◚ᦆ࡟ὀពࡋ࡞ࡀࡽูࡢ 24 well ࣉ࣮ࣞࢺ࡟ࣄࢺ⾲⓶ࣔࢹࣝࢆ⛣ືࡍࡿࠋMTT Ⰽ⣲ࢆྵࡴᇵ㣴ᾮࢆࣄࢺ⾲⓶ࣔࢹࣝࡢୗ᪉࡟ 0.3 mL ῧຍࡍࡿࠋ37ΥࠊCO2 ࢖࣮ࣥ࢟ࣗ࣋ࢱ୰࡟ 3 ᫬㛫㟼⨨ࡋࡓᚋࠊ௨ୗࡢᡭ㡰࡟࡚ᢳฟࢆ⾜࠺ࠋ࢖ࢯࣉ
ࣟࣃࣀ࣮ࣝࢆ 2 mL ῧຍࡋࠊ୍ᬌᐊ

ᨺ⨨ᚋࠊ96 well ࣉ࣮ࣞࢺ࡟ᢳฟᾮࢆ 200 μL ࡎࡘ⛣ࡋ㸦1 ≀
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㉁࠶ࡓࡾ 2㹼3 well㸧ࠊ࣐࢖ࢡࣟࣉ࣮ࣞࢺ࣮ࣜࢲ࣮ࢆ⏝࠸࡚ 540 nm ࠶ࡿ࠸ࡣ 570 nm 㡿ᇦ࡛ࡢ྾ග
ᗘࢆᐃࡍࡿࠋ࢖ࢯࣉࣟࣃࣀ࣮ࣝࡢࡳࢆຍ࠼ࡓ well ࢆࣈࣛࣥࢡ࡜ࡋࠊᐇ

್࡜ࣈࣛࣥࢡ್ࡢᕪࢆ

ồࡵࡿࠋ⁐፹ᑐ↷ࡢ྾ගᗘࢆ 100㸣࡜ࡋྛ᳨యࡢ 3 ࡲࡓࡣ 60 ศ㛫ฎ⌮᫬ࡢ྾ගᗘࢆ㸣࡜ࡋ࡚⟬ฟࡋ⣽⬊ࡢ⏕Ꮡ⋡࡜ࡍࡿࠋ3 ศ㛫ฎ⌮ࡋࡓ࡜ࡁࡢ⏕Ꮡ⋡ࡀ 50㸣ᮍ‶ࠊ࠶ࡿ࠸ࡣ 3 ศ㛫࡛ࡣ⏕Ꮡ⋡ࡀ
50㸣௨ୖ࡛࠶ࡿࡀ 60 ศ㛫ฎ⌮ࡋࡓ࡜ࡁ࡟ 15㸣ᮍ‶ࡢ⤖ᯝࢆ♧ࡍ≀㉁ࢆ“⭉㣗ᛶ”࡜ุᐃࡍࡿࠋ୍
᪉ࠊ3 ศ㛫ฎ⌮ࡋࡓ࡜ࡁ࡟⏕Ꮡ⋡ࡀ 50㸣௨ୖࠊ60 ศ㛫ฎ⌮ࡋࡓ࡜ࡁ࡟ 15㸣௨ୖࡢ≀㉁ࡣ“㠀⭉㣗ᛶ”࡜ุᐃࡍࡿࠋヨ㦂ࡣ㸯ᅇ࡜ࡋࠊwell 㛫࡛␗࡞ࡿ⤖ᯝࡀᚓࡽࢀࡓሙྜࡢࡼ࠺࡟᫂☜࡞ホ౯ࡀ࡛ࡁ࡞࠿ࡗࡓሙྜࡣ㏣ຍヨ㦂ࢆᐇ᪋ࡋࠊ᭱⤊ホ౯࡜ࡍࡿࠋ

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12

஦๓☜ㄆ

┤᥋ MTT 㑏ඖᛶࡢ

50 μL(ᾮయ)ࡶࡋࡃࡣ 20 mg(ᅛయ)࡟ 0.3 mg/mL MTT ⁐ᾮ 2 mL ࢆຍ࠼࡚ࠊ37ΥࠊCO2 ⃰ᗘ 5㸣ࠊ‵
ᗘ 95㸣࡛ 180±5 ศᇵ㣴ĺ⁐ᾮࡢⰍࡀ㟷/⣸࡟ኚࢃࡗࡓሙ
ྜࠊỈฎ⌮࡛ࣔࢹࣝᵓᡂ⣽⬊ࢆṚ⁛ࡉࡏࡓࡶࡢ࡟⿕㦂≀㉁ࢆฎ⨨ࡍࡿᑐ↷ࡶ࡜ࡿࠋ

ࣟ࢘ᵝ/⢓╔ᛶ≀㉁㸸ࢼ࢖࣓ࣟࣥ
ࢵࢩࣗࢆ⏝࠸࡚ 50±2 mg (131.6
mg/cm2)

650 μL(ᾮయ)ࡶࡋࡃࡣ 25 mg(ᅛయ)࡟ 1 mg/mL MTT ⁐ᾮ 1 mL ࢆຍ࠼࡚ࠊ37ΥࠊCO2 ⃰ᗘ 5㸣ࠊ‵ᗘ95㸣࡛ 60 ศᇵ㣴ĺ⁐ᾮࡢⰍࡀ㟷/⣸࡟ኚࢃࡗࡓሙ
ྜࠊ෾⤖ฎ⌮࡛ࣔࢹࣝᵓᡂ⣽⬊ࢆṚ⁛ࡉࡏࡓࡶࡢ࡟⿕㦂≀㉁ࢆ
ฎ⨨ࡍࡿᑐ↷ࡶ࡜ࡿࠋࣟ࢘ᵝ≀㸸15 μL ࡢỈ࡛‵ࡽࡏࡓ┤ᚄ⣙ 8 mm ࡢࣇࣛࢵࢺ࡞ࢹ࢕
ࢫࢡᵝࡢ∦ࢆୖ࡟஌ࡏࡿࠋ

ᅛయ㸸 25 mg (39.7 mg/cm )㸩25
μL ࡢỈ(ᚲせ࡛࠶ࢀࡤࡑࢀ௨ୖ)2

༙ᅛయ㸸50 μL (79.4 μL/cm )2ᾮయ㸸ࢼ࢖࣓ࣟࣥࢵࢩࣗࢆ⏝࠸ࡓ≧ែ࡛ࠊࡶࡋࡃࡣ⏝࠸࡞࠸ሙྜ࡛ࡶ 50 μL (79.4 μL/cm2)⿕㦂≀㉁࡜ࢼ࢖࣓ࣟࣥࢵࢩࣗ࡜ࡢぶ࿴ᛶࡣணヨ㦂࡛☜ㄆࡍࡿࠋᾮయ࠾ࡼࡧ⢓ᛶ≀㸸50±3 μL(131.6μL/cm 2)౑⏝㔞࡜㐺⏝

ᅛయ㸸20±2 mg (52.6 mg/cm2)+100±5 μL ࡢ NaCl ⁐ᾮ(9 g/L㸧᭚㟢᫬㛫ẖ࡟ 2-3᭚㟢᫬㛫ẖ࡟ᑡ࡞ࡃ࡜ࡶ 2

0.63 cm 2

0.38cm2ࣔࢹࣝ⾲㠃✚⤌⧊ᩘ

EpiDermTMSCT

EpiSkinTM

ࢸࢫࢺ᪉ἲ せ⣲±࡛♧ࡋࡓᩘᏐࡣࠊࡤࡽࡘࡁࡢチᐜ⠊ᅖࢆ♧ࡍࠋ⾲㸯㸬⓶⭵⭉㣗ᛶヨ㦂ࡢࡓࡵ࡟☜ㄆࡉࢀࡓ RhE ヨ㦂᪉ἲ࠿ࡽ࡞ࡿ୺࡞ヨ㦂᪉ἲ 3)

40 μL(ᾮయ)ࡶࡋࡃࡣ 20 mg(ᅛయ)࡟ 1 mg/mL MTT ⁐ᾮ 1 mL ࢆຍ࠼࡚ࠊ37ΥࠊCO2 ⃰ᗘ 5㸣ࠊ‵ᗘ95㸣࡛ 180±15 ศᇵ㣴ĺ⁐ᾮࡢⰍࡀ㟷/⣸࡟ኚࢃࡗࡓሙ
ྜࠊ෾⤖ฎ⌮࡛ࣔࢹࣝᵓᡂ⣽⬊ࢆṚ⁛ࡉࡏࡓࡶࡢ࡟⿕㦂≀㉁ࢆ
ฎ⨨ࡍࡿᑐ↷ࡶ࡜ࡿࠋࣟ࢘ᵝ/⢓╔ᛶ≀㉁㸸ࢼ࢖࣓ࣟࣥ
ࢵࢩࣗࢆ⏝࠸ࡓ≧ែ࡛ 20±3 mg(40 mg/cm2)

