⓶⭵ឤసᛶヨ㦂ホ౯ሗ࿌᭩human Cell Line Activation Test㸦h-CLAT㸧

JaCVAM ⓶⭵ឤసᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍

ᖹᡂ 28 ᖺ 9 ᭶ 19 ᪥

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JaCVAM ⓶⭵ឤసᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍
ጤဨ㛗 ⟄஭ᑦஂ㸦⏣㎶୕⳻〇⸆ᰴᘧ఍♫㸧ጤဨᏳ㐩⋹Ꮚ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧  㔠⃝⏤ᇶᏊ㸦୍⯡㈈ᅋἲே 㣗ရ⸆ရᏳ඲ࢭࣥࢱ࣮㸧  ᑠᓥᖾ୍㸦୍⯡㈈ᅋἲே 㣗ရ⸆ရᏳ඲ࢭࣥࢱ࣮㸧  బ⸨୍༤㸦ᅜ❧኱Ꮫἲே ⚟஭኱Ꮫ㸧  Ṋྜྷṇ༤㸦୍⯡㈈ᅋἲே ໬Ꮫ≀㉁ホ౯◊✲ᶵᵓ㸧  ᳃ᮏ㝯ྐ㸦ఫ཭໬Ꮫᰴᘧ఍♫㸧

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せ᪨
⓶⭵ឤసᛶࡣ໬Ꮫ≀㉁ࡢᏳ඲ᛶホ౯࡟࠾࠸࡚㔜せ࡞ホ౯㡯┠࡛࠶ࡾࠊᚑ᮶ࠊࣔࣝࣔࢵࢺࡸ
࣐࢘ࢫࢆ⏝࠸ࡓື≀ᐇ㦂࡟ࡼࡗ࡚ホ౯ࡉࢀ࡚ࡁࡓࠋ㏆ᖺ EU ࡟࠾ࡅࡿḢᕞ໬Ꮫရつไ࡛ࡣࠊᏳ඲ᛶホ౯ࡣࢥࣥࣆ࣮ࣗࢱ࣮ࢆ⏝࠸ࡓᐃ㔞ⓗᵓ㐀άᛶ┦㛵㸦 Quantitative Structure-Activity
Relationship: QSAR㸧ࣔࢹࣝࡸ in vitro ࡢ௦᭰ἲࡀ᥎ዡࡉࢀ࡚࠾ࡾࠊື≀ᐇ㦂࡟ࡼࡾᏳ඲ᛶࡀホ౯ࡉࢀࡓᡂศࢆྵࡴ໬⢝ရࡢ㈍኎ࡀ⚗Ṇࡉࢀࡓࡇ࡜࠿ࡽ㸦2013 ᖺ 3 ᭶඲㠃᪋⾜㸧ࠊື≀ࢆ⏝࠸࡞࠸ in vitro ヨ㦂ἲࡢ㛤Ⓨࡀᙉࡃᮃࡲࢀ࡚࠸ࡿࠋhuman Cell Line Activation Test
㸦h-CLAT㸧ࡣࠊከࡃࡢ⓶⭵ឤసᛶ≀㉁ࡀᶞ≧⣽⬊ࢆάᛶ໬ࡍࡿࡇ࡜ࢆ฼⏝ࡋࠊࣄࢺ༢⌫⣔ᇵ㣴⣽⬊࡛࠶
ࡿ THP-1 ⣽⬊ࢆ⏝࠸ࠊάᛶ໬࡟క࠸⣽⬊⾲㠃ୖࡢⓎ⌧㔞ࡀኚ໬ࡍࡿ CD86 ࡜ CD54 ࢆᐃࡍࡿࡇ࡜࡟ࡼࡾ⓶⭵ឤసᛶࡢ᭷↓ࢆุᐃࡍࡿヨ㦂ἲ࡛࠶ࡿࠋᮏሗ࿌᭩ࡣࠊࡇࡢ h-CLAT ࡟ࡘ
࠸࡚ࠊEuropean Union Reference Laboratory for Alternatives to Animal Testing㸦EURL ECVAM㸧
࡟ࡼࡾᐇ᪋ࡉࢀࡓࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩ࠊホ౯㸦ࣆ࢔ࣞࣅ࣮ࣗ㸧ሗ࿌᭩࠾ࡼࡧヨ㦂ἲ㛤Ⓨ⪅ࡢᢞ✏ㄽᩥ࡞࡝ࢆᇶ࡟ヨ㦂ᡭ㡰ࢆࡲ࡜ࡵࠊ᭷⏝ᛶ࡜㝈⏺ࢆホ౯ࡋࡓࡶࡢ࡛࠶ࡿࠋ
h-CLAT ࡣࠊឤసᛶⓎ⌧ᶵᗎ࡟࠾ࡅࡿ➨୕ẁ㝵ࡢ࢖࣋ࣥࢺ࡛࠶ࡿᶞ≧⣽⬊ࡀάᛶ໬ࡍࡿ㝿ࡢ⣽⬊⾲㠃ศᏊࡢⓎ⌧ஹ㐍ࢆ฼⏝ࡋࡓ in vitro ヨ㦂ἲ࡛࠶ࡾࠊ໬Ꮫ≀㉁ࡢឤసᛶࢆุ᩿ࡍࡿୖ࡛㔜せ࡞᝟ሗࢆ୚࠼࡚ࡃࢀࡿࠋ
h-CLAT ࡛ࡣࠊ࣐࢘ࢫࢆ⏝࠸ࡿ Local Lymph Node Assay LLNA ࡢ 1/5 ⛬ᗘࡢ⤒㈝࡛⿕㦂≀㉁ࡢ⓶⭵ឤసᛶࢆุᐃࡍࡿࡇ࡜ࡀྍ⬟࡜ヨ⟬ࡉࢀࠊヨ㦂ᮇ㛫ࡶ LLNA ࡟ẚ࡭▷ᮇ࡛࠶ࡿࡓࡵࠊ⤒῭ᛶ࣭㎿㏿ᛶࡢほⅬ࡛᭷⏝ᛶࡣ㧗࠸࡜ᛮࢃࢀࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊDirect Peptide Reactivity
Assay DPRA ࡸ ARE-Nrf2 Luciferase Test Method ࡟ẚ࡭࡚ࠊ᧯సࡀ↹㞧࡛࠶ࡾࠊไ⣙ࡀከ࠸ࠋࡲࡓࠊ㝧ᛶᑐ↷ࠊ㝜ᛶᑐ↷࠾ࡼࡧ፹యᑐ↷ࡢᐃࢆ⥅⥆ⓗ࡟ᐇ᪋ࡋࠊࣄࢫࢺࣜ࢝ࣝ࡞ࢹ࣮ࢱ

࣮࣋ࢫࢆసᡂࡍࡿࡇ࡜ࡀᚲせ࡜ࡉࢀ࡚࠸ࡿࠋ
ᮏヨ㦂ἲࡢ EURL ECVAM ࡟ࡼࡿࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟࠾࠸࡚ࠊ15 ≀㉁ࢆ⏝࠸࡚ᐇ᪋ࡉࢀࡓ᪋タෆ෌⌧ᛶࡣ 73.3㹼86.7%࡛࠶ࡾࠊ඲⯡ⓗ࡟㧗ࡃ࡞࠿ࡗࡓࠋࡓࡔࡋࠊOrganisation for
Economic Co-operation and Development OECD ᑓ㛛ᐙ఍㆟࡟࠾࠸࡚ࠊヨ㦂࡟౑⏝ࡍࡿ THP1 ⣽⬊ࡢ๓ᇵ㣴᮲௳࠾ࡼࡧ⿕㦂≀㉁ࡢ᭚㟢᫬㛫ࢆࡼࡾཝᐦ࡟⟶⌮ࡍࡿࡇ࡜࡟ࡼࡾࠊ᪋タෆ෌⌧ᛶࡢྥୖࡀᅗࢀࡿࡇ࡜ࡀ♧ࡉࢀࠊOECD ヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥࡢヨ㦂ᡭ㡰࡟ࡑࡢෆᐜࡀ཯
ᫎࡉࢀ࡚࠸ࡿࡇ࡜࠿ࡽࠊ᪋タෆ෌⌧ᛶࡢྥୖࡀᮇᚅ࡛ࡁࡿ࡜⪃࠼ࡿࠋ୍᪉ࠊ24 ≀㉁ࢆ౑࠸ 4᪋タ࡛ᐇ᪋ࡉࢀࡓ᪋タ㛫෌⌧ᛶࡣ 79.2㸣࡛࠶ࡗࡓࠋ
OECD ヨ㦂ἲ࢞࢖ࢻࣛ࢖࡛ࣥࡣࠊ142 ≀㉁ࡢᡂ⦼࠿ࡽṇ☜ᗘ 85㸣ࠊឤᗘ 93㸣ࠊ≉␗ᗘ 66㸣࡜グ㍕ࡉࢀ࡚࠸ࡿࡀࠊlog Kow ࡀ 3.5 ௨ୖ࡛㝜ᛶ࡜ุᐃࡉࢀࡓ≀㉁ࢆ㝖࠸ࡓୖ࡛ࡢᩘ್࡛࠶
ࡿࠋࡋࡓࡀࡗ࡚ࠊ≉࡟ log Kow ࡀ 3.5 ௨ୖࡢ≀㉁ࡢሙྜࡣഇ㝜ᛶࡢྍ⬟ᛶࢆ⪃៖ࡋࠊ⿵᏶ࡋ
ᚓࡿ௚ࡢヨ㦂ࡼࡾ☜ㄆࡋ࡞ࡅࢀࡤ࡞ࡽ࡞ࡃࠊᮏヨ㦂ἲࡢࡳ࡛⿕㦂≀㉁ࢆ⓶⭵ឤసᛶࡀ㝜ᛶ࡜ุᐃࡍࡿࡇ࡜ࡣ࡛ࡁ࡞࠸ࠋࡲࡓࠊ≉␗ᗘࡀ 66%࡛࠶ࡿࡓࡵഇ㝧ᛶ࡜ุᐃࡍࡿࡇ࡜ࡶ␃ពࡍࡿᚲせࡀ࠶ࡿࠋࡉࡽ࡟ࠊᮏヨ㦂ἲ࡟⏝࠸ࡿ⣽⬊ࡢ௦ㅰ⬟ࡣ㝈ᐃⓗ࡛࠶ࡿࡓࡵࠊάᛶ໬࡟௦
ㅰ⣔ࢆᚲせ࡜ࡍࡿ໬Ꮫ≀㉁࡛ࡣࠊṇࡋࡃࡑࡢ⓶⭵ឤసᛶࡀ᳨ฟࡉࢀ࡞࠸ྍ⬟ᛶࡀ࠶ࡿࠋ
39ᮏጤဨ఍ࡣࠊୖグࡢᮏヨ㦂ἲࡢᵝࠎ࡞㝈⏺ࢆ຺᱌ࡍࡿ࡜ࠊᮏヨ㦂ἲ༢⊂࡛ࡣ⓶⭵ឤసᛶࡢุᐃࡣ୙༑ศ࡛࠶ࡿ࡜⪃࠼ࠊドᣐࡢ㔜ࡳ௜ࡅࡸ௚ࡢヨ㦂ἲ࡜ࡢ⤌ྜࡏ࡛⏝࠸ࡿࡇ࡜ࢆ᥎
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1. ⥴ゝ ⓶⭵ឤసᛶࢆホ౯ࡍࡿࡇ࡜ࡣ໬Ꮫ≀㉁ࡢᏳ඲ᛶホ౯࡟࠾࠸࡚㔜せ࡛࠶ࡿࠋ໬Ꮫ≀㉁ࡢ⓶⭵࡛ࡢ᥋ゐឤసᛶࡢࣜࢫࢡࢆື≀࡛ணࡍࡿヨ㦂ἲ࡜ࡋ࡚ࣔࣝࣔࢵࢺࢆ⏝࠸ࡿ⓶⭵ឤసᛶ

ヨ㦂㸦Organisation for Economic Co-operation and Development: OECD Test Guideline: TG406㸧
ࡸ࣐࢘ࢫࢆ⏝࠸ࡿᒁᡤࣜࣥࣃ⠇ヨ㦂㸦Local Lymph Node Assay(LLNA), OECD TG429㸧ࡀ࠶ࡿࠋࡇࡢ[3H-Methyl]-thymidine ྲྀ㎸㔞ࢆࡏࡎ࡟ ATP 㔞ࢆᐃࡍࡿ LLNA ௨እ࡟ᨺᑕᛶྠ఩ඖ⣲㸦RI㸧ࢆ౑⏝ᐃࡍࡿ LLNA: DA㸦OECD TG442A㸧ࡸ Bromodeoxyuridine 㔞ࢆᐃࡍࡿ

