⓶⭵ឤసᛶヨ㦂ホ౯ሗ࿌᭩

ARE-Nrf2 Luciferase Test Methodゅ໬⣽⬊ᰴ࣏࣮ࣞࢱ࣮࢔ࢵࢭ࢖

ᖹᡂ 27 ᖺ 4 ᭶ 28 ᪥

JaCVAM ⓶⭵ឤసᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍

1
7

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

2
8

せ᪨
⓶⭵ឤసᛶࡣ໬Ꮫ≀㉁ࡢᏳ඲ᛶホ౯࡟࠾࠸࡚㔜せ࡞ホ౯㡯┠࡛࠶ࡾࠊᚑ᮶ࠊࣔࣝࣔࢵࢺ
ࡸ࣐࢘ࢫࢆ⏝࠸ࡓື≀ᐇ㦂࡟ࡼࡗ࡚ホ౯ࡉࢀ࡚ࡁࡓࠋ㏆ᖺ EU ࡟࠾ࡅࡿḢᕞ໬Ꮫရつไ࡛ࡣࠊᏳ ඲ ᛶ ホ ౯ ࡣ ࢥ ࣥ ࣆ ࣗ ࣮ ࢱ ࣮ ࢆ ⏝ ࠸ ࡓ ᐃ 㔞 ⓗ ᵓ 㐀 ά ᛶ ┦ 㛵 (QSAR: Quantitative
Structure-Activity Relationship)ࣔ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࠸ࡿࠋARE (Antioxidant response
element )-Nrf21 Luciferase Test Method ࡣࠊከࡃࡢ⓶⭵ឤసᛶ≀㉁ࡀ ARE ࡟ࡼࡾไᚚࡉࢀࡿ
㑇ఏᏊࡢⓎ⌧ࢆㄏᑟࡍࡿࡇ࡜ࢆ฼⏝ࡋࠊࡇࡢㄏᑟάᛶ࡟ࡘ࠸࡚ᇵ㣴⣽⬊ࢆ⏝࠸࡚࢔ࢵࢭ࢖
ࡍࡿヨ㦂ἲ࡛࠶ࡿࠋᮏሗ࿌᭩ࡣࠊࡇࡢ ARE-Nrf2 Luciferase Test Method ࡟ࡘ࠸࡚ࠊᮏヨ㦂ἲࢆ㛤Ⓨࡋࡓ Givaudan ♫ࡢඛ⾜ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡢᡂ⦼୪ࡧ࡟ EURL ECVAM㸦European
Union Reference Laboratory for Alternatives to Animal Testing㸧⛉Ꮫㅎၥጤဨ఍㸦ESAC㸧࡟ࡼࡾᐇ᪋ࡉࢀࡓ➨୕⪅ホ౯ሗ࿌᭩࡞࡝ࢆᇶ࡟ࡑࡢᡭ㡰ࢆ⧳ࡵࠊ᭷⏝ᛶ࡜㝈⏺ࢆホ౯ࡋࡓࡶࡢ࡛࠶
ࡿࠋ
ARE-Nrf2 Luciferase Test Method㸦௨ୗࠊᮏヨ㦂ἲ࡜グࡍ㸧ࡣࠊឤసᛶⓎ⌧ᶵᗎ࡟࠾ࡅࡿ➨஧ẁ㝵ࡢ࢖࣋ࣥࢺ࡛࠶ࡿࢣࣛࢳࣀࢧ࢖ࢺ࡟࠾ࡅࡿ⅖⑕཯ᛂ࠾ࡼࡧ Nrf2-Keap12-ARE pathwayࢆ฼⏝ࡋࡓヨ㦂ἲ࡛࠶ࡾࠊ໬Ꮫ≀㉁ࡢឤసᛶࢆุ᩿ࡍࡿୖ࡛㔜せ࡞᝟ሗࢆ୚࠼࡚ࡃࢀࡿࠋ
࣐࢘ࢫࢆ⏝࠸ࡿヨ㦂ἲ࡛࠶ࡿᒁᡤࣜࣥࣃ⠇ヨ㦂㸦LLNA:Local Lymph Node Assay㸧ࡢ⣙ 1/7⛬ᗘࡢࣛࣥࢽࣥࢢࢥࢫࢺ࡛ᐇ᪋ྍ⬟࡛࠶ࡾࠊin vitro ヨ㦂ἲ࡛࠶ࡿࡇ࡜࠿ࡽࠊ᭷⏝ᛶࡣ㧗࠸ࠋࡋ࠿ࡋ࡞ࡀࡽࠊ⌧᫬Ⅼ࡛ᮏヨ㦂ἲ࡟౑⏝࡛ࡁࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿ⣽⬊⣔ࡣ KeratinoSensTM
ࡢࡳ࡛࠶ࡾࠊࡑࡢ౑⏝࡟ࡣᮏ⣽⬊⣔ࢆᶞ❧ࡋࡓ Givaudan ♫࠿ࡽࣛ࢖ࢭࣥࢫࢆཷࡅࡿࡇ࡜ࡀᚲせ࡞ࡓࡵࠊ᪂つᑟධࡢᐜ᫆࡞ヨ㦂⣔࡜ࡣゝ࠸㞴࠸ࠋᮏヨ㦂ἲࡢඛ⾜ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟࠾ࡅࡿ᪋タෆ෌⌧ᛶࡣࠊ5 ᪋タ୰ 1 ᪋タ࡟࠾࠸࡚ࠊ
GHS(Globally Harmonized System of Classification and Labelling of Chemicals)༊ศ 1B ࡟ศ㢮ࡉࢀࡿ≀㉁㸦ᙅ࠸ឤసᛶ≀㉁㸧࠾ࡼࡧ㠀ឤసᛶ≀㉁࡛෌⌧ᛶࡀᚓࡽࢀ࡞࠿ࡗࡓࡓࡵࠊ┠Ᏻ࡜ࡋࡓ㐩ᡂᇶ‽㸦85㸣㸧࡟㐩ࡋ࡚࠾ࡽࡎࠊᙉ࠸ឤసᛶ≀㉁௨እ࡛ࡣุᐃࡀࡪࢀࡿᠱᛕࡀ࠶ࡿࠋ
୍᪉ࠊ᪋タ㛫෌⌧ᛶࡣࠊ┠Ᏻ࡜ࡋࡓ㐩ᡂᇶ‽㸦80㸣㸧ࢆୖᅇࡗࡓࠋ
ᮏヨ㦂ἲࡢឤᗘࡣࠊ⣙ 80㸣࡛࠶ࡾࠊ㝜ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓሙྜࡣࠊഇ㝜ᛶࡢྍ⬟ᛶࢆ⪃៖ࡋࠊ⿵᏶ࡋᚓࡿ௚ࡢヨ㦂࡟ࡼࡗ࡚☜ㄆࡋ࡞ࡅࢀࡤ࡞ࡽࡎࠊᮏヨ㦂ἲࡢࡳ࡛⓶⭵ឤసᛶࢆ㝜ᛶ࡜ุᐃࡍࡿࡇ࡜ࡣ࡛ࡁ࡞࠸ࠋࡲࡓࠊ≉␗ᗘࡶࠊ⣙ 80㸣࡛࠶ࡿࡇ࡜࠿ࡽࠊ㝧ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓሙྜ࡟ࡶࠊഇ㝧ᛶࡢ⤖ᯝࡀ⏕ࡌࡿྍ⬟ᛶࡀ࠶ࡿࡇ࡜࡟␃ពࡋ࡞ࡅࢀࡤ࡞ࡽ࡞࠸ࠋ
ᮏヨ㦂ἲ࡛ࡣࠊάᛶ໬࡟௦ㅰ⣔ࢆᚲせ࡜ࡍࡿ໬Ꮫ≀㉁ࡣࠊṇࡋࡃࡑࡢឤసᛶࡀ᳨ฟࡉࢀ
࡞࠸ྍ⬟ᛶࡀ࠶ࡿࠋࡲࡓࠊ⣽⬊⣔࡛࠶ࡿࡓࡵࠊ␯Ỉᛶࡢ㧗࠸≀㉁࡛ࡣつᐃࡉࢀ࡚࠸ࡿ᭱㧗1
2

Nrf2㸸Nuclear factor (erythroid-derived 2)-like 2
Keap1: Kelch-like ECH-associated protein 139⃰ᗘ㸦2000 μM㸧࡛ࡢホ౯ࡀ㞴ࡋࡃࠊ㝜ᛶุᐃࡀୗࡏ࡞࠸ሙྜࡀ࠶ࡿࠋ௨ ୖࢆ ㋃ࡲ࠼ ࠊᮏ ጤဨ఍ ࡣࠊ ᮏヨ㦂 ἲࡀ ⓶⭵ឤ సᛶ ホ౯࡟ ỗ⏝ ࡉࢀࡿ ࡓࡵ ࡟ ࡣ ࠊ
KeratinoSensTM ࡀᏳ౯࡟ධᡭ࡛ࡁࡿࡇ࡜ࡀ๓ᥦ࡜⪃࠼ࡿࠋࡲࡓࠊᮏヨ㦂ἲࡢᵝࠎ࡞㝈⏺ࢆ⪃៖ࡍࡿ࡜ࠊᮏヨ㦂ἲ༢⊂࡛ࡣ⓶⭵ឤసᛶࡢホ౯ࡣ୙༑ศ࡛࠶ࡾࠊドᣐࡢ㔜ࡳ௜ࡅࡸ௚ࡢヨ㦂ἲ㸦LLNAࠊࣔࣝࣔࢵࢺࢆ⏝࠸ࡿ⓶⭵ឤసᛶヨ㦂࡞࡝㸧࡜⤌ࡳྜࢃࡏ࡛ࡢホ౯ࢆ᥎ዡࡍࡿࠋ
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OECD TG406㸧ࡸ࣐࢘ࢫࢆ⏝࠸ࡿᒁᡤࣜࣥࣃ⠇ヨ㦂㸦LLNA: Local Lymph Node Assay,
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REACH: Registration, Evaluation, Authorisation and Restrictionof Chemicals㸧࡛ࡣࠊᏳ඲ᛶホ౯ࡣࢥࣥࣆ࣮ࣗࢱ࣮ࢆ⏝࠸ࡓᐃ㔞ⓗᵓ㐀άᛶ┦㛵㸦QSAR:
Quantitative Structure-Activity Relationship)ࣔࢹࣝࡸ in vitro ヨ㦂➼࡟ࡼࡿ௦᭰ἲࡀ᥎ዡࡉࢀ࡚࠾