ᅛయ㸸
20±3 mg (40 mg/cm2)㸩20±2 μL ࡢỈᾮయ࠾ࡼࡧ⢓ᛶ≀㸸ࢼ࢖࣓ࣟࣥ
ࢵࢩࣗࢆ౑⏝ࡋ࡚ 40±3 μL (80μL/cm2)⿕㦂≀㉁࡜ࢼ࢖࣓ࣟࣥࢵࢩࣗ࡜ࡢぶ࿴ᛶࡣணヨ㦂࡛☜ㄆࡍࡿࠋ᭚㟢᫬㛫ẖ࡟ᑡ࡞ࡃ࡜ࡶ 2

0.5 cm 2

SkinEthicTM RHE

50 μL(ᾮయ)ࡶࡋࡃࡣ 25 mg(ᅛయ)࡟ 1 mg/mL MTT ⁐ᾮ 1 mL ࢆຍ࠼࡚ 37ΥࠊCO2 ⃰ᗘ 5㸣ࠊ‵ᗘ95㸣࡛ 60 ศᇵ㣴ĺ⁐ᾮࡢⰍࡀ㟷/⣸࡟ኚࢃࡗࡓሙ
ྜࠊ෾⤖ฎ⌮࡛ࣔࢹࣝᵓᡂ⣽⬊ࢆṚ⁛ࡉࡏࡓࡶࡢ࡟⿕㦂≀㉁ࢆ
ฎ⨨ࡍࡿᑐ↷ࡶ࡜ࡿࠋࣟ࢘ᵝ≀㸸15 μL ࡢỈ࡛‵ࡽࡏࡓࣇࣛࢵࢺ࡞ࠊ┤ᚄ⣙ 8 mm ࡢࢡࢵ࣮࢟ᵝࡢ∦ࢆୖ࡟஌ࡏࡿࠋ

ᅛయ㸸25 mg (41.7 mg/cm2)+
25 μL ࡢỈ(ᚲせ࡛࠶ࢀࡤࡑࢀ௨
ୖ)༙ᅛయ㸸50 μL (83.3 μL/cm2)

ᾮయ㸸ࢼ࢖࣓ࣟࣥࢵࢩࣗࢆ⏝࠸ࡓ≧ែ࡛ 50 μL (83.3 μL/cm2)⿕㦂≀㉁࡜ࢼ࢖࣓ࣟࣥࢵࢩࣗ࡜ࡢぶ࿴ᛶࡣணヨ㦂࡛☜ㄆࡍࡿࠋ᭚㟢᫬㛫ẖ࡟ᑡ࡞ࡃ࡜ࡶ 2

0.6 cm 2

epiCS®

13

8 N Ỉ㓟໬࣒࢝ࣜ࢘ 40 μL ࡛ 1 ᫬㛫᭚㟢᫬ࡢࡳ

37ΥࠊCO2 ⃰ᗘ 5㸣ࠊ‵ᗘ 95㸣࡛
180 ศ(±15 ศ)
1.5 mL ࡢ࢖ࢯࣉࣟࣃࣀ࣮ࣝ
(࢖ࣥࢧ࣮ࢺ඲య࠿ࡽᢳฟࡍࡿ㸧

8 N Ỉ㓟໬࣒࢝ࣜ࢘ 50 μL ࡛᭚㟢᫬㛫ẖ࡟⃰ᗘ 1 mg/mL 300 μL
37ΥࠊCO2 ⃰ᗘ 5㸣ࠊ‵ᗘ 95㸣࡛180 ศ
2 mL ࡢ࢖ࢯࣉࣟࣃࣀ࣮ࣝ
(࢖ࣥࢧ࣮ࢺ඲య࠿ࡽᢳฟࡍࡿ㸧⃰ᗘ 0.3 mg/mL 2 mL

37ΥࠊCO2 ⃰ᗘ 5㸣ࠊ‵ᗘ 95㸣
࡛ 180 ศ(±15 ศ)

500 μL ࡢ㓟ᛶ໬࢖ࢯࣉࣟࣃࣀ
࣮ࣝ
(0.04 N ሷ㓟ࢆྵࡴ࢖ࢯࣉࣟࣃ
ࣀ࣮ࣝ)(ศ㞳ࡋࡓ⤌⧊ࢆ༑ศ࡟ᾐₕࡉࡏࡿ㸧MTT ⁐ᾮ

MTT ⁐ᾮ࡛ࡢᇵ㣴

ᢳฟ⁐፹

᫬㛫࠾ࡼࡧ

㝧ᛶᑐ↷

ᗘ

7

40 μL ࡢỈ᭚㟢᫬㛫ẖ࡟

50 μL ࡢỈ᭚㟢᫬㛫ẖ࡟

㝜ᛶᑐ↷⃰ᗘ 1 mg/mL 300 μL

PBS ࢆ୍ᐃࡋࡓᙅὶ࡛ 20 ᅇ

PBS ࢆ୍ᐃࡋࡓᙅὶ࡛ 20 ᅇ

PBS 25 mL(ࡍࡍࡂ୍ᅇẖ࡟ 2
mL)
50 μL ࡢሷ໬ࢼࢺ࣒ࣜ࢘⁐ᾮ
㸦9g/L)
᭚㟢᫬㛫ẖ࡟
50 μL ࡢị㓑㓟࡛ 4 ᫬㛫᭚㟢᫬ࡢࡳ

PBS ࡟ࡼࡿࡍࡍࡂ

(࢖ࣥࢧ࣮ࢺ඲య࠿ࡽᢳฟࡍࡿ㸧

2 mL ࡢ࢖ࢯࣉࣟࣃࣀ࣮ࣝ

37ΥࠊCO2 ⃰ᗘ 5㸣ࠊ‵ᗘ 95㸣࡛180 ศ

⃰ᗘ 1 mg/mL 300 μL

8 N Ỉ㓟໬࣒࢝ࣜ࢘ 5 0μL ࡛᭚㟢᫬㛫ẖ࡟

50 μL ࡢỈ᭚㟢᫬㛫ẖ࡟

PBS ࢆ୍ᐃࡋࡓᙅὶ࡛ 20 ᅇ

ᐊ ࡛ 3 ศࠊ࠾ࡼࡧ
37ΥࠊCO2 ⃰ᗘ 5㸣ࠊ‵ᗘ 95㸣࡛60 ศ

ᐊ 㸦18-28Υ㸧࡛ 3 ศࠊ60 ศ ᐊ ࡛ 3 ศࠊ࠾ࡼࡧ 37ΥࠊCO2
㸦±5 ศ㸧࠾ࡼࡧ 240 ศ㸦±10 ศ㸧 ⃰ᗘ 5㸣ࠊ‵ᗘ 95㸣࡛ 60 ศ᥮Ẽࡉࢀࡓ࢟ࣕࣅࢿࢵࢺෆ

᭚㟢᫬㛫࡜

ᗘ

50 μL(ᾮయ)ࡶࡋࡃࡣ 25 mg(ᅛయ)࡟ 300 μL ࡢỈࢆຍ࠼࡚ 37Υࠊ
CO2 ⃰ᗘ 5㸣ࠊ‵ᗘ 95㸣࡛ 60 ศᨩᢾࡍࡿࠋĺ⁐ᾮࡢⰍࡀ╔Ⰽࡋ࡚࠸ࡿሙ
ྜࠊMTT ࡢࡳࢆຍ࠼࡞࠸ᑐ↷ࢆ࡜ࡿࠋᐊ ࡛ 3 ศࠊ࠾ࡼࡧ
37ΥࠊCO2 ⃰ᗘ 5㸣ࠊ‵ᗘ 95㸣࡛
60 ศ

40 μL(ᾮయ)ࡶࡋࡃࡣ 20 mg(ᅛయ)࡟ 300 μL ࡢỈࢆຍ࠼࡚ᐊ ࡛ 60ศᨩᢾࡍࡿࠋ
ĺ⿕㦂≀㉁ࡢⰍࡀ╔Ⰽࡋ࡚࠸ࡿ
ሙྜࠊMTT ࡢࡳࢆຍ࠼࡞࠸ᑐ↷ࢆ࡜ࡿࠋ

50 μL(ᾮయ)ࡶࡋࡃࡣ 25 mg(ᅛయ)࡟ 300 μL ࡢỈࢆຍ࠼࡚ 37Υࠊ
CO2 ⃰ᗘ 5㸣ࠊ‵ᗘ 95㸣࡛ 60 ศᨩᢾࡍࡿࠋĺ⁐ᾮࡀ╔ⰍࡋࡓሙྜࠊMTT ࡢࡳࢆຍ࠼࡞࠸ᑐ↷ࢆ࡜ࡿࠋ