LLNA: BrdU-ELISA㸦OECD TG442B㸧ࡀ࠶ࡿࠋ EU ࡟ ࠾ ࡅ ࡿ Ḣ ᕞ ໬Ꮫ ရ つ ไ 㸦Registration, Evaluation, Authorization and Restriction ofChemicals: REACH㸧࡛ࡣࠊᏳ඲ᛶホ౯ࡣࢥࣥࣆ࣮ࣗࢱ࣮ࢆ⏝࠸ࡓᐃ㔞ⓗᵓ㐀άᛶ┦㛵㸦Quantitative Structure-Activity Relationship: QSAR㸧ࣔࢹࣝࡸ in vitro ➼࡟ࡼࡿ௦᭰ἲࡀ᥎ዡࡉ
ࢀ࡚࠾ࡾࠊື≀ᐇ㦂࡟ࡼࡾᏳ඲ᛶࡀホ౯ࡉࢀࡓᡂศࢆ౑⏝ࡋࡓ໬⢝ရࡢ㈍኎ࡀ⚗Ṇࡉࢀࡓ
㸦2013 ᖺ 3 ᭶඲㠃᪋⾜㸧ࠋࡑࡢࡓࡵࠊ໬Ꮫ〇ရࡢ⓶⭵ឤసᛶࢆุᐃࡍࡿ௦᭰ἲࡢ㛤Ⓨࡀᙉࡃồࡵࡽࢀ࡚࠸ࡿࠋ
 In chemico ヨ㦂ἲ࡜ࡋ࡚࣌ࣉࢳࢻ⤖ྜ཯ᛂࢆ฼⏝ࡋࡓ Direct Peptide Reactivity Assay㸦DPRA㸸OECD TG442C㸧࠾ࡼࡧ In vitro ヨ㦂ἲ࡜ࡋ࡚ࢣࣛࢳࣀࢧ࢖ࢺ⣽⬊⣔ࡢᶆⓗ㑇ఏᏊࢆ⏝࠸ࡓ ARE-Nrf2 Luciferase Test Method㸦OECD TG442D㸧ࡀ OECD ࠿ࡽヨ㦂ἲ࢞࢖ࢻࣛ࢖
ࣥ࡜ࡋ࡚බ⾲ࡉࢀ࡚࠸ࡿࠋࡲࡓࠊࡇࢀ௨እ࡟ࠊ༢⌫⣔⣽⬊ࡢάᛶ໬ࢆ฼⏝ࡋࡓ human Cell
Line Activation Test㸦h-CLAT㸧ࠊMyeloid U937 Skin Sensitization Test㸦U-SENS㸧࠾ࡼࡧ IL-8 Luc
assay ࡞࡝ࡢ⓶⭵ឤసᛶヨ㦂ࡢ in vitro ἲࡀᥦ᱌ࡉࢀ࡚࠾ࡾࠊEURL ECVAM㸦the European
Union Reference Laboratory for Alternatives to Animal Testing㸧➼࡟࠾࠸࡚ࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀ⾜ࢃࢀ࡚ࡁࡓࠋ
 h-CLAT ࡣࠊከࡃࡢ⓶⭵ឤసᛶ≀㉁ࡀᶞ≧⣽⬊ࢆάᛶ໬ࡍࡿࡇ࡜ࢆ฼⏝ࡋࠊࣄࢺ༢⌫⣔ᇵ㣴⣽⬊࡛࠶ࡿ THP-1 ⣽⬊ࢆ⏝࠸ࠊάᛶ໬࡟క࠸⣽⬊⾲㠃࡛ࡢⓎ⌧㔞ࡀኚ໬ࡍࡿ CD86 ࡜
CD54 ࢆ

ᐃࡍࡿヨ㦂ἲ࡛࠶ࡿࠋh-CLAT ࡢࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡢ⤖ᯝ࡟ࡘ࠸࡚ࡣࠊEURL

ECVAM ࡟ࡼࡿホ౯㸦ࣆ࢔ࣞࣅ࣮ࣗ㸧ࡀ᏶஢ࡋ 1)ࠊ2016 ᖺ 7 ᭶࡟ OECD ࡢヨ㦂ἲ࢞࢖ࢻࣛ࢖
ࣥࣜࢫࢺ࡟㏣ຍࡉࢀࡓ㸦TG442E㸧7)ࠋ
JaCVAM ⓶⭵ឤసᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍㸦௨ୗࠊጤဨ఍㸧ࡣࠊh-CLAT ࡢ⓶⭵ឤసᛶヨ㦂௦᭰ἲ࡜ࡋ࡚ࡢ⛉Ꮫⓗጇᙜᛶ࡟ࡘ࠸࡚ࠊ⌧ᅾࡲ࡛࡟බ㛤ࡉࢀ࡚࠸ࡿ᝟ሗࢆࡶ࡜࡟ホ౯ࡋࡓࡢ࡛ࠊࡑࡢ⤖ᯝࢆሗ࿌ࡍࡿࠋ
2㸬ヨ㦂ἲࡢཎ⌮⓶⭵ឤసᛶࡣࠊࣄࢺ࡛ࡣ᥋ゐ⓶⭵⅖ࠊື≀㸦㱎ṑ㢮㸧࡛ࡣ᥋ゐ㐣ᩄ⑕࡜ࡋ࡚▱ࡽࢀࡿ໬Ꮫ≀㉁ࡢẘᛶࡢ୍ࡘ࡛࠶ࡿࠋOECD ࡀࡲ࡜ࡵࡓ Adverse Outcome Pathway㸦AOP㸧࡛ࡣࠊ໬Ꮫ≀㉁࡟ࡼࡿ⓶⭵ឤసࡣḟࡢ 4 ࡘࡢ Key event ࠿ࡽᡂࡿ࡜ࡉࢀ࡚࠸ࡿࠋ
1) ໬Ꮫ≀㉁࡜ࢱࣥࣃࢡ㉁ࡢࢩࢫࢸ࢖ࣥṧᇶ࠶ࡿ࠸ࡣࣜࢪࣥṧᇶ࡜ࡢඹ᭷⤖ྜ5112) ࢣࣛࢳࣀࢧ࢖ࢺ࡟࠾ࡅࡿ⅖⑕ᛶᛂ⟅࠾ࡼࡧ Antioxidant/electrophile response element
㸦ARE㸧-dependent pathway ࡟ࡼࡿ㑇ఏᏊⓎ⌧3) ᶞ≧⣽⬊ࡢάᛶ໬㸦≉␗ⓗ⣽⬊⾲㠃࣐࣮࣮࢝ࡢⓎ⌧ࠊࢣࣔ࢝࢖ࣥࡸࢧ࢖ࢺ࢝࢖ࣥࡢ⏘⏕㸧4) ࣜࣥࣃ⠇࡟࠾ࡅࡿ T ⣽⬊ࡢቑṪh-CLAT ࡣୖグࡢ➨ 3 ࡢ Key event ࡟ᑐᛂࡍࡿヨ㦂ἲ࡛࠶ࡿࠋࡑࡢᇶᮏⓗཎ⌮ࡣࠊᶞ≧⣽⬊ࡢάᛶ໬᫬࡟Ⓨ⌧ࡀቑ኱ࡍࡿ⣽⬊⾲㠃ศᏊࡢᐃ࡛࠶ࡿࠋ ᶞ≧⣽⬊ࡢάᛶ໬᫬࡟ࡣࠊCD86ࠊCD54 ࡢࡼ࠺࡞⣽⬊⾲㠃ศᏊࡢⓎ⌧㔞ࡀቑ኱ࡍࡿࡇ࡜ࡀ
▱ࡽࢀ࡚࠸ࡿࠋCD86 ࡣࠊT ⣽⬊࡟ᢠཎᥦ♧ࡍࡿ㝿࡟ T ⣽⬊⾲㠃ࡢ CD28 ࡜┦஫స⏝ࡍࡿࡇ࡜࡟ࡼࡾ⿵ຓ่⃭ศᏊ࡜ࡋ࡚ᶵ⬟ࡍࡿࠋCD54 ࡣࠊูྡ Intercellular adhesion molecule-1
ICAM-1㸧࡜࠸࠺᥋╔ศᏊ࡛࠶ࡾࠊT ⣽⬊⾲㠃ࡢ Lymphocyte function-associated antigen 㸦
1 LFA1㸧࡜⤖ྜࡍࡿࠋ h-CLAT ࡛ࡣࠊᶞ≧⣽⬊ࡢࣔࢹࣝ࡜ࡋ࡚ࣄࢺ༢⌫⣔ᇵ㣴⣽⬊࡛࠶ࡿ THP-1 ⣽⬊ࢆ⏝࠸ࠊ໬Ꮫ≀㉁ࡀ THP-1 ⣽⬊ࢆάᛶ໬ࡍࡿ⬟ຊࢆホ౯ࡍࡿࠋTHP-1 ⣽⬊࡟᳨య࡛࠶ࡿ໬Ꮫ≀㉁ࢆῧ
ຍࡋ࡚ 24 ᫬㛫ᇵ㣴ࡋࡓᚋࠊCD86 ࠾ࡼࡧ CD54 ࡢⓎ⌧㔞ࢆࠊ⺯ගᶆ㆑ࡋࡓ≉␗ᢠయࢆ⏝࠸࡚ࣇ࣮ࣟࢧ࢖ࢺ࣓ࢺ࣮ࣜ࡟ࡼࡾ

ᐃࡍࡿࠋ⁐፹ࡢࡳࢆῧຍࡋࡓࢥࣥࢺ࣮ࣟࣝ࡟ᑐࡍࡿ┦ᑐ⺯ගᙉᗘࢆ⟬ฟࡋࠊ໬Ꮫ≀㉁࡟ࡼࡿᶞ≧⣽⬊ࡢάᛶ໬ࡢᣦᶆ࡜ࡍࡿࠋ
3㸬ヨ㦂ᡭ㡰㸭ุᐃh-CLAT ࡢヨ㦂ᡭ㡰ࡣࠊ⏝㔞タᐃヨ㦂࡜ CD86/CD54 Ⓨ⌧

