⓶⭵ឤసᛶヨ㦂ホ౯ሗ࿌᭩Direct Peptide Reactivity Assay (DPRA)㸸
࣌ࣉࢳࢻ⤖ྜᛶヨ㦂ᖹᡂ 27 ᖺ 2 ᭶ 6 ᪥

JaCVAM ⓶⭵ឤసᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍

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JaCVAM ⓶⭵ឤసᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍
ጤဨ㛗 ⟄஭ᑦஂ㸦⏣㎶୕⳻〇⸆ᰴᘧ఍♫㸧ጤ ဨ 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┠࡛࠶ࡾࠊᚑ᮶ࠊࣔࣝࣔࢵࢺ
ࡸ࣐࢘ࢫࢆ⏝࠸ࡿື≀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 ヨ㦂ࡢ௦᭰ἲࡀ᥎ዡࡉࢀ࡚࠾ࡾࠊື≀ᐇ㦂࡟ࡼࡾᏳ඲ᛶホ౯ࡉࢀࡓᡂศࢆྵࡴ໬⢝ရࡢ㍺ධ㈍኎ࡀ⚗Ṇࡉࢀࡓࡇ࡜࠿ࡽ㸦2013 ᖺ 3 ᭶඲㠃᪋⾜㸧ࠊື≀ࢆ⏝࠸࡞࠸ in vitro ヨ㦂ἲࡢ㛤Ⓨࡀᙉࡃᮃࡲࢀ࡚࠸ࡿࠋDirect Peptide Reactivity Assay㸦DPRA㸸࣌ࣉࢳࢻ⤖ྜᛶヨ㦂㸧ࡣឤసᛶᡂ❧ึᮇẁ㝵ࡢ཯ᛂ࡛࠶ࡿࢱࣥࣃࢡ㉁࡜໬Ꮫ≀㉁ࡢ⤖ྜ཯ᛂࢆᶵჾศᯒ࡟ࡼࡗ࡚ホ౯ࡍࡿᡭἲ࡛࠶ࡿࠋᮏሗ࿌᭩ࡣࠊࡇࡢ DPRA ࡟ࡘ࠸࡚ࠊ
ࡑࡢᡭ㡰ࡸ᭷⏝ᛶ࡜㝈⏺ࢆ The European Union Reference Laboratory for Alternatives to Animal
Testing㸦 EURL ECVAM㸧࡟ࡼࡾᐇ᪋ࡉࢀࡓࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩ཬࡧ EURL ECVAM

Scientific Advisory Committee㸦ESAC㸧➨୕⪅ホ౯㸦ࣆ࢔ࣞࣅ࣮ࣗ㸧ሗ࿌᭩࡞࡝ࢆᇶ࡟సᡂ
ࡋࡓࡶࡢ࡛࠶ࡿࠋDPRA ࡣࢩࢫࢸ࢖ࣥྵ᭷࣌ࣉࢳࢻ㸦Ac-RFAACAA-COOH㸧࡜⿕㦂≀㉁ཬࡧࣜࢪࣥྵ᭷࣌
ࣉࢳࢻ㸦Ac-RFAAKAA-COOH㸧࡜⿕㦂≀㉁ࢆࡑࢀࡒࢀΰྜ࣭཯ᛂࡉࡏࠊ24 ᫬㛫ᚋ࡟࠾ࡅࡿ
ᮍ཯ᛂࡢ࣌ࣉࢳࢻ㔞ࢆᇶ࡟⿕㦂≀㉁ࡢ཯ᛂᛶࢆศ㢮ࡍࡿᡭἲ࡛࠶ࡾࠊ㧗㏿ᾮయࢡ࣐ࣟࢺࢢ
ࣛࣇ࢕࣮㸦HPLC㸧ศᯒࡀྍ⬟࡞᪋タ࡛࠶ࢀࡤᐜ᫆࡟ᐇ᪋ࡍࡿࡇ࡜ࡀ࡛ࡁࡿࠋᮏヨ㦂ἲࡢඛ⾜ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟࠾ࡅࡿ᪋タෆ෌⌧ᛶࡣ 73%-100%࡛࠶ࡾࠊ3 ᪋タ୰ 1 ᪋タ࡟࠾࠸࡚ࠊ
GHS ༊ศ 1B㸦ᙅ࠸ឤసᛶ≀㉁㸧ࡢ≀㉁࡛෌⌧ᛶࡀప࠿ࡗࡓࡓࡵࠊ㐩ᡂᇶ‽㸦85㸣㸧࡟㐩ࡋ࡚࠾ࡽࡎࠊᙅ࠸ឤసᛶ≀㉁࡛ࡣุᐃࡀࡪࢀࡿᠱᛕࡀ࠶ࡗࡓࠋ୍᪉ࠊ᪋タ㛫෌⌧ᛶࡣ 75%࡛࠶ࡾࠊ㐩ᡂᇶ‽㸦80㸣㸧ࢆ‶ࡓࡉ࡞࠿ࡗࡓࡀࠊ㐺⏝⠊ᅖእࡢ㔠ᒓሷࢆ㝖࠸ࡓሙྜ࡟㐩ᡂᇶ‽ࢆୖᅇࡗࡓࠋ
᥋ゐ⓶⭵⅖ࡢࣜࢫࢡࢆື≀࡛ணࡍࡿヨ㦂ἲ࡜ࡋ࡚▱ࡽࢀ࡚࠸ࡿ࣐࢘ࢫࢆ⏝࠸ࡿᒁᡤࣜ

ࣥࣃ⠇ヨ㦂㸦Local Lymph Node Assay: LLNA㸧ࡢヨ㦂⤖ᯝࢆཧ↷ࡋࠊඛ⾜ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟࠾ࡅࡿṇ☜ᗘ㸦Accuracy㸧ࡣ 78㸣ࠊឤᗘ㸦Sensitivity㸧ࡣ 71㸣ࠊ≉␗ᗘ㸦Specificity㸧ࡣ
92%࡜ሗ࿌ࡉࢀ࡚࠸ࡿࠋᮏヨ㦂㐺⏝እࡢ㔠ᒓሷࢆ㝖࠸ࡓሙྜ࡟࡛ࡶឤᗘࡣ⣙ 75㸣࡛࠶ࡾࠊ㝜ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓሙྜࡣࠊഇ㝜ᛶࡢྍ⬟ᛶࢆ⪃៖ࡋࠊ⿵᏶ࡋᚓࡿ௚ࡢヨ㦂ἲ࡟ࡼࡾ☜ㄆࡋ࡞ࡅࢀࡤ࡞ࡽ࡞࠸ࠋDPRA ࡣឤసᛶⓎ⌧ᶵᗎ࡟࠾ࡅࡿึᮇࡢ㔜せ࡞࢖࣋ࣥࢺ࡛࠶ࡿࢱࣥࣃࢡ㉁࡜໬Ꮫ≀㉁ࡢ
⤖ྜ཯ᛂࢆ᳨ฟࡋ࡚࠾ࡾࠊ໬Ꮫ≀㉁ࡢឤసᛶࢆุ᩿ࡍࡿୖ࡛㔜せ࡞᝟ሗࢆ୚࠼࡚ࡃࢀࡿࠋ
LLNA ࡢ 1/10 ⛬ᗘࡢ⤒㈝࡛ᐇ᪋ྍ⬟࡛࠶ࡾࠊື≀ࢆ⏝࠸࡞࠸ in chemico ヨ㦂ἲ࡛࠶ࡿࡇ࡜
࠿ࡽࠊ᭷⏝ᛶࡣ㧗࠸ࠋࡋ࠿ࡋ࡞ࡀࡽࠊᮏἲࡣ௦ㅰ⣔ࢆḞࡃ໬Ꮫⓗヨ㦂ἲ࡛࠶ࡾࠊάᛶ໬࡟
௦ㅰ⣔ࡸ㠀⏕≀ⓗάᛶ໬ࢆᚲせ࡜ࡍࡿឤసᛶ≀㉁ࠊᙅ࠸ឤసᛶ≀㉁ࡸ㔠ᒓሷࠊ␯Ỉᛶࡢ㧗࠸≀㉁࡞࡝ࡣṇࡋࡃࡑࡢឤసᛶࡀ᳨ฟࡉࢀ࡞࠸ྍ⬟ᛶࡀ࠶ࡿࠋ௨ୖࡢ஦ᐇࢆ㋃ࡲ࠼ࠊ㈨ᩱ
⦅⧩ጤဨ఍ࡣࠊドᣐࡢ㔜ࡳ௜ࡅࡸ௚ࡢヨ㦂ἲ㸦LLNAࠊࣔࣝࣔࢵࢺࢆ⏝࠸ࡿ⓶⭵ឤసᛶヨ㦂㸧࡜⤌ࡳྜࢃࡏ࡛ࡢホ౯ࢆ᥎ዡࡍࡿࠋ

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㸯㸬⥴ゝ
 ⓶⭵ឤసᛶࢆホ౯ࡍࡿࡇ࡜ࡣ໬Ꮫ≀㉁ࡢᏳ඲ᛶホ౯࡟࠾࠸࡚㔜せ࡛࠶ࡿࠋ໬Ꮫ≀㉁ࡢ⓶⭵࡛ࡢ᥋ゐ⓶⭵⅖ࡢࣜࢫࢡࢆື≀࡛ணࡍࡿヨ㦂ἲ࡜ࡋ࡚ࣔࣝࣔࢵࢺࢆ⏝࠸ࡿ⓶⭵ឤసᛶ