10 μL(ᾮయ)ࡶࡋࡃࡣ 10 mg(ᅛయ)࡟ 90 μL ࡢỈࢆຍ࠼࡚ᐊ
࡛ 15 ศᨩᢾࡍࡿࠋ
ĺ⁐ᾮࡀ╔ⰍࡋࡓሙྜࠊMTTࡢࡳࢆຍ࠼࡞࠸ᑐ↷ࢆ࡜ࡿࠋ╔Ⰽ㞀ᐖࡢ஦๓☜ㄆ

epiCS®

SkinEthicTM RHE

EpiDermTMSCT

EpiSkinTM

ࢸࢫࢺ᪉ἲ せ⣲

14
1 ᅇࠊ᫂☜࡞⤖ᯝࡀᚓࡽࢀ࡞࠿ࡗ
ࡓ᫬ࡣ 2 ᅇ1 ᅇࠊ᫂☜࡞⤖ᯝࡀᚓࡽࢀ࡞࠿
ࡗࡓ᫬ࡣ 2 ᅇ

1.
1. 㝜ᛶᑐ↷(ሷ໬ࢼࢺ࣒ࣜ࢘⁐ᾮ)࡛ฎ⌮ࡉࢀࡓ⤌⧊ࡢ
OD ್ࡢᖹᆒࡣࠊ᭚㟢᫬㛫ẖ࡟ 0.6 ௨ୖ 1.5 ௨ୗࠋ
2. 㝧ᛶᑐ↷(ị㓑㓟)࡛ 4 ᫬㛫 2.
ฎ⨨ࡉࢀࡓ⤌⧊ࡢ⏕Ꮡ⋡ࡢ
ᖹᆒࡣ 20㸣௨ୗࠋ
3. ⏕Ꮡ⋡ࡢᖜࡀ 20-100㸣࠾ࡼࡧ OD ್ࡀ 0.3 ௨ୖࡢሙྜࠊ 3.
2 ࡘࡢ⤌⧊㛫ࡢ⏕Ꮡ⋡ࡢᕪࡣ 30㸣ࢆࡇ࠼࡞࠸ࠋヨ㦂ᅇᩘ㐺ྜุᐃᇶ‽

8㝜ᛶᑐ↷(Ỉ)࡛ฎ⨨ࡉࢀࡓ⤌⧊ࡢ OD ್ࡢᖹᆒࡣࠊ඲࡚ࡢ
᭚㟢᫬㛫࡟࠾࠸࡚ 0.8 ௨ୖ
2.8 ௨ୗࠋ
㝧ᛶᑐ↷㸦8N Ỉ㓟໬࣒࢝ࣜ࢘㸧࡟ 1 ᫬㛫᭚㟢ࡋࡓ⤌⧊」〇≀ࡢ ᐃ್ࡢ⏕Ꮡ⋡ࡣ
15㸣ᮍ‶ࠋ
⏕Ꮡ⋡ࡀ 20-100㸣ࡢሙྜ࡟࠾࠸࡚ࠊ⤌⧊㛫ࡢኚືಀᩘ(CV)ࡣ 30㸣௨ୗࠋ

4.08 ᫬㛫ӌET50ӌ8.7 ᫬㛫

1.0 mg/mLӌIC50ӌ3.0 mg/mL

1 ᅇࠊ᫂☜࡞⤖ᯝࡀᚓࡽࢀ࡞࠿ࡗ
ࡓ᫬ࡣ 2 ᅇ1. 㝜ᛶᑐ↷(Ỉ)࡛ฎ⨨ࡋࡓ⤌⧊ࡢ OD ್ࡢᖹᆒࡣࠊྛ᭚㟢᫬㛫ẖ࡟ 0.8 ௨ୖ 2.8 ௨ୗࠋ
2. 1 ᫬㛫(ࡶࡋྍ⬟࡛࠶ࢀࡤ 4 ᫬㛫)㝧ᛶᑐ↷(8N Ỉ㓟໬࣒࢝ࣜ࢘)࡛ฎ⨨ࡉࢀࡓ⤌⧊」〇≀ࡢ ᐃ್ࡢ⏕Ꮡ⋡ࡣ 15㸣ᮍ‶ࠋ
3. 20-100㸣ࡢ⏕Ꮡ⋡࠾ࡼࡧ 0.3
௨ୖࡢ OD ್࡟࠾࠸࡚ࠊ2 ࡘࡢ⤌⧊㛫ࡢ⏕Ꮡ⋡ࡢᕪࡣ
30㸣ࢆ㉸࠼࡞࠸ࠋ

1. 㝜ᛶᑐ↷(Ỉ)࡛ฎ⨨ࡉࢀࡓ⤌⧊ࡢ OD ್ࡢᖹᆒࡣࠊ᭚㟢᫬㛫ẖ࡟ 0.8 ௨ୖ 3.0 ௨ୗࠋ
2. 1 ᫬㛫(ࡶࡋྍ⬟࡛࠶ࢀࡤ 4 ᫬㛫)㝧ᛶᑐ↷(8N Ỉ㓟໬࣒࢝ࣜ࢘)࡛ฎ⨨ࡉࢀࡓ⤌⧊」〇≀ࡢ ᐃ್ࡢ⏕Ꮡ⋡ࡣ 15㸣ᮍ‶ࠋ
3. 20-100㸣ࡢ⏕Ꮡ⋡࠾ࡼࡧ 0.3
௨ୖࡢ OD ್࡟࠾࠸࡚ࠊ2 ࡘࡢ⤌⧊㛫ࡢ⏕Ꮡ⋡ࡢᕪࡣ
30㸣ࢆ㉸࠼࡞࠸ࠋ

2.0 ᫬㛫ӌET50ӌ7.0 ᫬㛫1㸣Triton X-100 ࡛ฎ⌮

540-570nmཧ↷ࣇ࢕ࣝࢱ࣮࡞ࡋ࡛

㸦㹼120rpm㸧120 ศࡃࡣᐊ ࡛᣺࡜࠺ࡋࡓ≧ែ࡛ᐊ ࡛᣺࡜࠺ࡏࡎ࡟୍ᬌࠊࡶࡋ

epiCS®

1 ᅇࠊ᫂☜࡞⤖ᯝࡀᚓࡽࢀ࡞࠿ࡗ
ࡓ᫬ࡣ 2 ᅇ4.0 ᫬㛫ӌET50ӌ10.0 ᫬㛫1㸣Triton X-100 ࡛ฎ⌮

1㸣Triton X-100 ࡛ฎ⌮

SDS ࡛ 18 ᫬㛫ฎ⌮

⤌⧊ࡢရ㉁☜ㄆ

570nm (545-600nm)

570nm (ࡶࡋࡃࡣ 540nm)ཧ↷ࣇ࢕ࣝࢱ࣮࡞ࡋ࡛

㸦㹼120rpm㸧120 ศ࡛᣺࡜࠺ࡋࡓ≧ែ࡛࡛᣺࡜࠺ࡏࡎ࡟୍ᬌࠊࡶࡋ

ࡃࡣᐊᐊ

570nm (545-595nm)

㸦㹼120rpm㸧120 ศ࡛᣺࡜࠺ࡋࡓ≧ែ࡛࡛᣺࡜࠺ࡏࡎ࡟୍ᬌࠊࡶࡋ

ࡃࡣᐊᐊ

SkinEthicTM RHE

ཧ↷ࣇ࢕ࣝࢱ࣮࡞ࡋ࡛

୍࡛ᬌ

EpiDermTMSCTཧ↷ࣇ࢕ࣝࢱ࣮࡞ࡋ࡛

OD

ᐃ᮲௳

㐽ගࡋࠊᐊ

ᢳฟ᫬㛫࠾ࡼࡧ

ᗘ

EpiSkinTM

ࢸࢫࢺ᪉ἲ せ⣲⾲ 2-1 EpiSkinTM ࡢண ࣔࢹࣝ 3)
3 ศࠊ60 ศ࠾ࡼࡧ 240 ศ᭚㟢ᚋ
ࡢ⏕Ꮡ⋡

ண

ᛶࡢホ౯

3 ศ᭚㟢ᚋࡢ⏕Ꮡ⋡ࡀ 35㸣ᮍ‶⭉㣗ᛶ࣭ᅜ㐃 GHS ⣽༊ศ 1A㸨

3 ศ᭚㟢ᚋࡢ⏕Ꮡ⋡ࡀ 35㸣௨ୖ࡛ࠊ࠿
ࡘ 60 ศ᭚㟢ᚋࡢ⏕Ꮡ⋡ࡀ 35㸣ᮍ‶ࡢሙྜࠊࡶࡋࡃࡣ
60 ศ᭚㟢ᚋࡢ⏕Ꮡ⋡ࡀ 35㸣௨ୖ࡛ࠊ࠿
ࡘ 240 ศ᭚㟢ᚋࡢ⏕Ꮡ⋡ࡀ 35㸣ᮍ‶
240 ศ᭚㟢ᚋࡢ⏕Ꮡ⋡ࡀ 35㸣௨ୖ⭉㣗ᛶ࣭ᅜ㐃 GHS ⣽༊ศ 1B ࠶ࡿ࠸ࡣ
1C

㠀⭉㣗ᛶ

*) ⭉㣗ᛶࡢ⣽༊ศ࡟࠾ࡅࡿ RhE ヨ㦂ἲࡢ᭷⏝ᛶࢆホ౯ࡍࡿࡓࡵ࡟సᡂࡋࡓࢹ࣮ࢱ࡟ࡼࡿ࡜ࠊEpiSkinTM ヨ㦂ἲ࡟ࡼࡾ༊ศ 1A ࡟ศ㢮ࡉࢀࡓ≀㉁㸭ΰྜ≀ࡢ⣙ 22%ࡀࠊᐇ㝿࡟ࡣ༊ศ 1B ࡲࡓࡣ 1C ࡟ᒓࡍࡿࡶࡢ࡛࠶ࡿྍ⬟ᛶࡀ࠶ࡿ 㸦ࡍ࡞ࢃࡕࠊ㐣኱ホ౯㸧ࠋ