ᐃࡢ 2 ࡘ࡟ศࡅࡽࢀࡿࠋ

3-1㸬⣽⬊ㄪ〇࠾ࡼࡧヨ⸆ h-CLAT ࡛ࡣࠊTHP-1 ⣽⬊ࢆ౑⏝ࡍࡿࠋ⣽⬊ࡣ American Type Culture Collection㸦ATCC㸧
࠿ࡽධᡭ㸦TIB-202TM㸧ࡍࡿࡇ࡜ࡀᮃࡲࡋ࠸ࠋ
 THP-1 ⣽⬊ࡣࠊᇵ㣴⏝ᇵᆅ㸦RPMI-1620 ࡢᇶ♏ᇵᆅ࡟ 10% Fetal bovine serum, 0.05 mM 2Mercaptoethanol, 100 unit/mL Penicillin, 100 ȝg/mL Streptomycin ࢆ ຍ ࠼ ࡓ ࡶ ࡢ 㸧 ࡟ ࡚ ࠊ
0.1~1.0×106 cells/mL ࡢ⣽⬊⃰ᗘ࡛᧛✀ࡋࠊ2~3 ᪥ࡈ࡜࡟⥅௦ࢆ⾜࠸ࠊ⥔ᣢࡍࡿࠋ⣽⬊⃰ᗘࡣ
0.1~0.8×106 cells/mL ࢆ⥔ᣢࡋࠊ1.0×106 cells/mL ࢆ㉸࠼࡞࠸ࡼ࠺࡟ࡍࡿࠋࡲࡓࠊATCC ࠿ࡽධᡭᚋࡢึᅇ⼥ゎ 2 㐌㛫ᚋ࡟ࠊ2,4-Dinitrochlorobenzene㸦DNCB, CAS No.97-00-7ࠊ⣧ᗘӍ99%
௨ୖ㸧ࠊNickel sulfate㸦CAS NO. 10101-97-0ࠊ⣧ᗘӍ99%௨ୖ㸧࠾ࡼࡧ㝜ᛶᑐ↷࡜ࡋ࡚ Lactic
acid㸦CAS NO. 50-21-5ࠊ⣧ᗘӍ85%௨ୖ㸧ࢆ⏝࠸࡚ࠊ཯ᛂᛶࡢ☜ㄆࢆ⾜࠺ࠋDNCB ࡜ Nickel
sulfate ࡛ࡣࠊCD86 ࡜ CD54 ࡢ୧᪉࡛㝧ᛶ࡜࡞ࡾࠊLactic acid ࡛ࡣࠊCD86 ࡜ CD54 ࡢ࠸ࡎࢀ࡛ࡶ㝜ᛶࡢ⤖ᯝ࡜࡞ࡿࠋ཯ᛂᛶヨ㦂࡛ྜ᱁ࡋࡓ⣽⬊ࡢࡳࢆᮏヨ㦂ἲ࡛⏝࠸ࡿࡇ࡜ࡀ࡛ࡁࡿࠋ
THP-1 ⣽⬊ࡣࠊ⼥ゎᚋ 2 ࣨ᭶ࡲ࡛⥅௦ࡉࡏࡿࡇ࡜ࡀ࡛ࡁࠊ⥅௦ᩘࡀ 30 ࢆ㉸࠼࡞࠸ࡼ࠺࡟ࡍ
࡭ࡁ࡛࠶ࡿࠋ
ヨ㦂ᐇ᪋๓࡟ࠊTHP-1 ⣽⬊ࢆ 0.1 ࡶࡋࡃࡣ 0.2×106 cells/mL ࡢ⣽⬊⃰ᗘ࡛ᇵ㣴⏝ࣇࣛࢫࢥ࡟᧛✀ࡋࠊࡑࢀࡒࢀ 72 ᫬㛫ࡶࡋࡃࡣ 48 ᫬㛫ᇵ㣴ࡍࡿࠋᐇ㦂᪥࡟ࠊ2×106 cells/mL ࡢ⃰ᗘ࡛612᪂ࡋ࠸ᇵ㣴⏝ᇵᆅ࡟෌ᠱ⃮ࡋࡓ THP-1 ⣽⬊ᠱ⃮ᾮࢆ 24 ✰ᖹᗏࣉ࣮ࣞࢺࡶࡋࡃࡣ 96 ✰ᖹᗏࣉ࣮ࣞࢺ࡟ࠊࡑࢀࡒࢀ 500 ȝL/well ࡶࡋࡃࡣ 80 ȝL/well ࡛ຍ࠼ࡿࠋ
3-2. ⏝㔞タᐃヨ㦂
⏝㔞タᐃヨ㦂ࡣࠊCV75㸦፹యᑐ↷࡜ẚ㍑ࡋ࡚ 75%ࡢ⣽⬊⏕Ꮡ⋡ࢆ♧ࡍ⿕㦂≀㉁⃰ᗘ㸧ࢆỴᐃࡍࡿࡓࡵ࡟ᐇ᪋ࡍࡿࠋCV75 ࡣ CD86 ࠾ࡼࡧ CD54 Ⓨ⌧㔞ᐃࡢ⃰ᗘタᐃ࡟฼⏝ࡉࢀࡿࠋ1) ⿕㦂≀㈨࠾ࡼࡧ㝜ᛶᑐ↷ࡢㄪ〇 ⿕㦂≀㉁࠾ࡼࡧ㝜ᛶᑐ↷ࡣࠊ⏝᫬ㄪ〇ࡍࡿࠋh-CLAT ࡛ࡣࠊ⿕㦂≀㉁ࡣ⁐ᾮࡶࡋࡃࡣᏳᐃ
࡞ᠱ⃮ᾮ࡟ㄪ〇ࡍࡿࠋ፹యࡣ⏕⌮㣗ሷỈ࠾ࡼࡧᇶ♏ᇵᆅࢆ➨୍ೃ⿵ࠊDimethyl sulfoxide㸦DMSO, ⣧ᗘӍ99%௨ୖ㸧ࢆ➨஧ೃ⿵࡜ࡋࠊ᭱⤊⃰ᗘࡣ⏕⌮㣗ሷỈ࠾ࡼࡧᇵᆅࡢሙྜࡣ 100
mg/mLࠊDMSO ࡢሙྜࡣ 500 mg/mL ࡜ࡍࡿࠋ100 mg/mL㸦⏕⌮㣗ሷỈ࠾ࡼࡧᇵᆅࡢሙྜ㸧࠾
ࡼࡧ 500 mg/mL㸦DMSO ࡢሙྜ㸧ࡢಖᏑ⁐ᾮࢆ⏝࠸࡚ࠊ௨ୗࡢᡭ㡰࡛ᕼ㔘᧯సࢆ⾜࠺ࠋ 㸫⏕⌮㣗ሷỈ࠾ࡼࡧᇶ♏ᇵᆅࢆ౑⏝ࡍࡿሙྜ㸸ࡑࢀࡒࢀࡢ፹యࢆ⏝࠸࡚බẚ 2 ࡛ᕼ㔘ࢆ⾜࠸ࠊ8 ⃰ᗘࡢಖᏑཎᾮࢆస〇ࡍࡿࠋࡇࢀࡽಖᏑཎᾮࡣࡑࢀࡒࢀᇵ㣴⏝ᇵᆅ࡛ࡉࡽ࡟ 50 ಸ࡟ᕼ㔘ࡍࡿ㸦౑⏝ᾮ㸧ࠋࡶࡋ᭱㧗⃰ᗘ࡛࠶ࡿ 1,000 ȝg/mL ࡛⣽⬊ẘᛶࡀ࡞࠸ሙྜࠊ᭱㧗⃰ᗘࢆୖࡆ࡚⣽⬊ẘᛶヨ㦂ࢆ෌ᐇ᪋ࡋ࡚Ỵᐃࡍࡿࡀࠊࣉ࣮ࣞࢺ࡟࠾ࡅࡿ᭱⤊⃰ᗘࡣࠊ5,000 ȝg/mLࢆ㉸࠼࡚ࡣ࠸ࡅ࡞࠸ࠋ
 㸫DMSO ࢆ౑⏝ࡍࡿሙྜ㸸DMSO ࢆ⏝࠸࡚බẚ 2 ࡛ᕼ㔘ࢆ⾜࠸ࠊ8 ⃰ᗘࡢಖᏑཎᾮࢆస
〇ࡍࡿࠋಖᏑཎᾮࡣࡑࡢᚋࠊᇵ㣴⏝ᇵᆅ࡛ࡉࡽ࡟ 250 ಸ࡟ᕼ㔘ࡍࡿ㸦౑⏝ᾮ㸧ࠋࡓ࡜࠼⣽⬊ẘᛶࡀ࡞ࡃ࡜ࡶࠊࣉ࣮ࣞࢺ୰ࡢ᭱⤊⃰ᗘࡣࠊ1,000 ȝg/mL ࢆ㉸࠼࡚ࡣ࠸ࡅ࡞࠸ࠋ ፹యᑐ↷࡟ࡣࠊ⿕㦂≀㉁ࡀ⏕⌮㣗ሷỈࡲࡓࡣᇶ♏ᇵᆅ࡛⁐ᾮࡶࡋࡃࡣᏳᐃ࡞ᠱ⃮ᾮ࡜࡞ࡿሙྜ࡟ࡣᇵ㣴⏝ᇵᆅࢆ⏝࠸ࠊ⿕㦂≀㉁ࡀ DMSO ࡛⁐ᾮࡶࡋࡃࡣᏳᐃ࡞ᠱ⃮ᾮ࡜࡞ࡿሙྜࡣ DMSO ࢆ⏝࠸࡚ࠊࡑࡢࣉ࣮ࣞࢺ࡟࠾ࡅࡿ᭱⤊⃰ᗘࡣ 0.2%࡜ࡍࡿࠋࡑࢀࡽࡣୖグࡢ౑⏝ᾮస〇ࡢᡭ㡰࡜ྠࡌᕼ㔘࡛⾜࠺ࠋ2) ⿕㦂≀㉁࠾ࡼࡧ㝜ᛶᑐ↷ࡢ᭚㟢 1) ࡛ㄪ〇ࡋࡓ⿕㦂≀㉁ࢆྵࡴ౑⏝ᾮ࠾ࡼࡧ፹యᑐ↷ࡣࠊ⣽⬊ᠱ⃮ᾮ࡜ 1:1 ࡛ΰྜࡋ㸦㏻ᖖࠊ᭱⤊⃰ᗘࡢ⠊ᅖࡣࠊ7.81~1,000 ȝg/mL㸧ࠊ24 ✰ᖹᗏࣉ࣮ࣞࢺࡶࡋࡃࡣ 96 ✰ᖹᗏࣉ࣮ࣞࢺ
࡟࡚ 37Υࠊ5%CO2 in air ࡛ 24 ᫬㛫㸦s0.5 ᫬㛫㸧ᇵ㣴ࡍࡿࠋ᥹Ⓨᛶࡢ㧗࠸⿕㦂≀㉁ࡢሙྜ
ࡣࠊ࢙࢘ࣝ㛫࡛⿕㦂≀㉁ࡢࢥࣥࢱ࣑ࡀ⏕ࡌࡿྍ⬟ᛶࡀ࠶ࡿࡢ࡛ࠊࣉ࣮ࣞࢺࢆࢩ࣮ࣝࡍࡿ࡞࡝
ࡢฎ⨨ࢆ᪋ࡍࡇ࡜ࡀᮃࡲࡋ࠸ࠋ
3)

Propidium iodide㸦PI㸧ᰁⰍ
24 ᫬㛫㸦s0.5 ᫬㛫㸧ࡢ᭚㟢ᚋࠊ⣽⬊ࡣࢧࣥࣉࣝࢳ࣮ࣗࣈ࡟⛣ࡋࠊ㐲ᚰ࡟ࡼࡾ⣽⬊ࢆ㞟ࡵࡿࠋୖ⃈ࡳࢆᤞ࡚ࡓᚋࠊ0.1%࢘ࢩ⾑Ύ࢔ࣝࣈ࣑ࣥ㸦Bovine Serum Albumin: BSA㸧ྵ᭷ࣜࣥ㓟⦆⾪ᾮ 200ȝL㸦96 ✰ࡢሙྜ㸧ࡶࡋࡃࡣ 600ȝL㸦24 ✰ࡢሙྜ㸧ࢆຍ࠼ࠊ෌ᠱ⃮ࡍࡿࠋࡇࡢ⣽⬊ᠱ⃮ᾮ 200ȝL ࡣࠊ96 ✰ࡢሙྜࡣ 96 ✰୸ᗏᆺࣉ࣮ࣞࢺ࡟ࠊ24 ✰ࡢሙྜࡣ࣐࢖ࢡࣟࢳ࣮ࣗ
713ࣈ࡟⛣ࡋࠊ96 ✰࡛ࡣ 200ȝL ࡢ 0.1% BSA ྵ᭷ࣜࣥ㓟⦆⾪ᾮ࡛ࠊ24 ✰࡛ࡣ 600ȝL ࡢ 0.1%BSAྵ᭷ࣜࣥ㓟⦆⾪ᾮ ࡛ 2 ᅇ௨ୖὙίࡍࡿࠋ᭱⤊ⓗ࡟ࠊ⣽⬊ࡣ 0.1㸣BSA ྵ᭷ࣜࣥ㓟⦆⾪ᾮ 㸦౛࠼ࡤࠊ400 ȝL㸧࡛෌ᠱ⃮ࡋࠊࡑࡇ࡟ 20 ȝL ࡢ PI ⁐ᾮ㸦PI ࡢ᭱⤊⃰ᗘࡣ 0.625 ȝg/mL㸧ࢆຍ࠼ࡿࠋ4) ⣽⬊ẘᛶࡢᐃ࡜ CV75 ࡢ⟬ฟPI ࡢྲྀࡾ㎸ࡳࡣࠊࣇ࣮ࣟࢧ࢖ࢺ࣓࣮ࢱ࣮࡛ศᯒࡍࡿࠋ⣽⬊ẘᛶࡣࠊ10,000 ࡢ⏕⣽⬊㸦PI ࢿ࢞ࢸ࢕ࣈ㸧ࡀ࢝࢘ࣥࢺ࡛ࡁࡿ᮲௳࡛ࠊ௨ୗࡢᘧ࡟ࡼࡾ⟬ฟࡍࡿࠋࡲࡓࠊ⣽⬊⏕Ꮡ⋡ࡀప࠸ሙྜ࡟ࡣࠊṚ⣽⬊ᩘࢆྵࡴ⣽⬊ᩘࢆ 30,000 ࡜ࡋࡓ᮲௳࡟࡚ࠊ⣽⬊⏕Ꮡ⋡㸦cell viability㸧ࢆ⟬ฟࡍࡿࠋࡲࡓࠊCV75㸦THP-1 ⣽⬊ࡢ 75%ࡀ⏕⣽⬊࡛࠶ࡿ⃰ᗘ㸧ࡣࠊḟᘧ࡟ࡼࡾ⟬ฟࡍࡿࠋ a㸸ヨ㦂ࡋࡓ⃰ᗘ࡟࠾࠸࡚⏕Ꮡ⋡ 75%ࢆ㉸࠼ࡿ᭱ᑠࡢ⏕Ꮡ⋡c㸸ヨ㦂ࡋࡓ⃰ᗘ࡟࠾࠸࡚⏕Ꮡ⋡ 75%ࢆ㉸࠼࡞࠸᭱኱ࡢ⏕Ꮡ⋡b㸸a ࡜࡞ࡿ⿕㦂≀㉁⃰ᗘ
d㸸c ࡜࡞ࡿ⿕㦂≀㉁⃰ᗘ
3-3㸬CD86 ࠾ࡼࡧ CD54 Ⓨ⌧ᐃ1) ⿕㦂≀㉁ࠊ㝜ᛶᑐ↷࠾ࡼࡧ㝧ᛶᑐ↷ࡢㄪ〇⿕㦂≀㉁ࢆ⁐ゎࡶࡋࡃࡣᏳᐃ࡞ᠱ⃮ᾮ࡜ࡉࡏࡿ፹య࡟ࡣࠊ⏕⌮㣗ሷỈࠊᇶ♏ᇵᆅ࠾ࡼࡧDMSO ࠿ࡽ㐺ษ࡞ࡶࡢࢆ౑⏝ࡍࡿࠋ⿕㦂≀㉁ࡢ᭱㧗⃰ᗘࡣ⏝㔞タᐃヨ㦂࡟࡚⟬ฟࡉࢀࡓ
CV75 ࡢ 1.2 ಸ⃰ᗘ࡜ࡍࡿࠋࡶࡋࠊCV75 ࡀồࡵࡽࢀ࡞࠿ࡗࡓሙྜ㸦౛࠼ࡤࠊ⏝㔞タᐃヨ㦂࡛༑ศ࡞⣽⬊ẘᛶࡀㄆࡵࡽࢀ࡞࠿ࡗࡓሙྜ㸧࡟ࡣࠊ⿕㦂≀㉁ࡢ᭱኱⁐ゎࡶࡋࡃࡣᏳᐃ࡞ᠱ⃮⃰ᗘࢆ㛤ጞ⃰ᗘ࡜ࡍࡿࡀࠊࣉ࣮ࣞࢺࡢ᭱⤊⃰ᗘࡣࠊ⏕⌮㣗ሷỈࡶࡋࡃࡣᇶ♏ᇵᆅ࡛ࡣ 5,000 ȝg/mLࠊDMSO ࡢሙྜࡣ 1,000 ȝg /mL ࢆ㉸࠼࡞࠸ࡼ࠺࡟ࡍࡿࠋࡑࡢᚋࠊྠࡌ፹యࢆ⏝࠸࡚ࠊබẚ 1.2 ࡛ᕼ㔘ࡍࡿࡇ࡜࡛ 8 ⃰ᗘࡢಖᏑཎᾮ㸦⃰ᗘᖜ㸸0.335CV75㹼1.2×CV75㸸፹యࡀ⏕⌮㣗ሷᾮ࠾ࡼࡧᇶ♏ᇵᆅࡢሙྜࡣࡇࡢ 100 ಸࡢ⃰ᗘࠊDMSO ࡢሙྜࡣࡇࡢ 500 ಸࡢ⃰ᗘ㸧ࢆస〇ࡍࡿࠋಖᏑཎᾮࡣࡑࡢᚋࠊᇵ㣴⏝ᇵᆅࢆ⏝࠸࡚ࠊ፹యࡀ⏕⌮㣗ሷỈ࠾ࡼࡧᇶ♏ᇵᆅࡢሙྜࡣ 50 ಸࠊ፹యࡀ DMSO ࡢሙྜࡣ 250 ಸ࡟ᕼ㔘ࡋࠊ౑⏝ᾮࢆస〇ࡍࡿࠋࡇࢀࡽ౑⏝ᾮࡣࠊ᭱⤊ⓗ࡟⣽⬊ᠱ⃮ᾮ࡛ࡉࡽ࡟ 2 ಸᕼ㔘ࡉࢀࠊ᭚㟢ࡉࢀࡿࡇ࡜࡜࡞ࡿࠋࡶࡋ⤖ᯝࡀࠊᚋ㏙ࡍࡿヨ㦂ᡂ❧ࡢ᮲௳࡟ྜࢃ࡞࠸᫬ࡣࠊṇ☜࡞ CV75 ࢆᚓࡿࡓࡵ࡟⏝㔞タᐃヨ㦂ࢆ෌ᗘᐇ᪋ࡋ࡚ࡶ
ࡼ࠸ࠋCD86 ࠾ࡼࡧ CD54 Ⓨ⌧