ࡾࠊື≀ᐇ㦂࡟ࡼࡾᏳ඲ᛶࡀホ౯ࡉࢀࡓᡂศࢆྵࢇࡔ໬⢝ရࡢ㍺ධ࠾ࡼࡧ㈍኎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㸧ࠊ༢⌫⣔⣽⬊ࡢάᛶ໬ࢆ฼⏝ࡋࡓ human Cell Line Activation Test㸦h-CLAT㸧࠾ࡼࡧ Myeloid U937 Skin
Sensitization Test㸦 MUSST 㸧ࠊ ࢣ ࣛࢳࣀ ࢧ ࢖ ࢺ⣽⬊ ⣔ ࡢ ᶆⓗ㑇 ఏ Ꮚ ࢆ⏝࠸ ࡓ ARE-Nrf2
Luciferase Test Method ࡞࡝ࡢ⓶⭵ឤసᛶヨ㦂ࡢື≀ࢆ⏝࠸࡞࠸ື≀ᐇ㦂௦᭰ἲࡀᥦ᱌ࡉࢀ
࡚࠾ࡾࠊEURL ECVAM㸦European Union Reference Laboratory for Alternatives to Animal Testing㸧➼࡟࠾࠸࡚ࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀ⾜ࢃࢀ࡚࠸ࡿࠋ
 ARE(Antioxidant response element )-Nrf2 Luciferase Test Method ࡣࠊከࡃࡢ⓶⭵ឤసᛶ≀㉁ࡀ ARE ࡟ࡼࡾไᚚࡉࢀࡿ㑇ఏᏊࡢⓎ⌧ࢆㄏᑟࡍࡿࡇ࡜ࢆ฼⏝ࡋࠊࡇࡢㄏᑟάᛶ࡟ࡘ࠸࡚ᇵ
㣴⣽⬊ࢆ⏝࠸࡚ホ౯ࡍࡿヨ㦂ἲ࡛࠶ࡿࠋKeratinoSens™ἲࡣᮏヨ㦂ἲࡢࡓࡵ࡟☜❧ࡉࢀࡓ⣽⬊ࢆ౑⏝ࡍࡿホ౯⣔࡛࠶ࡿࠋKeratinoSens™ἲࢆ⏝࠸࡚ᐇ᪋ࡉࢀࡓᮏヨ㦂ἲࡢࣂࣜࢹ࣮ࢩࣙ
ࣥ◊✲ࡢ⤖ᯝ࡟ࡘ࠸࡚ࡣࠊEURL ECVAM ⛉Ꮫㅎၥጤဨ఍㸦ESAC㸧࡟ࡼࡿ➨୕⪅ホ౯ࡀ᏶஢ࡋ࡚࠸ࡿ 1)ࠋJaCVAM ⓶⭵ឤసᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍㸦௨ୗࠊᮏጤဨ఍㸧ࡀ ARE-Nrf2 Luciferase Test
Method ࡢ⓶⭵ឤసᛶヨ㦂௦᭰ἲ࡜ࡋ࡚ࡢ⛉Ꮫⓗጇᙜᛶ࡟ࡘ࠸࡚ࠊKeratinoSens™ἲࢆ⏝࠸࡚ᐇ᪋ࡉࢀࡓᮏヨ㦂ἲࡢࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡢ⤖ᯝ➼ࠊ⌧ᅾࡲ࡛࡟බ㛤ࡉࢀ࡚࠸ࡿ᝟ሗࢆࡶ࡜࡟ホ౯ࡋࡓࡢ࡛ࠊࡑࡢ⤖ᯝࢆሗ࿌ࡍࡿࠋ
410

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 (AOP)࡛ࡣࠊ໬Ꮫ≀㉁࡟ࡼࡿ⓶⭵ឤసᛶࡣḟࡢ 4 ࡘࡢ Key event ࠿ࡽᡂࡿ࡜ࡉࢀ࡚࠸ࡿ 1)ࠋ
1) ໬Ꮫ≀㉁࡜ࢱࣥࣃࢡ㉁ࡢࢩࢫࢸ࢖ࣥṧᇶ࠶ࡿ࠸ࡣࣜࢪࣥṧᇶ࡜ࡢඹ᭷⤖ྜ2) ࢣࣛࢳࣀࢧ࢖ࢺ࡟࠾ࡅࡿ⅖⑕ᛶᛂ⟅࠾ࡼࡧ ARE-dependent pathway ࡟ࡼࡿ㑇ఏᏊⓎ⌧3) ᶞ≧⣽⬊ࡢάᛶ໬㸦≉␗ⓗ⣽⬊⾲㠃࣐࣮࣮࢝ࡢⓎ⌧ࠊࢣࣔ࢝࢖ࣥࡸࢧ࢖ࢺ࢝࢖ࣥࡢ⏘⏕㸧4) ࣜࣥࣃ⠇࡟࠾ࡅࡿ T ⣽⬊ࡢቑṪ
ARE-Nrf23 Luciferase Test Method ࡣୖグࡢ➨ 2 ࡢ Key event ࡟ᑐᛂࡍࡿヨ㦂ἲ࡛࠶ࡿࠋࡑ
ࡢᇶᮏⓗཎ⌮ࡣ Nrf2-Keap14-ARE pathway㸦ᅗ 1㸧ࢆ฼⏝ࡋࡓ࣏࣮ࣞࢱ࣮࢔ࢵࢭ࢖࡛࠶ࡿ 1)ࠋ
ᅗ 1㸬Nrf2-Keap1-ARE pathway ࡢᶍᘧᅗNrf2-Keap1-ARE pathway ࡣࠊ㌿෗ᅉᏊ Nrf2ࠊNrf2 ࡢᢚไᅉᏊ࡛࠶ࡿ Keap1 ࠾ࡼࡧ AREࡀ㛵ಀࡍࡿ㑇ఏᏊⓎ⌧⤒㊰࡛࠶ࡿࠋNrf2 ࡣ Keap1 ࡜⤖ྜࡋࠊARE ࡟౫Ꮡࡋ࡚Ⓨ⌧ࡍࡿ㑇ఏᏊ⩌ࡢⓎ⌧㔞ࢆไᚚࡋ࡚࠸ࡿࠋKeap1 ࡢࢩࢫࢸ࢖ࣥṧᇶ࡟ồ㟁Ꮚᛶࡢ໬Ꮫ≀㉁ࡀ⤖ྜࡍࡿ࡜ࠊ
Nrf2 ࡣ Keap1 ࠿ࡽゎ㞳ࡋࠊ᰾ෆ࡬⛣⾜ࡋ࡚ DNA ୖࡢ ARE ࡟⤖ྜࡍࡿࠋࡑࡢ⤖ᯝࠊୗὶࡢ
㑇ఏᏊ⩌ࡢⓎ⌧ࡀㄏᑟࡉࢀࠊ໬Ꮫ≀㉁࡟ࡼࡿ㞀ᐖ࠿ࡽ⣽⬊ࢆಖㆤࡍࡿࡓࡵ࡟ᶵ⬟ࡍࡿࠋከࡃࡢ⓶⭵ឤసᛶ≀㉁ࡀ Nrf2-Keap1-ARE pathway ࢆάᛶ໬ࡍࡿࠋ
ARE-Nrf2 Luciferase Test Method ࡛ࡣࠊAKR1C2 㑇ఏᏊ㸦ᶞ≧⣽⬊࡟࠾࠸࡚⓶⭵ឤసᛶ≀㉁࡟ࡼࡾⓎ⌧ㄏᑟࡉࢀࡿ㑇ఏᏊࡢ 1 ࡘ㸧ࡢ ARE ࢆ⼥ྜࡉࡏࡓ SV40 ࣉ࣮ࣟࣔࢱ࣮ࢆ᭷ࡍࡿ
ࣝࢩࣇ࢙࣮ࣛࢮ㑇ఏᏊࡢࣉࣛࢫ࣑ࢻࢆᏳᐃⓗ࡟ᑟධࡋࡓ HaCaT ⣽⬊㸦ࣄࢺࢣࣛࢳࣀࢧ࢖ࢺ
3
4

Nrf2 :Nuclear factor-erythroid 2-related factor 2
Keap1 : Kelch-like ECH-associated protein 1511⣔ᇵ㣴⣽⬊ᰴ㸧ࢆ⏝࠸ࡿࠋ໬Ꮫ≀㉁࡟ࡼࡾ Nrf2-Keap1-ARE pathway ࡀάᛶ໬ࡉࢀࡿ࡜ࣝࢩ
ࣇ࢙࣮ࣛࢮ㑇ఏᏊࡀⓎ⌧ࡍࡿࠋᇶ㉁ࢆῧຍࡋࠊࣝࢩࣇ࢙࣮ࣛࢮࡀゐ፹ࡍࡿ཯ᛂࡢⓎගᙉᗘࢆᐃࡍࡿࡇ࡜࡟ࡼࡾࠊ໬Ꮫ≀㉁ࡢ⓶⭵ឤసᛶࢆホ౯ࡍࡿࠋ3㸬ヨ㦂ᡭ㡰㸭ุᐃヨ㦂ᡭ㡰ࡣ≉࡟♧ࡉ࡞࠸㝈ࡾ KeratinoSensTM ࢆ⏝࠸ࡓሙྜࡢグ㍕࡛࠶ࡿࠋࢣࣛࢳࣀࢧ࢖ࢺ
⏤᮶ ARE-Nrf2 ࣝࢩࣇ࢙࣮ࣛࢮ࣏࣮ࣞࢱ࣮㑇ఏᏊ㸦keratinocyte-based ARE-Nrf2 luciferase
reporter gene㸧ࢆ⏝࠸ࡓᮏヨ㦂ἲࡢ⣽⬊࡜ࡋ࡚ KeratinoSensTM ௨እࡢ⣽⬊ࢆ⏝࠸ࡿሙྜࡣࠊ
OECD సᡂࡢ ARE-Nrf2 LuciferaseTest Method ࡟㛵ࡍࡿ Performance Standard㸦᱌㸧2)࡟ᚑ࠸
KeratinoSensTM ࢆ⏝࠸ࡓሙྜ࡜ྠ➼࠿࠶ࡿ࠸ࡣඃࡿಙ㢗ᛶࠊṇ☜ᗘࠊឤᗘࠊ≉␗ᗘ࡞࡝ࢆ♧ࡍࡇ࡜ࢆ☜ㄆࡋࡓࡢࡕ࡟౑⏝ࡋ࡞ࡅࢀࡤ࡞ࡽ࡞࠸ࠋ࠸ࡎࢀࡢሙྜࡶ᪥ᖖⓗ࡟⏝࠸ࡿ๓࡟OECD ࢞࢖ࢻࣛ࢖ࣥ㸦᱌㸧3)ࡢ Annex 2 ࡟ᚑ࠸ᢏ⾡ⓗ⇍㐩ᗘࢆ☜ㄆࡍࡿࡇ࡜ࢆ᥎ዡࡍࡿࠋ
3-1. ⣽⬊ࡢㄪ〇
ARE ไᚚୗࡢࣝࢩࣇ࢙࣮ࣛࢮ࣏࣮ࣞࢱ࣮㑇ఏᏊࢆᏳᐃⓗ࡟ྲྀࡾ㎸ࢇࡔࢺࣛࣥࢫࢪ࢙ࢽࢵ
ࢡ⣽⬊⣔ࢆ⏝࠸ࡿࠋ㸦⌧ᅾࡣࠊKeratinoSensTM ࡢࡳ࡛࠶ࡿࡓࡵࠊヨ㦂ἲࡢ㛤Ⓨ⪅࡜౑⏝チㅙዎ⣙ࢆ⤖ࢇࡔ࠺࠼࡛ࡇࡢ⣽⬊⣔ࢆධᡭࡋ࡚౑⏝ࡍࡿࡇ࡜࡟࡞ࡿ㸧ࠋᣦᐃࡉࢀࡓ⥅௦ᩘࡢ⣽⬊㸦2 ࠿ࡽ 4 ௦㸧ࢆቑṪࡋࠊศ๭ࡋ࡚ಖ⟶ࡋࠊࡇࢀࢆ୺ࢫࢺࢵࢡ⣽⬊࡜ࡍࡿࠋ୺ࢫࢺࢵࢡ⣽⬊࠿ࡽቑṪࡉࡏࠊᣦᐃࡉࢀࡓ⥅௦ᩘ㸦25 ௦㸧௨ෆ࡛ヨ㦂࡟౑⏝ࡍࡿࠋヨ㦂๓᪥࡟ 80㹼90㸣ࢥࣥࣇ࢚ࣝࣥࢺ࡟࡞ࡗࡓᇵ㣴ࣇࣛࢫࢥ࠿ࡽㄪ〇ࡋࡓᆒ୍࡞⣽⬊ᾮࢆ 4 ᯛ㸦3 ᯛࡣࣝࢩࣇ࢙࣮ࣛࢮάᛶ