⾲ 2-2 EpiDermTMSCTࠊSkinEthicTMRHE ࠾ࡼࡧ epiCS®ࡢண ࣔࢹࣝ 3)
3 ศ࠾ࡼࡧ 60 ศ᭚㟢ᚋࡢ⏕Ꮡ⋡ண ᛶࡢホ౯
3 ศ᭚㟢ᚋࡢ⏕Ꮡ⋡ࡀ 50㸣ᮍ‶
3 ศ᭚㟢ᚋࡢ⏕Ꮡ⋡ࡀ 50㸣௨ୖ࡛ࠊ࠿
ࡘ 60 ศ᭚㟢ᚋࡢ⏕Ꮡ⋡ࡀ 15㸣ᮍ‶
3 ศ᭚㟢ᚋࡢ⏕Ꮡ⋡ࡀ 50㸣௨ୖ࡛ࠊ࠿
ࡘ 60 ศ᭚㟢ᚋࡢ⏕Ꮡ⋡ࡀ 15㸣௨ୖ⭉㣗ᛶ࣭ᅜ㐃 GHS ⣽༊ศ 1A㸨
⭉㣗ᛶ࣭ᅜ㐃 GHS ⣽༊ศ 1B ࠶ࡿ࠸ࡣ1C㠀⭉㣗ᛶ *) ⭉㣗ᛶࡢ⣽༊ศ࡟࠾ࡅࡿ RhE ヨ㦂ἲࡢ᭷⏝ᛶࢆホ౯ࡍࡿࡓࡵ࡟సᡂࡋࡓࢹ࣮ࢱ࡟ࡼࡿ࡜ࠊEpiSkinTM ヨ㦂ἲ࡟ࡼࡾ༊ศ 1A ࡟ศ㢮ࡉࢀࡓ≀㉁㸭ΰྜ≀ࡢ⣙ 22%ࡀࠊᐇ㝿࡟ࡣ༊ศ 1B ࡲࡓࡣ 1C ࡟ᒓࡍࡿࡶࡢ࡛࠶ࡿྍ⬟ᛶࡀ࠶ࡿ 㸦ࡍ࡞ࢃࡕࠊ㐣኱ホ౯㸧ࠋ

3. 㛤Ⓨ࠾ࡼࡧホ౯࡟౑ࢃࢀࡓ≀㉁ࡢศ㢮ࠊ㑅ᢥ⌮⏤ࡢጇᙜᛶࠊin vitro ࠾ࡼࡧཧ↷ࢹ࣮ࢱࡢ᭷↓EpiSkinTM ࡢ෌⌧ᛶࡣࠊ60 ࡢ⿕㦂≀㉁࡟ࡼࡾㄪ࡭ࡽࢀ࡚࠸ࡿࠋࡑࢀࡽࡢ≀㉁ࡀᥖ㍕ࡉࢀࡓㄽᩥࡢᢤ
⢋ࢆ ANNEX 1 ࡟♧ࡍ 4)ࠋEpiDerm™ࡢ෌⌧ᛶࡣࠊ24 ࡢ⿕㦂≀㉁࡟ࡼࡾㄪ࡭ࡽࢀ࡚࠸ࡿࠋࡑࢀࡽࡢ≀㉁
ࡀᥖ㍕ࡉࢀࡓㄽᩥࡢᢤ⢋ࢆ ANNEX 2 ࡟♧ࡍ 5)ࠋSkinEthic™ࡢ෌⌧ᛶࡣࠊ12 ࡢ⿕㦂≀㉁࡟ࡼࡾㄪ࡭ࡽ
ࢀ࡚࠸ࡿࠋࡑࢀࡽࡢ≀㉁ࡀᥖ㍕ࡉࢀࡓㄽᩥࡢᢤ⢋ࢆ ANNEX 3 ࡟♧ࡍ 6)ࠋEpiCS®ࡢ෌⌧ᛶࡣࠊ12 ࡢ⿕㦂≀㉁࡟ࡼࡾㄪ࡭ࡽࢀ࡚࠸ࡿࠋࡑࢀࡽࡢ≀㉁ࡢᢤ⢋ࢆ ANNEX 4 ࡟♧ࡍ 2㸧ࠋࡉࡽ࡟ࠊୖグ㸲ࡘࡢࣔࢹࣝࡢண

ᛶࡀ 80 ⿕㦂≀㉁࡛ㄪ࡭ࡽࢀ࡚࠸ࡿࠋࡑࢀࡽࡢ≀㉁ࡀᥖ㍕ࡉࢀࡓㄽᩥࡢᢤ⢋ࢆ ANNEX 5 ࡟♧

ࡍ 7㸧ࠋホ౯࡟౑⏝ࡉࢀࡓ⿕㦂≀㉁ࡢከࡃࡣḢᕞ௦᭰ἲホ౯ࢭࣥࢱ࣮㸦ECVAM㸸European Centre for the
Validation of Alternative Methods㸧୺ᑟࡢ⓶⭵⭉㣗ᛶヨ㦂ࣂࣜࢹ࣮ࢩ࡛ࣙࣥ౑⏝ࡉࢀࡓ≀㉁࡛࠶ࡿࠋࡇ
ࢀࡽ⿕㦂≀㉁ࡢホ౯⤖ᯝࡣୗグࡢ㈨ᩱ࡜ࡋ࡚ᥦฟࡉࢀ࡚࠸ࡿࠋ
Liebsh et al., ATLA 20005), Barratt et al., Toxicol. In Vitro 19988), Fentem et al, Toxicol. In Vitro 19984),
Worth et al., ATLA 19989), Botham et al., ATLA 199510) , ICCVAM(1999) NIH Publication No:99-449511),
ICCVAM (2002) NIH Publication No: 02-450212)

915

4. ヨ㦂ἲࡢṇ☜ᛶ㸦෌⌧ᛶ㸧
EpiSkinTM ࡟࠾࠸࡚ࡣࠊ60 ≀㉁ࢆ⏝࠸ࡓ 3 ᪋タ࡛ࡢ 2-way ࡢ ANOVA ゎᯒࢆ⏝࠸ࠊ᪋タෆ࠾ࡼࡧ᪋タ㛫ࡢኚື࡟ࡘ࠸࡚ࡣࠊࡑࢀࡽࡢ㛫࡟᭷ព࡞ᕪࡣ࡞࠸࡜ุ᩿ࡉࢀࡓ(Fentem et al 1998)4)ࠋ60 ≀㉁㸦27⭉㣗ᛶ≀㉁࠾ࡼࡧ 33 㠀⭉㣗ᛶ≀㉁㸧ࡢ࠺ࡕࠊ42 ≀㉁ࡣ 3 ᪋タ࡜ࡶ᪋タෆ࠾ࡼࡧ᪋タ㛫෌⌧ᛶࡀⰋዲ࡛࠶ࡗࡓࠋṧࡿ 18 ≀㉁࡛ࡣఱ➼࠿ࡢ⤖ᯝࡀ␗࡞ࡗ࡚࠸ࡓࡀࠊECVAM ࡣᮏヨ㦂ἲࡢಙ㢗ᛶ࡜෌⌧ᛶࡣ㧗࠸࡜ุ᩿ࡋࡓ 13 㸧ࠋࡇࡢ⤖ㄽࡣࠊ ECVAM ⛉Ꮫㅎၥ఍㆟㸦ESAC: ECVAM Scientific Advisory
Committee 13 㸧 ࠾ࡼࡧ⡿ᅜࡢ௦᭰ἲ࡟㛵ࡍࡿ┬ᗇ㛫㐃⤡఍㆟㸦ICCVAM㸸Interagency Coordinating
Committee on the Validation of Alternative Methods 12㸧࡛ࡢホ౯࡟࠾࠸࡚ࡶ☜ㄆࡉࢀࡓࠋ
EpiDerm™࡟࠾࠸࡚ࡣࠊ24 ≀㉁ࢆ⏝࠸ࡓ 3 ᪋タ࡛ࡢ 2 ᅇࡢᐇ㦂࡟࠾࠸࡚ࠊ21 ≀㉁ࡢ⭉㣗ᛶࢆ 3 ᪋タࡍ࡭࡚࡛ṇࡋࡃண