ᐃ࡛ࡣࠊ24 well ࡢࡳࡀ౑ࢃࢀࡿࡇ࡜࡟ὀពࡍࡿᚲせࡀ࠶
8

14

ࡿࠋ
㝜ᛶᑐ↷/፹యᑐ↷ࡣࠊ⏝㔞タᐃヨ㦂࡜ྠᵝ࡟ㄪ〇ࡍࡿࠋ㝧ᛶᑐ↷࡟ࡣ DNCB ࢆ౑⏝ࡍࡿࠋ፹య࡟ DMSO ࢆ౑⏝ࡋࠊୖẁ࡟♧ࡍ࡜࠾ࡾࠊಖᏑཎᾮࢆస〇ࡍࡿࠋDNCB ࡣࠊCD86 ࠾ࡼࡧ
CD54 Ⓨ⌧ᐃࡢ㝧ᛶᑐ↷࡜ࡋ࡚ࠊ1 ⃰ᗘ㸦୍⯡ⓗ࡟ࡣࠊࣉ࣮ࣞࢺ୰ࡢ᭱⤊⃰ᗘࡀ 4 ȝg/mL㸧ࡀ౑ࢃࢀࡿࠋ4 ȝg/mL ࡜ࡍࡿࡓࡵ࡟ࠊDMSO ࢆ፹య࡟ࡋࡓ 2 mg/mL ࡢಖᏑཎᾮࢆᇵ㣴⏝ᇵ
ᆅ࡛ 250 ಸ࡟ᕼ㔘ࡋ࡚ࠊ8 ȝg/mL ࡢ౑⏝ᾮࢆస〇ࡍࡿࠋDNCB ⃰ᗘ࡟㛵ࡋ࡚ࡣࠊྛ᪋タ࡛Ỵᐃࡉࢀࡓ CV75 ࢆ㝧ᛶᑐ↷⃰ᗘ࡜ࡋ࡚౑⏝ࡍࡿࡇ࡜ࡶྍ⬟࡛࠶ࡿࠋ㝧ᛶᑐ↷࡟࠾࠸࡚ࡶࣉ࣮ࣞࢺࡢ᭱⤊⃰ᗘࡣࠊ⏕⌮㣗ሷỈࡶࡋࡃࡣᇶ♏ᇵᆅ࡛ࡣ 5,000
ȝg/mLࠊDMSO ࡢሙྜࡣ 1,000 ȝg/mL ࢆ㉸࠼࡞࠸ࡼ࠺࡟ࡋࠊ㝧ᛶᑐ↷ࡣ 1 ⃰ᗘ࡛࠶ࡿࡇ࡜࠿
ࡽᚋ㏙ࡍࡿ⿕㦂≀㉁ࡢヨ㦂ᡂ❧ࡢ᮲௳ࡢ࠺ࡕࠊ᭱⤊㡯┠ࡢࡳᑐ㇟እ࡜࡞ࡿࠋ
2) ⿕㦂≀㉁࠾ࡼࡧ㝜ᛶᑐ↷ࡢ᭚㟢⿕㦂≀㉁ࡢ⓶⭵ឤసᛶࡢண
ࡢ CD86 ࠾ࡼࡧ CD54 Ⓨ⌧⤖ᯝ㸦㝧ᛶࡶࡋࡃࡣ㝜ᛶ㸧ࢆᚓࡿࡓࡵ࡟ࡣࠊᑡ࡞ࡃ࡜ࡶ 2 ᅇᐃࢆ⧞ࡾ㏉ࡍᚲせࡀ࠶ࡿࠋ2 ᅇࡢྠ᪥ࡢ࡝ࡕࡽ࡛ࡶⰋ࠸ࡀࠊࡑࢀࡒࢀࡢ

ᐃࡣࠊ␗࡞ࡿ᪥ࡶࡋࡃࡣࠊ

ᐃ࡛ಖᏑཎᾮ, ⿕㦂≀㉁ࡢ౑⏝ᾮ࠾ࡼࡧᰁⰍ⏝ᢠయ⁐ᾮࡣูࠎ࡟ㄪ〇ࡉࢀࡿࡇ࡜ࠊ౑⏝ࡉࢀࡿ⣽⬊ሢࢆศࡅࡿ㸦౛࠼ࡤࠊ⣽⬊ࡣ␗࡞ࡿࣇࣛࢫࢥ
࠿ࡽ㞟ࡵࡿ࡞࡝㸧ࡇ࡜ࡀᚲせ࡛࠶ࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊ⣽⬊ࡣྠࡌ⥅௦ᩘ⏤᮶࡛ࡶᵓࢃ࡞࠸ࠋ
౑⏝ᾮ࡜ࡋ࡚ㄪ〇ࡋࡓ⿕㦂≀㉁࠾ࡼࡧ㝜ᛶᑐ↷ࡣࠊࡑࢀࡒࢀ⣽⬊ᠱ⃮ᾮ࡜ 1:1 ࡢ๭ྜ࡛ΰྜ
ࡋࠊ37Υࠊ5%CO2 in airࠊ24 ᫬㛫s0.5 ᫬㛫ࠊᇵ㣴ࡍࡿࠋᑡ࡞ࡃ࡜ࡶ 2 ᅇࡢ࡛ࠊࡑࢀࡒࢀࡢ

ᐃࢆᐇ᪋ࡍࡿࡢ

ᐃ࡛ࡣࠊ ᐃෆࡢ⿕㦂≀㉁ࡢྛ⃰ᗘ࠾ࡼࡧ㝜ᛶᑐ↷ࡣࠊn=1 ࡛࠶ࡗ࡚ࡶၥ㢟࡞࠸ࠋ3) ⣽⬊ࡢᰁⰍ࠾ࡼࡧศᯒ
24 ᫬㛫ࡢ᭚㟢ᚋࠊ⣽⬊ᾮࢆࢧࣥࣉࣝࢳ࣮ࣗࣈ࡟⛣ࡋࠊ㐲ᚰ࡟ࡼࡾ⣽⬊ࢆ㞟ࡵࠊ1 mL ࡢ
0.1%BSA ྵ᭷ࣜࣥ㓟⦆⾪ᾮ㸦pH 7.4㸧࡛ 2 ᅇὙίࡍࡿࠋὙίᚋࠊ600 ȝL ࡢ 0.1%BSA ྵ᭷ࣜࣥ㓟⦆⾪ᾮ࡟ 0.01%㸦w/v㸧ࡢ⃰ᗘ࡛ࢢࣟࣈࣜࣥ㸦Cohn fraction II, III, Human; SIGMA, #238810G㸧ࢆຍ࠼ࡓࡶࡢࢆຍ࠼ࠊ4Υ࡛ 15 ศࠊ࢖࣮ࣥ࢟ࣗ࣋ࢺࡋ࡚ࠊࣈࣟࢵࢡࢆ⾜࠺ࠋࡑࡢᚋࠊ
⣽⬊ࡣࠊ180 ȝL ࡎࡘ 3 ࡘ࡟➼ศࡋࠊ୸ᗏ 96 ✰ࣉ࣮ࣞࢺࡶࡋࡃࡣ࣐࢖ࢡࣟࢳ࣮ࣗࣈ࡟⛣ࡍࠋ㐲ᚰᚋࠊ⣽⬊ࡣࠊ50 ȝL ࡢ Fluorescein isothiocyanate㸦FITC㸧ᶆ㆑ᢠ CD86 ᢠయࠊFITC ᶆ㆑ᢠ CD54 ᢠయࡶࡋࡃࡣ FITC ᶆ㆑࣐࢘ࢫ IgG1 ᢠయ㸦࢔࢖ࢯࢱ࢖ࣉ㸧࡟࡚ࠊ4Υ࡛ 30 ศ㛫ࠊᰁⰍࡍࡿࠋh-CLAT DB-ALM ࣉࣟࢺࢥ࣮ࣝ 2)࡛グ㍕ࡉࢀ࡚࠸ࡿᢠయ࡛ࡣࠊᢠ CD86 ᢠయ㸦BDPharMingen #555657; Clone:Fun-1㸧࡛ࡣ 3:25 ࡢ๭ྜ࡛ᢠయ࡜ 0.1%BSA ྵ᭷ࣜࣥ㓟⦆⾪ᾮࢆΰྜࡋࠊᢠ CD54 ᢠయ㸦DAKO, #F7143; Clone: 6.5B5㸧࠾ࡼࡧ IgG1㸦DAKOࠊ#X0927㸧࡛ࡣ
3:50 ࡢ๭ྜ࡛ᢠయ࡜ 0.1%BSA ྵ᭷ࣜࣥ㓟⦆⾪ᾮࢆΰྜࡍࡿࠋ
ᢠయᰁⰍᚋࡢ⣽⬊ࡣ 200ȝL ࡢ 0.1%BSA ྵ᭷ࣜࣥ㓟⦆⾪ᾮ࡛ 3 ᅇὙίᚋࠊ400 ȝL ࡢ
0.1%BSA ྵ᭷ࣜࣥ㓟⦆⾪ᾮ࡛෌ᠱ⃮ࡋࠊࡑࡇ࡟ PI ⁐ᾮ㸦PI ⃰ᗘࡀ 0.625 ȝg/mL ࡛࠶ࢀࡤ20 ȝL㸧ࡶࡋࡃࡣࡑࡢ௚ࡢ⣽⬊ẘᛶ࣐࣮࣮࢝ᾮࢆῧຍࡍࡿࠋCD86 ࠾ࡼࡧ CD54 ࡢⓎ⌧ࠊ⣽⬊⏕Ꮡ⋡ࡣࣇ࣮ࣟࢧ࢖ࢺ࣓࣮ࢱ࣮ࢆ⏝࠸࡚

ᐃࡍࡿࠋ
9
15

3-4 ࢹ࣮ࢱ࠾ࡼࡧሗ࿌
1) ࢹ࣮ࢱࡢホ౯
CD86 ࡜ CD54 ࡢⓎ⌧ࡣࠊࢳࣕࣥࢿࣝ FL-1 ࢆ᭷ࡍࡿࣇ࣮ࣟࢧ࢖ࢺ࣓࣮ࢱ࣮࡛

ᐃࡍࡿࠋᖹᆒ⺯ගᙉᗘ㸦Mean Fluorescence Intensity: MFI㸧ࢆᇶ࡟ࠊ௨ୗࡢᘧ࠿ࡽࠊ㝧ᛶᑐ↷࠾ࡼࡧ⿕㦂≀㉁࡛ฎ⌮ࡋࡓ⣽⬊࡟࠾ࡅࡿ CD86 ࠾ࡼࡧ CD54 ࡢ┦ᑐ⺯ගᙉᗘ㸦Relative Fluorescence
Intensity: RFI㸧ࢆồࡵࡿࠋ࢔࢖ࢯࢱ࢖ࣉࢥࣥࢺ࣮ࣟࣝ㸦࣐࢘ࢫ IgG1 ᢠయ࡛ᰁⰍࡋࡓࡶࡢ㸧ࡢ⣽⬊⏕Ꮡ⋡ࡣࠊ3-2 4)ࡢ
ᘧࢆ⏝࠸࡚ィ⟬ࡍࡿࠋ
2) ண

᪉ἲ

CD86 ࠾ࡼࡧ CD54 Ⓨ⌧ᐃ࡛ࡣࠊࡑࢀࡒࢀࡢ⿕㦂≀㉁ࡢண 㸦㝧ᛶࡶࡋࡃࡣ㝜ᛶ㸧ࢆ⾜࠺ࡓࡵ࡟ࠊᑡ࡞ࡃ࡜ࡶ 2 ᅇࡢ⊂❧ࡋࡓࡢᐃࡢ࠺ࡕ 2 ᅇࠊࡶࡋࡃࡣ 3 ᅇࡢᐃࢆ⾜࠺ᚲせࡀ࠶ࡿࠋ௨ୗࡢ≧ἣࡢ࠺ࡕ㸯ࡘࡀ 2 ᅇᐃࡢ࠺ࡕ 2 ᅇ࡛ㄆࡵࡽࢀࡓሙྜࠊh-CLAT ࡟ࡼࡿணࡣࠊ㝧ᛶ࡜ุᐃࡍࡿࠋ
࣭⣽⬊⏕Ꮡ⋡ 50%௨ୖ࡜࡞ࡿ࠸ࡎࢀ࠿ࡢ⃰ᗘ࡟࠾࠸࡚ࠊCD86 ࡢ┦ᑐ⺯ගᙉᗘࡀ 150%௨
ୖ࡜࡞ࡿሙྜ
࣭⣽⬊⏕Ꮡ⋡ 50%௨ୖ࡜࡞ࡿ࠸ࡎࢀ࠿ࡢ⃰ᗘ࡟࠾࠸࡚ࠊCD54 ࡢ┦ᑐ⺯ගᙉᗘࡀ 200%௨
ୖ࡜࡞ࡿሙྜ ᭱ึࡢ 2 ᅇࡢணᐃ࡟࠾࠸࡚ࠊCD86 ࠾ࡼࡧ/ࡶࡋࡃࡣ CD54 ࡀ࡜ࡶ࡟㝧ᛶ࡛࠶ࢀࡤࠊh-CLATࡣ㝧ᛶ࡜ุᐃࡋࠊ3 ᅇ┠ࡢᐃࡣ୙せ࡛࠶ࡿࠋྠᵝ࡟᭱ึࡢ 2 ᅇࡢ