ᐃࠊ1 ᯛࡣ⏕⣽⬊ᩘᐃ㸧ࡢ 96 ࢚࢘ࣝࣉ࣮ࣞࢺ࡟᧛✀㸦10,000 cells / well㸧ࡍࡿࠋ

3-2. ⿕㦂≀㉁࠾ࡼࡧᑐ↷≀㉁ࡢㄪ〇ཎ๎࡜ࡋ࡚ࠊヨ㦂ᙜ᪥ࡢㄪ〇࡜ࡍࡿࠋ⿕㦂≀㉁ࡣ DMSO ࡟⁐ゎࡋ࡚ 200 mM ࡢ⁐ᾮࢆㄪ〇ࡍࡿࠋDMSO ࡟୙⁐ࡢሙྜࡣ⁛⳦Ỉ࠶ࡿ࠸ࡣᇵ㣴ᾮ࡟࡚ྠᵝ࡟ㄪ〇ࡋࠊࡑࡢ⁐ᾮࡣࠊ⁛⳦㸦౛࠼ࡤℐ㐣㸧ࡍࡿࠋศᏊ㔞ࡀ୙᫂ࡢ⿕㦂≀㉁ࡢሙྜࡣ 40 mg/mL ࠶ࡿ࠸ࡣ 4㸣㸦W/V㸧ࡢ⁐ᾮ࡜ࡍࡿࠋࡇࢀࡽࡢ⁐ᾮࢆ DMSO㸦୙⁐ࡢሙྜࡣ⁛⳦Ỉࡲࡓࡣ⁛⳦ᇵ㣴ᾮ㸧࡛ಸࠎᕼ㔘ࡋ࡚ 12 ẁ㝵ࡢ⃰ᗘ㸦0.098㹼200 mM㸧
⁐ᾮࢆㄪ〇ࡍࡿࠋ
㝜ᛶᑐ↷㸦፹య㸧ࡣ 1 ࣉ࣮ࣞࢺ࠶ࡓࡾ 6 ࢚࢘ࣝศࢆྠᵝ࡟ㄪ〇ࡍࡿࠋ㝧 ᛶ ᑐ ↷ ࡣ Cinnamic Aldehyde 㸦 CAS No: 14371-10-9, trans-3-Phenyl-2-propenal,trans-Cinnamaldehyde㸧ࢆ⏝࠸ࠊDMSO ࡟⁐ゎࡋ࡚ 200 mM ࡢ⁐ᾮࢆㄪ〇ࡋࠊࡉࡽ࡟ DMSO࡛ᕼ㔘ࡋ࡚ 6.4 mM ࡜ࡍࡿࠋࡇࡢ⁐ᾮ࠿ࡽ DMSO ࡛ಸࠎᕼ㔘ࢆࡋ࡚ 5 ẁ㝵ࡢ⃰ᗘ㸦0.4㹼6.4
mM㸧⁐ᾮࢆㄪ〇ࡍࡿࠋࡉࡽ࡟ࠊࡍ࡭࡚ࡢ⁐ᾮࢆ⾑Ύྵ᭷ᇵ㣴ᾮ࡛ 25 ಸᕼ㔘ࡍࡿࠋࡇࢀࡽࡢㄪ〇⁐ᾮࢆྛ࢚࢘ࣝ
࡟ຍ࠼ࡿ㸦ࠕ⿕㦂≀㉁➼ࡢ㐺⏝ࠖཧ↷㸧࡜ࠊ᭱⤊⃰ᗘࡣ⿕㦂≀㉁࡛ 0.98㹼2000 μMࠊ㝧ᛶᑐ
612

↷࡛ 4㹼64 μM ࡜࡞ࡿࠋ㝜ᛶᑐ↷ࡢ DMSO ࡢ᭱⤊⃰ᗘࡣ௚ࡢㄪ〇ᾮ࡜ྠࡌ 1%࡜࡞ࡿࠋ ᑡ࡞ࡃ࡜ࡶ 2 ᅇࡢ⧞ࡾ㏉ࡋࡋᐃࢆ⾜࠺ࡀࠊ3 ᅇ┠ࢆ⾜࠺ሙྜࡶྵࡵ࡚ࠊࡑࢀࡒࢀࡢ⧞ࡾ㏉ᐃࡣ᪥ࢆኚ࠼࡚⾜࠸ࠊ⿕㦂≀㉁ࡢ⁐ᾮㄪ〇ࠊ⣽⬊ࡢ๓ᇵ㣴㸦⥅௦ᩘࡣྠࡌࡶࡢ㸧ࡶ⧞ࡾ㏉ࡋ

ᐃࡈ࡜࡟⾜࠺ࠋ

3-3. ⿕㦂≀㉁➼ࡢ㐺⏝24 ᫬㛫ᇵ㣴ᚋࡢ 4 ᯛࡢࣉ࣮ࣞࢺࡢᇵ㣴ᾮࢆᤞ࡚ࠊ1 ࢚࢘ࣝ࠶ࡓࡾ 150 μL ࡢ⾑Ύྵ᭷ᇵᆅ
㸦ᢠ⏕≀㉁୙ྵ㸧࡛⨨ࡁ᥮࠼ࡿࠋㄪ〇ࡋࡓ⿕㦂≀㉁⁐ᾮ➼ࢆ 50 μL ࡎࡘྛ࢚࢘ࣝ࡟ຍ࠼ࠊ48
᫬㛫ࠊ37±1Υࠊ5㸣CO2 ࢖࣮ࣥ࢟ࣗ࣋ࢱෆ࡛ᇵ㣴ࡍࡿࠋࡓࡔࡋࠊ1 ࢚࢘ࣝࡣ↓ฎ⨨㸦↓⣽⬊ࠊ✵࢚࢘ࣝ㸧࡜ࡍࡿࠋ࢚࢘ࣝ࠿ࡽࡢ⵨Ⓨࡸ஺ᕪởᰁࢆ㑊ࡅࡿࡓࡵ࡟ࣉ࣮ࣞࢺࡈ࡜࡟㐽ⶸࡍࡿࠋ
3-4. ࣝࢩࣇ࢙࣮ࣛࢮάᛶࡢ

ᐃ㐺ษ࡞ࣝࢩࣇ࢙࣮ࣛࢮάᛶࡢ

ᐃ࡟ࡣࠊ1) ឤᗘࡢⰋ࠸࣑ࣝࣀ࣓࣮ࢱࠊ2) ගࡢ஺ᕪ࡟ࡼࡿ

ᐃࡢጉᐖࢆ㜵ࡄ࡟༑ศ࡞࢚࢘ࣝࡢ㧗ࡉࢆᣢࡗࡓࣉ࣮ࣞࢺࠊ3) ༑ศ࡞ឤᗘ࡜ࣂࣛࡘࡁࡢప
࠸ᐃ್ࢆᚓࡿࡓࡵࡢࣝࢩࣇ࢙࣮ࣛࢮᇶ㉁ࡢ㑅ᢥࡀ㔜せ࡛࠶ࡿࠋࡇࢀࡽࢆ☜ㄆࡍࡿࡓࡵ࡟

Annex 3 3)࡟♧ࡉࢀࡓࢭࢵࢺ࢔ࢵࣉ᪉ἲࢆヨ㦂๓࡟☜ㄆࡍࡿࡇ࡜ࢆ່ࡵࡿࠋ
ᇵ㣴⤊஢ᚋࠊୖΎࢆᤞ࡚ࠊࣜࣥ㓟⦆⾪໬⏕⌮㣗ሷỈ୍࡛ᗘὙ࠺ࠋ⣽⬊⁐ゎ⏝⦆⾪ᾮࢆྛ࢚࢘ࣝ࡟ຍ࠼ࠊᐊ

࡛ 20 ศ㛫ฎ⌮ࡍࡿࠋ⣽⬊⁐ゎ≀ࢆྵࡴࣉ࣮ࣞࢺࢆ࣑ࣝࣀ࣓࣮ࢱ࡛

ᐃࡍࡿࡓࡵࠊྛ࢚࢘ࣝ࡟ࣝࢩࣇ࢙࣮ࣛࢮࡢᇶ㉁ᾮ 50 μL ࢆຍ࠼ࠊ1 ⛊ᚅࡕࠊ2 ⛊㛫ࡢⓎග㔞ࢆ✚⟬ࡍࡿࠋ
3-5. ⣽⬊⏕Ꮡ⋡ࡢᐃ⣽ ⬊ ࡢ ⏕ Ꮡ ⋡ ࢆᐃ ࡍ ࡿ ࣉ ࣞ ࣮ ࢺ ࡣ ࠊ ᇵ 㣴 ⤊ ஢ ᚋ ࡟ ᇵ ᆅ ࢆ MTT

㸦 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium
bromide; CAS No. 298-93-1㸧ྵ᭷᪂㩭ᇵᆅ࡟஺᥮ࡍࡿࠋ4 ᫬㛫ࠊ37Υࠊ5㸣CO2 ࢖࣮ࣥ࢟ࣗ࣋
ࢱෆ࡛ᇵ㣴ࡍࡿࠋᇵ㣴ᚋࠊMTT ྵ᭷ᇵᆅࢆᤞ࡚ࠊ10%SDS(Sodium Dodecyl Sulphate)⁐ᾮ࡞࡝୍࡛ኪ⣽⬊⁐ゎᚋࠊ600 nm ࡢ྾ගᗘࢆ
3-6.