࡛ࡁࡓ㸦Liebsch et al 2000㸧5)ࠋECVAM ࡣᮏヨ㦂ἲࡢಙ㢗ᛶ࡜෌⌧ᛶࡣ㧗࠸࡜ุ᩿ࡋࡓࠋࡇࡢ⤖ㄽࡣࠊESAC ࡢホ౯ᚋ 14㸧ࠊICCVAM ࡟࠾࠸࡚ࡶ☜ㄆࡉࢀࡓ 11,12㸧ࠋSkinEthic™࡟࠾࠸࡚ࡣࠊ12 ≀㉁ࢆ⏝࠸ࡓ 3 ᪋タ࡛ࡢ 3 ᅇࡢᐇ㦂࡟࠾࠸࡚ࠊ93.2㸣ࡢ⤖ᯝࡀ୍⮴ࡋ
Kandarova et al 2006 㸴㸧ࠊESAC ࡟࠾࠸࡚᪋タෆ࠾ࡼࡧ᪋タ㛫෌⌧ᛶࡀ㧗࠸࡜ุ᩿ࡉࢀࡓ 15㸧ࠋ
EpiCS®࡟࠾࠸࡚ࡣࠊTG431㸦2004㸧ࡢཧ↷ 12 ≀㉁ࢆ⏝࠸ࡓ 4 ᪋タ࡛ࡢ 3 ᅇࡢᐇ㦂࡟࠾࠸࡚ 2㸧ࠊࢸࢺࣛࢡ࢚ࣟࣟࢳࣞࣥࢆ㝖ࡃ 11 ≀㉁ࡢ⤖ᯝࡀ୍⮴ࡋࠊESAC ࡟࠾࠸࡚᪋タෆ࠾ࡼࡧ᪋タ㛫෌⌧ᛶࡀㄆࡵࡽ
ࢀࡓ 16㸧ࠋ
5. ヨ㦂ἲࡢಙ㢗ᛶ 7ࠊ17ࠊ18㸧 ࠸ࡎࢀࡢࣄࢺ⾲⓶ࣔࢹࣝࡶ⭉㣗ᛶࡢ᭷↓ࡢணᛶࡣ㧗ࡃࠊṇ☜ᗘ1ࡣ 84㹼90%ࠊឤᗘ2ࡣ 95㹼100㸣ࠊ≉␗ᗘ3ࡣ 72㹼79㸣࡛࠶ࡾࠊഇ㝜ᛶ⋡ࡣ 0㹼5%࡜ప࠸࡛ࣞ࣋ࣝ࠶ࡗࡓ㸦⾲ 3㸧ࠋࡇࢀࡽࡢ್࠿ࡽࠊOECDࡢᛶ⬟ᶆ‽࡛ࡣṇ☜ᗘࠊឤᗘ࠾ࡼࡧ≉␗ᗘࢆࣂࣜࢹ࣮ࢩࣙࣥ࡟ᚲせ࡞ᇶ‽࡜ࡋ࡚ 82.5ࠊ95 ࠾ࡼࡧ 70㸣࡜ᐃࡵ࡚࠸ࡿࠋ ࡉࡽ࡟ࠊྛࣔࢹࣝࡢ⣽༊ศࡢணᛶࢆ⾲ 4 ࡲ࡜ࡵࡓࠋ80 ≀㉁ࢆ 2 ࡲࡓࡣ 3 ᅇᐇ㦂ࡋ࡚ᚓࡽࢀࡓ್ࢆ

ぢࡿ㝈ࡾࠊ⣽༊ศ࡛࠶ࡿ㸯B/1C ࢆ 80㸣⛬ᗘ࡛ホ౯࡛ࡁ࡚࠸ࡿࣔࢹࣝࡣ EpiSkinTM ࡢࡳ࡛࠶ࡗࡓࠋࡑࡢ
௚ࡢࣔࢹࣝࡣᅜ㐃 GHS ⣽༊ศࡢホ౯࡟ࡣ฼⏝࡛ࡁ࡞࠸࡜ุ᩿ࡋࡓࠋ
ࡇࢀࡽࣔࢹࣝࡣ MTT 㑏ඖ≀㉁࡬ࡢᑐᛂ᪉ἲࡶ TG431 ࡟グ㍕ࡉࢀ࡚࠸ࡿࠋ㐺⏝࡛ࡁ࡞࠸≀㉁࡜ࡋ࡚
ࡣ࢞ࢫࠊ࢚࢔ࣟࢰ࣮ࣝࡢࡳࡀグ㍕ࡉࢀ࡚࠸ࡿ㸦ࣂࣜࢹ࣮ࢩࣙࣥᮍᐇ᪋㸧ࠋࡇࢀࡽࣔࢹࣝࡣ≀⌮≧ែ㸦ᾮయ࣭ᅛయ➼㸧࠾ࡼࡧỈ⁐ᛶࡢ᭷↓࡟࠿࠿ࢃࡽࡎ㐺⏝ྍ⬟࡛࠶ࡾࠊ࢞ࢫࠊ࢚࢔ࣟࢰ࣮ࣝࢆ㝖ࡃΰྜ≀࡛ࡶ㐺⏝ྍ⬟࡜ࡉࢀ࡚࠸ࡿࠋ࡞࠾ࠊ≉ᐃࡢ✀㢮ࡢ≀㉁ࡸΰྜ≀࡟࠾࠸࡚ࡇࢀࡽࣔࢹࣝࡢ㐺⏝ᛶࢆྰᐃࡍ
ࡿࡼ࠺࡞᫂☜࡞᰿ᣐࡀᚓࡽࢀࡓሙྜ࡟ࡣࠊ㐺⏝⠊ᅖ࠿ࡽ㝖እࡍࡿ࡭ࡁ࡛࠶ࡿ࡜ࡉࢀ࡚࠸ࡿࠋ

1ṇ☜ᗘ㸸⿕㦂≀㉁࡟ࡘ࠸࡚ヨ㦂ἲ࡛ᚓࡽࢀࡿ⤖ᯝࡀࠊ⿕㦂≀㉁ࡢ᪤ᐃࡢཧ↷್࡜㢮ఝࡋ࡚࠸ࡿ⛬ᗘࠋឤᗘ㸸ヨ㦂ࡉࢀࡓ㝧ᛶ≀㉁ࡢ୰࡛ࠊヨ㦂ἲ࡛ṇࡋࡃ㝧ᛶ࡜ุᐃࡉࢀࡓࡶࡢࡢ๭ྜࠋ
3 ≉␗ᗘ㸸ヨ㦂ࡉࢀࡓ㝜ᛶ≀㉁ࡢ୰࡛ࠊヨ㦂ἲ࡛ṇࡋࡃ㝜ᛶ࡜ุᐃࡉࢀࡓࡶࡢࡢ๭ྜࠋ
10
2

16

⾲ 3㸬OECD ࡛㞟ィࡋࡓ඲ࢭࢵࢺࡢ໬Ꮫ≀㉁ࢆ⏝࠸ࡓண ᛶࡢィ⟬⤖ᯝ㸦⭉㣗ᛶ [ᅜ㐃 GHS ༊ศ㸯] ࡢ᭷↓㸧17㸧
EpiDermTM SkinEthicTM
epiCS®
EpiSkinTM
87.9%
ṇ☜ᗘ
89.6㸣
84.6㸣
84.3㸣
100%
ឤᗘ
98.5㸣
94.6㸣
95.3㸣
73.9%
≉␗ᗘ
79.3㸣
73.0㸣
71.6㸣
20.7%
26.1%
27.0%
28.4%
ഇ㝧ᛶ⋡
1.5%
0%
5.4%
4.7%
ഇ㝜ᛶ⋡

⾲ 4. OECD ࡛㞟ィࡋࡓ඲ࢭࢵࢺࡢ໬Ꮫ≀㉁ࢆ⏝࠸ࡓண
㸯Aࠊ㸯B/1Cࠊ㠀⭉㣗ᛶ㸧3㸧ᛶࡢィ⟬⤖ᯝ㸦⣽༊ศ㸸ᅜ㐃 GHS ༊ศ໬Ꮫရ඲యࡢ⤫ィࢹ࣮ࢱ㸦㸣㸧㸦80 ✀㢮ࡢ໬Ꮫရࢆ 2 ࡲࡓࡣ 3 ᅇヨ㦂ࠊࡍ࡞ࢃࡕࠊ159㸨ࡲࡓࡣ 24 ᅇࡢศ㢮⤖ᯝ㸧㸨
1 ရ┠ࡣࠊධᡭ୙ྍ⬟࡛࠶ࡗࡓࡓࡵ 1 ᅇࡢࡳヨ㦂

