࣌ࣉࢳࢻ⤖ྜᛶヨ㦂㸦Direct Peptide Reactivity Assay㸸DPRA㸧ࡢホ౯఍㆟ሗ࿌᭩

JaCVAM ホ౯఍㆟

ᖹᡂ 27 ᖺ㸦2015 ᖺ㸧2 ᭶ 6 ᪥11JaCVAM ホ౯఍㆟኱㔝Ὀ㞝㸦㐠Ⴀጤဨ఍᥎⸀㸧㸸ᗙ㛗஬༑ᔒⰋ᫂ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ ⏕ά⾨⏕໬Ꮫ㒊㸧୍㨣 ຮ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ᖹ㈡⚽᫂㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧∾ ᰤ஧㸦᪥ᮏච␿ẘᛶᏛ఍㸧ቑ⏣ග㍤㸦ᗙ㛗᥎⸀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఍㸧ྜྷ⏣ ⥳ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ⑓⌮㒊㸧ྜྷᮧ ຌ㸦ᗙ㛗᥎⸀㸧Ώ㒊୍ே㸦᪥ᮏ〇⸆ᕤᴗ༠఍㸧                  ௵ᮇ㸸ᖹᡂ 26 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 28 ᖺ 3 ᭶ 31 ᪥22࣌ࣉࢳࢻ⤖ྜᛶヨ㦂㸦Direct Peptide Reactivity Assay 㸸DPRA) ࡣࠊ໬Ꮫ≀㉁ࡢ⓶⭵ឤసᛶࢆணࡍࡿヨ㦂ἲ࡛࠶ࡾࠊᚑ᮶ࡢࣔࣝࣔࢵࢺࡢ⓶⭵ឤసᛶヨ㦂㸦OECD TG406㸧ࠊ࣐࢘ࢫࡢᒁᡤࣜࣥࣃ⠇ヨ㦂㸦Local Lymph Node Assay: LLNAࠊOECD TG429㸧࠾ࡼࡧ LLNA ࡢኚἲ࡛࠶ࡿ LLNA:DA (OECDTG442A)࡞ࡽࡧ࡟ LLNA:BrdU-ELISA (OECD TG442B)࡜ẚ࡭ࠊື≀ࢆ⏝࠸࡞࠸࡜࠸࠺≉ᚩࢆ᭷ࡍࡿࠋDPRA ࡣࠊ⓶⭵ឤసᛶᡂ❧ࡢึᮇẁ㝵ࡢ཯ᛂ࡛࠶ࡿࣁࣉࢸࣥ࡜ࢱࣥࣃࢡ㉁ࡢ⤖ྜᛶࢆㄪ࡭ࡿࡇ࡜࡟ࡼࡾࠊ⓶⭵ឤసᛶࡢ᭷↓ࢆணࡍࡿヨ㦂ἲ࡛࠶ࡿࠋEuropean Union Reference Laboratory for