ᐃࡍࡿࠋ

ᐃ್࠿ࡽ௨ୗࡢࣃ࣓࣮ࣛࢱ࣮ࢆồࡵࡿࠋ ࣭⿕㦂≀㉁࠾ࡼࡧ㝧ᛶᑐ↷࡛ほᐹࡉࢀࡓࣝࢩࣇ࢙࣮ࣛࢮάᛶࡢ᭱኱ㄏᑟಸ⋡㸸Imax ࣭ࣝࢩࣇ࢙࣮ࣛࢮάᛶࡢㄏᑟࡀ⁐፹ᑐ↷ࡢ 1.5 ಸࡢ㜈್㸦ࣝࢩࣇ࢙࣮ࣛࢮάᛶࡀ 50%ቑຍ㸧ࢆ㉸࠼ࡓ⃰ᗘ㸸EC1.5
 ࣭⣽⬊⏕Ꮡ⋡ࡀ 50㸣࠾ࡼࡧ 30%ῶ࡜࡞ࡿ⃰ᗘ㸸IC50 ࠾ࡼࡧ IC30
ࣝࢩࣇ࢙࣮ࣛࢮάᛶࡢㄏᑟಸ⋡㸦Fold induction㸧ࡣᘧ 1 ࠿ࡽồࡵࡿࠋ඲యࡢ᭱኱ㄏᑟಸ
⋡ࡣࠊಶࠎࡢ⧞ࡾ㏉ࡋᐃࡢᖹᆒ࡜ࡋ࡚ồࡵࡿࠋᘧ㸯  Fold induction 㸻 (Lsample㸫Lblank) / (Lsolvent㸫Lblank)713   Lsample 㸸⿕㦂≀㉁ࡢⓎගᙉᗘ   Lblank  㸸↓⣽⬊ࠊ↓ฎ⨨ࡢᑐ↷࢚࢘ࣝࡢⓎගᙉᗘ   Lsolvent 㸹⣽⬊࡜⁐፹࠿ࡽ࡞ࡿ࢚࢘ࣝࡢᖹᆒⓎගᙉᗘ
EC1.5 ࡣᘧ 2 ࡟ࡼࡾ⥺ᙧ⿵㛫࡟ᇶ࡙࠸࡚ồࡵࡿࠋ඲యࡢ EC1.5 ࡣࠊಶࠎࡢ⧞ࡾ㏉ࡋᐃࡢᗄఱᏛⓗᖹᆒ࡛ồࡵࡿࠋ
ᘧ 2  EC1.5 = (Cb㸫Ca) × {(1.5㸫Ia) / (Ib㸫Ia)} 㸩Ca   Ca㸸1.5-fold induction ࢆ㉸࠼ࡓ᭱ప⃰ᗘ㸦μM㸧   Cb㸸1.5-fold induction ᮍ‶ࡢ᭱㧗⃰ᗘ㸦μM㸧   Ia㸸1.5-fold induction ࢆ㉸࠼ࡓ᭱ప⃰ᗘ࡛ࡢ fold induction㸦3 ᅇ   Ib㸸1.5-fold induction ᮍ‶ࡢ᭱㧗⃰ᗘ࡛ࡢ fold induction㸦3 ᅇ

ᐃࡢᖹᆒ㸧

ᐃࡢᖹᆒ㸧

⏕Ꮡ⋡㸦Viability㸧ࡣᘧ 3 ࠿ࡽồࡵࡿࠋ
ᘧ 3  Viability = (Vsample㸫Vblank) / (Vsolvent㸫Vblank) × 100   Vsample㸸⿕㦂≀㉁ࡢ࢚࢘ࣝࡢ MTT ྾ගᗘ   Vblank㸸↓⣽⬊ࠊ↓ฎ⨨ࡢᑐ↷࢚࢘ࣝࡢ MTT ྾ගᗘ   Vsolvent㸸⣽⬊࡜⁐፹࠿ࡽ࡞ࡿ࢚࢘ࣝࡢᖹᆒ MTT ྾ගᗘ
IC50 ࡜ IC30 ࡣᘧ 4 ࡟ࡼࡾ⥺ᙧ⿵㛫࡟ᇶ࡙࠸࡚ồࡵࡿࠋ඲యࡢ IC50 ࡜ IC30 ࡣࠊಶࠎࡢ⧞ࡾ
㏉ࡋ

ᐃࡢᗄఱᏛⓗᖹᆒ࡛ồࡵࡿࠋ

ᘧ 4  ICx 㸻 (Cb㸫Ca)×[{(100㸫x)㸫Va} / (Vb㸫Va)]㸩Ca   㹶㸸⃰ᗘࢆồࡵࡿῶᑡ⋡   Ca㸸⣽⬊⏕Ꮡ⋡㹶㸣ῶࢆ㉸࠼ࡓ᭱ప⃰ᗘ㸦μM㸧   Cb㸸⣽⬊⏕Ꮡ⋡㹶㸣ῶᮍ‶ࡢ᭱㧗⃰ᗘ㸦μM㸧   Va㸸⣽⬊⏕Ꮡ⋡㹶㸣ῶࢆ㉸࠼ࡓ᭱ప⃰ᗘ࡟࠾ࡅࡿ⏕Ꮡ⋡   Vb㸸⣽⬊⏕Ꮡ⋡㹶㸣ῶᮍ‶ࡢ᭱㧗⃰ᗘ࡟࠾ࡅࡿ⏕Ꮡ⋡
1.5-fold induction ࢆ㉸࠼ࡓྛ⃰ᗘ࡟ࡘ࠸࡚ࠊࣝࢩࣇ࢙࣮ࣛࢮάᛶࡢㄏᑟࡀ⤫ィᏛⓗ࡟㝜ᛶᑐ↷࡟ᑐࡋ࡚᭷ព㸦p㸺0.05㸧࡛࠶ࡿ࠿ࢆ᳨ドࡍࡿࠋࢢࣛࣇࢆసᡂࡋ࡚どぬⓗ࡟☜ㄆࡍࡿࡇ࡜ࡶ᥎ዡࡍࡿࠋ᫂ࡽ࠿࡞⃰ᗘ౫Ꮡᛶࡀㄆࡵࡽࢀ࡞࠸ሙྜࠊ⃰ᗘ཯ᛂ᭤⥺ࡀ஧┦ᛶࢆ♧ࡍሙྜ࡟ࡣࠊᐃࢆ⧞ࡾ㏉ࡋࠊ⿕㦂≀㉁≉␗ⓗ࠿ࠊᐃ࢚࣮ࣛ࡞ࡢ࠿࡞࡝ࢆ☜ㄆࡍࡿࠋ஧┦ᛶ࡛࠶ࡿࡇ࡜ࡀ☜ㄆ࡛ࡁࡓሙྜࡣࠊࡼࡾప࠸ EC1.5 ್ࢆ㑅ᢥࡍࡿࠋ
3-7.