ࡧ CD54 ࡀඹ࡟㝜ᛶ࡛࠶ࢀࡤࠊ3 ᅇ┠ࡢ
2 ᅇࡢ

ᐃࡣ୙せ࡛ h-CLAT ண

ᐃ࡛ࠊCD86 ࠾ࡼࡣ㝜ᛶ࡜ุᐃࡍࡿࠋ୍᪉ࠊ

ᐃ࡛஧ࡘࡢ࣐࣮࣮࢝ࡢ࠺ࡕ࠸ࡎࢀࡶ㐃⥆ࡋ࡚㝧ᛶࢆ♧ࡉ࡞࠿ࡗࡓሙྜࠊ࠶ࡿ࠸ࡣ୧

⪅࡜ࡶ㐃⥆ࡋ࡚㝜ᛶ࡜࡞ࡽ࡞࠿ࡗࡓሙྜࠊ3 ᅇ┠ࡢᐃࡀᚲせ࡛ࠊ᭱⤊ࡢண

ࡣࠊ3 ᅇࡢ

ᐃࡢከᩘ⤖ᯝࢆࡶࡗ࡚Ỵᐃࡍࡿࠋ2 ᅇࡢ┠ࡢᐃࢆ⾜࠸ࠊ1 ᅇ┠ࡣ CD86 ࡀ㝧ᛶࠊ2 ᅇ┠

ᐃࡀᚲせ࡛࠶ࡿࠋࡑࡢ⤖ᯝࠊ3 ᅇ┠ุᐃࡍࡿࠋ୍᪉ࠊ3 ᅇ┠ࡢᐃ࡛ CD54 ࡀ㝧ᛶ࡜࡞ࡗࡓሙྜࠊ3 ᅇᐃࡀ㝜ᛶ࡜࡞ࡗࡓሙྜࠊh-CLAT ணࡣ㝜ᛶ࡜ᐃ࡛ࠊ࡝ࡕࡽ࠿ࡢ࣐࣮࣮࢝㸦CD86 ࡶࡋࡃࡣ CD54㸧ࡀ㝧ᛶࠊࡶࡋࡃࡣ୧࣐࣮࣮࢝ࡀ㝧ᛶ࡜࡞ࡗࡓሙྜࠊh-CLAT ணࡣ㝧ᛶ࡜ุᐃࡍࡿࠋ

ḟ࣮࣌ࢪ࡟ࣇ࣮ࣟᅗ㸦ᅗ 1㸧ࢆ♧ࡍࠋ
3-5㸬ヨ㦂ᡂ❧ࡢ᮲௳h-CLAT ࡢሙྜࠊ௨ୗࡢ᮲௳ࡀᡂ❧ࡍࡿᚲせࡀ࠶ࡿࠋ࣭ᇶ♏ᇵᆅᑐ↷࠾ࡼࡧ፹యᑐ↷ࡢ⣽⬊⏕Ꮡ⋡ࡀࠊ90%௨ୖ࡛࠶ࡿࠋ
࣭፹యᑐ↷࡟࠾ࡅࡿ CD86 ࠾ࡼࡧ CD54 ࡢ୧᪉ࡢ┦ᑐ⺯ගᙉᗘ್ࡀ㝧ᛶᇶ‽್㸦CD86 ࡛
1016

ࡣ 150㸣ࠊCD54 ࡛ࡣ 200㸣㸧ࢆ㉸࠼࡞࠸ࠋ⁐፹ᑐ↷ࡢ┦ᑐ⺯ගᙉᗘ್ࡣࠊ3-4

1)࡟グ㍕ࡢ

ᘧ࡟ࡼࡾィ⟬ࡉࢀࡿ㸦ࡑࡢ㝿ࠊࠕMFI of chemicalࠖࡣࠕMFI of solvent/vehicleࠖ࡟ࠊࠕMFI ofsolvent/vehicleࠖࡣࠕMFI of (medium) controlࠖ࡟ㄞࡳ᥮࠼ࡿᚲせࡀ࠶ࡿࠋ㸧࣭ᇶ♏ᇵᆅᑐ↷࠾ࡼࡧ፹యᑐ↷ࡢ୧᪉࡟࠾࠸࡚ࠊ࢔࢖ࢯࢱ࢖ࣉ࡟ᑐࡍࡿ CD86 ࡜ CD54 ࡢ୧᪉ࡢᖹᆒ⺯ගᙉᗘࡢ๭ྜࡀ 105%ࡼࡾ኱ࡁࡃ࡞ࡿࠋ
࣭㝧ᛶᑐ↷㸦DNCB㸧࡟࠾࠸࡚ࠊ⣽⬊⏕Ꮡ⋡ࡀ 50%௨ୖࢆ♧ࡋࠊCD86 ࠾ࡼࡧ CD54 ࡢ┦ᑐ⺯ගᙉᗘࡀ࠸ࡎࢀࡶ㝧ᛶᇶ‽࡜࡞ࡿࠋ࣭⿕㦂≀㉁࡟࠾࠸࡚ࠊࡑࢀࡒࢀࡢ

ᐃ࡛ヨ㦂ࡉࢀࡓᑡ࡞ࡃ࡜ࡶ 4 ⃰ᗘ࡟࠾࠸࡚ࠊ⣽⬊⏕Ꮡ⋡ࡀ 50㸣ࢆ㉸࠼ࡿ᭱㧗⃰ᗘ㸦1.2CV75㸧࡛ࡢ⣽⬊⏕Ꮡ⋡ࡀ 90㸣ࡼࡾᑠࡉ࠸ሙྜࠊ㝜ᛶ⤖ᯝࡣཷࡅධࢀࡽ
ࢀࡿࡀࠊ1.2CV75 ࡢ⣽⬊⏕Ꮡ⋡ࡀ 90㸣௨ୖ࡛࠶ࡗࡓሙྜ࡟ࡣࠊ㝜ᛶ⤖ᯝࡣཷࡅධࢀࡽࢀ
࡞࠸ࠋࡑࡢࡼ࠺࡞ሙྜ࡟ࡣࠊ෌ᗘ CV75 ࢆỴᐃࡍࡿࡇ࡜࡛ࠊ⃰ᗘタᐃࢆࡸࡾ┤ࡍࡇ࡜ࡀᮃ
ࡲࡋ࠸ࠋࡓࡔࡋࠊ፹య࡟⏕⌮㣗ሷỈࠊᇶ♏ᇵᆅ࠾ࡼࡧࡑࡢ௚ࡢᇵᆅࢆ౑⏝ࡋࡓ 5,000
ȝg/mLࠊDMSO ࢆ౑⏝ࡋࡓ 1,000 ȝg/mL ࠾ࡼࡧ᭱኱⁐ゎ⃰ᗘࡀヨ㦂ࡢ᭱㧗⃰ᗘ࡛࠶ࡿሙ
ྜࠊ⏕Ꮡ⋡ࡀ 90%௨ୖ࡛࠶ࡗ࡚ࡶࠊ㝜ᛶ⤖ᯝࡣチᐜࡉࢀࡿࠋ

1117

ᅗ 1 ண᪉ἲP1㸸CD86 ࡢࡳࡀ㝧ᛶࡢሙྜࠊP2㸸CD54 ࡢࡳࡀ㝧ᛶࡢሙྜࠊP12㸸CD86 ࡜ CD54 ࡀ࡜ࡶ࡟㝧ᛶࡢሙྜࠊN㸸CD86 ࡜ CD54 ࡀ࡜ࡶ࡟㝜ᛶࡢሙྜࠊ*㸸᭱ึࡢ 2 ᅇࡢࡳྜࢃࡏࠊ㸡㸸᭱ึࡢ 2 ᅇࡢᐃ⤖ᯝ࡟ᇶ࡙࠸࡚ᐇ᪋ࡉࢀࡓ 3 ᅇ┠ࡢ

ࢃࡏ 

12
18

ᐃ⤖ᯝࡢ⤌

ᐃ⤖ᯝࡢ⤌ࡳྜ

4㸬⢭ᗘ
EURL ECVAM ࡟ࡼࡾᐇ᪋ࡉࢀࡓࣂࣜࢹ࣮ࢩࣙࣥヨ㦂 3)࡟ࡣ 4 ᪋タ㸦Kao Corporation㸦୺ᑟ᪋タ㸧ࠊShiseido Quality Assessment Center㸦୺ᑟ᪋タ㸧ࠊEURL ECVAMࠊBioassay GmbH㸧ࡀཧຍࡋࠊᢏ⾡⛣㌿ᛶࠊ᪋タෆ෌⌧ᛶ࠾ࡼࡧ᪋タ㛫෌⌧ᛶࡀ᳨ウࡉࢀ࡚࠸ࡿࠋ
4-1. ᢏ⾡⛣㌿ᛶ5 ≀㉁㸦3 ឤసᛶ≀㉁㸸DNCBࠊNickel Sulfateࠊ Phenylacetaldehyde ࠾ࡼࡧ 2 㠀ឤసᛶ≀㉁㸸
Sodium lauryl sulfateࠊLactic acid㸧ࡢࢹ࣮ࢱྲྀᚓࢆ⾜࠸ࠊ୺ᑟ᪋タ࡛࠶ࡿ Kao ࡜ Shiseido ࠿ࡽ EURL ECVAM ࡜ Bioassay ࡢ 2 ᪋タ࡬ࡢᢏ⾡⛣㌿ᛶ࡟ࡘ࠸࡚ホ౯ࡀ⾜ࢃࢀࠊࣂࣜࢹ࣮ࢩ
ࣙࣥሗ࿌᭩࡛ࡣࠊᢏ⾡⛣㌿ᛶ࡟ၥ㢟࡞࠸࡜グ㍕ࡉࢀ࡚࠸ࡿࠋࡓࡔࡋࠊOECD ヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥ 7)࡛ࡣࠊᮏἲࢆ౑⏝ࡍࡿ๓࡟ヨ㦂᪋タ࡟࠾࠸࡚ᢏ⬟⩦⇍⏝ 10 ≀㉁࠾ࡼࡧ㝧ᛶᑐ↷ࠊ㝜ᛶᑐ↷ࠊ፹యᑐ↷ࢆ⏝࠸࡚཯ᛂᛶࢆ☜ㄆࡋࠊᢏ⾡ࡀ⩦⇍ࡋ࡚࠸ࡿࡇ࡜ࢆ♧ࡍࡇ࡜ࢆồࡵ࡚࠸ࡿࠋຍ࠼࡚ࠊ㝧ᛶᑐ↷ࠊ㝜ᛶᑐ↷࠾ࡼࡧ፹యᑐ↷ࡢᐃࢆ⥅⥆ⓗ࡟ᐇ᪋ࡋࠊࣄࢫࢺࣜ࢝ࣝ࡞ࢹ࣮ࢱ࣮࣋ࢫࡢసᡂࢆồࡵ࡚࠸ࡿࠋ
4-2. ᪋タෆ෌⌧ᛶ㸦⾲ 1㸧 ┣᳨ୗ࡛ホ౯ࡉࢀࡓ 15 ≀㉁࡟ࡘ࠸࡚ࠊ4 ᪋タࡢ᪋タෆ෌⌧ᛶ㸦3 ᅇࡢ⧞ࡾ㏉ࡋࡢヨ㦂࡛ྠࡌ⤖ᯝ㸧ࡣࠊKao㸸86.7%㸦୍⮴ࡋ࡞࠿ࡗࡓ≀㉁㸸BenzylsalicylateࠊMethylsalicylate㸧ࠊShiseido㸸
80.0%㸦୍⮴ࡋ࡞࠿ࡗࡓ≀㉁㸸Kathon CGࠊBeryllium sulfateࠊFormaldehyde㸧ࠊBioassay㸸73.3%
㸦୍⮴ࡋ࡞࠿ࡗࡓ≀㉁㸸FormaldehydeࠊBenzylsalicylateࠊMethylsalicylateࠊXylene㸧ࠊECVAM㸸
80.0%㸦୍⮴ࡋ࡞࠿ࡗࡓ≀㉁㸸Chlorpromazine HClࠊBenzylcinnamateࠊDimethylisophthalate㸧
࡛࠶ࡗࡓࠋ
⾲ 1

᪋タෆ෌⌧ᛶࡢホ౯ᡂ⦼

ID

Chemical name

10 Kathon CG
11 Beryllium sulfate
12 Formaldehyde
13 Chloramine T
14 Chlorpromazine HCl
15 2-mercaptobenzothiazole
16 Benzylsalicylate
17 Benzylcinnamate
18 R(+) Limonene
19 M ethylsalicylate
20 Isopropanol
21 Dimethylisophtalate
22 4-aminobenzoic acid
23 Nickel chloride
24 Xylene

GHS
potency
category

LLNA potency

LLNA
Kao

Shiseido

Bioassay

ECVAM

26172-55-4

1A

extreme

P

3/3

2/3

3/3

3/3

7787-56-6

1A

extreme

P

0/3

2/3

0/3

3/3

50-00-0

1A

strong

P

3/3

2/3

2/3

3/3

CAS NoPositve with EC 150 or EC200

149358-73-6

1A

strong

P

3/3

3/3

3/3

3/3

69-09-0

1A

strong

P

3/3

3/3

3/3

2/3

149-30-4

1A

moderate

P

3/3

3/3

3/3

3/3

118-58-1

1B

moderate

P

1/3

0/3

1/3

3/3

103-41-3

1B

weak

P

0/3

3/3

0/3

1/3

5989-27-5

1B

weak

P

3/3

3/3

3/3

3/3

119-36-8

1B

weak

N

2/3

0/3

1/3

3/3

67-63-0

NC

NC

N

0/3

0/3

0/3

0/3
1/3

1459-93-4

NC

NC

N

0/3

0/3

0/3

150-13-0

NC

NC

N

0/3

0/3

0/3

0/3

7718-54-9

1B

NC (false neg)