ヨ㦂㸦OECD TG406㸧ࡸ࣐࢘ࢫࢆ⏝࠸ࡿᒁᡤࣜࣥࣃ⠇ヨ㦂㸦Local Lymph Node Assay: LLNA,
OECD TG429㸧ࡀ࠶ࡿࠋࡇࡢ[3H-Methyl]-thymidine ྲྀ㎸㔞ࢆ఩ඖ⣲㸦RI㸧ࢆ⏝࠸ࡎ ATP 㔞ࢆ㔞ࢆᐃࡍࡿ LLNA ௨እ࡟ᨺᑕᛶྠ

ᐃࡍࡿ LLNA:DA㸦OECD TG442A㸧ࡸ Bromodeoxyuridine

ᐃࡍࡿ LLNA:BrdU-ELISA㸦OECD TG442B㸧ࡀ࠶ࡿࠋࡇࡢࡼ࠺࡟ࠊ⌧ᅾ OECD ࠿ࡽ࢞࢖ࢻࣛ࢖ࣥ࡜ࡋ࡚බ⾲ࡉࢀ࡚࠸ࡿヨ㦂ἲࡣࠊື≀ࢆ⏝࠸ࡓ in vivo ࡢヨ㦂ἲࡢࡳ࡛࠶ࡿࠋ
 EU ࡟࠾ࡅࡿḢᕞ໬Ꮫရつ๎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 ヨ㦂➼࡟ࡼࡿ௦᭰ἲࡀ᥎
ዡࡉࢀ࡚࠾ࡾࠊື≀ᐇ㦂࡟ࡼࡾᏳ඲ᛶࡀホ౯ࡉࢀࡓᡂศࢆྵࢇࡔ໬⢝ရࡢ㍺ධཬࡧ㈍኎ࡀ
⚗Ṇࡉࢀࡓ㸦2013 ᖺ 3 ᭶඲㠃᪋⾜㸧ࠋࡑࡢࡓࡵࠊ໬Ꮫ≀㉁ࡢ⓶⭵ឤసᛶࢆホ౯ࡍࡿ௦᭰ἲࡢ
㛤Ⓨࡀᙉࡃồࡵࡽࢀ࡚࠸ࡿࠋ ⌧ᅾࠊ࣌ࣉࢳࢻ࡜ࡢ⤖ྜ཯ᛂࢆ฼⏝ࡋࡓ Direct Peptide Reactivity Assay㸦DPRA㸸࣌ࣉࢳࢻ⤖ྜᛶヨ㦂㸧
ࠊ༢⌫⣔⣽⬊ࡢάᛶ໬ࢆ฼⏝ࡋࡓ human Cell Line Activation Test㸦h-CLAT㸧ཬࡧ Myeloid U937 Skin Sensitization Test㸦MUSST㸧ࠊࢣࣛࢳࣀࢧ࢖ࢺ⣽⬊⣔ࡢᶆⓗ㑇ఏᏊࢆ⏝࠸ࡓ ARE-Nrf2 Luciferase Test Method㸦KeratinoSensτἲ㸧࡞࡝ࡢ⓶⭵ឤసᛶヨ㦂ࡢື≀ࢆ⏝࠸ ࡞ ࠸ື ≀ᐇ 㦂 ௦᭰ ἲࡀ ᥦ ᱌ࡉ ࢀ࡚ ࠾ ࡾࠊ The European Union Reference Laboratory for
Alternatives to Animal Testing㸦EURL ECVAM㸧➼࡟࠾࠸࡚ඛ⾜ࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀ⾜ࢃࢀ࡚࠸ࡿࠋ DPRA ࡣࠊ໬Ꮫ≀㉁ࡀ࣌ࣉࢳࢻ࡜ඹ᭷⤖ྜࡋච␿ᛂ⟅ࢆᘬࡁ㉳ࡇࡍᢠཎ࡜࡞ࡿᛶ㉁ࢆ฼⏝ࡋࠊᮍ཯ᛂࡢࢩࢫࢸ࢖ࣥᡈ࠸ࡣࣜࢪࣥࢆྵࡴ࣌ࣉࢳࢻࢆ㧗㏿ᾮయࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮
㸦HPLC㸧࡛ᐃࡍࡿヨ㦂ἲ࡛࠶ࡿࠋᮏἲࡢඛ⾜ࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡢ⤖ᯝ࡟ࡘ࠸࡚ࡣࠊ

EURL ECVAM Scientific Advisory Committee㸦ESAC㸧࡟ࡼࡿ➨୕⪅ホ౯㸦ࣆ࢔ࣞࣅ࣮ࣗ㸧ࡀ᏶஢ࡋ࡚࠸ࡿࠋJaCVAM ⓶⭵ឤసᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍㸦௨ୗࠊጤဨ఍㸧ࡀ DPRA ࡢ⓶⭵ឤసᛶヨ㦂௦᭰ἲ࡜ࡋ࡚ࡢ⛉Ꮫⓗጇᙜᛶ࡟ࡘ࠸࡚ࠊ⌧ᅾࡲ࡛࡟බ㛤ࡉࢀ࡚࠸ࡿ᝟ሗࢆࡶ࡜࡟ホ౯ࡋࡓࡢ࡛ࠊࡑࡢ⤖ᯝࢆሗ࿌ࡍࡿࠋ
㸰㸬ヨ㦂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⭵ឤసᛶࡣḟࡢ 4 ࡘࡢ Key event ࠿ࡽᡂࡿ࡜ࡉࢀ࡚࠸ࡿ 1)ࠋ
1㸧໬Ꮫ≀㉁࡜ࢱࣥࣃࢡ㉁ࡢࢩࢫࢸ࢖ࣥṧᇶ࠶ࡿ࠸ࡣࣜࢪࣥṧᇶ࡜ࡢඹ᭷⤖ྜ
2 㸧 ࢣ ࣛ ࢳ ࣀ ࢧ ࢖ ࢺ ࡟ ࠾ ࡅ ࡿ ⅖ ⑕ ᛶ ᛂ ⟅ ཬ ࡧ Antioxidant/electrophile response element(ARE)-dependent pathway ࡟ࡼࡿ㑇ఏᏊⓎ⌧
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3㸧ᶞ≧⣽⬊ࡢάᛶ໬㸦≉␗ⓗ⣽⬊⾲㠃࣐࣮࣮࢝ࡢⓎ⌧ࠊࢣࣔ࢝࢖ࣥࡸࢧ࢖ࢺ࢝࢖ࣥࡢ⏘⏕㸧
4㸧ࣜࣥࣃ⠇࡟࠾ࡅࡿ T ⣽⬊ࡢቑṪ
DPRA ࡣࠊୖ㏙ࡋࡓ⓶⭵ឤసᛶࡢ AOP ࡟࠾ࡅࡿࠗ໬Ꮫ≀㉁࡜ࢱࣥࣃࢡ㉁ࡢඹ᭷⤖ྜ࠘࡟ᑐᛂࡋࡓື≀ࢆ⏝࠸࡞࠸ in chemico ヨ㦂࡛࠶ࡿࠋ௚࡟ࡶ in chemico ࡢ⓶⭵ឤసᛶヨ㦂ࡀ࠸ࡃࡘ࠿ሗ࿌ࡉࢀ࡚࠸ࡿ 2)-4)ࠋ
DPRA ࡛ࡣࠊ⓶⭵ෆࡢࢱࣥࣃࢡ㉁ࡢ௦ࢃࡾ࡟ྜᡂ࣌ࣉࢳࢻ࡛࠶ࡿࢩࢫࢸ࢖ࣥྵ᭷࣌ࣉࢳࢻ
㸦Ac-RFAACAA-COOH㸧࡜ࣜࢪࣥྵ᭷࣌ࣉࢳࢻ㸦Ac-RFAAKAA-COOH㸧ࡢ 2 ✀㢮ࢆ౑⏝ࡍࡿࠋ໬Ꮫ≀㉁࡜ࡑࢀࡒࢀࡢ࣌ࣉࢳࢻࢆΰྜࡋࠊ཯ᛂࡉࡏࠊΰྜ 24 ᫬㛫ᚋ࡟࠾ࡅࡿᮍ཯ᛂࡢ
࣌ࣉࢳࢻ㔞ࢆ㧗㏿ᾮయࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮㸦HPLC㸧࡛ศ㞳ᐃ㔞ࡍࡿࠋࡑࡢ⤖ᯝࢆᇶ࡟ࠊ໬
Ꮫ≀㉁ࡢ཯ᛂᛶࢆ 4 ẁ㝵㸦High, Moderate, Low, Minimal㸧࡟ศ㢮ࡍࡿࠋ㸱㸬ヨ㦂ᡭ㡰㸭ุᐃ 5)
3-1. ౑⏝ࡍࡿ࣌ࣉࢳࢻ࠾ࡼࡧ㝧ᛶᑐ↷ࡢㄪ〇࣭ࢩࢫࢸ࢖ࣥྵ᭷࣌ࣉࢳࢻ/ࣜࣥ㓟⦆⾪⁐ᾮ㸦ࢩࢫࢸ࢖ࣥྵ᭷࣌ࣉࢳࢻ⁐ᾮ㸧ࢩࢫࢸ࢖ࣥྵ᭷࣌ࣉࢳࢻ㸦Ac-RFAACAA-COOHࠊ⣧ᗘ㸸90-95%㸧ࡣࠊࣜࣥ㓟⦆⾪ᾮ
㸦pH7.5㸧࡟ 0.667 mM ࡢ⃰ᗘ࡟࡞ࡿࡼ࠺࡟⁐ゎࡉࡏࡿࠋ࣭ࣜࢪࣥྵ᭷࣌ࣉࢳࢻ/㓑㓟࢔ࣥࣔࢽ࣒࢘⦆⾪⁐ᾮ㸦ࣜࢪࣥྵ᭷࣌ࣉࢳࢻ⁐ᾮ㸧ࣜࢪࣥྵ᭷࣌ࣉࢳࢻ㸦Ac-RFAAKAA-COOHࠊ⣧ᗘ㸸90-95%㸧ࡣࠊ㓑㓟࢔ࣥࣔࢽ࣒࢘⦆
⾪ᾮ㸦pH10.2㸧࡟ 0.667 mM ࡢ⃰ᗘ࡟࡞ࡿࡼ࠺࡟⁐ゎࡉࡏࡿࠋ࣭㝧ᛶᑐ↷㸸Cinnamic Aldehyde㸦CAS No:104-55-2ࠊ⣧ᗘӍ95%㸧
  Cinnamic Aldehyde ࢆࠊAcetonitrile ࡟ 100 mM ࡢ⃰ᗘ࡛⁐ゎࡉࡏࠊࡑࡢᚋ⿕㦂≀㉁࡜ྠ
ᵝ࡟୧࣌ࣉࢳࢻ⁐ᾮ࡜ΰྜࡍࡿࠋ
3-2. ᪉ἲ௨ୗࡢࣇ࣮ࣟࢳ࣮ࣕࢺ࡟ᚑ࠸ࠊᐇ᪋ࡍࡿࠋ