ᐃᡂ❧᮲௳

௨ୗࡢ 3 ᮲௳ࢆࡍ࡭࡚‶ࡓࡍሙྜ࡟ᡂ❧ࡍࡿࠋ1) 㝧ᛶᑐ↷ࡢ Cinnamic Aldehyde ࡣ㝧ᛶ࡛࡞ࡃ࡚ࡣ࡞ࡽ࡞࠸ࠋࡍ࡞ࢃࡕࠊ㝧ᛶᑐ↷ࡢㄏ8
14ᑟࡣᑡ࡞ࡃ࡜ࡶ 1 ⃰ᗘ࡛ 1.5 ࡢ㜈್௨ୖ࡛⤫ィᏛⓗ࡟᭷ព࡛࡞ࡃ࡚ࡣ࡞ࡽ࡞࠸ࠋ
2㸧Cinnamic Aldehyde ࡢ EC1.5 ್ࡣࣄࢫࢺࣜ࢝ࣝࢹ࣮ࢱ㸦7 μM ࡜ 30 μM ࡢ㛫ࡢࣂࣜࢹ࣮ࢩ
ࣙࣥࢹ࣮ࢱ࡟ᇶ࡙ࡁᐃᮇⓗ࡟᭦᪂ࡍࡿࡇ࡜㸧ࡢᖹᆒࡢ 2 ᶆ‽೫ᕪ್௨ෆ࡛࠶ࡿࡇ࡜࡜ࠊ
64 μM ࡢ Cinnamic Aldehyde ࡢ 3 ࣉ࣮ࣞࢺࡢᖹᆒࡢ Fold induction ࡣ 2 ࠿ࡽ 8 ࡢ㛫࡟࠶ࡿࡇ࡜ࢆ☜ㄆࡍࡿࠋᚋ⪅ࡀ‶ࡓࡉࢀ࡞࠸ሙྜࡣࠊCinnamic Aldehyde ࡟ࡼࡿࣝࢩࣇ࢙ࣛ
࣮ࢮάᛶࡢㄏᑟ࡜⃰ᗘ౫Ꮡᛶࡢ㛵㐃ࢆៅ㔜࡟☜ㄆࡋࠊ⃰ᗘ౫Ꮡᛶࡀ᫂ࡽ࠿࡞ሙྜ࡟ཷ
ࡅධࢀࡽࢀࡿࠋ
3) 3 ࣉ࣮ࣞࢺࡢ 6 ࢚࢘ࣝࡢ⁐፹ᑐ↷㸦ྜィ 18 ࢚࢘ࣝ㸧ࡢᖹᆒኚືಀᩘࡀ 20㸣௨ୗ࡛࠶ࡿࡇ࡜ࡀᚲせ࡛ࠊࡇࢀࡼࡾࡶ㧗࠸ሙྜࡣ↓ຠ࡜ࡍࡿࠋ
3-8. 㝧ᛶࡢุᐃ
2 ᅇࡢ⧞ࡾ㏉ࡋᐇ㦂ࡢ 2 ᅇ࠶ࡿ࠸ࡣ 3 ᅇࡢ⧞ࡾ㏉ࡋᐇ㦂ࡢ 2 ᅇ࡛ࠊ௨ୗࡢࡍ࡭࡚ࡢ᮲௳࡟
ྜ⮴ࡋࡓሙྜ࡟⿕㦂≀㉁ࡣឤసᛶ≀㉁࡜ุ᩿ࡍࡿࠋ
1) Imax ್ࡀ 1.5 ಸㄏᑟࡼࡾࡶ኱ࡁࡃ⁐፹ᑐ↷࡟ẚ㍑ࡋ࡚⤫ィᏛⓗ࡟᭷ព࡛࠶ࡿࡇ࡜ࠋ
2) 1.5 ಸ௨ୖࡢࣝࢩࣇ࢙࣮ࣛࢮάᛶࡢㄏᑟࢆ㉳ࡇࡋࡓ᭱ప⃰ᗘ࡟࠾࠸࡚ࠊ⣽⬊⏕Ꮡ⋡ࡣ
70㸣௨ୖ࡛࠶ࡿࡇ࡜ࠋ
3) EC1.5 ್ࡀ 1000 μM ᮍ‶㸦ศᏊ㔞ᮍ▱ࡢሙྜࡣ 200 mg/mL ᮍ‶㸧࡛࠶ࡿࡇ࡜ࠋ
4) ࣝࢩࣇ࢙࣮ࣛࢮࡢㄏᑟ࡟᫂ࡽ࠿࡞඲యⓗ⃰ᗘ౫Ꮡᛶࡀ࠶ࡿࡇ࡜ࠋ
1㸧࠿ࡽ 3㸧ࡢ࠸ࡎࢀࡶࡀ‶ࡓࡉࢀࡓࡀࠊࣝࢩࣇ࢙࣮ࣛࢮࡢㄏᑟ࡟᫂ࡽ࠿࡞⃰ᗘ౫Ꮡᛶࡀ
ㄆࡵࡽࢀ࡞࠸࡜ࡁࠊ⤖ㄽࡣୗࡏࡎࠊ ࡉࡽ࡟⧞ࡾ㏉ࡋࡢᐇ㦂ࡀᚲせ࡜࡞ࡿࠋ1000 μM ᮍ‶㸦ศᏊ㔞ᮍ▱ࡢሙྜࡣ 200 mg/mL ᮍ‶㸧࡛㝜ᛶࡢሙྜࡶ⤖ㄽࡣୗࡏ࡞࠸ࠋ⣽⬊ẘᛶࢆ♧ࡍ⃰ᗘ㡿ᇦ࡛ࣝࢩࣇ࢙࣮ࣛࢮάᛶࡢㄏᑟࢆ♧ࡍ≀㉁ࡣࠊࢃࡎ࠿࡞⃰ᗘኚ໬࡛㝧ᛶࡢุᐃࡀそࡿ౛ࡀࡲࢀ࡟࠶ࡿࠋࡇࡢࡼ࠺࡞≀㉁ࡣࠊࡼࡾ⊃࠸⃰ᗘ⠊ᅖ࡛ࡼࡾᑠࡉ࠸ᕼ㔘⣔ิ㸦౛࠼ࡤ 1.33
࠶ࡿ࠸ࡣ 1.41㸧ࢆ⏝࠸࡚ࠊㄏᑟࡀ⣽⬊ẘᛶ⃰ᗘ࡛㉳ࡇࡿࡢ࠿ྰ࠿ࢆỴࡵࡿࡇ࡜ࡀᚲせ࡛࠶ࡿࠋ4㸬⢭ᗘGivaudan ♫ࡢඛ⾜ࣂࣜࢹ࣮ࢩࣙࣥ◊✲ 4)࡟࠾࠸࡚ࠊᢏ⾡⛣㌿ᛶࠊ᪋タෆ෌⌧ᛶ࠾ࡼࡧ᪋タ㛫෌⌧ᛶࡀ᳨ウࡉࢀ࡚࠸ࡿࠋ
4-1. ᢏ⾡⛣㌿ᛶ㸦⾲ 1㸧
 7 ≀㉁ࢆ⏝࠸࡚୺ᑟ᪋タࡢ Givaudan ♫࠿ࡽ Procter&Gamble ♫ࠊBeiersdorf ♫ࠊInstitute forin vitro science㸦IIVS㸧࠾ࡼࡧ BASF ࡢ 4 ᪋タ࡬ࡢᢏ⾡⛣㌿ᛶ࡟ࡘ࠸࡚ホ౯ࡀ⾜ࢃࢀࡓࠋࡑ
ࡢ⤖ᯝࠊ┤᥋ࡢᢏ⾡ᣦᑟࢆࡏࡎ࡟ SOP ࡢᥦ౪ࡔࡅ࡛ᢏ⾡⛣㌿ࡣྍ⬟࡛࠶ࡗࡓࠋ
GHS(Globally Harmonized System of Classification and Labelling of Chemicals)࡛༊ศ 1A ࡟ศ㢮ࡉࢀࡿ 2,4-Dinitrochlorobenzene ࠾ࡼࡧ CitralࠊGHS ࡛༊ศ 1B ࡟ศ㢮ࡉࢀࡿ Ethylene glycol
915dimethacrylate ࡣࠊ඲ 5 ᪋タ 3 ᅇ⧞ࡾ㏉ࡋ⾜ࡗࡓヨ㦂ࡢ⤖ᯝࡣࡍ࡭࡚㝧ᛶ࡛࠶ࡗࡓࠋ୍᪉ࠊ
GHS ࡛༊ศ 1B ࡟ศ㢮ࡉࢀࡿ Hexyl cinnamic aldehyde ࡣ 2 ᪋タ࡛ 3 ᅇ୰ 3 ᅇ㝧ᛶ࡛࠶ࡗࡓࡀࠊ୺ᑟ᪋タࢆྵࡴ 3 ᪋タ࡛ࡣࡍ࡭࡚㝜ᛶ࠶ࡿ࠸ࡣࠊ㝧ᛶ 1 ᅇࠊ㝜ᛶ 2 ᅇࡢΰᅾࡋࡓุᐃ⤖ᯝࢆ♧ࡋࡓࠋ㠀ឤసᛶ≀㉁࡛࠶ࡿ ChlorobenzeneࠊMethyl salicylate ࠾ࡼࡧ Sulfanilamide ࡣࡍ࡭࡚ࡢ᪋タ࡛㝜ᛶ࡛࠶ࡗࡓࡀࠊ3 ᅇ⧞ࡾ㏉ࡋ୰ 1 ᅇ㝧ᛶࡢุᐃࢆ♧ࡍ᪋タࡀ࠶ࡗࡓࠋࡇࢀࡽࡢ
⤖ᯝࡣࠊ୺ᑟ᪋タࡀ᝿ᐃࡋࡓ⠊ᅖෆࡢ⤖ᯝ࡛࠶ࡿ࡜᥎

ࡉࢀࡿࡓࡵࠊᢏ⾡⛣㌿ᛶ࡟ၥ㢟ࡣ࡞࠸࡜⪃࠼ࡽࢀࡿࠋ

⾲ 1ᢏ⾡⛣㌿ᛶࡢホ౯ᡂ⦼
CAS

Positve with EC 1.5 up to 1000 PM

GHS
potency
category

Lead Lab.
Histolocal
Data

No.

Chemical Name

1

2,4-Dinitrochlorobenzene

97-00-7

3 of 33 of 33 of 35392-40-5

2 of 22 of 23 of 3

Citral1A
1A

3 of 32

3 of 33 of 33 of 33 of 33 of 33456Ethylne glycol dimethacrylate
Hexyl cinnamic aldehyde
Chlorobenzene
M ethyl salicylate

97-90-5
101-86-0
108-90-7
119-36-8

1B
1B
No
No

2 of 22 of 20 of 20 of 23 of 3
1 of 30 of 30 of 33 of 30 of 30 of 3
1 of 33 of 3
1 of 30 of 30 of 33 of 33 of 30 of 3
1 of 33 of 33 of 3
1 of 3
1 of 37

Sulfanilamide

64-74-1No

0 of 20 of 30 of 30 of 3

1 of 30 of 3

Lead䚷lab.

Lab.1

Lab.2

Lab.3

Lab.4

4-2. ᪋タෆ෌⌧ᛶ㸦⾲ 2㸧 ࣈࣛ࢖ࣥࢻୗ࡛ホ౯ࡉࢀࡓ 21 ≀㉁࡟࠾࠸࡚ࠊ5 ᪋タࡢ᪋タෆ෌⌧ᛶ㸦3 ᅇࡢ⧞ࡾ㏉ࡋࡢ
ヨ㦂࡛ྠࡌ⤖ᯝ㸧ࡣࠊ୺ᑟ᪋タ㸸90.5%ࠊヨ㦂᪋タ 1㸸90.5%ࠊヨ㦂᪋タ 2㸸95.2%ࠊヨ㦂᪋
タ 3㸸95.2%ࠊヨ㦂᪋タ 4㸸81.0%࡛࠶ࡗࡓࠋᮏヨ㦂࡛ࡣࠊ㐩ᡂᇶ‽ࡣタᐃࡉࢀ࡚࠸࡞࠸ࡓࡵࠊDPRA ࡢࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡛᥇⏝ࡉࢀࡓ 85㸣ࢆ┠Ᏻ࡟⪃࠼ࡓሙྜࠊ5 ᪋タ୰ 4 ᪋タࡀࡇࡢᇶ‽ࢆୖᅇࡗࡓࠋᇶ‽࡟㐩ࡋ࡞࠿ࡗࡓヨ㦂᪋タ 4 ࡟࠾࠸࡚෌⌧ᛶࡢᚓࡽࢀ࡞࠸≀㉁ࡣࠊGHS
࡛༊ศ 1B ࡟ศ㢮ࡉࢀࡿ EugenolࠊPhenyl benzoateࠊ࠾ࡼࡧ㠀ឤసᛶ≀㉁࡛࠶ࡿ Diethyl phthalate
࠾ࡼࡧ Sodium lauryl sulfate ࡛࠶ࡗࡓࠋ ࡓࡔࡋࠊᮏホ౯࡟౑⏝ࡉࢀࡓ⓶⭵ឤసᛶ㝧ᛶ≀㉁ࡢෆヂࡣࠊGHS ࡛ 1A ࡟ศ㢮ࡉࢀࡿ≀㉁ࡀ 11 ≀㉁࡟ᑐࡋࠊ᪋タෆ࡛෌⌧ᛶࡀᚓࡽࢀ࡞࠸ሙྜࡢ࠶ࡿ 1B ࡟ศ㢮ࡉࢀࡿ≀㉁ࡀ 4 ≀㉁࡜೫ࡾࡀ࠶ࡿࡇ࡜ࡣ␃ពࡍ࡭ࡁⅬ࡜⪃࠼ࡿࠋ
4-3. ᪋タ㛫෌⌧ᛶ㸦⾲ 2㸧 ࣈࣛ࢖ࣥࢻୗ࡛ホ౯ࡉࢀࡓ 21 ≀㉁ࡢ 5 ᪋タࡢ᪋タ㛫෌⌧ᛶ㸦5 ᪋タ࡛ྠࡌ⤖ᯝ㸧ࡣ 85.7%
࡛࠶ࡗࡓࠋᮏඛ⾜ࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡛ࡣࠊ㐩ᡂᇶ‽ࡣタᐃࡉࢀ࡚࠸࡞࠸ࡓࡵࠊDPRA ࡢࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡛᥇⏝ࡉࢀࡓ 80㸣ࢆ┠Ᏻ࡟⪃࠼ࡓሙྜࠊࡇࡢᇶ‽ࢆୖᅇࡗࡓࠋ᪋タෆ෌⌧ᛶ࡜ྠᵝ࡟ࠊᮏホ౯࡟౑⏝ࡉࢀࡓ⓶⭵ឤసᛶ㝧ᛶ≀㉁ࡢෆヂ࡟೫ࡾࡀ࠶ࡿࡇ
࡜ࡣ␃ពࡍ࡭ࡁⅬ࡜⪃࠼ࡿࠋ
10
16

⾲ 2
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

᪋タ㛫ኚື࠾ࡼࡧ᪋タෆኚືࡢホ౯ᡂ⦼
Chemical Name

CAS

2-M ercaptobenzothiazole
149-30-4
4-M ethylaminophenol sulphate 55-55-0
4-Nitrobenzylbromide
100-11-8
4-Phenylenediamine
106-50-3(5-Chloro)26172-55-4
methylisothiazolinone
Cinnamic aldehyde
104-55-2
Isoeugenol
97-54-1
Glyoxal107-22-2
M ethyldibromo glutaronitrile 35691-65-7
Oxazolone
15646-46-5
Tetramethylthiuramdisulfide
137-26-8
Cinnamyl alcohol
104-54-1
Eugenol
97-53-0
Imidazolidinyl urea
39236-46-9
Phenyl benzoate
93-99-2
Diethyl phthalate
84-66-2Isopropanol
67-63-0Glycerol
56-81-5Lactic acid50-21-5
Salicylic acid
69-72-7

21 Sodium lauryl sulfate

151-21-3

GHS
potency
category
1A
1A
1A
1A
1A
1A
1A
1A
1A
1A
1A
1B
1B
1B
1B
No
No
No
No
No
No

Within Laboratory Reproducibility

Positve with EC 1.5 up to 1000 PM
Lead䚷lab.