EpiSkinTM

EpiDermTM

SkinEthicTM

EpiCSR

㐣኱ホ౯









༊ศ 1BC ࡢ໬Ꮫရࡢ㸯A ࡬ࡢ㐣኱ホ౯

21.5

29.0

31.2

32.8

༊ศ NC ࡢ໬Ꮫရࡢ㸯B/㸯C ࡬ࡢ㐣኱ホ౯

20.7

23.4

27.0

28.4

༊ศ NC ࡢ໬Ꮫရࡢ㸯A ࡬ࡢ㐣኱ホ౯

0.0

2.7

0.0

0.0

㐣኱ホ౯ࡉࢀࡓ༊ศ NC ໬Ꮫရ

20.7

26.1

27.0

28.4

඲༊ศ࡛ࡢ㐣኱ホ౯⋡

17.9

23.3

24.5

25.8









༊ศ㸯A ࡢ㸯B/1C ࡬ࡢ㐣ᑠホ౯

16.7

16.7

16.7

12.5

༊ศ㸯A ࡢ NC ࡬ࡢ㐣ᑠホ౯

0.0

0.0

0.0

0.0

༊ศ㸯B/1C ࡢ NC ࡬ࡢ㐣ᑠホ౯

2.2

0.0

7.5

6.6

඲༊ศ࡛ࡢ㐣ᑠホ౯⋡

3.3

2.5

5.4

4.4

ṇ☜࡞ศ㢮









ṇࡋࡃศ㢮ࡉࢀࡓ㸯A ໬Ꮫရ

83.3

83.3

83.3

87.5

ṇࡋࡃศ㢮ࡉࢀࡓ㸯B/1C ໬Ꮫရ

76.3

71.0

61.3

60.7

ṇࡋࡃศ㢮ࡉࢀࡓ NC ໬Ꮫရ

79.3

73.9

73.0

71.6

୍⮴ᗘ(ண

78.8

74.2

70.0

69.8

㐣ᑠホ౯

⬟㸧

NC:㠀⭉㣗ᛶ

6. ௚ࡢ⛉Ꮫⓗ࡞ሗ࿌࡜ࡢẚ㍑ࡢ᭷↓ OECD ࡢ⭉㣗ᛶヨ㦂௦᭰ἲ࢞࢖ࢻࣛ࢖ࣥ࡜ࡋ࡚ࠊTG431 ࡢ௚࡟ࠕTG430 TER㸦Transcutaneous Electrical
Resistance Test Method㸸⤒⓶㟁Ẽ᢬ᢠᛶヨ㦂 ࠖ19㸧࠾ࡼࡧࠕTG435

In Vitro Membrane Barrier Test Method

for Skin Corrosion : in vitro ⭷ࣂࣜ࢔ヨ㦂ࠖ20㸧ࡀᢎㄆࡉࢀ࡚࠸ࡿࠋࡇࢀࡽࡣ࠸ࡎࢀࡶ ECVAM ࡟࡚ࣂࣜ1117ࢹ࣮ࢩࣙࣥ◊✲ࡀᐇ᪋ࡉࢀࠊICCVAM ࡣࡇࢀࡽࡢヨ㦂ἲ Rat Skin TER, EpiSkin™, EpiDerm™࠾ࡼࡧ
Corrositex® ࡢṇ☜ᗘࠊឤᗘ࠾ࡼࡧ≉␗ᗘ࡟ࡘ࠸࡚ẚ㍑ࡋ࡚࠸ࡿ㸦⾲ 5㸧11,12㸧ࠋࡇࢀࡽࡢẚ㍑࡟࠾࠸࡚ࡣྠࡌ≀㉁ࢆ⏝࠸࡚ホ౯ࡉࢀ࡚࠾ࡽࡎࠊヨ㦂≀㉁ࡢᩘ㔞ࡸ㑅ᢥ≀㉁ࡢ✀㢮ࡀ␗࡞ࡗ࡚࠸ࡿࡓࡵ⤖ᯝࡢᩘ್ࡔࡅࢆࡶࡗ࡚ࠊ༢⣧࡟ࣄࢺ⾲⓶ࣔࢹࣝࡢඃ㉺ᛶࢆẚ㍑ホ౯ࡍࡿࡇ࡜ࡣᅔ
㞴࡛࠶ࡿࡀࠊ࠸ࡎࢀࡢヨ㦂ἲࡶྠ➼ࡢணᛶࢆ᭷ࡍࡿ࡜ᛮࢃࢀࡿࠋ

⾲ 5㸬ヨ㦂ἲࡢẚ㍑⤖ᯝ 11,12㸧

≀㉁ᩘ
ṇ☜ᗘ
ឤᗘ
≉␗ᗘ

TER
122
81% (99/122)
94%(51/54)
71%(48/68)

EpiSkin
60
83% (50/60)
82%(23/28)
84%(27/32)

EpiDerm
24
92% (22/24)
92%(11/12)
83%(10/12)

Corrositex
163
79% (128/163)
85%(76/89)
72%(52/74)

7. 3Rs ཎ๎࡜ࡢ㛵ಀ㸦ື≀⚟♴㠃࠿ࡽࡢጇᙜᛶ㸧 ࠸ࡎࢀࡢ⾲⓶ࣔࢹࣝࡶື≀ࢆ౑⏝ࡋ࡚࠾ࡽࡎࠊື≀⚟♴㠃࠿ࡽ௦᭰ἲ࡜ࡋ࡚ጇᙜ࡛࠶ࡿࠋ

8. ヨ㦂ἲࡢ᭷⏝ᛶ࡜㝈⏺㸦ࢥࢫࢺࠊ᫬㛫࠿ࡽࡢጇᙜᛶ࡞࡝㸧ᙉ࠸㓟ᛶ㸦pH 2.0 ௨ୗ㸧ࡲࡓࡣ࢔ࣝ࢝ࣜᛶ㸦pH 11.5 ௨ୖ㸧≀㉁ࡣࠊᙉ࠸ᒁᡤస⏝ࢆ᭷ࡍࡿྍ⬟ᛶࡀ㧗࠸ࡇ࡜࠿ࡽࠊ⓶⭵⭉㣗ᛶ࡜ุ᩿ࡋ࡚ࡶⰋ࠸ࡇ࡜࡟࡞ࡗ࡚࠸ࡿ 21㸧ࠋࡋ࠿ࡋ࡞ࡀࡽࠊࡇࢀࡣ⭉㣗ᛶ࡟ࡘ࠸࡚ࡢ᝟ሗࡀ௚࡟࡞࠸ሙྜ࡟⾜ࢃࢀࡿ࣮࣡ࢫࢺࢣ࣮ࢫ࡜ࡋ࡚ࡢุ᩿࡛࠶ࡾࠊ౛࠼ࡤ㓟ࡸሷᇶࡢῧຍ࡟
ࡼࡾ pH ࡀኚࢃࡾ᫆࠸≀㉁ࡸΰྜ≀ࡢሙྜࡣഇ㝧ᛶࡢุ᩿࡜࡞ࡿྍ⬟ᛶࡶ⪃࠼ࡽࢀࡿ 22㸧ࠋࡇࡢࡓࡵࠊ
ࡇࡢࡼ࠺࡞≀㉁࡟ᑐࡋ࡚ື≀ࢆ⏝࠸ࡿᚲせࡢ࡞࠸ TG431 ࡛⓶⭵⭉㣗ᛶࢆホ౯ࡍࡿࡇ࡜ࡣ᭷⏝࡛࠶ࡿࠋ⾲ 6 ࡟♧ࡍ⩦⇍ᗘ☜ㄆ≀㉁ࢆṇࡋࡃศ㢮࡛ࡁࡿ࠿ྰ࠿ヨ㦂ࡍࡿࡇ࡜࡟ࡼࡾࠊᑓ㛛ᢏ⾡ࡢ⩦⇍࡟ࡘ࠸࡚☜ㄆࡍࡿࡇ࡜ࡀ࡛ࡁࡿ࡜ࡢグ㍕ࡀ TG431 ࡟࠶ࡿࠋ9. ࡑࡢ௚
 OECD ࡟ᢎㄆࡉࢀࡓࣄࢺ⾲⓶ࣔࢹࣝࡣᾏእ࡛㛤Ⓨࡉࢀࡓ〇ရ࡛࠶ࡾࠊࢥࢫࢺ㠃࡛ࡸࡸ๭㧗࡛࠶ࡿࠋ᪥ᮏ〇ࡢࣄࢺ⾲⓶ࣔࢹࣝ࡜ࡋ࡚ࠊࡇࢀࡲ࡛࡟ LabCyte EPI-Model24㸧ࡸ Vitrolife-Skin ࡀሗ࿌ࡉࢀ࡚࠸ࡿ
25㸧

ࠋ≉࡟ࠊVitrolife-Skin ࡣཌ⏕ປാ⛉Ꮫ◊✲⿵ຓ㔠஦ᴗ࡛ࣂࣜࢹ࣮ࢺࡉࢀ 26㸧ࠊJaCVAM ࡟࠾࠸࡚ࡶホ౯ࡀ࡞ࡉࢀ࡚࠸ࡿ 27㸧ࠋࡓࡔࡋࠊOECD ࡢᛶ⬟ᶆ‽࡟‽ࡌࡓࣂࣜࢹ࣮ࢩࣙࣥࡣᐇ᪋ࡉࢀ࡚࠾ࡽࡎࠊOECD
࡟᪂ࡓ࡞ヨ㦂ἲ࡜ࡋ࡚᥎ዡ࡛ࡁࡿࣔࢹ࡛ࣝࡣ࡞࠸ࠋࡼࡗ࡚ࠊࡇࢀࡽࡢࣔࢹࣝࡢホ౯ࡣᮏホ౯᭩࡛ࡣ⾜ࢃ࡞࠿ࡗࡓࠋ ≉チ࡟ࡘ࠸࡚ࡢ᝟ሗࡣ௒ᅇࡢ᳨ウ㈨ᩱ࡟♧ࡉࢀ࡚࠸࡞࠸ࠋ࡞࠾ࠊୖグࡋࡓࣄࢺ⾲⓶ࣔࢹࣝࡣ᪤࡟ᕷ㈍ࡉࢀ࡚࠾ࡾࠊ࠸ࡎࢀࡶ㉎ධྍ⬟࡛࠶ࡿࠋ