Alternatives to Animal Testing (EURL ECVAM)࡟ࡼࡿࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࢆᇶ࡟ࡋࡓ ESAC㸦ECVAM
Scientific Advisory Committee㸧 ࡟ࡼࡿ➨୕⪅ホ౯ࡀ⤊஢ࡋ OECD ࡟ࡼࡿᑓ㛛ᐙ఍㆟ࢆ⤒࡚ࠊOECDTG442C ࡜ࡋ࡚ 2015 ᖺ 2 ᭶ 5 ᪥࡟᥇ᢥࡉࢀࡓ 1)ࠋJaCVAM ホ౯఍㆟ࡣࠊ⓶⭵ឤసᛶヨ㦂㈨ᩱ⦅⧩ጤ
ဨ఍ࡣࠊୖグ㈨ᩱࢆࡶ࡜࡟సᡂࡋࡓ⓶⭵ឤసᛶヨ㦂ホ౯ሗ࿌᭩ࢆ⏝࠸࡚ࠊDPRA ࡢጇᙜᛶ࡟ࡘ࠸᳨࡚ウࡋࡓࠋ㸯㸬ヨ㦂ἲࡢᐃ⩏ྡ⛠㸸 ࣌ࣉࢳࢻ⤖ྜᛶヨ㦂㸦Direct Peptide Reactivity Assay 㸸DPRA)
௦᭰ࡍࡿᑐ㇟ẘᛶヨ㦂㸸 ࣔࣝࣔࢵࢺࡢ⓶⭵ឤసᛶヨ㦂㸦OECD TG406㸧࠾ࡼࡧ࣐࢘ࢫࡢᒁᡤࣜࣥ
ࣃ⠇ヨ㦂 [Local Lymph Node Assay: LLNA (OECD TG429)ࠊLLNA:DA (OECD TG442A)ࠊ
LLNA:BrdU-ELISA (OECD TG442B)]
ヨ㦂ἲࡢᴫ␎㸸 DPRA ࡣࠊ⏕య࡟࠾ࡅࡿ⓶⭵ឤసᛶⓎ⌧ࡲ࡛ࡢ㐣⛬ࡢ໬Ꮫ≀㉁࡜ࢱࣥࣃࢡ㉁࡜ࡢ
Ᏻᐃ࡞⤖ྜ≀ࡢᙧᡂᛶࢆホ౯ࡍࡿ͆ື≀ࢆ⏝࠸࡞࠸ in chemico ヨ㦂͇࡛࠶ࡿࠋDPRA ࡛ࡣࠊ⓶⭵ෆࡢࢱࣥࣃࢡ㉁ࡢ௦ࢃࡾ࡟ 7 ಶࡢ࢔࣑ࣀ㓟࠿ࡽᡂࡿྜᡂ࣌ࣉࢳࢻࡢࢩࢫࢸ࢖ࣥྵ᭷࣌ࣉࢳࢻ࡜ࣜࢪࣥྵ᭷࣌ࣉࢳࢻࡢ 2 ✀㢮㸦࠸ࡎࢀࡶᕷ㈍ရ㸧ࢆ౑⏝ࡍࡿࠋ໬Ꮫ≀㉁࡜ࡑࢀࡒࢀࡢ࣌ࣉࢳࢻࢆ
ΰྜࡍࡿࡇ࡜࡟ࡼࡾ཯ᛂࡉࡏࠊΰྜ 24 ᫬㛫ᚋ࡟࠾ࡅࡿᮍ཯ᛂࡢ࣌ࣉࢳࢻ㔞ࢆ HPLC ࡛ศ㞳ᐃ㔞ࡍࡿࠋヨ㦂ᡂ❧᮲௳ࢆ‶ࡓࡋࡓ໬Ꮫ≀㉁ࡢ཯ᛂᛶࡣࠊ ᐃࡈ࡜ࡢ࣌ࣉࢳࢻῶᑡ⋡࠿ࡽᖹᆒ್ࢆ⟬ฟࡋࠊணࣔࢹࣝ࡟ᚑࡗ࡚ HighࠊModerateࠊLow ࠾ࡼࡧ Minimal ࡢ 4 ẁ㝵࡟ศ㢮ࡉࢀࡿࠋࡑࡢ⤖ᯝࠊ཯ᛂᛶࡀ LowࠊModerate ࠾ࡼࡧ High ࡟ศ㢮ࡉࢀࡿ໬Ꮫ≀㉁ࡣ⓶⭵ឤసᛶ࠶ࡾࠊMinimal࡟ศ㢮ࡉࢀࡿ໬Ꮫ≀㉁ࡣ⓶⭵ឤసᛶ࡞ࡋ࡜ணࡉࢀࡿࠋ㸰㸬ホ౯࡟⏝࠸ࡓ㈨ᩱ࠾ࡼࡧホ౯ෆᐜࡢ⛉Ꮫⓗጇᙜᛶ