511

1. ⿕㦂≀㉁ࢆ100mMࡢ⃰ᗘ࡛௨ୗࡢ࠸ࡎࢀ࠿ࡢ፹య࡟⁐ゎࡉࡏࡿ㸦⿕㦂≀㉁⁐ᾮ㸧ࠋ࠙፹యࠚAcetonitrileࠊWaterࠊ Acetonitrile 㸸Water(1:1)ࠊIsopropanolࠊAcetoneࠊ
Acetone : Acetonitrile(1:1)ࠊDMSO: Acetonitrile(1:9)ࠊ
DMSO: Acetonitrile(1:1)
2. ⿕㦂≀㉁⁐ᾮ࡜㺫㺽㺪㺽㺟㺢㺼⁐ᾮࢆΰྜࡍࡿ
2-A. ⿕㦂≀㉁⁐ᾮ࡜㺚㺛㺡㺐㺻ྵ᭷㺫㺽㺪㺽㺟㺢㺼⁐ᾮࢆ10:1࡛ΰྜ(n=3)࣭Sample:⿕㦂≀㉁⁐ᾮ㸩㺚㺛㺡㺐㺻ྵ᭷㺫㺽㺪㺽㺟ࢻ⁐ᾮ࣭Co-elusion Control:⿕㦂≀㉁⁐ᾮ㸩㺶㺻㓟⦆⾪ᾮ࣭Reference Control: ፹య㸩㺚㺛㺡㺐㺻ྵ᭷㺫㺽㺪㺽㺟ࢻ⁐ᾮ
2-B.⿕㦂≀㉁⁐ᾮ࡜ 㺶㺚㺼㺻ྵ᭷㺫㺽㺪㺽㺟㺢㺼⁐ᾮࢆ50:1࡛ΰྜ(n=3)࣭Sample:⿕㦂≀㉁⁐ᾮ㸩㺶㺚㺼㺻ྵ᭷㺫㺽㺪㺽㺟ࢻ⁐ᾮ࣭Co-elusion Control:⿕㦂≀㉁⁐ᾮ㸩㓑㓟㺏㺻㺱㺤㺑㺯⦆⾪ᾮ࣭Reference Control: ፹య㸩㺶㺚㺼㺻ྵ᭷㺫㺽㺪㺽㺟ࢻ⁐ᾮ
3. ྛΰྜᾮࢆ24s2᫬㛫㺐㺻㺕㺋㺫㺼㺎㺢㸦ᬯᐊࠊ25s2.5Υ㸧ࡍࡿ
4. HPLC࡛ศᯒࡍࡿ*5. ᮍ཯ᛂ㺫㺽㺪㺽㺟㺢㺼ࡢPeak㠃✚࠿ࡽࠊ௨ୗࡢᘧ࡟ࡼࡾ
㺫㺽㺪㺽㺟㺢㺼ῶᑡ⋡㸦Percent Peptide Depletion㸧ࢆ⟬ฟ ࡍࡿ
Percent Peptide Depletion=

1--

Peptide Peak Area in Sample
Mean Peptide Peak Area in Reference Controls

100*ศᯒ᮲௳㸦᥎ዡ㸧᥎ዡ࣒࢝ࣛ㸸Zorbax SB-C18 㸦3.5 ȣm, 2.1mm×100mm㸧➼ᗘ㸸30Υ
UV ᳨ฟἼ㛗㸸220 nm
ὶ㏿㸸0.35 mL/min⛣ື┦㸸 㸦A㸧0.1%ࢺࣜࣇࣝ࢜ࣟ㓑㓟Ỉ
㸦B㸧0.085%ࢺࣜࣇࣝ࢜ࣟ㓑㓟࢔ࢭࢺࢽࢺࣜࣝ
᫬㛫㸦ศ㸧

A%

B%

0

90

10

10

75

25

11

10

90

13

10

90

13.5

90

10

3-3. ヨ㦂ᡂ❧ࡢ᮲௳1) ヨ㦂ᡂ❧࡟ࡣࠊ௨ୗࡢ᮲௳ࢆ‶ࡓࡉ࡞ࡅࢀࡤ࡞ࡽ࡞࠸ࠋ
࣭ࢩࢫࢸ࢖ࣥྵ᭷࣌ࣉࢳࢻ࠾ࡼࡧࣜࢪࣥྵ᭷࣌ࣉࢳࢻࡢࡑࢀࡒࢀࢆ⏝࠸࡚ࠊ
0.0167~0.534mM ࡢ⠊ᅖࡢ 6 ⃰ᗘ࡟࡚ᶆ‽᭤⥺ࡢసᡂࢆ⾜࠸ࠊࡑࡢ┦㛵ಀᩘࡀ 0.99 ࡼࡾ኱ࡁࡃ࡞ࡅࢀࡤ࡞ࡽ࡞࠸ࠋ
࣭㝧ᛶᑐ↷࡛࠶ࡿ Cinnamic aldehyde ࡢ⤖ᯝࡣࠊࢩࢫࢸ࢖ࣥྵ᭷࣌ࣉࢳࢻ࡛ࡣ 3 ᅇࡢ⧞ࡾ㏉ࡋ࡟ࡼࡿᖹᆒ࣌ࣉࢳࢻῶᑡ⋡ࡣ 60.8%~100%࡛ࠊᶆ‽೫ᕪࡣ 14.9%ࡼࡾᑠࡉࡃࠊࣜࢪࣥྵ
612

᭷࣌ࣉࢳࢻ࡛ࡣ 3 ᅇ⧞ࡾ㏉ࡋ࡟ࡼࡿᖹᆒ࣌ࣉࢳࢻῶᑡ⋡ࡣ 40.2%~69.0%࡛ࠊᶆ‽೫ᕪࡣ
11.6%ࡼࡾᑠࡉࡃ࡞ࡅࢀࡤ࡞ࡽ࡞࠸ࠋ
࣭ࡑࢀࡒࢀࡢ࣌ࣉࢳࢻࡈ࡜࡟ 3 ✀㢮ࡢ Reference Control
㸦A, B, C㸧ࢆタࡅࡿࠋReference Control
A㸦n=3㸧ࡣࠊศᯒ๓ࡢ HPLC ࢩࢫࢸ࣒㐺ྜᛶࡢ☜ㄆࡢࡓࡵࡢࡶࡢࠊ Reference Control B㸦n=6㸧ࡣศᯒ᫬㛫୰ࡢ Reference Control ࡢᏳᐃᛶࢆ☜ㄆࡍࡿࡓࡵࡢࡶࡢࠊ Reference
Control C㸦n=3㸧ࡣ౑⏝ࡉࢀࡓ⁐፹ࡀ࣌ࣉࢳࢻࡢῶᑡ࡟ᙳ㡪ࡋ࡞࠸ࡇ࡜ࢆ☜ㄆࡍࡿࡓࡵࡢ
ࡶࡢ࡛࠶ࡿࠋReference Control A ࡢᖹᆒ࣌ࣉࢳࢻ⃰ᗘࡣ 0.50s0.05mM ࡜࡞ࡾࠊ9 ࡘࡢ
Reference Control B ࠾ࡼࡧ C ࡛ࡣ࣌ࣉࢳࢻࣆ࣮ࢡ㠃✚ࡢኚືಀᩘࡀ 15.0%ࡼࡾᑠࡉࡃ࡞ࡅ
ࢀࡤ࡞ࡽ࡞࠸ࠋ
2) ⿕㦂≀㉁ࢆྵࡴ Sample ࡢ⤖ᯝ࡛ࡣࠊ௨ୗࡢ᮲௳ࡀ‶ࡓࡉࢀ࡞ࡅࢀࡤ࡞ࡽ࡞࠸
࣭⧞ࡾ㏉ࡋ࡛⾜࠺ศᯒ್ࡢ᭱኱ᶆ‽೫ᕪࡣࠊࢩࢫࢸ࢖ࣥྵ᭷࣌ࣉࢳࢻῶᑡ⋡࡛ࡣ 14.9%ࡼ
ࡾᑠࡉࡃࠊࣜࢪࣥྵ᭷࣌ࣉࢳࢻῶᑡ⋡࡛ࡣ 11.6%ࡼࡾᑠࡉࡃ࡞ࡅࢀࡤ࡞ࡽ࡞࠸ࠋ
࣭3 ࡘࡢ Reference Control C ࡢᖹᆒ⃰ᗘࡣ 0.50s0.05mM ࡛࡞ࡅࢀࡤ࡞ࡽ࡞࠸ࠋ
3-4. ホ౯⿕㦂≀㉁ࡢ཯ᛂᛶࡣࠊ