1218

10. ⤖ㄽ ಙ㢗ᛶ࡜ጇᙜᛶ࡜࠸࠺どⅬ࡟࠾࠸࡚ࠊࣄࢺ⾲⓶ࣔࢹࣝࢆ⏝࠸ࡓ⓶⭵⭉㣗ᛶヨ㦂ࢆホ౯ࡋࡓ⤖ᯝࠊ⿕㦂≀㉁ࡢ⓶⭵⭉㣗ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡜ࡋ࡚ EpiSkin™ࠊEpiDerm™ࠊSkinEthic™࠾ࡼࡧ epiCS®ࡀ᥎ዡ࡛ࡁࡿࣔࢹࣝ࡜ࡋ࡚ᣲࡆࡽࢀࡓࠋࡓࡔࡋࠊᅜ㐃 GHS ⣽༊ศࢆ⪃៖ࡍࡿሙྜࡣࠊEpiSkin™ࡀ⓶⭵⭉㣗ᛶヨ㦂ࡢ௦᭰ἲ࡜ࡋ࡚ࡶࡗ࡜ࡶ᭷⏝࡛࠶ࡿ࡜⤖ㄽࡋࡓࠋ1319

⾲ 6㸬⩦⇍ᗘ☜ㄆ≀㉁ 3㸧໬Ꮫ≀㉁ 1

CASRN

໬Ꮫ≀㉁
ศ㢮 2

UN GHS
VRM㸦inMTT㸦in vivo ヨ㦂㸧 vitro ヨ㦂㸧࡟
5
㑏ඖ๣
3
࡟ࡼࡿ༊ศ
ࡼࡿ༊ศ 4≀⌮ⓗ≧ែ༊ศ 1A ࡢ in vivo ⭉㣗ᛶ≀㉁
79-08-3

᭷ᶵ㓟

1A(3) 1A--ᅛయ

3 ࣇࢵ໬࣎ࣟࣥ
஧Ỉ࿴≀13319-75-0

↓ᶵ㓟

1A

(3) 1A

--

ᾮయ

ࣇ࢙ࣀ࣮ࣝ

108-95-2

ࣇ࢙ࣀ࣮ࣝ㢮

1A(3) 1A--ᅛయࢪࢡࣟࣟ࢔ࢭࢳࣝ
ࢡࣟࣜࢻ

79-36-7

ồ㟁Ꮚ๣

1A(3) 1A

--

ᾮయ

ࣈࣟࣔ㓑㓟

༊ศ 1B/1C ࡢ in vivo ⭉㣗ᛶ≀ࢢࣜ࢜࢟ࢩࣝ㓟୍Ỉ࿴≀

563-96-2

᭷ᶵ㓟

1B/1C

(3) 1B/1C

--

ᅛయ

ங㓟

598-82-3

᭷ᶵ㓟

1B/1C

(3) 1B/1C--

ᾮయ࢚ࢱࣀ࣮ࣝ࢔࣑ࣥ

141-43-5᭷ᶵሷᇶ

1B(3) 1B/1C

ሷ㓟㸦14.4%㸧

7647-01-0

↓ᶵ㓟

1B/1C

(3) 1B/1C

⢓⛻ᛶ
--

ᾮయ

in vivo 㠀⭉㣗ᛶ≀㉁⮯໬ࣇ࢙ࢿࢳࣝ

103-63-9ồ㟁Ꮚ๣NC(3) NC

4-࢔࣑ࣀ-1,2,4ࢺࣜ࢔ࢰ࣮ࣝ

584-13-4

᭷ᶵሷᇶNC(3) NC

4-㸦࣓ࢳࣝࢳ࢜㸧࣋ࣥࢬ࢔ࣝࢹࣄࢻ

3446-89-7

ồ㟁Ꮚ๣

NC

(3) NCࣛ࢘ࣜࣥ㓟

143-07-7᭷ᶵ㓟NC(3) NC

ᾮయ
--

ᅛయ
ᾮయ

--

ᅛయ

␎ㄒ㸸CASRN㸻CAS Ⓩ㘓␒ྕ㸹UN GHS㸻ᅜ㐃່࿌ࠕ໬Ꮫရࡢศ㢮࠾ࡼࡧ⾲♧࡟㛵ࡍࡿୡ⏺ㄪ࿴ࢩࢫࢸ࣒ࠖ(1)㸹VRM
㸻ࣂࣜࢹ࣮ࢩࣙࣥ῭ࡳᶆ‽ヨ㦂ἲ㸹NC㸻㠀⭉㣗ᛶ
1ࡇࢀࡽࡢ໬Ꮫ≀㉁ࡣࠊࡲࡎ⭉㣗ᛶ࡛࠶ࡿ࠿㠀⭉㣗ᛶ࡛࠶ࡿ࠿࡟ࡼࡾศ㢮ࡋࠊࡉࡽ࡟⭉㣗ᛶࡢ⣽༊ศ࠾ࡼࡧ໬Ꮫ≀㉁ࡢ✀㢮࡟ࡼࡾ⣽ศ㢮ࡋࡓࠋ⾲࡟グ㍕ࡋࡓ໬Ꮫ≀㉁ࡣࠊECVAM ࡟ࡼࡿ EpiSkinTM ࠾ࡼࡧ EpiDermTM ࡢࣂࣜࢹ࣮ࢩࣙ
ࣥヨ㦂࡛౑⏝ࡉࢀࡓ≀㉁ 4,5,8)ࠊ࡞ࡽࡧ࡟ EpiSkinTM 23㸧ࠊEpiDermTMࠊSkinEthicTM ࠾ࡼࡧ epiCS® 7)ࡢ㛤Ⓨ⪅࡟ࡼࡾᥦ౪ࡉ
ࢀࡓࢹ࣮ࢱ࡟ᇶ࡙࠸ࡓࣂࣜࢹ࣮ࢩࣙࣥᚋヨ㦂ࡼࡾ㑅ᢥࡋࡓࡶࡢ࡛࠶ࡿࠋ≉࡟グ㍕ࡢ࡞࠸㝈ࡾࠊᕷ㈍ࡉࢀ࡚࠸ࡿ໬Ꮫ≀㉁ࡢ㉎ධ᫬ࡢ⣧ᗘ࡟࠾࠸࡚ヨ㦂ࢆ⾜ࡗࡓ 5,8)ࠋࡇࡢ㑅ᢥ࡟࠶ࡓࡗ࡚ࡣࠊྍ⬟࡞㝈ࡾ௨ୗࡢࡼ࠺࡞≀㉁ࢆྵࡵࡿࡇ࡜
࡜ࡋࡓ㸸(i) VRM ࡟ࡼࡾ ᐃࡲࡓࡣண ྍ⬟࡞⭉㣗ᛶ཯ᛂࡢ⠊ᅖ㸦౛࠼ࡤࠊ㠀⭉㣗ᛶࠊᙅ⭉㣗ᛶ࡞࠸ࡋᙉ⭉㣗ᛶ㸧ࢆ௦⾲ࡍࡿ໬Ꮫ≀㉁ࠊ(ii)ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡛⏝࠸ࡽࢀࡓ໬Ꮫ≀㉁ศ㢮ࢆ௦⾲ࡍࡿ≀㉁ࠊ
(iii)໬Ꮫᵓ㐀ࡀ᫂☜࡟ྠᐃࡉࢀ࡚࠸ࡿ≀㉁ࠊ(iv)VRM ࡟࠾࠸࡚෌⌧ᛶࡢ㧗࠸⤖ᯝࡀᚓࡽࢀࡿ≀㉁ࠊ(v)in vivo ࡛ࡢᶆ‽ヨ㦂ἲ࡟࠾࠸࡚☜ᐇ࡞⤖ᯝࡀᚓࡽࢀࡿ≀㉁ࠊ(vi)ᕷ㈍ࡉࢀ࡚࠸ࡿ≀㉁ࠊ࡞ࡽࡧ࡟(vii) 㠀ᖖ࡟㧗㢠࡞ᗫᲠࢥࢫࢺࡀ࠿࠿
ࡽ࡞࠸≀㉁ࠋ
2
Barratt ࡽ(1998)࡟ࡼࡿ໬Ꮫ≀㉁ศ㢮 8)ࠋ
3
UN GHS ࡢ⣽༊ศ 1Aࠊ1B ࠾ࡼࡧ 1C ࡟ࡣࠊᅜ㐃ໟ⿦➼⣭ IࠊII ࠾ࡼࡧ III ࡀࡑࢀࡒࢀᑐᛂࡍࡿࠋ
4⾲࡟グ㍕ࡋࡓ VRM ࡟ࡼࡿ in vitro ࡛ࡢศ㢮ண ࡣࠊヨ㦂ἲࡢ㛤Ⓨ⪅ࡀ⾜ࡗࡓࣂࣜࢹ࣮ࢩࣙࣥᚋヨ㦂࡟࠾࠸࡚
EpiSkinTM ࠾ࡼࡧ EpiDermTM ヨ㦂ἲ㸦VRM㸧࡟ࡼࡾᚓࡽࢀࡓࡶࡢ࡛࠶ࡿࠋ
5
ECVAM ࡟ࡼࡿ⓶⭵⭉㣗ᛶヨ㦂࡛ᚓࡽࢀࡓ⏕Ꮡ⋡ࡢ್ࡣࠊ┤᥋ⓗ࡞ MTT 㑏ඖࢆ⪃៖ࡋࡓ⿵ṇࢆ⾜ࡗ࡚࠸࡞࠸ࠋ
ࣂࣜࢹ࣮ࢩࣙࣥᚋヨ㦂ࡢࢹ࣮ࢱࡣࠊ㐺ษ࡞ᑐ↷ࢆ⏝࠸࡚ᚓࡓࡶࡢ࡛࠶ࡿࠋ