DPRA ࡣࠊEURL ECVAM ୺ᑟࡢࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡜ࡑࢀ࡟⥆ࡃ ESAC ࡟ࡼࡿ⊂❧ࡋࡓ➨୕⪅ホ౯࡟࠾࠸࡚ホ౯ࡉࢀࠊ௦᭰ヨ㦂ἲ࡜ࡋ࡚⛉Ꮫⓗ࡟ጇᙜ࡛࠶ࡿ࡜ሗ࿌ࡉࢀ࡚࠸ࡿ 2)ࠋᮏ㑥࡟࠾࠸࡚ࡣࠊJaCVAM ⓶⭵ឤసᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍ࡀࠊDPRA ࡢ⓶⭵ឤసᛶヨ㦂௦᭰ἲ࡜ࡋ࡚ࡢ⛉Ꮫⓗጇᙜᛶ࡟ࡘ࠸࡚ࠊOECD TG442C㸯㸧ࠊEURL ECVAM ࡢࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩ 3)ࡢࡳ࡞ࡽࡎ⌧ᅾࡲ࡛
33බ㛤ࡉࢀ࡚࠸ࡿ᝟ሗ 4)ࢆᇶ࡟᳨ドࡋࡓࠋ᳨ドࡢ⤖ᯝ 5)ࠊDPRA ࡟ࡣឤసᛶⓎ⌧ᶵᗎ࡟࠾ࡅࡿึᮇࡢ㔜せ࡞࢖࣋ࣥࢺ࡛࠶ࡿࢱࣥࣃࢡ㉁࡜໬Ꮫ≀㉁ࡢ⤖ྜ཯ᛂࢆ᳨ฟࡍࡿ࡜࠸࠺⛉Ꮫᛶࡀ࠶ࡾࠊinchemico ࡢ⓶⭵ឤసᛶヨ㦂௦᭰ἲ࡜ࡋ࡚ጇᙜ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡓࠋ㸱㸬ᮏヨ㦂ἲࡢ᭷⏝ᛶ࡜㐺⏝㝈⏺
DPRA ࡣࠊື≀ࢆ⏝࠸࡞࠸ in chemico ࡢヨ㦂ἲ࡛ࠊ3Rs ࡢ⢭⚄࡟ྜ⮴ࡋ࡚࠸ࡿࠋࡲࡓࠊ㸯࢔ࢵࢭ࢖ᙜࡓࡾࡢ⤒㈝ࡶࠊLLNA ࡜ẚ㍑ࡋ࡚ 1/10 ⛬ᗘ࡛ᐇ᪋ྍ⬟࡛࠶ࡾࠊヨ㦂ᮇ㛫ࡶ LLNA ࡼࡾ▷ᮇ㛫࡛࠶ࡿࡇ࡜࠿ࡽࠊᮏヨ㦂ἲࡣ⡆౽ᛶ࠾ࡼࡧ⤒῭ᛶࡢ㠃࠿ࡽ᭷⏝࡜࠸࠼ࡿࠋ᭦࡟ࠊDPRA ࡣࠊឤసᛶⓎ⌧ᶵᗎ࡟࠾ࡅࡿึᮇࡢ㔜せ࡞࢖࣋ࣥࢺ࡛࠶ࡿࢱࣥࣃࢡ㉁࡜໬Ꮫ≀㉁ࡢ⤖ྜ཯ᛂࢆ᳨ฟࡍࡿࡶࡢ࡛࠶ࡾࠊ໬Ꮫ≀㉁ࡢឤసᛶࢆุ᩿ࡍࡿୖ࡛㔜せ࡞᝟ሗࢆ୚࠼ࡿࠋ