N

3/3

3/3

3/3

3/3

1330-20-7NC

weak (false pos)P

Number of concordancy
Concordancy rate (%)

0/3

0/3

1/3

3/3

13/15
86.7

12/15
80.0

11/15
73.3

12/15
80.0

13
19

ᮏヨ㦂࡛ࡣࠊ㐩ᡂᇶ‽ࢆ 85㸣࡟タᐃࡋ࡚࠸ࡓࡀࠊࡇࡢᇶ‽ࢆୖᅇࡗࡓࡢࡣ 4 ᪋タ୰ 1 ᪋タࡢࡳ࡛࠶ࡗࡓࠋ෌⌧ᛶࡢᚓࡽࢀ࡞࠸≀㉁ࡣࠊUN GHS 1A ศ㢮ࡀ 4 ≀㉁ࠊ1B ศ㢮ࡀ 3 ≀㉁ࠊศ㢮୙ྍ≀㉁ࡀ 2 ≀㉁ࡢ 9 ≀㉁࡛࠶ࡗࡓࠋࡲࡓࠊ୍ࡘࡢ᪋タ࡛ࡶഇ㝜ᛶࢆ♧ࡋࡓ≀㉁
ࡣࠊUN GHS 1A ศ㢮ࡀ 1 ≀㉁㸦Beryllium sulfate㸧ࠊ1B ศ㢮ࡀ 3 ≀㉁㸦Benzylsalicylateࠊ
BenzylcinnamateࠊMethylsalicylate㸧ࡢ 4 ≀㉁࡛࠶ࡗࡓࠋࡓࡔࡋࠊOECD ᑓ㛛ᐙ఍㆟࡟࠾࠸࡚ࠊヨ㦂࡟౑⏝ࡍࡿ THP-1 ⣽⬊ࡢ๓ᇵ㣴᮲௳࠾ࡼࡧ⿕㦂≀㉁ࡢ᭚㟢᫬㛫ࢆࡼࡾཝᐦ࡟⟶⌮ࡍࡿࡇ࡜࡟ࡼࡾࠊ᪋タෆ෌⌧ᛶࡢྥୖࡀᅗࢀࡿࡇ࡜ࡀ
♧ࡉࢀ㸦ῧ௜㈨ᩱ㸧ࠊOECD ヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥ 7)ࡢヨ㦂ᡭ㡰࡟ࡑࡢෆᐜࡀ཯ᫎࡉࢀ࡚࠸ࡿࠋ
4-3. ᪋タ㛫෌⌧ᛶ㸦⾲ 2㸧 ┣᳨ୗ࡛ホ౯ࡉࢀࡓ 24 ≀㉁ࡢ 4 ᪋タࡢ᪋タ㛫෌⌧ᛶ㸦4 ᪋タ࡛ྠࡌ⤖ᯝ㸧ࡣ 79.2%㸦୍⮴ࡋ࡞࠿ࡗࡓ≀㉁㸸 Beryllium sulfateࠊBenzylsalicylateࠊBenzylcinnamateࠊMethylsalicylateࠊ
Xylene㸧࡛࠶ࡾࠊ㐩ᡂᇶ‽ 80㸣ࢆୗᅇࡗࡓࠋ୍⮴ࡋ࡞࠿ࡗࡓ≀㉁ࡣ᪋タෆ෌⌧ᛶࡢヨ㦂࡟࠾࠸୍࡚⮴ࡋ࡞࠿ࡗࡓ 9 ≀㉁࡟ྵࡲࢀࡿࠋࡋࡓࡀࡗ࡚ࠊ᪋タෆ෌⌧ᛶ࡜ྠᵝ࡟ࠊᮏἲ࡛ࡣ GHS
1A ศ㢮ࡢ≀㉁ࡀ㐺ษ࡟ุᐃࡉࢀ࡞࠸ሙྜࡢ࠶ࡿࡇ࡜ࡀ♧ࡉࢀࡓࠋ⾲ 2ID᪋タ㛫෌⌧ᛶࡢホ౯ᡂ⦼
Chemical name

1 Benzoquinone
2 4-phenylenediamine
3 Dihydroeugenol
4 1-thioglycerol
5 Imidazolidinylurea
6 Methylmethacrylate
7 Glycerol
8 2,4-dichloronitrobenzene
9 Benzyl alcohol
10 Kathon CG
11 Beryllium sulfate
12 Formaldehyde
13 Chloramine T
14 Chlorpromazine HCl
15 2-mercaptobenzothiazole
16 Benzylsalicylate
17 Benzylcinnamate
18 R(+) Limonene
19 Methylsalicylate
20 Isopropanol
21 Dimethylisophtalate
22 4-aminobenzoic acid
23 Nickel chloride
24 Xylene

LLNA
potency

LLNA
Kao

Shiseido106-51-4

1A

extreme

P

1/1

1/1

1/1

1/1

Y

106-50-3

1A

strong

P

1/1

1/1

1/1

1/1

Y

2785-87-7

1B

moderate

P

1/1

1/1

1/1

1/1

Y

96-27-5

1B

moderate

P

1/1

1/1

1/1

1/1

Y

39236-46-9

1B

weak

P

1/1

1/1

1/1

1/1

Y

80-62-6

1B

weak

P

0/1

0/1

0/1

0/1

Y

56-81-5

NC

NC

N

0/1

0/1

0/1

0/1

Y

611-06-3

NC

NC

N

1/1

1/1

1/1

1/1

Y

100-51-6

NC

NC

N

1/1

1/1

1/1

1/1

Y

26172-55-4

1A

extremeP

7787-56-6

1A

extreme

P

3/3
0/3

2/3
2/3

3/3
0/3

3/3
3/3

Y
N

50-00-0

1A

strong

P

3/3

2/3

2/3

3/3

Y

149358-73-6

1A

strong

P

3/3

3/3

3/3

3/3

Y

69-09-0

1A

strong

P

3/3

3/3

3/3

2/3

Y

149-30-4

1A

moderateP

118-58-1

1B

moderate

P

3/3
1/3

3/3
0/3

3/3
1/3

3/3
3/3

Y
N

CAS NoPositve with EC 150 or EC200
Bioassay

103-41-3

1B

weak

P

0/3

3/3

0/3

1/3

N

5989-27-5

1B

weak

P

119-36-8

1B

weak

N

3/3
2/3

3/3
0/3

3/3
1/3

3/3
3/3

Y
N

67-63-0

NC

NC

N

0/3

0/3

0/3

0/3

Y

1459-93-4

NC

NC

N

0/3

0/3

0/3

1/3

Y

150-13-0

NC

NC

N

0/3

0/3

0/3

0/3

Y

3/3
0/3

3/3
0/3

3/3
1/3

3/3
3/3

Y
N

7718-54-9

1B

NC (false neg)N1330-20-7NC

weak (false pos)P

Number of concordancy
Concordancy rate (%)

1420

Between4 Laboratory
Reproducibili
ECVAM
ty

GHS
potency
category

19/24
79.2

ᮏヨ㦂ࡣࠊ2 ẁ㝵࡛ᐇ᪋ࡉࢀ࡚࠸ࡿࠋ1 ẁ㝵┠ࡢ ID No. 1-9 ࡢ≀㉁࡟ࡼࡿヨ㦂ࡣ 1 ᅇࡢࡳࡢᐇ᪋࡛࠶ࡿࠋ2 ẁ㝵┠ࡢ ID No. 10-24 ࡢ≀㉁ࢆ⏝࠸ࡓヨ㦂ࡣ 3 ᅇᐇ᪋ࡋࠊ2 ᅇ࠶ࡿ࠸ࡣ 3ᅇྠࡌ࡛࠶ࡗࡓ⤖ᯝࢆ᥇⏝ࡋ࡚࠸ࡿࠋࡑࡢ⤖ᯝࠊn=3 ࡛ᐇ᪋ࡋࡓ 15 ≀㉁࡛ࡢ᪋タ㛫෌⌧ᛶࡣ 66.7㸣࡜࡞ࡾࠊn=1 ࡛ࡢホ౯ࢆྵࡵࡓ 79.2㸣ࢆୗᅇࡿ⤖ᯝ࡜࡞ࡗࡓࠋࡇࢀࡣ᪋タෆ෌⌧ᛶࡢヨ㦂࡟࠾࠸࡚ࠊn=3 ࡛ࡢ⧞ࡾ㏉ࡋࡢ㝿ࠊ3 ᅇ࡜ࡶྠࡌ⤖ᯝࢆ♧ࡉ࡞࠸ࢣ࣮ࢫࡀ 60 ᅇ୰ 12ᅇ㸦20.0㸣㸧ྵࡲࢀࡿࡇ࡜ࡀཎᅉ࡜⪃࠼ࡽࢀࡓࠋ
5㸬ṇ☜ᗘ㸦ឤᗘ࠾ࡼࡧ≉␗ᗘ㸧
EURL ECVAMࡀ24≀㉁㸦ឤసᛶ≀㉁㸸㠀ឤసᛶ≀㉁㸻16㸸8㸧࡛⾜ࡗࡓࣂࣜࢹ࣮ࢩࣙࣥ
ヨ㦂2)࡛ࡣࠊṇ☜ᗘ76㸣ࠊឤᗘ81㸣ࠊ≉␗ᗘ66㸣࡛࠶ࡗࡓࠋࡇࡢᡂ⦼ࡣࠊAshikagaࡽ4)࡟ࡼ
ࡿ100≀㉁㸦ឤసᛶ≀㉁㸸㠀ឤసᛶ≀㉁㸻72㸸28㸧ࡢᡂ⦼ṇ☜ᗘ84㸣ࠊឤᗘ88㸣ࠊ≉␗ᗘ
75㸣㸧࡜ẚ㍑ࡋ࡚ప࠸ࡶࡢ࡛࠶ࡗࡓࠋ
Takenouchiࡽ5)ࡀỈ࡬ࡢ⁐ゎᛶࡀప࠸≀㉁ࡀഇ㝜ᛶࢆ♧ࡍᙳ㡪ࢆㄪ࡭ࡿࡓࡵ࡟ᐇ᪋ࡋࡓ37≀㉁㸦ឤసᛶ≀㉁㸸㠀ឤసᛶ≀㉁㸻30㸸7㸧ࡢ⤖ᯝ࡛ࡣࠊṇ☜ᗘ68%ࠊឤᗘ70%ࠊ≉␗ᗘ
57%࡛࠶ࡾࠊ30ࡢឤసᛶ≀㉁ࡢ࠺ࡕ9≀㉁ࡀ㝜ᛶ࡜ุᐃࡉࢀࡓࠋࡇࡢ୰ࡢ2≀㉁ࡣᇵ㣴ᾮ୰࡟ỿẊࡀほᐹࡉࢀࠊ5≀㉁࡛ࡣἜ⁲ࡀほᐹࡉࢀࡓࠋࡇࡢ9≀㉁ࡢlog Kowࡣࠊ3.5௨ୖ࡛࠶ࡗ
ࡓࠋࡉࡽ࡟ࠊNukadaࡽ6)࡟ࡼࡿ106≀㉁㸦ឤసᛶ≀㉁㸸㠀ឤసᛶ≀㉁㸻75㸸31㸧ࡢᡂ⦼࡜ྜ
ࢃࡏྜィ143≀㉁㸦ឤసᛶ≀㉁㸸㠀ឤసᛶ≀㉁㸻105㸸38㸧᳨࡛ウࡋࡓ⤖ᯝࠊṇ☜ᗘࡣ
80%ࠊឤᗘࡣ83%ࠊ≉␗ᗘࡣ71%࡛࠶ࡾࠊ8≀㉁ࡀഇ㝜ᛶࢆ♧ࡋࡓࠋࡇࡢ࠺ࡕࡢ5≀㉁ࡣ3.5
ᮍ‶ࡢlog Kow࡛࠶ࡾࠊࡑࡢ࠺ࡕ2≀㉁ࡣࣉࣟࣁࣉࢸࣥ/ࣉࣞࣁࣉࢸ࡛ࣥ࠶ࡗࡓࠋࡲࡓࠊ11≀㉁ࡀഇ㝧ᛶࢆ♧ࡋࡓࠋ
OECD ヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥ 7)࡟ࡣࠊEURL ECVAM3)ࡢ 24 ≀㉁࡜ࠊTakenouchi ࡽ 5)ࡢ 143 ≀㉁ࡢ⤖ᯝࢆ⤫ྜࡋࠊ142 ≀㉁㸦ឤసᛶ≀㉁㸸㠀ឤసᛶ≀㉁㸻101㸸41㸧ࡢᡂ⦼ࢆࡶ࡜࡟ࠊṇ☜ᗘࠊឤᗘࠊ≉␗ᗘࢆồࡵࠊࡑࢀࡒࢀࠊ85㸣ࠊ93㸣ࠊ66㸣࡜⾲グࡉࢀ࡚࠸ࡿࠋࡇࡢ୰࡛ࠊ୧ሗ
࿌࡛㔜」ࡍࡿ 10 ≀㉁࡟ࡘ࠸࡚ࡣ EURL ECVAM3)ࡢࢹ࣮ࢱࢆ᥇⏝ࡋࠊTakenouchi ࡽ 5)ࡢࢹ࣮
ࢱ࠿ࡽࡣࠊࡇࡢ 10 ≀㉁࡜ log Kow ࡀ 3.5 ௨ୖࡢ 14 ≀㉁࡜ΰྜ≀ 1 ✀ࢆ㝖ࡃ 118 ≀㉁ࡢࢹ࣮
ࢱࢆ᥇⏝ࡋ࡚࠸ࡿࠋ୍᪉ࠊOECD ヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥ 7)ࡢ 142 ≀㉁࡟ Takenouchi ࡽ 5)ࡢㄽᩥ
ࡢ log Kow ࡀ 3.5 ௨ୖࡢ 14 ≀㉁ࢆຍ࠼ࡓ 156 ≀㉁㸦ឤసᛶ≀㉁㸸㠀ឤసᛶ≀㉁㸻113㸸43㸧࡛ࡢṇ☜ᗘࠊឤᗘࠊ≉␗ᗘࡣࠊࡑࢀࡒࢀ 83㸣ࠊ68㸣ࠊ79㸣࡜࡞ࡾࠊឤᗘࡀపୗࡍࡿഴྥࡀࡳ
ࡽࢀࡓࠋ
6. ホ౯ྍ⬟࡞≀㉁ࡢ⠊ᅖOECD ヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥ 7)࡟グ㏙ࡢ࠶ࡿ 142 ≀㉁࡟ࡼࡿホ౯࡛ࡣࠊ⾲ 3 ࡟♧ࡍ㏻ࡾࠊ
ᵝࠎ࡞໬Ꮫ≀㉁ࡢ⓶⭵ឤసᛶࡢண