ᐃࡈ࡜ࡢ࣌ࣉࢳࢻῶᑡ⋡㸦Percent Peptide Depletion㸧࠿ࡽࠊᖹᆒ

್ࢆ⟬ฟࡋࠊ௨ୗࡢ DPRA ศ㢮ணࣔࢹࣝ㸦⾲ 1㸧࡟ᚑࡗ࡚཯ᛂᛶࢆศ㢮ࡍࡿ 5)ࠋ཯ᛂᛶࡢ

ศ㢮࡛ࠊLowࠊModerate ࠾ࡼࡧ High ࡟ศ㢮ࡉࢀࡿ໬Ꮫ≀㉁ࡣ㝧ᛶࠊNo or Minimal ࡟ศ㢮ࡉࢀࡿ໬Ꮫ≀㉁ࡣ㝜ᛶ࡜ணࡍࡿ 6)ࠋࡕ࡞ࡳ࡟ࠊ࣌ࣉࢳࢻ࡜⿕㦂≀㉁ࡢ⁐ฟ᫬㛫ࡀ㔜࡞ࡗࡓሙྜ㸦Co-elution㸧ࠊ࣌ࣉࢳࢻῶᑡ⋡㸦Percent Peptide Depletion㸧ࡢ⟬ฟࡀ୙ྍ⬟࡜࡞ࡿࡀࠊࡑ
ࡢ⁐ฟ᫬㛫ࡢ㔜࡞ࡾࡀࠊࣜࢪࣥྵ᭷࣌ࣉࢳࢻ࡛ࡢࡳㄆࡵࡓሙྜ࡟ࡣࠊࢩࢫࢸ࢖ࣥྵ᭷࣌ࣉ
ࢳࢻࡢ⤖ᯝ࠿ࡽࠕࢩࢫࢸ࢖ࣥ 1:10 ࡢࡳࡢண⾲1ศ㢮ணࣔࢹࣝࠖ࡟ᚑ࠸ࠊ཯ᛂᛶࢆศ㢮ࡋࠊண

ࡍࡿࠋ

ࣔࢹࣝ

ࢩࢫࢸ࢖ࣥ 1:10 ࠾ࡼࡧࣜࢪࣥ 1:50 ࡢணࣔࢹࣝࢩࢫࢸ࢖ࣥࡢῶᑡ⋡࡜ࣜࢪࣥࡢῶᑡ⋡ࡢᖹᆒ್

DPRA ண

཯ᛂᛶࡢศ㢮

0%ӌῶᑡ⋡ࡢᖹᆒ್ӌ6.38%

No or Minimal㝜ᛶ

6.38%<ῶᑡ⋡ࡢᖹᆒ್ӌ22.62%

Low

㝧ᛶ

22.62%<ῶᑡ⋡ࡢᖹᆒ್ӌ42.47%

Moderate

42.47%<ῶᑡ⋡ࡢᖹᆒ್ӌ100%

Highࢩࢫࢸ࢖ࣥ 1:10 ࡢࡳࡢணࣔࢹࣝࢩࢫࢸ࢖ࣥࡢῶᑡ⋡DPRA ண

཯ᛂᛶࡢศ㢮

0%ӌῶᑡ⋡ӌ13.89%

No or Minimal㝜ᛶ

13.89%<ῶᑡ⋡ӌ23.09%

Low

㝧ᛶ

23.09%<ῶᑡ⋡ӌ98.24%

Moderate

98.24%<ᖹᆒῶᑡ⋡ӌ100%

High

7

13

㸲㸬⢭ᗘ
ᮏἲࡣࠊEURL ECVAM ࡟࠾࠸࡚ࠊ⾲ 2 ࡟♧ࡍ 24 ≀㉁ࢆ⏝࠸࡚ඛ⾜ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡀᐇ᪋ࡉࢀࠊᢏ⾡⛣㌿ᛶࠊ᪋タෆ෌⌧ᛶཬࡧ᪋タ㛫෌⌧ᛶࡀホ౯ࡉࢀ࡚࠸ࡿࠋ
4-1. ᢏ⾡⛣㌿ᛶ
 16 ≀㉁ࢆ⏝࠸࡚୺ᑟ᪋タࡢ P㸤G ♫࠿ࡽ Ricerca ♫࡜ IVMU㸦EURL ECVAM ࡢ◊✲ࣘࢽࢵࢺ㸧࡬ࡢᢏ⾡⛣㌿ᛶ࡟ࡘ࠸࡚ホ౯ࡀ⾜ࢃࢀ࡚࠸ࡿࠋࡑࡢ⤖ᯝࠊタഛࡀᩚࡗ࡚࠸࡚カ⦎ࡉࢀࡓࢫࢱࢵࣇࡀᒃࢀࡤ 3 ᪥㛫ࡢࢺ࣮ࣞࢽࣥࢢ࡛ P㸤G ♫࠿ࡽ Ricerca ♫࡜ IVMU ࡬ࡢ DPRAࡢᢏ⾡⛣㌿ࡀྍ⬟࡛࠶ࡗࡓࠋࡲࡓࠊDPRA ࡣ㞴ࡋ࠸᧯సᡭ㡰ࢆᚲせ࡜ࡏࡎࠊ⿦⨨ࡸヨ⸆ࡣ㉎ධྍ⬟࡛࠶ࡾࠊᶆ‽᧯సᡭ㡰᭩㸦Standard Operating Procedure: SOP㸧ࡀヲ⣽࡟సᡂࡉࢀ࡚࠸ࡿ
ࡇ࡜࠿ࡽࠊጤဨ఍ࡣࠊᮏ㑥࡛ࡣỗ⏝ HPLC ཬࡧࡑࡢᢏ⾡ࢆಖ᭷ࡍࡿ᪋タ࡛ᐜ᫆࡟ᐇ᪋ྍ⬟࡜⪃࠼ࡿࠋ
4-2. ᪋タෆ෌⌧ᛶ㸦⾲ 3-1㸧
 15 ≀㉁ࢆ⏝࠸ࡓ 3 ᪋タࡢ᪋タෆ࡛ࡢ⓶⭵ឤసᛶ㸦S㸧࡜㠀ឤసᛶ㸦NS㸧ࡢ୍⮴ᗘࡣࠊP&G♫㸸73.3%, Ricerca ♫㸸100%, IVMU㸸86.7%࡛࠶ࡗࡓࠋ㐩ᡂᇶ‽ࡣ 85㸣࡟タᐃࡉࢀ࡚࠾ࡾࠊ
P㸤G ♫ࡣࡇࡢ್ࢆୗᅇࡗࡓࠋP&G ♫ࡣࠊGHS ༊ศ 1A ࡢ 6 ≀㉁ࡣࡍ࡭࡚෌⌧ᛶࡢ࠶ࡿ⤖ᯝ࡛࠶ࡗࡓࡀࠊ1B ࡢ 3 ≀㉁୰ 2 ≀㉁㸦Benzyl Salicylate ཬࡧ R(+)Limonene㸧࡛෌⌧ᛶࡀᚓࡽࢀ
࡞࠿ࡗࡓࡓࡵࠊ㐩ᡂᇶ‽ࢆୗᅇࡗࡓ࡜⪃࠼ࡽࢀࡓࠋࡲࡓࠊMinimal, Low, Moderate, High ࡢ 4ࢡࣛࢫࡢ୍⮴ᗘࡣ P&G ♫㸸66.7㸣ࠊRicerca ♫㸸100㸣ࠊIVMU㸸73.3%࡛࠶ࡗࡓࠋ
4-3. ᪋タ㛫෌⌧ᛶ㸦⾲ 3-2㸧
 24 ≀㉁ࡢ 3 ᪋タࡢ᪋タ㛫෌⌧ᛶࡣ⓶⭵ឤసᛶ㸦S㸧࡜㠀ឤసᛶ㸦NS㸧ࡢ୍⮴ᗘ࡛ࡣ 75%࡜࡞ࡾ㐩ᡂᇶ‽ࡢ 80%ࢆୗᅇࡗࡓࠋ24 ≀㉁ࡢ୰࡟ࡣ㔠ᒓሷ࡛࠶ࡿ Beryllium sulfate ཬࡧ
Nickel chloride ࡀྵࡲࢀ࡚࠾ࡾࠊ㔠ᒓሷࡣᑐ㇟࣌ࣉࢳࢻ࡜ࡢඹ᭷⤖ྜࢆ⏕ࡌ࠺ࡿᐁ⬟ᇶࢆᣢࡓ࡞࠸ࡇ࡜࠿ࡽࠊᮏἲࡢ㐺⏝⠊ᅖእ࡜ุ᩿ࡉࢀࡿࡓࡵ㺂ࡇࢀࡽ 2 ≀㉁ࢆ㝖࠸ࡓሙྜࡢ᪋タ㛫෌⌧ᛶࡣ 82%࡛࠶ࡗࡓࠋࡲࡓࠊ24 ≀㉁࡛ࡢ Minimal, Low, Moderate, High ࡢ 4 ࢡࣛࢫ࡛ࡣࠊ᪋タ㛫෌⌧ᛶࡣ 62.5%࡛࠶ࡗࡓࠋ
㸳㸬ṇ☜ᗘ㸦ឤᗘཬࡧ≉␗ᗘ㸧⢭ᗘ࡜ྠᵝ࡟ࠊ⾲ 2 ࡟♧ࡍ 24 ≀㉁ࢆ⏝࠸ࡓ EURL ECVAM ࡢඛ⾜ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡢᡂ⦼ࢆᇶ࡟᪤▱ࡢ LLNA ࡢᡂ⦼࡜ࡢ୍⮴ᗘࠊឤᗘཬࡧ≉␗ᗘࡀホ౯ࡉࢀ࡚࠸ࡿࠋ
 24 ≀㉁඲࡚ࡢᡂ⦼ࢆᇶ࡟ホ౯ࢆ⾜ࡗࡓሙྜࠊᐇ᪋ 3 ᪋タࡢᡂ⦼ࡢ✚⟬࡟ࡼࡿឤᗘ
㸦Sensitivity㸧ࡣ 70.8%ࠊ≉␗ᗘ㸦Specificity㸧ࡣ 91.7%ࠊṇ☜ᗘ㸦Accuracy㸧ࡣ 77.8%࡛࠶ࡗࡓࠋヨ㦂᪋タẖࡢᡂ⦼ࡣࠊヨ㦂᪋タ 1㸦P&G㸧࡛ࡣឤᗘࡣ 68.8%ࠊ≉␗ᗘࡣ 100%ࠊṇ☜ᗘ
ࡣ 79.2㸣ࠊヨ㦂᪋タ 2㸦Ricerca㸧࡛ࡣឤᗘࡣ 68.8%ࠊ≉␗ᗘࡣ 100%ࠊṇ☜ᗘࡣ 79.2㸣ࠊヨ㦂᪋タ 3㸦IVMU㸧࡛ࡣឤᗘࡣ 75㸣ࠊ≉␗ᗘࡣ 75%ࠊṇ☜ᗘࡣ 75㸣࡛࠶ࡗࡓࠋࡲࡓࠊᮏἲ
ࡢṇ☜ᗘ࡟㛵ࡋ࡚㛤Ⓨ᪋タ࡛࠶ࡿ P&G ࠿ࡽᥦฟࡉࢀࡓࣄࢫࢺࣜ࢝ࣝࢹ࣮ࢱ࡛ࡣ 86㸣ࠊ᪤ሗ8