ࡋ࠿ࡋ࡞ࡀࡽࠊDPRA ࡢ᪋タෆ෌⌧ᛶ࡟࠾࠸࡚ࠊUnited Nations (UN) Globally Harmonized System ofClassification and Labelling of Chemicals (GHS) ༊ศ 1B ࡢ≀㉁㸦ᙅ࠸ឤసᛶ≀㉁㸧࡛ࡣ෌⌧ᛶࡀᚓࡽࢀ

࡞࠿ࡗࡓࡓࡵ㐩ᡂᇶ‽㸦85㸣㸧ࢆ‶ࡓࡏࡎࠊDPRA ࡛ࡣᙅ࠸ឤసᛶ≀㉁࡟ᑐࡋ࡚ࡣุᐃࢆㄗࡿᠱᛕ
ࡀ࠶ࡿࠋࡲࡓࠊ᪋タ㛫෌⌧ᛶ࡟࠾࠸࡚ࡣࠊ࣌ࣉࢳࢻ࡜ඹ᭷⤖ྜࢆ⏕ࡌ࠺ࡿᐁ⬟ᇶࢆᣢࡓ࡞࠸㔠ᒓሷࢆ㝖ࡃࡇ࡜࡟ࡼࡾ෌⌧ᛶࡣ 82%࡜࡞ࡾࠊ㐩ᡂᇶ‽ (80%) ࢆୖᅇࡿࠋᚑࡗ࡚ࠊDPRA ࡟࠾࠸࡚㔠ᒓሷࡣ㐺⏝⠊ᅖእ࡜ࡍ࡭ࡁ࡛࠶ࡿࠋDPRA ࡣᾮ┦୰࡛཯ᛂࢆ⾜࠺ࡓࡵࠊ໬Ꮫ≀㉁ࡣᣦᐃࡉࢀࡓᩘ✀㢮ࡢ⁐፹࡟⁐ゎࡍࡿᚲせࡀ࠶ࡾࠊ␯Ỉᛶࡢ㧗࠸໬Ꮫ≀㉁ࡣṇࡋࡃุᐃࡉࢀ࡞࠸ྍ⬟ᛶࡀ࠶ࡿࠋࡲࡓࠊDPRA ࡢ᳨ドヨ㦂࡟࠾ࡅࡿឤᗘࡣࠊヨ㦂㐺⏝እ࡜⪃࠼ࡽࢀࡿ㔠ᒓሷࢆ㝖࠸ࡓሙྜ࡛ࡶ 75%࡛࠶ࡾࠊ
DPRA ࡛㝜ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓሙྜࡣࠊഇ㝜ᛶࡢྍ⬟ᛶࢆ⪃៖ࡋࠊ⿵᏶ࡋᚓࡿ௚ࡢヨ㦂ἲ࡜ࡢ⤌ࡳྜࢃࡏ࡛☜ㄆࡍࡿᚲせࡀ࠶ࡿࠋࡇࢀࡲ࡛ࡢ⤖ᯝ࠿ࡽࠊ⓶⭵ឤసᛶࡀ Moderate ࡲࡓࡣ Weak ࡢ໬Ꮫ≀㉁ࠊឤసᛶࡢ⋓ᚓ࡟௦ㅰⓗ࠶ࡿ࠸ࡣ㠀௦ㅰⓗάᛶ໬ࢆᚲせ࡜ࡍࡿࣉࣟࣁࣉࢸࣥ࠶ࡿ࠸ࡣࣉࣞࣁࣉࢸࣥࠊ࠾ࡼࡧ␯Ỉᛶࡢ㧗࠸໬Ꮫ≀㉁ࡣഇ㝜ᛶࢆ♧ࡍྍ⬟ᛶࡀ㧗࠸ࡓࡵࠊヨ㦂⤖ᯝࡢゎ㔘࡟ࡣὀពࡀᚲせ࡛࠶ࡿ㸲㸬┠ⓗ࡜ࡍࡿ≀㉁ཪࡣ〇ရࡢẘᛶࢆホ౯ࡍࡿヨ㦂ἲ࡜ࡋ࡚ࡢࠊ⾜ᨻୖࡢ฼⏝࠾ࡼࡧ♫఍ⓗཷࡅධࢀࡢྍ⬟ᛶ ♫఍ⓗཷࡅධࢀᛶ㸸DPRA ࡣࠊHPLC ศᯒࡀྍ⬟࡞᪋タ࡛࠶ࢀࡤᐜ᫆࡟ᐇ᪋࡛ࡁࡿヨ㦂ἲ࡛࠶ࡾࠊࡲࡓ໬Ꮫ≀㉁࡜
ࡢ཯ᛂ࡟⏝࠸ࡿ 2 ✀㢮ࡢྜᡂ࣌ࣉࢳࢻࡶᕷ㈍ࡉࢀ࡚࠾ࡾࠊධᡭ࡟ไ㝈ࡣ࡞࠸ࠋDPRA ࡢᐇ᪋࡟ᙜࡓࡗ࡚ࡣࠊ໬Ꮫ≀㉁ࡢᛶ㉁ࢆぢᴟࡵࠊᚓࡽࢀࡓ⤖ᯝࡢゎ㔘࡟ὀពࢆᡶ࠺ᚲせࡀ࠶ࡿࠋࡋ࠿ࡋࠊDPRA ࡣ⓶⭵ឤసᛶᡂ❧ࡢึᮇẁ㝵ࡢ໬Ꮫ≀㉁࡜ࢱࣥࣃࢡ㉁ࡢ⤖ྜ཯ᛂࢆ᳨ฟࡋ࡚࠾ࡾࠊ໬Ꮫ≀㉁ࡢឤసᛶࢆ⪃࠼ࡿୖ࡛㔜せ࡞᝟ሗࢆ୚࠼࡚࠸ࡿࡇ࡜ࠊࡲࡓࠊ⡆౽࠿ࡘᏳ౯࡞ヨ㦂ἲ࡛࠶ࡿࡇ࡜࠿ࡽࠊDPRA ࡢ♫఍ⓗཷࡅධࢀᛶࡣ㧗࠸ࡶࡢ࡜⪃࠼ࡿࠋ