ࡀྍ⬟࡛࠶ࡿࡇ࡜ࡀ♧ࡉࢀ࡚࠸ࡿࠋ

15
21

⾲3
LLNA
Chemical Name
Benzo(a)pyrene
Diphenylcyclopropenone
Oxazolone
Dinitrofluorobenzene
Tetrachlorosalicylanilide
Bandrowskis base
1-Benzoylacetone
4-Nitrobenzyl bromide
2,4-Dinitrochlorobenzene
Potassium dichromate
Beryllium sulfate
Kathon CG (1.2% CM I)
Benzoquinone
Glutaraldehyde
1,4-Dihydroquinone
Phthalic anhydride
M aleic anhydride
Hexyl salicylate
Benzyl bromide
Benzyl peroxide
Lauryl gallate
Propyl gallate
2-Aminophenol
2-Nitro-4-phenylenediamine
Cobalt chloride
Chloramine T (CAS No. 149358-73-6)
CD-3 (CAS No. 25646-71-3)
Iodopropynyl butylcarbamate
1,2-Dibromo-2,4-dicyanobutane
Chlorpromazine HCl
4-Phenylenediamine
Chloramine T (CAS No. 127-65-1)
Formaldehyde
Isoeugenol
1-Naphtol
1-Phenyl-1,2-propanedione
2-Hydroxyethyl acrylate
Glyoxal
Bisphenol A-diglycidyl ether
Vinyl pyridine
2-M ethyl-2H-isothiazolone
3-Dimethylaminopropylamine
Ethylene diamine
1,2-Benzisothiazoline-3-one
M ethyl-2-nonynoate
Phenylacetaldehyde
Cinamic aldehyde
3-Aminophenol
Diethyl sulphate
3-Propylidenephthalide
Benzylidene acetone
2,4-Heptadienal
5-M ethyl-2-phenyl-2-hexanal
Alpha-methyl cinnamic aldehyde
Nickel sulfate
Tetramethylthiuramdisulfide
Trans-2-hexenal
Resorcinol
3,4-Dihydrocoumarin
Diethyl maleate2-M ethoxy-4-methyl-phenol
Diethylenetriamine
2-Phenylpropionaldehyde
4-Chloroaniline-damascone
Perillaaldehyde
Trimellitic anhydride2-M ercaptobenzothiazole
Benzylsalicylate
1-Thioglycerol
Dihydroeugenol

h-CLAT

Potency
EC3 (%) Results
CD86
Category
Extreme
0.00009P+
Extreme
0.003
P
Extreme
0.003
P+
Extreme
0.03
P+
Extreme
0.04
P
Extreme
0.04
P+
Extreme
0.04
P
Extreme
0.05
P+
Extreme
0.05
P+
Extreme
0.08
P+
Extreme0.001
N
Extreme
0.009
P+
Extreme0.0099P+
Strong0.1
P+Strong0.11
P+Strong0.16
NStrong0.16
PStrong0.18
PStrong0.2
PStrong0.22
NStrong0.3
P+Strong0.32
PStrong0.4
P+Strong0.5
PStrong0.6
PStrong0.6
P+Strong0.6
P+Strong0.9
PStrong0.9
P+Strong0.14
PStrong0.11
P+Strong0.4
P+Strong0.61
P+
M oderate
1.2
N
M oderate
1.3
P+
M oderate
1.3
P+
M oderate
1.4
P+
M oderate
1.4
P
M oderate
1.5
P+
M oderate
1.6
P+
M oderate
1.9
P+
M oderate
2.2
P+
M oderate
2.2
P+
M oderate
2.3
P
M oderate
2.5
P+
M oderate
3.0
P+
M oderate
3.0
P+
M oderate
3.2
P
M oderate
3.3
P+
M oderate
3.7
P+
M oderate
3.7
P+
M oderate
4.0
P+
M oderate
4.4
P
M oderate
4.5
P+
M oderate
4.8
P+
M oderate
5.2
P+
M oderate
5.5
P+
M oderate
5.5
P
M oderate
5.6
P
M oderate
5.8
P+
M oderate
5.8
P+
M oderate
5.8
N
M oderate
6.3
P+
M oderate
6.5
P+
M oderate
6.7
P+
M oderate
8.1
P+
M oderate
9.2
P+
M oderate
1.7
P
M oderate
2.9
N
M oderate
3.6
P
M oderate
6.8
P: Takenouchi
2013: h-CLAT Validation Study Report 2012

LLNA
CD54
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-

Chemical Name
2-Ethylhexyl acrylate
Amyl cinnamic aldehyde
2,3-Butanedion
CitralEugenolOxalic acid
Lyral (CAS No. 31906-04-4)
4-Allylanisole
Lilial (CAS No. 80-54-6)
Pentachlorophenol
Phenyl benzoate
Cinnamic alcohol-iso-methylionone
Benzocaine
Geraniol
5-M ethyl-2,3-hexanedione
2,2'-dihydroxyazobenzene
Ethyleneglycol dimethacrylate
Penicillin G
Linalool
Butyl glycidyl ether
Hydroxycitronellal
Pyridine
AnilineNonanoic acid
Benzylcinnamate
Imidazolidinylurea
R(+) Limonene
M ethylmethacrylate
Furil (CAS No. 109-65-9)
1-Butanol
1-Iodohexane
2-Acetylcyclohexanone
2-Hydroxypropyl methacrylate
4-Hydroxybenzoic acid
6-M ethylcoumarin
Acetanisole
Benzalchonium chloride
Benzaldehyde
Benzoic acid
Chlorobenzene
Clofibrate
Coumarin
DextranDiethylphthalate
Dimethyl formamide
Ethyl benzoylacetate
Ethyl vanillin
Furil (CAS No. 492-94-4)
Kanamycin
Lactic acid
Octanoic acid
Propyl paraben
Propylene glycol
Saccharin
Salicylic acid
Streptomycin sulfate
Sulfanilamide
Tween 80
Vanillin
Zinc sulfate
Glycerol
2,4-Dichloronitrobenzene
Benzyl alcohol
M ethylsalicylate
Isopropanol
Dimethylisophthalate
4-Aminobenzoic acid
Sodium lauryl sulfate
Nickel chloride
XylenePotency
EC3 (%) Results
Category
Weak
10
P
Weak
11
P
Weak
11
P
Weak
13
P
Weak
13
P
Weak
15
P
Weak
17
P
Weak
18
P
Weak
19
P
Weak
20
P
Weak
20
P
Weak
21
P
Weak
21.8
P
Weak
22
P
Weak
26
P
Weak
26
P
Weak
27.9
P
Weak
28
P
Weak
30
P
Weak
30
P
Weak
31
N
Weak
33
P
Weak
72
P
Weak
89
P
Weak
21
P
Weak
18.4
N
Weak
24
P
Weak
69
P
Weak
90
N
Non-sensitizer
ND
P
Non-sensitizer
ND
N
Non-sensitizer
ND
P
Non-sensitizer
ND
P
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
P
Non-sensitizer
ND
N
Non-sensitizer
ND
P
Non-sensitizer
ND
P
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
P
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
P
Non-sensitizer
ND
P
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
P
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
P
Non-sensitizer
ND
N
Non-sensitizer
ND
P
Non-sensitizer
ND
P
Non-sensitizer
ND
P
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Others
14
N
OthersND
P
Others
95.8
Nh-CLAT
CD86

CD54

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

-

-

7. ᭷⏝ᛶ࡜㝈⏺
ᮏἲࡣ⣽⬊ᐇ㦂࡜ࣇ࣮ࣟࢧ࢖ࢺ࣓ࢺ࣮ࣜࢆ⤌ࡳྜࢃࡏࡓヨ㦂ἲ࡛࠶ࡾࠊ⣽⬊ᇵ㣴ᶵჾ࠾ࡼࡧࣇ࣮ࣟࢧ࢖ࢺ࣓࣮ࢱ࣮ࢆಖ᭷ࡋࠊࡑࡢᢏ⾡࡟⩦⇍ࡋࡓ᪋タ࡛࠶ࢀࡤᐇ᪋ྍ⬟࡜ᛮࢃࢀࡿ ࠋ ࡲ ࡓ ࠊ ᮏ ἲ ࡣ ື ≀ ࢆ ⏝ ࠸ ࡞ ࠸ in vitro ࡢ ᡭ ἲ ࡛ ࠶ ࡾ ࠊ ⛉ Ꮫ ⓗ ┠ ⓗ 1622

     ࡢࡓࡵ࡟ᐇ᪋ࡉࢀࡿື≀ᐇ㦂࡟㛵ࡋࠊࠕື≀ࡢឡㆤཬࡧ⟶⌮࡟㛵ࡍࡿἲᚊࠖ࠾ࡼࡧ 3R ࡢ⢭⚄࡜ྜ⮴ࡋ࡚࠸ࡿࠋࡉࡽ࡟ h-CLAT ࠾ࡼࡧ LLNA ᐇ᪋ࡢ㝿࡟ᚲせ࡜࡞ࡿᾘ⪖ရ㈝ࡣࠊLLNA
࡛ࡣ 1 ࢔ࢵࢭ࢖ᙜࡓࡾ⣙ 10 ୓෇࡛࠶ࡿࡢ࡟ᑐࡋࠊh-CLAT ࡛ࡣ⣙ 2 ୓෇࡛࠶ࡾ 1/5 ⛬ᗘࡢ⤒㈝࡛ᐇ᪋ྍ⬟࡜ヨ⟬ࡉࢀࡓࠋヨ㦂ᮇ㛫ࡶ LLNA ࡼࡾ▷ᮇ㛫࡛ᐇ᪋ྍ⬟࡛࠶ࡿࡇ࡜࠿ࡽࠊヨ㦂ἲ࡜ࡋ࡚㎿㏿ᛶ࣭⤒῭ᛶࡢ㠃࠿ࡽ᭷⏝࡜ᛮࢃࢀࡿࠋ
ᢏ⾡ⓗ㝈⏺࡜ࡋ࡚ࠊᾮ┦࡛ࡢ཯ᛂࢆᚲせ࡜ࡍࡿヨ㦂⣔࡛࠶ࡿࡓࡵࠊ⏕⌮㣗ሷᾮ࠶ࡿ࠸ࡣᇵ
ᆅ࡟ 100 mg/mLࠊࡇࢀࡽ࡟ᡤᐃࡢ⃰ᗘ࡛୙⁐ࡢሙྜࡣࠊDMSO ࡟ 500 mg/mL ࡢ⃰ᗘ࡛⁐ゎ࠶ࡿ࠸ࡣᏳᐃⓗ࡟ศᩓࡍࡿᚲせࡀ࠶ࡿ㸦ࡓࡔࡋࠊ⛉Ꮫⓗ᰿ᣐࡀ࠶ࢀࡤ௚ࡢ⁐፹ࡶ౑⏝ྍ⬟࡜ࡉࢀ࡚࠸ࡿ㸧
ࠋࡲࡓࠊᮏἲࡣࣇ࣮ࣟࢧ࢖ࢺ࣓࣮ࢱ࣮ࢆ⏝࠸ࡿᡭἲ࡛࠶ࡾࠊ⺯ගࢆ᭷ࡍࡿ≀㉁ࡶホ౯ࡣྍ⬟࡛࠶ࡿࡀࠊFITC ࡸ PI ࡜ྠ୍Ἴ㛗࡟ᙉ࠸⺯ගࢆ᭷ࡍࡿ≀㉁ࡣ