14

࡛ࡣ 80㸣㹼89㸣࡜ሗ࿌ࡉࢀ࡚࠸ࡿࠋ
24 ≀㉁ࡢヨ㦂≀㉁ࡢ୰࡟ࡣ㔠ᒓሷ࡛࠶ࡿ Beryllium sulfate ཬࡧ Nickel chloride ࡀྵࡲࢀ࡚
࠾ࡾࠊ㔠ᒓሷࡣᑐ㇟࣌ࣉࢳࢻ࡜ࡢඹ᭷⤖ྜࢆ⏕ࡌ࠺ࡿᐁ⬟ᇶࢆᣢࡓ࡞࠸ࡇ࡜࠿ࡽࠊᮏἲࡢ
㐺⏝⠊ᅖእ࡜ุ᩿ࡉࢀࡿࡓࡵ㺂ࡇࢀࡽࢆ㝖ࡃ 22 ≀㉁࡟ࡼࡿホ౯ࢆ⾜ࡗࡓሙྜࠊᐇ᪋ 3 ᪋タࡢᡂ⦼ࡢ✚⟬࡟ࡼࡿឤᗘࡣ 76.2%ࠊ≉␗ᗘࡣ 91.7%ࠊṇ☜ᗘࡣ 81.8%࡜࡞ࡿࠋ㐺⏝⠊ᅖእ࡜⪃࠼ࡽࢀࡿ㔠ᒓሷࢆ㝖እࡋࡓሙྜ࡛ࡶࠊ඲࡚࠶ࡿ࠸ࡣ୍㒊ࡢ᪋タ࡛ 5 ≀㉁㸦DihydroeugenolࠊChlorpromazine HClࠊBenzylsalicylateࠊBenzylcinnamateࠊR㸦+㸧-Limonene㸧ࡀഇ㝜ᛶ࡜ホ౯ࡉࢀࡿࡀࠊࡇࢀࡽࡢ≀㉁ࡢ୰࡛ Dihydroeugenol ཬࡧ Limonene ࡣࣉࣟࣁࣉࢸ
ࣥ࠶ࡿ࠸ࡣࣉࣞࣁࣉࢸࣥ࡜ࡋ࡚▱ࡽࢀ࡚࠾ࡾࠊᮏヨ㦂⣔ࡀ௦ㅰ⣔ࢆ᭷ࡉ࡞࠸༢⣧࡞໬Ꮫ཯ᛂࢆ᳨ฟࡍࡿヨ㦂⣔࡛࠶ࡿࡇ࡜ࡀഇ㝜ᛶ࡜࡞ࡿ୍ᅉ࡜ᛮࢃࢀࡿࠋ
࡞࠾ࠊ඲࡚ᡈ࠸ࡣ୍㒊ࡢ᪋タ࡛ഇ㝜ᛶ࡜ุᐃࡉࢀࡿ Chlorpromazine HClࠊBenzylsalicylate
ཬࡧ Benzylcinnamate ࡢ 3 ≀㉁ࠊ୍㒊ࡢ᪋タ࡛ഇ㝧ᛶ࡜ุᐃࡉࢀࡿ Benzyl alcohol ཬࡧMethylsalicylate ࡢ 2 ≀㉁࡟ࡘ࠸࡚ࠊࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩࡛ࡣࠊࡑࡢ⌮⏤࡟ࡘ࠸࡚᫂☜࡞ㄝ᫂ࡣ࡞ࡉࢀ࡚࠸࡞࠸ࠋࡋ࠿ࡋࠊࡇࢀࡽࡢ୰࡛ഇ㝜ᛶ࡜ุᐃࡉࢀࡿ 3 ≀㉁ࡢ cLogP ࡢ್ࡣ࠸ࡎࢀࡶ 3.2 ௨ୖ࡛࠶ࡾࠊࡑࡢ␯Ỉᛶࡢ㧗ࡉࡀỈ⣔࡛ࡢ཯ᛂࢆᚲせ࡜ࡍࡿᮏἲ࡛ഇ㝜ᛶ࡜࡞ࡿ୍せᅉ࡜᥎ᐹࡉࢀࡿࠋ୍᪉࡛ 2-Mercaptobenzothiazole㸦cLogP㸸3.42㸧ࡣ㧗␯Ỉᛶ࡟ࡶ࠿࠿ࢃ
ࡽࡎṇࡋࡃ㝧ᛶ࡜ุᐃࡉࢀ࡚࠸ࡿࡀࠊࡇࢀࡣᮏ≀㉁ࡢᣢࡘ SH ᇶࡀࣜࢪࣥṧᇶ࡜㧗࠸཯ᛂᛶࢆ᭷ࡍࡿࡇ࡜࡟㉳ᅉࡍࡿ࡜ᛮࢃࢀࡿࠋ

915

⾲ 2 ౪ヨ≀㉁ࣜࢫࢺ 6)
No.

Chemical NameCAS

S tatecLogP*

LLNA

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24

Benzoquinone
4-Phenylenediamine
Kathon CG (1.2% CM I)
Beryllium sulfate
Formaldehyde
Chloramine T
Chlorpromazine HCl
2-M ercaptobenzothiazole
Dihydroeugenol
1-Thioglycerol
Imidazolidinylurea
M ethylmethacrylate
Benzylsalicylate
Nickel chloride
Benzylcinnamate
R(+)-Limonene
Glycerol
2,4-Dichloronitrobenzene
Benzyl alcohol
M ethylsalicylate
Isopropanol
Dimethylisophthalate
4-Aminobenzoic acid
Xylene106-51-4
106-50-3
26172-55-4
7787-56-6
50-00-0
127-65-1
69-09-0
149-30-4
2785-87-7
96-27-539236-46-9
80-62-6
118-58-1
7718-54-9
103-41-3
5989-27-5
56-81-5
611-06-3
100-51-6
119-36-8
67-63-01459-93-4
150-13-0
1330-20-7

Solid
Solid
LiquidSolid
Liquid
Solid
Solid
Solid
LiquidLiquidSolid
LiquidLiquidSolid
Solid
LiquidLiquidSolid
LiquidLiquidLiquidSolid
Solid
Liquid

0.96
0.43
-0.69
4.89
3.42
2.84
-0.5
-1.28
1.14
3.2
3.89
3.01
-1.33
3.06
1.02
1.46
0.38
2.12
0.78
3.01

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

LLNA
potency
category**
extremestrongextremeextremestrongstrongstrongmoderate
moderate
moderateweak
weak
moderateno category
weak
weak
no categoryno categoryno categoryno categoryno categoryno categoryno category
weak

GHS
potency
category
1A
1A
1A
1A
1A
1A
1A
1A
1B
1B
1B
1B
1B
1B
1B
1B
NC
NC
NC
NC
NC
NC
NC
NC

+㸸LLNA 㝧ᛶࠊ-㸸LLNA 㝜ᛶࠊNC㸸GHS ༊ศእࠋ*㸸ChemBio Draw Ultra 11.0 (Cambridge Soft) ࢆ⏝࠸࡚⟬ฟࠋ**㸸ECETOC Technical Report #87 ࡢศ㢮࡟ࡼࡿࠋDirect Peptide Reactivity Assay (DPRA) , ECVAM Validation Study Report 㸦 EUROPEAN
COMMISSION, 2012㸧グ㍕ࡢ᝟ሗࢆᇶ࡟⦅㞟ࠋ

1016

⾲ 3-1 ᪋タෆ෌⌧ᛶᐇ㦂ᡂ⦼ 6)
ChemicalKathon CG (1.2% CM I)
Beryllium sulfate
Formaldehyde
Chloramine T
Chlorpromazine HCl
2-M ercaptobenzothiazole
Benzylsalicylate
Nickel chloride
Benzylcinnamate
R(+)-Limonene
M ethylsalicylate
Isopropanol
Dimethylisophthalate
4-Aminobenzoic acid
XyleneReference result(GHS category)
+(1A)
+(1A)
+(1A)
+(1A)
+(1A)
+(1A)
+(1B)
+(1B)
+(1B)
+(1B)
-(NC)
-(NC)
-(NC)
-(NC)
-(NC)