Lab.1

Lab.2

Lab.3

Lab.4

BetweenLaboratory
Reproducibility

3 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 3YYY
Y

3 of 33 of 33 of 33 of 33 of 3Y

3 of 33 of 33 of 33 of 33 of 33 of 33 of 3
1 of 32 of 30 of 30 of 30 of 30 of 30 of 30 of 33 of 33 of 33 of 33 of 33 of 33 of 32 of 3
1 of 33 of 30 of 30 of 30 of 30 of 30 of 30 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 30 of 3
1 of 30 of 30 of 30 of 30 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 30 of 30 of 30 of 30 of 30 of 30 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 3
1 of 33 of 3
1 of 32 of 30 of 30 of 30 of 30 of 3

Y
Y
Y
Y
Y
Y
Y
N
Y
Y
N
Y
Y
Y
Y

3 of 3

N

3 at
1 at
1 at
1 at
cytotox,conc. cytotox,conc. cytotox,conc. cytotox,conc.

90.5%(19/21) 90.5%(19/21) 95.2%(20/21) 100%(21/21) 85.7%(18/21)

85.7%(18/21)

5㸬ṇ☜ᗘ㸦ឤᗘ࠾ࡼࡧ≉␗ᗘ㸧
Givaudan ♫ࡢඛ⾜ࣂࣜࢹ࣮ࢩࣙࣥ◊✲ 4)࡟࠾࠸࡚ࣈࣛ࢖ࣥࢻୗ࡛ホ౯ࡉࢀࡓ 21 ≀㉁ࢆᇶ
࡟ GHS ศ㢮࡜ࡢ୍⮴ᗘࠊឤᗘ࠾ࡼࡧ≉␗ᗘ࡟㛵ࡍࡿホ౯ࢆ⾜ࡗࡓࠋ ᐇ᪋ 5 ᪋タࡢᡂ⦼ࡢ✚⟬࡟ࡼࡿឤᗘ㸦Sensitivity㸧ࡣ 89.3%ࠊ≉␗ᗘ㸦Specificity㸧ࡣ 93.3%ࠊ
ṇ☜ᗘ㸦Accuracy㸧ࡣ 90.5%࡛࠶ࡗࡓࠋヨ㦂᪋タẖࡢᡂ⦼ࡣࠊ୺ᑟ᪋タ࠾ࡼࡧヨ㦂᪋タ 1㸸
ឤᗘ 86.7%ࠊ≉␗ᗘ 100%ࠊṇ☜ᗘ 90.5㸣ࠊヨ㦂᪋タ 2 ࠾ࡼࡧ 3㸸ឤᗘ 93.3%ࠊ≉␗ᗘ 100%ࠊṇ☜ᗘ 95.2㸣ࠊヨ㦂᪋タ 4㸸ឤᗘ 86.7%ࠊ≉␗ᗘ 66.7%ࠊṇ☜ᗘ 81.0㸣࡛࠶ࡗࡓࠋ
ᮏヨ㦂ἲࡢṇ☜ᗘ࡟㛵ࡋ࡚㛤Ⓨ᪋タ࡛࠶ࡿ Givaudan ♫࠿ࡽᥦฟࡉࢀࡓࣄࢫࢺࣜ࢝ࣝࢹ࣮
ࢱ࡛ࡣ 92.9㸣࡜ሗ࿌ࡉࢀ࡚࠸ࡿࠋࡲࡓࠊึᮇࡢホ౯≀㉁ᩘࡀ୙༑ศ࡛࠶ࡿ࡜ࡢ ESAC ࡢࢥ࣓ࣥࢺࢆཷࡅࠊGivaudan ♫ࡀᐇ᪋ࡋࡓ 67 ≀㉁ 㸦ឤసᛶ≀㉁ 44ࠊ㠀ឤసᛶ≀㉁ 23㸧࡜ࠊࡉ
ࡽ࡟㏣ຍ࡛ᐇ᪋ࡋࡓ≀㉁ࢆྵࡴ 114 ≀㉁㸦ឤసᛶ≀㉁ 86ࠊ㠀ឤసᛶ≀㉁ 28㸧࡛ࡢホ౯⤖ᯝࡣ 4)ࠊ67 ≀㉁㸸ឤᗘ 86.4㸣ࠊ≉␗ᗘ 82.6㸣ࠊṇ☜ᗘ 85.1%ࠊ114 ≀㉁㸸ឤᗘ 76.7㸣ࠊ≉␗ᗘ
82.1㸣ࠊṇ☜ᗘ 78.1㸣࡛࠶ࡗࡓࠋࡉࡽ࡟ࠊNatsch ࡽ 5)ࡢ 145 ≀㉁ࢆ⏝࠸ࡓヨ㦂࡛ࡣࠊឤᗘ 77%ࠊ≉␗ᗘ 79%ࠊṇ☜ᗘ 72%࡛࠶ࡗࡓࠋࡲࡓࠊ⾲ 3 ࡟♧ࡍ㏻ࡾࠊGHS ࡛ 1A ࡟ศ㢮ࡉࢀࡿ≀㉁ࢆㄗࡗ࡚㝜ᛶ࡜ุ᩿ࡍࡿࡇ࡜ࡣ࡞࠿ࡗࡓࠋ

1117

⾲ 3 GHS ༊ศ 1A ≀㉁ࡢᡂ⦼
Imax

GHS
ChemicalCas no.
ศ㢮20086)

20107)

20118)INVITTOX4)

Citral5392-40-5

1A

9.8

96.4

22.3-104.4

96.4

Tetramethylthiuram disulfide

137-26-8

1A

ND

6.8

8.1-67.9

6.8

Cinnamic aldehyde

104-55-2

1A

31.6

16.2

9.8-44.7

16.2

2-Mercaptobenzothiazole

149-30-4

1A

10.9

8.8

4.9-64.1

8.8

Glyoxal107-22-2

1A

ND

28.2

14.5-195.0

28.2

Isoeugenol

97-54-1

1A

60.2

6.4

9.5-56.8

6.4

1,2-Dibromo-2,4-dicyanobutane

35691-65-7

1A

ND

4.0

2.1-7.8

4.0

4-(Methylamino)phenol sulfate

55-55-0

1A

32.4

5.9

4.5-12.2

10.3

1,4-Phenylenediamine

106-50-3

1A

12.7

26.8

19.4-45.2

26.8

4-Nitrobenzyl bromide

100-11-8

1A

ND

6.9

4.7-14.0

6.9

1-Chloro-2,4-dinitrobenzene

97-00-7

1A

12.3

14.8

4.3-19.5

14.8

5-Chloro-2-methyl-4-isothiazolin-3-one

26172-55-4

1A

7.2

7.2

4.8-13.5

7.2

Oxazolone

15646-46-5

1A

2.3

2.4

6.4-46.3

2.4

ND: Not determined

6. ホ౯ྍ⬟࡞≀㉁ࡢ⠊ᅖ
Emter ࡽࡀሗ࿌ࡋ࡚࠸ࡿࡼ࠺࡟ 6)ࠊᵝࠎ࡞ᵓ㐀ࢆ᭷ࡍࡿ໬Ꮫ≀㉁ࡢ⓶⭵ឤసᛶࡢண

ࡀྍ⬟࡛࠶ࡿ㸦⾲ 4㸧ࠋࡓࡔࡋࠊ⁐ゎᛶࡸ⁐፹୰࡛ࡢᏳᐃᛶ㸦౛࠼ࡤࠊ⁐፹୰࡛ࡢຍỈศゎ㸧ࡀၥ㢟࡛ホ౯୙ྍ⬟࡜࡞ࡿ≀㉁ࡀᏑᅾࡍࡿࠋලయⓗ࡟ࡣࠊLogP ࡀ 7 ௨ୖࡢᙉ␯ỈᛶศᏊࡣDMSO ࡜Ỉ࡬ࡢ⁐ゎ≉ᛶ࠿ࡽヨ㦂ࡀ࡛ࡁ࡞࠸ࠋ୍᪉ࠊLogP ࡀ 5 ࡲ࡛ࡢ໬Ꮫ≀㉁ࡣࠊỈ࠶ࡿ࠸ࡣ DMSO ࡟ྍ⁐࡞ࡓࡵࠊᐜ᫆࡟ヨ㦂ࡀྍ⬟࡛࠶ࡿࠋ㓟↓Ỉ≀ࡣࠊࢩࢫࢸ࢖ࣥṧᇶ࡛ࡣ࡞ࡃࣜࢪࣥṧᇶ࡜཯ᛂ≉ᚩࡀ࠶ࡾࠊࢩࢫࢸ࢖ࣥṧᇶ࡜ࡢ཯ᛂࡀᚲせ࡞ Nrf2 pathway ࢆㄏᑟࡋ࡞࠸ࡇ࡜ࡀ᥎ᐹࡉࢀࠊഇ㝜ᛶ࡜ุᐃࡉࢀࡿࡇ࡜ࡀ⪃࠼ࡽࢀࡿࠋࡉࡽ࡟ࠊ㓟໬཯ᛂࡸ㓟໬ⓗ⬺࢔࣑ࣀ཯ᛂࢆᚲせ࡜ࡍࡿከࡃࡢࣉࣞࣁࣉࢸࣥࢆṇࡋࡃุᐃ࡛ࡁࡿࡀࠊP450 ࡟ࡼࡿάᛶ໬
ࡀᚲせ࡜᥎ᐃࡉࢀࡿࣉࣟࣁࣉࢸࣥࡣ᳨ฟ࡛ࡁ࡞࠸㸦KeratinoSensTM ࡢ⏤᮶⣽⬊࡛࠶ࡿ HaCaT⣽⬊ࡣ⸆≀௦ㅰ㓝⣲άᛶࢆ᭷ࡍࡿࡀ㝈ᐃⓗ࡛࠶ࡿ㸧ࠋࣝࢩࣇ࢙࣮ࣛࢮ㓝⣲࡟ᖸ΅ࡍࡿ໬Ꮫ≀㉁ࡶホ౯࡟ᙳ㡪ࡍࡿ 3)ࠋ