ᐃࢆጉᐖࡍࡿྍ⬟ᛶࡀ࠶ࡿࠋ

᭦࡟ࠊ㐣ᗘࡢ⣽⬊ẘᛶࢆ᭷ࡍࡿ≀㉁ࡣ⣽⬊ࡢᵓ㐀ኚ໬ࢆᘬࡁ㉳ࡇࡋṇࡋࡃホ౯ࡉࢀ࡞࠸ྍ
⬟ᛶࡀ࠶ࡾࠊ௚ࡢ⣽⬊ࢆ⏝࠸ࡿヨ㦂⣔࡜ྠᵝ࡟ࠊ᥹Ⓨᛶ≀㉁ࡣ㣕ᩓ࡟ࡼࡿ≀㉁ࡢࣟࢫࡸ㏆㞄ࡢ࢙࢘ࣝ࡬ࡢࢡࣟࢫࢥࣥࢱ࣑ࢆ㉳ࡇࡍࡓࡵࠊ㐺ษ࡟ホ౯ࡉࢀ࡞࠸ྍ⬟ᛶࡀ࠶ࡿࠋ
≀ᛶ࡟㛵ࡍࡿ㐺⏝㝈⏺࡜ࡋ࡚ࡣ log Kow ࡀ 3.5 ࢆ㉸࠼ࡿ≀㉁ࡣഇ㝜ᛶࢆ⏕ࡌࡸࡍ࠸ഴྥ࡟
࠶ࡿࡀࠊヱᙜ≀㉁࡟ࡼࡿ㝧ᛶࡢ⤖ᯝࡣ࠶ࡿ⛬ᗘಙ㢗࡛ࡁࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋ
ᮏ⣽⬊ࡢ௦ㅰ⬟ࡣ㝈ᐃⓗ࡛࠶ࡿࡓࡵࠊࣉࣟࣁࣉࢸࣥ㸦P450 ➼࡟ࡼࡿ௦ㅰάᛶ໬ࢆᚲせ࡜ࡍࡿ≀㉁㸧ࡸࣉࣞࣁࣉࢸࣥ㸦㓟໬࡟ࡼࡾάᛶ໬ࡉࢀࡿ≀㉁㸧ࡣഇ㝜ᛶࢆ⏕ࡌࡿྍ⬟ᛶࡀ࠶ࡿࠋ
ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡛ホ౯ࡋࡓ㔠ᒓሷ࡛ࡣ United Nations (UN) Globally Harmonized Systemof Classification and Labelling of Chemicals 㸦GHS㸧 1A ࡟ศ㢮ࡉࢀࡿ Beryllium sulfate ࡀࠊഇ㝜ᛶ࡜࡞ࡗࡓࡀࠊ᪤ሗ 4)࡟ࡼࡿ࡜ Potassium dichromateࠊCobalt chlorideࠊNickel sulfate ࡣṇࡋࡃホ౯ࡉࢀ࡚࠸ࡿࡓࡵࠊ㔠ᒓሷࢆ㐺⏝⠊ᅖእ࡜ࡍࡿ᰿ᣐࡣ࡞࠸ࠋ
ᮏἲ࡟ࡼࡿഇ㝜ᛶࡢุᐃࡣࠊᙉᗘឤసᛶ≀㉁㸦UN GHS 1A ศ㢮㸧ࡼࡾࡶ㍍ᗘ㹼୰➼ᗘឤసᛶ≀㉁㸦UN GHS 1B ศ㢮㸧࡛⏕ࡌࡸࡍ࠸ഴྥ࡟࠶ࡿࠋࡇࢀࡽࡢ⤖ᯝ࠿ࡽࠊ㝧ᛶ࡜ุᐃࡉ
ࢀࡓሙྜࡣឤసᛶ㝧ᛶ࡜ุ᩿ࡍࡿࡇ࡜ࡣྍ⬟࡜⪃࠼ࡿࡀࠊ1 ๭⛬ᗘࡣഇ㝧ᛶࡢྍ⬟ᛶࡀ࠶ࡿࡇ࡜࡟␃ពࡍࡿᚲせࡀ࠶ࡿࠋ ࡲࡓࠊᮏἲࡣᢏ⾡ⓗ࡟ࡣΰྜ≀࡟ࡶᛂ⏝ྍ⬟࡜ࡉࢀ࡚࠸ࡿࡀࠊΰྜ≀࡛ࡢᐇ㦂౛ࡣᴟࡵ࡚
㝈ࡽࢀ࡚࠸ࡿࠋ
ᚑࡗ࡚ࠊ⌧᫬Ⅼ࡛ࡣ௨ୗࡢ≀㉁⩌࡬ࡢ㐺⏝ᛶࡣ᫂☜࡛࡞࠸࡜⪃࠼ࡿࠋ㸫ヨ㦂⁐፹࡟⁐ゎࡋ࡞࠸≀㉁࠾ࡼࡧヨ㦂⁐፹୰࡛୙Ᏻᐃ࡞≀㉁㸫ࣉࣟࣁࣉࢸࣥࠊࣉࣞࣁࣉࢸࣥ
㸫᥹Ⓨᛶ≀㉁㸫FITC ࡸ PI ࡜ྠࡌἼ㛗ᇦ࡟ᙉ࠸⺯ගࢆ᭷ࡍࡿ≀㉁࡞࠾ࠊࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡢᡂ⦼࠿ࡽࡣࠊᮏἲࡢឤసᛶᙉᗘศ㢮ࡸ UN GHS ࡢࢧࣈ࢝ࢸ
ࢦ࣮ࣜศ㢮࡬ࡢᛂ⏝࡟ࡣ㐺ࡉ࡞࠸࡜⪃࠼ࡽࢀࡿࠋ

1723

8. ⤖ㄽ
h-CLAT ࡣࠊឤసᛶⓎ⌧ᶵᗎ࡟࠾ࡅࡿ➨୕ẁ㝵ࡢ࢖࣋ࣥࢺ࡛࠶ࡿᶞ≧⣽⬊ࡀάᛶ໬ࡍࡿ㝿ࡢ⣽⬊⾲㠃ศᏊࡢⓎ⌧ஹ㐍ࢆ฼⏝ࡋࡓ in vitro ヨ㦂ἲ࡛࠶ࡾࠊ໬Ꮫ≀㉁ࡢឤసᛶࢆุ᩿ࡍࡿୖ࡛㔜せ࡞᝟ሗࢆ୚࠼࡚ࡃࢀࡿࠋ
࣐࢘ࢫࢆ⏝࠸ࡿ LLNA ࡢ 1/5 ⛬ᗘࡢ⤒㈝࡛ᐇ᪋ྍ⬟࡜ヨ⟬ࡉࢀࠊヨ㦂ᮇ㛫ࡶ LLNA ࡟ẚ࡭▷ᮇ࡛࠶ࡿࡓࡵࠊ᭷⏝ᛶࡣ㧗࠸࡜ᛮࢃࢀࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊ⣽⬊ᐦᗘ࡜ᇵ㣴᫬㛫ࢆཝᏲࡍࡿࡇ࡜ࠊබẚ 1.2 ࡢ⃰ᗘタᐃࡀᚲせ࡞ࡓࡵࠊDPRA ࡸ ARE-Nrf2 Luciferase Test Method ࡟ẚ࡭࡚ࠊ᧯సࡀ↹㞧࡛࠶ࡾࠊไ⣙ࡀከ࠸ࠋࡲࡓࠊ㝧ᛶᑐ↷ࠊ㝜ᛶᑐ↷࠾ࡼࡧ፹యᑐ↷ࡢᐃࢆ⥅⥆ⓗ࡟ᐇ᪋ࡋࠊࣄࢫࢺࣜ࢝ࣝ࡞ࢹ࣮ࢱ࣮࣋ࢫࢆసᡂࡍࡿࡇ࡜ࡀᚲせ࡜ࡉࢀ࡚࠸ࡿࠋ
ᮏヨ㦂ἲࡢࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟࠾࠸࡚ࠊ15 ≀㉁ࢆ⏝࠸࡚ᐇ᪋ࡉࢀࡓ᪋タෆ෌⌧ᛶࡣࠊ
73.3㹼86.7%࡛࠶ࡾࠊ඲⯡ⓗ࡟㧗ࡃ࡞࠿ࡗࡓࠋࡓࡔࡋࠊOECD ᑓ㛛ᐙ఍㆟࡟࠾࠸࡚ࠊヨ㦂࡟౑⏝ࡍࡿ THP-1 ⣽⬊ࡢ๓ᇵ㣴᮲௳࠾ࡼࡧ⿕㦂≀㉁ࡢ᭚㟢᫬㛫ࢆࡼࡾཝᐦ࡟⟶⌮ࡍࡿࡇ࡜࡟
ࡼࡾࠊ᪋タෆ෌⌧ᛶࡢྥୖࡀᅗࢀࡿࡇ࡜ࡀ♧ࡉࢀࠊOECD ヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥࡢヨ㦂ᡭ㡰࡟ࡑࡢෆᐜࡀ཯ᫎࡉࢀ࡚࠸ࡿࡇ࡜࠿ࡽࠊ᪋タෆ෌⌧ᛶࡢྥୖࡀᮇᚅ࡛ࡁࡿ࡜⪃࠼ࡿࠋ୍᪉ࠊ24≀㉁ࢆ⏝࠸࡚ᐇ᪋ࡉࢀࡓ᪋タ㛫෌⌧ᛶࡣࠊ79.2㸣࡛࠶ࡗࡓࠋ
OECD ヨ㦂ἲ࢞࢖ࢻࣛ࢖࡛ࣥࡣࠊ142 ≀㉁ࡢᡂ⦼࠿ࡽṇ☜ᗘ 85㸣ࠊឤᗘ 93㸣ࠊ≉␗ᗘ 66㸣࡜グ㍕ࡉࢀ࡚࠸ࡿࡀࠊlog Kow ࡀ 3.5 ௨ୖ࡛㝜ᛶ࡜ุᐃࡉࢀࡓ≀㉁ࢆ㝖࠸ࡓୖ࡛ࡢᩘ್࡛࠶
ࡿࠋࡲࡓࠊOECD ヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥࡢ 142 ≀㉁࡟ log Kow ࡀ 3.5 ௨ୖࡢ 14 ≀㉁ࢆຍ࠼ࡓ
156 ≀㉁ࡢṇ☜ᗘࠊឤᗘࠊ≉␗ᗘࡣࠊࡑࢀࡒࢀ 83㸣ࠊ68㸣ࠊ79㸣࡜࡞ࡾࠊឤᗘࡀపୗࡍࡿഴ
ྥࡀࡳࡽࢀࡓࠋࡋࡓࡀࡗ࡚ࠊ≉࡟ log Kow ࡀ 3.5 ௨ୖࡢ≀㉁ࡢሙྜࡣഇ㝜ᛶࡢྍ⬟ᛶࢆ⪃៖ࡋࠊ⿵᏶ࡋᚓࡿ௚ࡢヨ㦂ࡼࡾ☜ㄆࡋ࡞ࡅࢀࡤ࡞ࡽ࡞ࡃࠊᮏヨ㦂ἲࡢࡳ࡛⓶⭵ឤసᛶࢆ㝜ᛶ࡜ุᐃࡍࡿࡇ࡜ࡣ࡛ࡁ࡞࠸࡜⪃࠼ࡿࠋࡲࡓࠊ≉␗ᗘࡀ 66%࡛࠶ࡿࡓࡵഇ㝧ᛶ࡜ุᐃࡍࡿࡇ࡜
ࡶ␃ពࡍࡿᚲせࡀ࠶ࡿࠋࡉࡽ࡟ࠊUN GHS ศ㢮ࡢ≀㉁࡛ࠊ㍍ᗘឤసᛶ≀㉁࡛ࡣഇ㝜ᛶࡀ⏕ࡌࡸࡍࡃࠊᙉᗘឤసᛶ≀㉁࡛ࡣࡑࡢẚ⋡ࡣప࠸ࠋࡇࡢࡇ࡜࠿ࡽࠊᮏヨ㦂ἲࡣࠊ≀㉁ࡢឤసᛶᙉᗘศ㢮ࡸ UN GHS ࡢࢧࣈ࢝ࢸࢦ࣮ࣜศ㢮࡬ࡢᛂ⏝࡟ࡣ㐺ࡉ࡞࠸࡜⪃࠼ࡿࠋࡲࡓࠊᮏヨ㦂ἲ࡟⏝࠸ࡿ⣽⬊ࡢ௦ㅰ⬟ࡣ㝈ᐃⓗ࡛࠶ࡿࡓࡵࠊάᛶ໬࡟௦ㅰ⣔ࢆᚲせ࡜ࡍࡿ໬Ꮫ≀㉁࡛ࡣࠊṇࡋࡃࡑࡢឤసᛶࡀ᳨ฟ
ࡉࢀ࡞࠸ྍ⬟ᛶࡀ࠶ࡿࠋ
ᮏጤဨ఍ࡣࠊୖグࡢᮏヨ㦂ἲࡢᵝࠎ࡞㝈⏺ࢆ຺᱌ࡍࡿ࡜ࠊᮏヨ㦂ἲ༢⊂࡛ࡣ⓶⭵ឤసᛶࡢุᐃࡣ୙༑ศ࡛᭷ࡾࠊドᣐࡢ㔜ࡳ௜ࡅࡸ௚ࡢヨ㦂ἲ࡜ࡢ⤌ྜࡏ࡛⏝࠸ࡿࡇ࡜ࢆ᥎ዡࡍ
ࡿࠋ

18
24

ᘬ⏝ᩥ⊩
1㸬EURL ECVAM (2015) Recommendation on the human Cell Line Activation Test (h-CLAT) forskin sensitization testing. Accessible at: https://eurl-ecvam.jrc.ec.europa.eu/news/news_docs/eurlecvam-recommendation-on-the-human-cell-line-activation-test-h-clat-for-skin-sensitisation2㸬DB-ALM (INVITTOX) (2014) Protocol 158: human Cell Line Activation Test (h-CLAT), 23pp.
Accessible at: https://ecvam-dbalm.jrc.ec.europa.eu/methods-and-protocols/search/h-clat
3㸬EURL ECVAM (2013) human Cell Line Activation Teat (h-CLAT) Validation Study Report.
Accessible at: https://eurl-ecvam.jrc.ec.europa.eu/news/news_docs/h-clat-validation-study-report
4. Ashikaga T, Sakaguchi H, Sono S, Kosaka N, Ishikawa M, Nukada Y,Miyazawa M, Ito Y,
Nishiyama N, Itagaki H. (2010) A comparative evaluation of in vitro skin sensitization tests: thehuman cell-line activation test (h-CLAT) versus the local lymph node assay (LLNA). Altern. Lab.
Anim., 38, 275-284.
5. Takenouchi O, Miyazawa M, Saito K, Ashikaga T, Sakaguchi H. (2013) Predictive performance ofthe human Cell Line Activation Test (h-CLAT) for lipophilic chemicals with high octanol-waterpartition coefficients. J. Toxicol. Sci., 38, 599-609.
6. Nukada Y, Ashikaga T, Miyazawa M, Hirota M, Sakaguchi H, Sasa H, Nishiyama N. (2012)
Prediction of skin sensitization potency of chemicals by human Cell Line Activation Test (h-CLAT)
and an attempt at classifying skin sensitization potency. Toxcol. In Vitro, 26, 1150-60.
7. OECD (2016) OECD Guideline for the Testing of Chemicals No. 442E: In Vitro Skin Sensitization:
human

Cell

Line

Activation

Test(h-CLAT).

Availableat:

http://www.oecd-

ilibrary.org/environment/test-no-442e-in-vitro-skin-sensitisation_9789264264359-en

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