Ricerca
P&G
Exp 1 Exp 2 Exp 3 Exp 1 Exp 2 Exp 3
SLYS
SLYS
S
S
S
SLYS
S
S
S
NS
NS
NS
S
S
S
S
S
S
SLYS
SLYS
SLYS
SLYS
SLYS
SLYS
NS
NS
NS
NS
NS
NS
SLYS
SLYS
SLYS
S
S
S
NS
NS
NS
S
S
S
NS
NS
S
NS
NS
NS
NS
NS
NS
NS
NS
NS
S
NS
S
S
S
S
NS
NS
S
NS
NS
NS
NS
NS
NS
NS
NS
NS
NSLYS NSLYS NSLYS NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
S
NS
NS
NS

IVMU
Exp 1 Exp 2 Exp 3
S
S
S
NS
NS
NS
S
S
S
S
S
S
NS
NS
NS
S
S
S
NS
NS
NS
S
NS
S
S
NS
NS
S
S
S
S
S
S
NS
NS
NS
NSLYS NSLYS NSLYS
NS
NS
NS
NS
NS
NS

+㸸LLNA 㝧ᛶࠊ-㸸LLNA 㝜ᛶࠊNC㸸GHS ༊ศእࠋSLYS ᡈ࠸ࡣ NSLYS㸸ࣜࢪࣥྵ᭷࣌ࣉࢳࢻ࡟࡚ Co-elution ࡀ⏕ࡌࡓࡓࡵࠊࢩࢫࢸ࢖ࣥྵ᭷࣌ࣉ
ࢳࢻࡢ⤖ᯝࡢࡳ࠿ࡽࠊุᐃ
S:⓶⭵ឤసᛶ࠶ࡾ࡜ุᐃࠊNS:⓶⭵ឤసᛶ࡞ࡋ࡜ุᐃ
Direct Peptide Reactivity Assay (DPRA) , ECVAM Validation Study Report 㸦 EUROPEAN
COMMISSION, 2012㸧グ㍕ࡢ᝟ሗࢆᇶ࡟⦅㞟ࠋ

1117

⾲ 3-2 ᪋タ㛫෌⌧ᛶᐇ㦂ᡂ⦼ 6)
Chemical

Reference result(GHS category)

P&G

Ricerca

IVMU

Benzoquinone

+(1A)

S

SLYS

S

4-Phenylenediamine

+(1A)

S

SCL

S

Kathon CG (1.2% CMI)

+(1A)

S

S

S

Beryllium sulfate

+(1A)

S

NS

NS

Formaldehyde

+(1A)

S

S

S

Chloramine T

+(1A)

S

S

S

Chlorpromazine HCl

+(1A)

NS*

NS

NS

2-Mercaptobenzothiazole

+(1A)

S

S

S

Dihydroeugenol

+(1B)

NS

NSLYS

S

1-Thioglycerol

+(1B)

SLYS

SCL

SCL

Imidazolidinylurea

+(1B)

S

S

S

Methylmethacrylate

+(1B)

SCL

SCL

S

Benzylsalicylate

+(1B)

NS

NS

S

Nickel chloride

+(1B)

NS

S

NS

Benzylcinnamate

+(1B)

NS

NS

NS

R(+)-Limonene

+(1B)

S

S

S

Glycerol

-(NC)

NS

NS

NS

2,4-Dichloronitrobenzene

-(NC)

NS

NS

NS

Benzyl alcohol

-(NC)

NSLYS

NS

S

Methylsalicylate

-(NC)

NS

NS

S

Isopropanol

-(NC)

NS

NS

NS

Dimethylisophthalate

-(NC)

NS

NS

NS

4-Aminobenzoic acid

-(NC)

NS

NS

NS

Xylene

-(NC)

NS

NS

NS

+㸸LLNA 㝧ᛶࠊ-㸸LLNA 㝜ᛶࠊNC㸸GHS ༊ศእࠋLYS ᡈ࠸ࡣ CL㸸Co-elution ࡀ⏕ࡌࡓࡇ࡜ࢆ♧ࡍࠋ
S:⓶⭵ឤసᛶ࠶ࡾ࡜ุᐃࠊNS:⓶⭵ឤసᛶ࡞ࡋ࡜ุᐃ
*ኴᏐࡢᡂ⦼ࡣ๓ฟࡢ᪋タෆ෌⌧ᛶࢹ࣮ࢱࡢከᩘỴุᐃ࡟ࡼࡾỴᐃࡉࢀࡓࡶࡢࠋ
Direct Peptide Reactivity Assay (DPRA) , ECVAM Validation Study Report 㸦 EUROPEAN
COMMISSION, 2012㸧グ㍕ࡢ᝟ሗࢆᇶ࡟⦅㞟ࠋ

1218

㸴㸬ホ౯ྍ⬟࡞≀㉁ࡢ⠊ᅖGerberick ࡽࡢ 82 ໬Ꮫ≀㉁ࢆ⏝࠸ࡓホ౯࡛ࡣࠊ⾲ 4 ࡟♧ࡍ㏻ࡾࠊᵝࠎ࡞໬Ꮫ≀㉁ࡢ⓶⭵ឤసᛶࡢணࡀྍ⬟࡛࠶ࡿࡇ࡜ࡀ♧ࡉࢀ࡚࠸ࡿࠋࡓࡔࡋࠊLLNA ࡛ Weak sensitizer ࡟ศ㢮ࡉࢀ

ࡿ 5 ≀ ㉁ 㸦 D-Hexylcinnamaldehyde, D-Amylcinnamaldehyde, Oxalic acid, Benzyl benzoate,
2,2,6,6-Tetramethyl-3,5-heptanedione㸧 ࡜ Modelate sensitizer ࡟ศ㢮ࡉࢀࡿ 1 ≀㉁㸦Nonanoyl
chloride㸧ࡣࠊDPRA ࡛ࡣ Minimal ࡜࡞ࡾࠊ㝜ᛶ࡟ศ㢮ࡉࢀ࡚࠸ࡿࠋࡋࡓࡀࡗ࡚ࠊឤసᛶ࣏
ࢸࣥࢩࣕࣝࡢᙅ࠸≀㉁ࡣࠊഇ㝜ᛶ࡜ุᐃࡉࢀࡿሙྜࡀ࠶ࡿࡇ࡜࡟␃ពࡍࡿᚲせࡀ࠶ࡿࠋ୍㒊ࡢࣉࣞࣁࣉࢸࣥ㸦౛ࠊ4-Phenylenediamine㸧ࡣṇࡋࡃุᐃࡉࢀࡿࡀࠊࣉࣞࣁࣉࢸࣥཬ
ࡧࣉࣟࣁࣉࢸࣥࡢࡍ࡭࡚ࢆホ౯ྍ⬟࡞≀㉁࡜ࡍࡿ᰿ᣐࡣ༑ศ࡟ࡣ࡞࠸ࠋࡲࡓࠊᮏヨ㦂ἲࡣࠊ୺࡟ࢩࢫࢸ࢖ࣥᇶࡸࣜࢪࣥᇶ࡜཯ᛂࡍࡿ໬Ꮫ≀㉁ࢆᑐ㇟࡜ࡍࡿࠋ୍᪉ࠊࢱࣥࣃࢡ㉁࡜㓄఩⤖ྜࢆᙧᡂࡍࡿ㔠ᒓ㢮ࡣࠊホ౯ྍ⬟࡞≀㉁ࡢ⠊ᅖ࠿ࡽእࢀࡿࠋ
⾲ 4 82 ໬Ꮫ≀㉁ࡢホ౯⤖ᯝ 7)
Chemical Name
Diphenylcyclopropenone
Oxazolone
Benzoyl peroxide
Kathon CG
Bandrowski's base
5-Chloro-2-methyl-4-isothiazolin-3-one
p -Benzoquinone
Tetrachlorosalicylanilide
2,4-Dinitrochlorobenzene
Glutaraldehyde
Fluorescein isothiocynate
Phthalic anhydride
Lauryl gallate
Propyl gallate
CD3
Trimellitic anhydride
Formaldehyde
M etol2-Hydroxyethyl acrylate
GlyoxalVinyl pyridine
2-M ercaptobenzothiazole
Nonanoyl chloride
2-M ethyl-2H-isothiazol-3-one
1,2-Benzisothiazolin-3-one
M ethyl-2-nonynoate
Cinnamaldehyde
Phenylacetaldehyde
Benzylideneacetone
2,4-Heptadienal
Squaric acidTrans-2-hexanal
Diethyl maleate
2-Phenylpropionaldehyde
Perillaldehyde
Palmitoyl chloride
1-(4-M ethoxyphenyl)-1-penten-3-one
a-Hexylcinnamaldehyde
a-Amylcinnamaldehyde
2,3-Butanedione
Farnesal

EC3 value
0.0003
0.003
0.004
0.008
0.008
0.009
0.0099
0.04
0.05
0.1
0.14
0.16
0.3
0.32
0.6
0.6
0.61
0.8
1.4
1.4
1.6
1.7
1.8
1.9
2.3
2.5
3
3
3.7
4
4.3
5.5
5.8
6.3
8.1
8.8
9.3
11
11
11
12

LLNA
categoly
ExtremeExtremeExtremeExtremeExtremeExtremeExtremeExtremeExtremeStrongStrongStrongStrongStrongStrongStrongStrongStrongM oderate
M oderate
M oderate
M oderate
M oderate
M oderate
M oderate
M oderate
M oderate
M oderate
M oderate
M oderate
M oderate
M oderate
M oderate
M oderate
M oderate
M oderate
M oderate
Weak
Weak
Weak
Weak