12
18

⾲ 4

Emter ࡽࡢሗ࿌Chemical Name

Sensitizers
Oxazolone
Benzoquinone(5-Chloro)-M ethylisothiazolinone
2,4-Dinitrochlorobenzene
4-nitrobenzylbromide
4-phenylenediamine
Glutaraldehyde
Benzoyl peroxide
Glyoxal4-M ethylaminophenol sulphate
Formaldehyde
M ethyldibromo glutaronitrile
Cinnamic aldehyde
2-Hydroxyethyl acrylate
Isoeugenol
Ethylenediamine
Benzylidene Acetone
M ethyl-2-nonynoate
2-M ercaptobenzothiazole
Benzyl salicylate
Tetramethylthiuramdisulfide
Diethylenetriamine
Thioglycerol
Phenylacetaldehyde
Resorcinol
Dihydroeugenol
Benzoisothiazolione
CitralHexyl cinnamic aldehyde
Eugenol
Abietic acidPhenyl benzoate
Lyral HM PCC
Benzocaine
Benzyl cinnamate
2,4-Dichloronitrobenzene
Cinnamyl alcohol
Hydroxycitronellal
Imidazolidinyl urea
Butyl glycidyl ether
Ethylene glycol dimethacrylate
Cobalt chloride
Nickel sulfate

LLNA EC3
䚷

KeratinoS ens results
ARE I max

ARE EC1.5

0.003
0.01
0.01
0.05
0.05
0.11
0.12
0.22
0.75
0.80
0.84
0.90
1.3
1.4
1.5
2.2
2.2
2.5
2.5
2.9
3.1
3.3
3.5
4.5

䚷
2.4
15.2
7.2
14.8
6.9
26.8
80.7
1.4
28.2
5.9
16.9
4.0
16.2
54.9
6.4
13.2
503.9
33.1
8.8
5.5
6.8
1.7
1.5
11.3

175.5
6.5
8.7
2.5
1.3
5.0
24.3
n.i.
89.1
9.4
63.2
7.8
16.1
32.3
16.1
99.9
9.7
1.8
48.1
8.4
0.8
1259.4
n.i.
28.5

5.9
6.8
7.8
9.8
9.9
10.1
11.6
13.6
17.1
17.1
18.4
20.0
21.0
23.0
24.0
30.9
32.9
Pos.
var.

1.0
1.5
24.0
96.4
2.7
1.3
11.4
1.3
16.1
3.0
8.7
2.9
1.7
137.1
2.9
340.7
188.4
23.3
4.2

n.i.
462.0
3.2
23.2
17.3
n.i.
16.6
n.i.
79.6
18.2
11.0
68.3
123.6
79.4
45.4
218.5
57.4
298.6
329.0

ARE IC50

Chemical Name

LLNA EC3

KeratinoS ens results
ARE I max

ARE EC1.5 ARE IC50
Non-sensitizers
1370.9 Sodium lauryl sulfate
var.
1.2
n.i.
44.7
104.5 Salicylic acid
var.
1.1
n.i.
>2000
7.1 M ethyl salicylate
var.
1.2
n.i.
>2000
8.2 Sulfanilamide
NC
1.4
n.i.
>2000
9.1 Diethyl phthalate
>100%
1.1
n.i.
>2000
438.9 Glycerol
>100%
1.2
n.i.
>2000
242.6 Propylene glycol
>100%
1.2
n.i.
>2000
567.6 Benzoic acid
>20%
1.1
n.i.
>2000
677.9 1-Butanol
>20%
1.1
n.i.
>2000
11.7 4-Hydroxybenzoic acid
>25%
1.1
n.i.
>2000
201.6 Sulfanilic acid
>25%
1.3
n.i.
>1000
25.6 Tartaric acid
>25%
1.2
n.i.
>2000
194.4 Propylparaben
>25%
9.7
14.5
813.1
207.2 Ethyl vanillin
>50%
5.4
161.7
>2000
731.4 Isopropanol
>50%
1.2
n.i.
>2000
>2000 Benzyl alcohol
>50%
1.2
n.i.
>2000
174.5 Dimethylisophtalate
NC
2.1
694.9
>2000
121.9 Dextran
NC
1.5
n.i.
>2000
1003.1 Tween 80
NC
2.7
19.3
399.8
111.0 Chlorobenzene
Neg.
1.2
n.i.
>2000
39.1 Lactic acid
Neg.
1.3
n.i.
>2000
>2000 Phenol
Neg.
1.3
n.i.
>2000
>2000 Benzaldehyde
>25
2.3
443.1
>2000
116.2 Octanoic acid
>50
1.1
n.i.
>2000
n.i.: no significant induction above threshold
>2000
759.2
ver.: Variable results
50.9
NC: Level not specified
Neg: Negative reference according to D. Basketter. Food Chem. Toxicol. 37, 1167-1174
182.8
26.3
1505.7
104.6
191.6
355.4
>2000
>2000
816.0
774.6
>2000
90.4
>2000
1655.81330.2998.7

7. ᭷⏝ᛶ࡜㝈⏺
ᮏヨ㦂ἲࡣࠊ⣽⬊ᇵ㣴ࡢᢏ⾡࡜ 96 ࢚࢘ࣝᑐᛂࡢ࣑ࣝࣀ࣓࣮ࢱ࣮ࡢ౑⏝ᢏ⾡ࡀ࠶ࢀࡤᐜ᫆
࡟ᐇ᪋ྍ⬟࡛࠶ࡿࠋKeratinoSensTM ἲ࠾ࡼࡧ LLNA ࡟㛵ࡍࡿ㈝⏝ࡢヨ⟬࡛ࡣࠊLLNA ࡛ࡣ 1≀㉁ᙜࡓࡾࡢࣛࣥࢽࣥࢢࢥࢫࢺࡀ⣙ 10 ୓෇࡛࠶ࡿࡢ࡟ᑐࡋࠊKeratinoSensTM ἲ࡛ࡣ 1 ≀㉁࡟ࡘ࠸࡚ 12 ⃰ᗘ࡛୕㔜ᐃࠊ࠿ࡘ 3 ᅇࡢ⧞ࡾ㏉ࡋᐇ㦂ࡢሙྜࡢࣛࣥࢽࣥࢢࢥࢫࢺࡣ⣙ 1.3

࠿ࡽ 1.5 ୓෇࡛࠶ࡿࠋᐇ㦂ᮇ㛫ࡶ LLNA ࡼࡾ▷ᮇ㛫࡛ᐇ᪋ྍ⬟࡛࠶ࡿࡇ࡜࠿ࡽࠊ⤒῭ᛶ࣭⡆౽ᛶࡢ㠃࠿ࡽ᭷⏝࡜⪃࠼ࡽࢀࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊ⌧᫬Ⅼ࡛ࡇࡢ
TMࡽࢀ࡚࠸ࡿ⣽⬊⣔ࡣ KeratinoSensᐃ࡟౑⏝࡛ࡁࡿࡇ࡜ࡀ▱ࡢࡳ࡛࠶ࡿࠋࡑࡢ౑⏝࡟ࡣࠊᮏ⣽⬊⣔ࢆᶞ❧ࡋࡓ