1420

11㸬ᩥ⊩
1) OECD (2015) Guideline for the testing of chemicals. 404, Acute Dermal Irritation/Corrosion
2) OECD (2004) Guideline for the testing of chemicals. 431, in vitro Skin Corrosion: Human skin model
test.
3) OECD (2016) Guideline for the testing of chemicals. 431, in vitro Skin Corrosion: Human skin model
test.
4) Fentem, J.H. et al. (1998) The ECVAM International Validation Study on in vitro tests for skincorrosivity. 2. Results and evaluation by the management team. Toxicology in Vitro, 12, 483-524.
5) Liebsch, M., et al. (2000) The ECVAM prevalidation study on the use of EpiDerm for skin corrosivity
testing. ATLA, 28, 371-401.
6) Kandarova, H. et al. (2006) Assessment of the human epidermis model SkinEthic RHE for in vitro
skin corrosion testing of chemicals according to new OECD TG 431, Toxicology in Vitro, 20,
547-559.
7) Desprez, B. et al. (2015) Two novel prediction models improve prediction of skin corrosive
sub-category by test methods of OECD Test Guideline No. 431, Toxicol. In Vitro, 29,2055-2080.
8)

Barratt, M.D. et al. (1998) The ECVAM International Validation Study on in vitro tests for skincorrosivity. 1. Selection and Distribution of the test chemicals. Toxicology in Vitro, 12, 471-482.

9)

Worth, A.P. et al. (1998) An evaluation of the proposed OECD testing strategy for skin corrosion.
ATLA, 26, 709-720.

10)Botham, P.A. et al. (1995) A pre-validation study on in vitro skin corrosivity testing: The report andrecommendations of ECVAM workshop 6. ATLA, 23, 219-255.

11) ICCVAM (1999) NIH Publication No.99-4495. Corrositex: An in vitro test method for assessing
dermal corrosivity potential of chemicals.
12) ICCVAM (2002) NIH Publication No.02-4502. ICCVAM Evaluation of EPISKIN, and EpiDerm(EPI-200) and rat skin transcutaneous electrical resistance (TER) assay: in vitro test method forassessing dermal corrosivity potential of chemicals.
13) ECVAM (1998) Statement on the scientific validity of the Episkin test (An in vitro test for skincorrosivity)14) ECVAM (2000) Statement on the application of the EpiDermTM human skin model for skin corrosivity
testing
15)

ECVAM (2006) Statement on the application of the SkinEthicTM human skin model for skincorrosivity testing

16)

ECVAM (2009) ESAC Statement on the scientific validity of an in-vitro test for skin corrosivity
testing

17)

OECD (2015) Series on Testing & Assessment No. 219, Performance Standards for the assessmentof proposed similar or modified in vitro reconstructed human epidermis (RhE) test methods for skincorrosion testing as described in TG 431

18) OECD (2013) Series on Testing & Assessment No. 190, Summary document on the Statistical
Performance of Methods in OECD Test Guideline 431 for Sub-Categorisation
19) OECD (2015) Guideline for the testing of chemicals. 430, in vitro Skin Corrosion: Transcutaneouselectrical resistance test (TER).
15

2120) OECD (2015) Guideline for the testing of chemicals. 435, in vitro membrane barrier test method for
Skin Corrosion.
21) OECD (2014) Series on Testing and Assessment No. 203, Guidance document on an Integrated
Approach on Testing and Assessment (IATA) for skin corrosion and irritation
22) Sheel J. et.al. (2011) Classification and labeling of industrial products with extreme pH by making useof in vitro methods for the assessment of skin and eye irritation and corrosion in a weight of evidence
approach, Toxicology in Vitro, 25, 1435-1447
23) Alépée N., Grandidier M.H., and Cotovio J. (2014). Sub-Categorisation of Skin Corrosive

Chemicals by the EpiSkinTM Reconstructed Human Epidermis Skin Corrosion Test Method
according to UNGHS: Revision of OECD Test Guideline 431. Toxicol. In Vitro 28, 131-145.
24) Katoh, M., et al. (2010) Assessment of the human epidermal model LabCyte EPI-MODEL for In vitro
skin corrosion testing according to the OECD test guideline 431. J Toxicol Sci., 35(3):411-7.
25) Morikawa, N., et al. (2005) Experimental study on a novel chemical application procedure for invitro skin corrosivity testing using the Vitrolife-Skin TM human skin model, AATEX, 11, 1, 68-78.
26) Kojima H et al. (2008) Validation of human skin models for skin corrosivity tests in Japan, AATEX
13(1), 36-44.
27) JaCVAM (2008) ࣄࢺ⓶⭵ࣔࢹࣝࢆ⏝࠸ࡓ⓶⭵⭉㣗ᛶヨ㦂௦᭰ἲࡢ➨୕⪅ホ౯ሗ࿌᭩㸦Vitrolif-SkinTM㸧

16

22

ANNEX
1) Fentem J.H. et al. (1998), The ECVAM International Validation Study on in vitro tests for skincorrosivity. 2. Results and evaluation by the management team. Toxicology in Vitro, 12, 483-524 ᢤ⢋2) Liebsch, M. et al. (2000), The ECVAM prevalidation study on the use of EpiDerm for skin corrosivity
testing. ATLA, 28, 371-401 ᢤ⢋
3) Kandarova, H. et al. (2006), Assessment of the human epidermis model SkinEthic RHE for in vitro skincorrosion testing of chemicals according to new OECD TG 431, Toxicology in Vitro, 20, 547-559ᢤ⢋4) OECD (2004) Guideline for the testing of chemicals. 431, in vitro Skin Corrosion: Human skin model
test. ᢤ⢋5) OECD (2013) GD190 Summary document on the Statistical Performance of Methods in OECD Test
Guideline 431 for Sub-Categorisation ᢤ⢋6) Desprez, B. et al. (2015), Two novel prediction models improve prediction of skin corrosive
sub-category by test methods of OECD Test Guideline No. 431, Toxicol. In Vitro, 29,2055-2080 ᢤ⢋

17

23

ANNEX㸯㸸Fentem et al (1998)4)ࡢࣂࣜࢹ࣮ࢩ࡛ࣙࣥ౑ࢃࢀࡓ 60 ≀㉁

18

24

ANNEX 2: Liebsch et al (2000)5)ࡢࣂࣜࢹ࣮ࢩ࡛ࣙࣥ౑ࢃࢀࡓ 24 ≀㉁

19

25

ANNEX 3: Kandarova et al (2006)6)ࡢࣂࣜࢹ࣮ࢩ࡛ࣙࣥ౑ࢃࢀࡓ 12 ≀㉁

ANNEX 4:OECDTG431 (2004)3)ࡢཧ↷≀㉁

Table 1: Reference Chemicals
1,2-Diaminopropane

CAS-No. 78-90-0

Severely Corrosive

Acrylic Acid
2-tert-Butylphenol

CAS-No. 79-10-7

Severely Corrosive

CAS-No. 88-18-6

Corrosive

Potassium hydroxide (10%)

CAS-No. 1310-58-3

Corrosive

Sulfuric acid (10%)

CAS-No. 7664-93-9

Corrosive

Octanoic acid (caprylic acid)

CAS-No. 124-07-02

Corrosive

4-Amino-1,2,4-triazole

CAS-No. 584-13-4

Not corrosive

Eugenol

CAS-No. 97-53-0

Not corrosive

Phenethyl bromide

CAS-No. 103-63-9

Not corrosive

Tetrachloroethylene

CAS-No. 127-18-4

Not corrosive

Isostearic acid

CAS-No. 30399-84-9

Not corrosive

4-(Methylthio)-benzaldehyde

CAS-No. 3446-89-7

Not corrosive

20

26

2

1

27

ANNEX 5: Desprez et al(2015)7) 80 ≀㉁ᢤ⢋

21

1

28
22

1

29

23

4

3

2

1

30
24

ANNEX 6: OECD (2013)18)ண

ᛶࢹ࣮ࢱᢤ⢋

⾲ A-1㸬EpiDermTM ࡢ⭉㣗ᛶ᭷↓ࡢண

ᛶ

25

31

⾲ A-2㸬EpiSkinTM ࡢ⭉㣗ᛶ᭷↓ࡢண

ᛶ

26

32

⾲ A-3㸬SkinEthicTM ࡢ⭉㣗ᛶ᭷↓ࡢண⾲ A-4㸬EpiCS®⭉㣗ᛶ᭷↓ࡢண

ᛶ

ᛶ

27

33