Reactivity based onCys(1:10) andLys(1:50) data
High
High
High
High
High
High
High
M oderate
High
High
High
M oderate
High
High
High
Low
M oderate
High
High
High
M oderateHigh
M inimal
High
High
High
High
M oderateHighHigh
M oderateHighHigh
M oderate
M oderate
M oderate
Low
M inimalM inimalHigh
Low

Chemical Name
Oxalic acid
Benzyl benzoate
4-Allylanisole
LilialCyclamen aldehyde
Imidazolidinyl urea
5-M ethyl-2,3-hexanedione
2,2,6,6-Tetramethyl-3,5-heptanedione
Ethyleneglycol dimethacrylate
Ethyl acrylate
Hydroxycitronellal
GlycerolHexaneDiethyl phthalate
Octanoic acid
2-Hydroxypropyl methacrylate
1-Butanol
4-Hydroxybenzoic acid
6-M ethyl coumarin
M ethyl salicylate
Chlorobenzene
Lactic acid
1-Bromobutane
2-Acetylcyclohexanone
4-M ethoxyacetophenone
Ethylbenzoylacetate
Ethyl vanillin
Isopropanol
Propylene glycol
Sulfanilamide
Isopropyl myristate
Benzaldehyde
M ethylparaben
Nonanoic acid
Propyl paraben
Rsorcinol
Salicylic acid
Sulphanilic acid
Vanillin
Coumarin
Vinylidene dichloride

EC3 value

LLNA
categoly

15
17
18
19
22
24
26
27
28
28
33
Not calculated
Not calculated
Not calculated
Not calculated
Not calculated
Not calculated
Not calculated
Not calculated
Not calculated
Not calculated
Not calculated
Not calculated
Not calculated
Not calculated
Not calculated
Not calculated
Not calculated
Not calculated
Not calculated
Not calculated
Not calculated
Not calculated
21 (False +)Not calculated
Not calculated
Not calculated
Not calculated
Not calculated
Not calculated
Not calculated

Weak
Weak
Weak
Weak
Weak
Weak
Weak
Weak
Weak
Weak
Weak
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer

Reactivity based onCys(1:10) andLys(1:50) data
M inimalM inimal
Low
Low
Low
M oderate
Low
M inimal
High
High
Low
M inimalM inimalM inimalM inimalLow
M inimalM inimalM inimalM inimalM inimalM inimalLow
Low
M inimalM inimalM inimalM inimalM inimalM inimalM inimalM inimalM inimalM inimalM inimalM inimalM inimalM inimalM inimalM inimal㸵㸬᭷⏝ᛶ࡜㝈⏺ᮏἲࡣỗ⏝ HPLC ཬࡧࡑࡢᢏ⾡ࢆಖ᭷ࡍࡿ᪋タ࡛ᐜ᫆࡟ᐇ᪋ྍ⬟࡛࠶ࡿࠋࡲࡓࠊᮏἲࡣ
ື≀ࢆ⏝࠸࡞࠸ in chemico ࡢᡭἲ࡛࠶ࡾࠊ⛉Ꮫⓗ࡞┠ⓗࡢࡓࡵ࡟ᐇ᪋ࡉࢀࡿື≀ᐇ㦂࡟㛵ࡋࠊࠕື≀ࡢឡㆤཬࡧ⟶⌮࡟㛵ࡍࡿἲᚊࠖཬࡧ 3Rs ࡢ⢭⚄࡜ྜ⮴ࡋ࡚࠸ࡿࠋࡉࡽ࡟ DPRA ཬࡧ
LLNA ࡟ࡘ࠸࡚౑⏝ࡍࡿᾘ⪖ရ㈝ࢆヨ⟬ࡋࡓ࡜ࡇࢁࠊ1 ࢔ࢵࢭ࢖ᙜࡓࡾࡢᾘ⪖ရ㈝ࡣ LLNA
1319࡛⣙ 10 ୓෇࡛࠶ࡿࡢ࡟ᑐࡋࠊDPRA ࡛ࡣ⣙ 1 ୓෇࡜ 1/10 ⛬ᗘࡢ⤒㈝࡛ᐇ᪋ྍ⬟࡛࠶ࡾࠊᐇ
㦂ᮇ㛫ࡶ LLNA ࡼࡾ▷ᮇ㛫࡛ᐇ᪋ྍ⬟࡛࠶ࡿࡇ࡜࠿ࡽࠊヨ㦂ἲ࡜ࡋ࡚⡆౽ᛶ࣭⤒῭ᛶࡢ㠃࠿ࡽ᭷⏝࡜ᛮࢃࢀࡿࠋ
ࡋ࠿ࡋ࡞ࡀࡽࠊᮏἲࡣᾮ┦୰࡛ࡢ཯ᛂࢆᚲせ࡜ࡍࡿࡓࡵࠊ⿕㦂≀㉁ࡣᑡ࡞ࡃ࡜ࡶ㐺ษ࡞⁐፹㸦AcetonitrileࠊWaterࠊAcetonitrile:Water (1:1)ࠊIsopropanolࠊAcetoneࠊAcetone:Acetonitrile(1:1)ࠊDMSO:Acetonitrile (1:9) ࠊDMSO:Acetonitrile (1:1)㸧࡟ 100mM ࡢ⃰ᗘ࡛⁐ゎࡍࡿᚲせࡀ࠶ࡿࠋࡲࡓࠊᮏἲࡢឤᗘࡣヨ㦂㐺⏝እ࡜⪃࠼ࡽࢀࡿ㔠ᒓሷࢆ㝖࠸ࡓሙྜ࡛ࡶ⣙ 75㸣࡛࠶ࡾࠊ1/4 ࡢ໬ྜ≀ࡀഇ㝜ᛶ࡜ホ౯ࡉࢀࡿࡓࡵࠊࡇࡢヨ㦂ἲ༢⊂࡛⓶⭵ឤసᛶࡢ௦᭰ἲ࡜ࡋ࡚
⪃࠼ࡿࡢࡣ㞴ࡋ࠸ࠋ≉࡟ୗグࡢ໬ྜ≀ࡀഇ㝜ᛶ࡜࡞ࡿྍ⬟ᛶࡀ㧗࠸ࡓࡵヨ㦂⤖ᯝࡢゎ㔘࡟ࡣὀពࡀᚲせ࡜⪃࠼ࡽࢀࡿࠋ
1㸧LLNA ࡛ moderate ࡲࡓࡣ weak ࡢ໬ྜ≀
LLNA

࡛

weak

sensitizer࡟ ศ 㢮 ࡉ ࢀ ࡿ

5≀ ㉁ 㸦 D-Hexylcinnamaldehyde,

D-Amylcinnamaldehyde, Oxalic acid, Benzyl benzoate, 2,2,6,6-Tetramethyl-3,5-heptanedione㸧 ࡜Modelate sensitizer ࡟ศ㢮ࡉࢀࡿ 1 ≀㉁㸦Nonanoyl chloride㸧ࡣࠊDPRA ࡛ࡣ Minimal ࡜࡞ࡾࠊ㝜ᛶ࡟ศ㢮ࡉࢀࡿࠋ
2㸧ࣉࣟࣁࣉࢸࣥࠊࣉࣞࣁࣉࢸࣥ
ᮏヨ㦂⣔ࡣ௦ㅰ⣔ࢆ᭷ࡉ࡞࠸༢⣧࡞໬Ꮫ཯ᛂࢆ᳨ฟࡍࡿヨ㦂⣔࡛࠶ࡿࡇ࡜࠿ࡽࠊឤసᛶ
ࡢ⋓ᚓ࡟௦ㅰⓗᡈ࠸ࡣ㠀௦ㅰⓗάᛶ໬ࢆᚲせ࡜ࡍࡿࣉࣟࣁࣉࢸࣥᡈ࠸ࡣࣉࣞࣁࣉࢸࣥ
㸦౛㸸Dihydroeugenol ཬࡧ Limonene㸧ࡣഇ㝜ᛶ࡜ホ౯ࡉࢀࡿྍ⬟ᛶࡀ࠶ࡿࠋ
3㸧␯Ỉᛶࡢ㧗࠸≀㉁➼ṇ☜ᗘ㸦ឤᗘཬࡧ≉␗ᗘ㸧࡛㏙࡭ࡓ࡜࠾ࡾࠊỈ⣔࡛ࡢ཯ᛂࢆᚲせ࡜ࡍࡿᮏἲ࡛ࡣ␯Ỉᛶࡢ㧗࠸≀㉁㸦౛㸸Chlorpromazine HCl㸧ࡣഇ㝜ᛶ࡜ุᐃࡉࢀࡿሙྜࡀ࠶ࡿࠋࡉࡽ࡟ࢩࢫࢸ࢖
ࣥࡸࣜࢪࣥ௨እࡢ࢔࣑ࣀ㓟࡜ඃඛⓗ࡟཯ᛂࡍࡿࡼ࠺࡞ồ㟁Ꮚṧᇶࢆ᭷ࡍࡿ≀㉁࡟㛵ࡋ࡚ࡶṇࡋࡃุᐃࡉࢀ࡞࠸ྍ⬟ᛶࡀ࠶ࡿࠋ
௨ୖࡢࡇ࡜࠿ࡽࠊDPRA ࡟ࡼࡾឤసᛶ㝜ᛶ࡜ุ᩿ࡉࢀࡓሙྜࡣࠊࡑࡢ≀ᛶ➼࡟ࡼࡾഇ㝜ᛶ࡜࡞ࡿྍ⬟ᛶࢆ⪃៖ࡋࠊ⿵᏶ࡋᚓࡿ௚ࡢヨ㦂ἲ࡟ࡼࡾ☜ㄆࡍࡿᚲせࡀ࠶ࡿࠋඛ⾜ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡢᡂ⦼࠿ࡽ DPRA ࡟ࡼࡾ㝧ᛶ࡜ุ᩿ࡉࢀࡓሙྜࡣࠊឤసᛶ㝧ᛶ࡜ุ᩿ࡍࡿࡇ࡜ࡣྍ⬟࡜⪃࠼ࡿࡀࠊᮏἲࡢ≉␗ᗘࡣ⣙ 90㸣࡛࠶ࡾࠊᕼ࡟ഇ㝧ᛶࡢ⤖ᯝࡀ⏕ࡌࡿྍ⬟ᛶࡀ࠶ࡿࡇ࡜࡟ࡶ␃ពࡍࡿᚲせࡀ࠶ࡿࠋ࡞࠾ࠊᮏἲࡣ⤖ྜᙉᗘࢆ Minimal, Low,
Moderate, High ࡢ 4 ࢡࣛࢫࡢศ㢮ࡍࡿ᪉ἲࡶᥦ᱌ࡉࢀ࡚࠸ࡿࡀࠊ᪋タ㛫෌⌧ᛶࡣ 62.5%࡜ప
ࡃࠊ⤖ྜᙉᗘࡢศ㢮࡟ࡣ㐺ࡉ࡞࠸࡜⪃࠼ࡿࠋ୍᪉ࠊDPRA ࡣឤసᛶⓎ⌧ᶵᗎ࡟࠾ࡅࡿึᮇࡢ㔜せ࡞࢖࣋ࣥࢺ࡛࠶ࡿࢱࣥࣃࢡ㉁࡜໬Ꮫ
≀㉁ࡢ⤖ྜ཯ᛂࢆ᳨ฟࡋ࡚࠾ࡾࠊ໬Ꮫ≀㉁ࡢឤసᛶࢆุ᩿ࡍࡿୖ࡛㔜せ࡞᝟ሗࢆ୚࠼ࡿࡇ࡜࠿ࡽࠊドᣐࡢ㔜ࡳ௜ࡅࡸ௚ࡢヨ㦂ἲ࡜⤌ࡳྜࢃࡏ࡛ࡢホ౯ࢆ᥎ዡࡍࡿࠋ
14