Givaudan ♫࠿ࡽࣛ࢖ࢭࣥࢫࢆཷࡅࡿࡇ࡜ࡀᚲせ࡜⪃࠼ࡽࢀࡿࡀࠊ㈝⏝ࡶྵࡵࡑࡢ᮲௳ࡢヲ⣽ࡣࢃ࠿ࡗ࡚࠸࡞࠸ࠋ୍᪉ࠊKeratinoSensTM ࢆ౑⏝ࡋ࡞࠸ሙྜ࡟ࡣࠊྠ➼ࡢ⣽⬊⣔ࢆᶞ❧ࡋࠊ
OECD సᡂࡢᮏヨ㦂ἲ࡟㛵ࡍࡿ performance standard㸦᱌㸧2)࡟ᚑ࠸ࠊጇᙜᛶࢆホ౯ࡋࡓୖ࡛
౑⏝ࡋ࡞ࡅࢀࡤ࡞ࡽ࡞ࡃࠊ┦ᛂࡢ㈝⏝࡜᫬㛫ࡀᚲせ࡞ࡇ࡜ࡀᐜ᫆࡟᝿ീࡉࢀࡿࠋ
⣽⬊ᇵ㣴ᢏ⾡࡟⇍⦎ࡋࡓ◊✲⪅࡛࠶ࢀࡤࠊ⮬ື໬ࡋࡓᶵჾࢆ౑⏝ࡋ࡞ࡃ࡚ࡶࠊᑡ࡞ࡃ࡜
ࡶ 1 ≀㉁ 12 ẁ㝵ࡢ⃰ᗘ࡛ 42 ≀㉁ࡢ୕㔜ᐃࡢᐇ㦂ࢆ 1 㐌㛫࡛ᐇ᪋࡛ࡁࠊࡉࡽ࡟ࡇࢀࢆ 313
19ᅇࡢ⧞ࡾ㏉ࡋ࡛ᐇ᪋ࡍࡿ࡜ࠊ42 ≀㉁࡟ࡘ࠸࡚ 3 㐌㛫࡛᏶඲࡞⤖ᯝࢆᚓࡿࡇ࡜ࡀ࡛ࡁࡿࡓࡵࠊ
ࣁ࢖ࢫ࣮ࣝࣉࢵࢺࡢヨ㦂ἲ࡜⪃࠼ࡽࢀࡿࠋ⮬ື໬ࡉࢀࡓᶵჾࢆ⏝࠸ࢀࡤ᭦࡞ࡿຠ⋡໬ࡶྍ⬟࡛࠶ࡿࠋ
EURL ECVAM ࡀᐇ᪋ࡋࡓ LLNA ࡜ࡢឤసᛶࡢ᭷↓ࡢẚ㍑㸵)࡛ࡣࠊṇ☜ᗘࡀ 77㸣㸦155/201㸧ࠊ
ឤᗘ 78㸣㸦71/91㸧ࠊ≉␗ᗘ 76㸣㸦84/110㸧࡛࠶ࡾࠊNatsch ࡽ 6)ࡢ 145 ≀㉁ࢆ⏝࠸ࡓヨ㦂࡛ࡶྠᵝࡢ⤖ᯝ㸦77%, 79%, 72%㸧ࡀᚓࡽࢀ࡚࠸ࡿࠋᮏヨ㦂ἲࡣឤసᛶ≀㉁࡜㠀ឤసᛶ≀㉁ࢆศ
ูࡍࡿࡢ࡟ຍ࠼࡚ࠊ⏝㔞-཯ᛂ᝟ሗࢆᚓࡿࡢ࡟ࡶᐤ୚ࡍࡿྍ⬟ᛶࡀ࠶ࡿ࡜ࡢሗ࿌ࡀ࠶ࡿࡀࠊ
⌧᫬Ⅼ࡛ࡑࡢ᰿ᣐࡣ༑ศ࡜ࡣゝ࠼࡞࠸ࠋᮏヨ㦂ἲࡣከᡂศ࠶ࡿ࠸ࡣΰྜ≀࡛ࡶᢏ⾡ⓗ࡟ࡣ㐺⏝ྍ⬟࡛࠶ࡿࡀࠊᐇ᪋౛ࡀ࡞࠸ࡓࡵࠊ
ៅ㔜࡞ุ᩿ࡀᚲせ࡛࠶ࡿࠋࡲࡓࠊ2000 μM ࢆ᭱㧗⃰ᗘ࡜ࡋ࡚ヨ㦂ࢆᐇ᪋ࡍࡿࡀࠊ㈋⁐ゎᛶࢆ♧ࡍ≀㉁࡟࠾࠸࡚ࠊࡇࡢ᭱㧗⃰ᗘ࡛ࡢホ౯ࡀᅔ㞴࡛ 1000 μM ᮍ‶࡛ᚓࡽࢀࡓ㝜ᛶࡢ⤖ᯝࢆᇶ࡟ࠊឤసᛶࡀ㝜ᛶ࡜ࡣุᐃ࡛ࡁ࡞࠸ࠋ
8. ⤖ㄽ
ARE-Nrf2 Luciferase Test Method ࡣࠊឤసᛶⓎ⌧ᶵᗎ࡟࠾ࡅࡿ➨஧ẁ㝵ࡢ࢖࣋ࣥࢺ࡛࠶ࡿ
ࢣࣛࢳࣀࢧ࢖ࢺ࡟࠾ࡅࡿ⅖⑕཯ᛂ࠾ࡼࡧ Nrf2-Keap1-ARE pathway ࢆ฼⏝ࡋࡓ࣏࣮ࣞࢱ࣮࢔
ࢵࢭ࢖࡛࠶ࡾࠊ໬Ꮫ≀㉁ࡢឤసᛶࢆุ᩿ࡍࡿୖ࡛㔜せ࡞᝟ሗࢆ୚࠼࡚ࡃࢀࡿࠋ
࣐࢘ࢫࢆ⏝࠸ࡿ LLNA ࡢ 1/7 ⛬ᗘࡢࣛࣥࢽࣥࢢࢥࢫࢺ࡛ᐇ᪋ྍ⬟࡛࠶ࡾࠊin vitro ヨ㦂ἲ࡛࠶ࡿࡇ࡜࠿ࡽ᭷⏝ᛶࡣ㧗࠸ࠋࡋ࠿ࡋ࡞ࡀࡽࠊ⌧᫬Ⅼ࡛ᮏヨ㦂ἲ࡟౑⏝࡛ࡁࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿ⣽⬊⣔ࡣ KeratinoSensTM ࡢࡳ࡛࠶ࡾࠊࡑࡢ౑⏝࡟ࡣ⣽⬊⣔ࢆᶞ❧ࡋࡓ Givaudan ♫࠿ࡽࣛ࢖ࢭࣥࢫࢆཷࡅࡿࡇ࡜ࡀᚲせ࡞ࡓࡵࠊᑟධࡢᐜ᫆࡞ヨ㦂⣔࡜ࡣゝ࠸㞴࠸ࠋᮏヨ㦂ἲࡢඛ⾜ࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡟࠾ࡅࡿ᪋タෆ෌⌧ᛶࡣࠊ5 ᪋タ୰ 1 ᪋タ࡟࠾࠸࡚ࠊ
GHS ༊ศ 1B ࡟ศ㢮ࡉࢀࡿ≀㉁㸦ᙅ࠸ឤసᛶ≀㉁㸧࠾ࡼࡧ㠀ឤసᛶ≀㉁࡛෌⌧ᛶࡀᚓࡽࢀ࡞
࠿ࡗࡓࡓࡵࠊ┠Ᏻ࡜ࡋࡓ㐩ᡂᇶ‽㸦85㸣㸧࡟㐩ࡋ࡚࠾ࡽࡎࠊᙉ࠸ឤసᛶ≀㉁௨እ࡛ࡣุᐃ
ࡀࡪࢀࡿᠱᛕࡀ࠶ࡿࠋ୍᪉ࠊ᪋タ㛫෌⌧ᛶࡣࠊ┠Ᏻ࡜ࡋࡓ㐩ᡂᇶ‽㸦80㸣㸧ࢆୖᅇࡗࡓࡶࡢࡢࠊᮏホ౯࡟౑⏝ࡉࢀࡓ⓶⭵ឤసᛶ㝧ᛶ≀㉁ࡢෆヂࡣࠊGHS ࡛༊ศ 1A ࡟ศ㢮ࡉࢀࡿ≀㉁ࡀ 11 ≀㉁࡟ᑐࡋࠊ᪋タෆ࡛෌⌧ᛶࡀᚓࡽࢀ࡞࠸ሙྜࡢ࠶ࡿ GHS ࡛༊ศ 1B ࡟ศ㢮ࡉࢀࡿ≀㉁ࡀ 4 ≀㉁࡜೫ࡾࡀ࠶ࡿࡇ࡜ࡣ␃ពⅬ࡜⪃࠼ࡿࠋ
100 ≀㉁௨ୖࢆホ౯ࡋࡓ஧ࡘࡢሗ࿌࡟࠾ࡅࡿᮏヨ㦂ἲࡢឤᗘࡣࠊ76.7㸣࡜ 77㸣࡛࠶ࡾࠊ㝜ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓሙྜࡣࠊഇ㝜ᛶࡢྍ⬟ᛶࢆ⪃៖ࡋࠊ⿵᏶ࡋᚓࡿ௚ࡢヨ㦂ࡼࡾ☜ㄆࡋ࡞ࡅࢀࡤ࡞ࡽ࡞ࡃࠊᮏヨ㦂ἲࡢࡳ࡛⓶⭵ឤసᛶࢆ㝜ᛶ࡜ุᐃࡍࡿࡇ࡜ࡣ࡛ࡁ࡞࠸ࠋࡲࡓࠊ≉␗ᗘࡶࠊ82.1%࡜ 79%࡛࠶ࡿࡇ࡜࠿ࡽࠊ㝧ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓሙྜ࡟ࡶࠊഇ㝧ᛶࡢ⤖ᯝࡀ⏕ࡌࡿྍ⬟ᛶࡀ࠶ࡿࡇ࡜࡟␃ពࡋ࡞ࡅࢀࡤ࡞ࡽ࡞࠸ࠋ
ᮏヨ㦂ἲ࡛ࡣࠊάᛶ໬࡟௦ㅰ⣔ࢆᚲせ࡜ࡍࡿ໬Ꮫ≀㉁ࡣࠊṇࡋࡃࡑࡢឤసᛶࡀ᳨ฟࡉࢀ
࡞࠸ྍ⬟ᛶࡀ࠶ࡿࠋࡲࡓࠊ⣽⬊ࢆ⏝࠸ࡓホ౯⣔࡛࠶ࡿࡓࡵࠊ⣽⬊ẘᛶࡸ␯Ỉᛶࡢ㧗࠸≀㉁࡛ࡣつᐃࡉࢀ࡚࠸ࡿ᭱㧗⃰ᗘ㸦2000 μM㸧࡛ࡢホ౯ࡀ㞴ࡋࡃࠊ㝜ᛶุᐃࡀୗࡏ࡞࠸ሙྜࡀ࠶
14
20

ࡿࠋ
௨ ୖࢆ ㋃ࡲ࠼ ࠊᮏ ጤဨ఍ ࡣࠊ ᮏヨ㦂 ἲࡀ ⓶⭵ឤ సᛶ ホ౯࡟ ỗ⏝ ࡉࢀࡿ ࡓࡵ ࡟ ࡣ ࠊ
KeratinoSensTM ࡀᏳ౯࡟ධᡭ࡛ࡁࡿࡇ࡜ࡀ๓ᥦ࡜⪃࠼ࡿࠋࡲࡓࠊᮏヨ㦂ἲࡢᵝࠎ࡞㝈⏺ࢆ⪃៖ࡍࡿ࡜ࠊᮏヨ㦂ἲ༢⊂࡛ࡣ⓶⭵ឤసᛶࡢホ౯ࡣ୙༑ศ࡛࠶ࡾࠊドᣐࡢ㔜ࡳ௜ࡅࡸ௚ࡢヨ㦂ἲ㸦LLNAࠊࣔࣝࣔࢵࢺࢆ⏝࠸ࡿ⓶⭵ឤసᛶヨ㦂࡞࡝㸧࡜⤌ࡳྜࢃࡏ࡛ࡢホ౯ࢆ᥎ዡࡍࡿࠋ

15
21

ᘬ⏝ᩥ⊩
1) EURL ECVAM (2012) ESAC Working Group Peer Review Consensus Report on
Givaudan-coordinated study transferability and reliability of the KeratinoSens assay for skinsensitisation testing2) OECD (2014, in preparation) Performance Standards for the assessment of proposed similar or
modified in vitro skin sensitization ARE-Nrf2 luciferase test methods in TG xxx. OECD
Environment, Health and Safety publications, Series on Testing and Assessment N.XXX, OECD,
Paris.
3) OECD (2015) In Vitro Skin Sensitization: ARE-Nrf2 Luciferase Test Method, OECD Guidelines
for the Testing of Chemicals No. 442D.
4) DB-ALM (INVITTOX) (2013) Protocol 155: KeratinoSens™
5) Natsch A., Ryan C.A., Foertsch L., Emter R., Jaworska J., Gerberick F. and Kern P. (2013) A
dataset on 145 chemicals tested in alternative assays for skin sensitization undergoingprevalidation. Journal of Applied Toxicology, 33, 1337-1352.
6) Natsch A. and Emter R. (2008) Skin sensitizers induce antioxidant response element dependent
genes: application to the in vitro testing of the sensitization potential of chemicals. Toxicological
Science, 102, 110-119.
7) Emter R., Ellis G. and Natsch A. (2010) Performance of a novel keratinocyte-based reporter
cell line to screen skin sensitizers in vitro. Toxicology and Applied Pharmacology, 245,
281-290.
8) Natsch A., Bauch C., Foerttsch L., Gerberick F., Norman K., Hilberer A., Inglis H.,
Landsiedel R., Onken S., Reuter H., Schepky A. and Emter R. (2011) The intra- andinter-laboratory reproducibility and predictivity of the KeratinoSens assay to predict skinsensitizers in vitro: results of a ring-study in five laboratories. Toxicology in Vitro, 25,
733-744.
9) EURL ECVAM (2014). Recommendation on the KerationSensTM assay for skin sensitization
testing, 42 pp.
http://ihcp.jrc.ec.europa.eu/our_labs/eurl-ecvam/eurl-ecvam-recommendations/recommendationkeratinosens-skin-sensitisation.

16
22