20

㸶㸬⤖ㄽ
DPRA ࡣࠊ⡆౽ᛶ࣭⤒῭ᛶࡢ㠃࠿ࡽ᭷⏝࡞ື≀ᐇ㦂௦᭰ἲ࡛࠶ࡿࠋ
ᮏヨ㦂ἲࡢඛ⾜ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟࠾ࡅࡿ᪋タෆ෌⌧ᛶࡣࠊ3 ᪋タ୰ 1 ᪋タ࡟࠾࠸࡚ࠊ
GHS ༊ศ 1B㸦ᙅ࠸ឤసᛶ≀㉁㸧ࡢ≀㉁࡛෌⌧ᛶࡀᚓࡽࢀ࡞࠿ࡗࡓࡓࡵ㐩ᡂᇶ‽࡟㐩ࡋ࡚࠾ࡽࡎࠊᙅ࠸ឤసᛶ≀㉁࡛ࡣุᐃࡀࡪࢀࡿᠱᛕࡀ࠶ࡿࠋ୍᪉ࠊ᪋タ㛫෌⌧ᛶࡣࠊ㐺⏝⠊ᅖእࡢ㔠ᒓሷࢆ㝖࠸ࡓሙྜ࡟㐩ᡂᇶ‽ࢆୖᅇࡗࡓࠋ
ᮏヨ㦂ἲ㐺⏝እࡢ㔠ᒓሷࢆ㝖࠸ࡓሙྜࡢඛ⾜ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟࠾ࡅࡿឤᗘࡣ⣙ 75㸣࡛࠶ࡿࡓࡵࠊ㝜ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓሙྜࡣࠊഇ㝜ᛶࡢྍ⬟ᛶࢆ⪃៖ࡋࠊ⿵᏶ࡋᚓࡿ௚ࡢヨ㦂ἲ࡟ࡼࡾ☜ㄆࡋ࡞ࡅࢀࡤ࡞ࡽ࡞ࡃࠊDPRA ࡢࡳ࡛⓶⭵ឤసᛶࢆ㝜ᛶ࡜ุᐃࡍࡿࡇ࡜ࡣ࡛ࡁ
࡞࠸ࠋඛ⾜ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟࠾ࡅࡿᮏἲࡢ≉␗ᗘࡣ⣙ 90㸣࡛࠶ࡾࠊ㝧ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓ
ሙྜࡣࠊឤసᛶ㝧ᛶ࡜ุ᩿ࡍࡿࡇ࡜ࡣྍ⬟ࡔࡀࠊᕼ࡟ഇ㝧ᛶࡢ⤖ᯝࡀ⏕ࡌࡿྍ⬟ᛶࡀ࠶ࡿࡇ࡜࡟␃ពࡋ࡞ࡅࢀࡤ࡞ࡽ࡞࠸ࠋ
࡞࠾ࠊᮏἲࡣ⤖ྜᙉᗘࢆ Minimal, Low, Moderate, High ࡢ 4 ࢡࣛࢫ࡟ศ㢮ࡍࡿ᪉ἲࡶᥦ᱌
ࡉࢀ࡚࠸ࡿࡀࠊ᪋タ㛫෌⌧ᛶࡣ 62.5%࡜పࡃࠊ⤖ྜᙉᗘࡢศ㢮࡟ࡣ㐺ࡉ࡞࠸ࠋ
DPRA ࡣឤసᛶⓎ⌧ᶵᗎ࡟࠾ࡅࡿึᮇࡢ㔜せ࡞࢖࣋ࣥࢺ࡛࠶ࡿࢱࣥࣃࢡ㉁࡜໬Ꮫ≀㉁ࡢ
⤖ྜ཯ᛂࢆ᳨ฟࡋ࡚࠾ࡾࠊ໬Ꮫ≀㉁ࡢឤసᛶࢆุ᩿ࡍࡿୖ࡛㔜せ࡞᝟ሗࢆ୚࠼࡚ࡃࢀࡿࠋ
LLNA ࡢ 1/10 ⛬ᗘࡢ⤒㈝࡛ᐇ᪋ྍ⬟࡛࠶ࡾࠊື≀ࢆ⏝࠸࡞࠸ in chemico ヨ㦂ἲ࡛࠶ࡿࡇ࡜
࠿ࡽࠊ᭷⏝ᛶࡣ㧗࠸ࠋࡋ࠿ࡋ࡞ࡀࡽࠊᮏἲࡣ௦ㅰ⣔ࢆḞࡃ໬Ꮫⓗヨ㦂ἲ࡛࠶ࡾࠊάᛶ໬࡟
௦ㅰ⣔ࡸ㠀⏕≀ⓗάᛶ໬ࢆᚲせ࡜ࡍࡿឤసᛶ≀㉁ࠊᙅ࠸ឤసᛶ≀㉁ࡸ㔠ᒓሷࠊ␯Ỉᛶࡢ㧗࠸≀㉁࡞࡝ࡣṇࡋࡃࡑࡢឤసᛶࡀ᳨ฟࡉࢀ࡞࠸ྍ⬟ᛶࡀ࠶ࡿࠋ௨ୖࡢ஦ᐇࢆ㋃ࡲ࠼ࠊጤဨ఍ࡣࠊドᣐࡢ㔜ࡳ௜ࡅࡸ௚ࡢヨ㦂ἲ㸦LLNAࠊࣔࣝࣔࢵࢺࢆ⏝࠸ࡿ⓶⭵ឤసᛶヨ㦂㸧࡜⤌ࡳྜࢃࡏ࡛ࡢホ౯ࢆ᥎ዡࡍࡿࠋ

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ᘬ⏝ᩥ⊩
1) OECD ENV/JM/MONO (2012) /Part1, The adverse outcome pathway for skin sensitization
Initiated by covalent binding to proteins : Scientific evidence.
2) Kato H., Okamoto M., Yamashita K., Nakamura Y., Fukumori Y., Nakai K. and Kaneko H. (2003)
Peptide binding assessment using mass spectrometory as a new screening method for skinsensitization. J. Toxicol. Sci. 28(1), 19-24.
3) Natsch, A. and Gfeller H. (2008) LC-MS-based characterization of the peptide reactivity ofchemicals to improve the in vitro prediction of the skin sensitization potential. Toxicol. Sci.
106(2), 464-478
4) Chipinda I., Ajibola R. O., Marakinyo M. K., Ruwona T. B. Simoyi R. H. and Siegel P. D. (2010)
Rapid and simple kinetics screening assay for electrophillic dermal sensitizers using
nitrobenzenethiol. Chem. Res. Toxicol. 23(5) 918-925.
5) OECD (2015) In Chemico Skin Sensitization: Direct Peptide Reactivity Assay (DPRA). OECD
Guideline for the Testing of Chemicals No. 442C.
6) EUROPEAN COMMISSION (2012) Direct Peptide Reactivity Assay (DPRA) , ECVAM
Validation Study Report
http://ihcp.jrc.ec.europa.eu/our_labs/eurl-ecvam/eurl-ecvam-recommendations/files-dpra/DPRA%20Validation%20Study%20Report.pdf
7) Gerberick F., Vassallo J.D., Foertsch L.M., Price B. B., Chaney, J. G., Lepoittevin, J-P. (2007)
Quantification of chemical peptide reactivity for screening contact allergens: a classification treemodel approach. Toxicol. Sci. 97(2), 417-427

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