44 ⾜ᨻୖࡢ฼⏝ᛶ㸸
DPRA ࡛㝧ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓሙྜࠊࡑࡢ໬Ꮫ≀㉁ࢆ UN GHS ༊ศ㸯࡟ศ㢮㸦ᙉ࠸ឤసᛶ≀㉁㸧
ࡍࡿࡇ࡜ࡣ⾜ᨻୖྍ⬟࡛࠶ࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊࡲࢀ࡟ഇ㝧ᛶࡢ⤖ᯝࡀ⏕ࡌࡿࡇ࡜࡟␃ពࡋ࡞ࡅࢀࡤ
࡞ࡽ࡞࠸ࠋ୍᪉ࠊDPRA ࡢ㝜ᛶ⤖ᯝࡀᚓࡽࢀࡓሙྜࠊࡑࡢ໬Ꮫ≀㉁ࡢ⓶⭵ឤసᛶࡣഇ㝜ᛶࡢྍ⬟ᛶ
ࡀ࠶ࡾࠊDPRA ༢⊂࡛ࡑࡢ⓶⭵ឤసᛶࢆุᐃࡍࡿࡇ࡜ࡣ㞴ࡋ࠸ࠋIntegrated Approaches to Testing andAssessment: IATA)* ࢆᵓᡂࡍࡿࡑࡢ௚ࡢ᝟ሗ※࡜⤌ࡳྜࢃࡏ࡚ホ౯ࡍࡿࡇ࡜࡟ࡼࡾࠊDPRA ࡣ⾜ᨻⓗ࡟฼⏝ྍ⬟࡛࠶ࡿ࡜⪃࠼ࡿࠋ
*:OECD ࡟࡚࢞࢖ࢲࣥࢫ᱌ࡀ᳨ウ୰ཧ⪃ᩥ⊩1) OECD Test Guideline 442C, In Chemico Skin Sensitisation, Direct Peptide Reactivity Assay (DPRA)(2015)
http://www.oecd-ilibrary.org/environment/test-no-442c-in-chemico-skin-sensitisation_97892642297
09-en;jsessionid=2d30155etbl8v.x-oecd-live-022) EC EURL-ECVAM (2013). Recommendation on the Direct Peptide Reactivity Assay (DPRA) for skinsensitization testing.
3) 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%20V
alidation%20Study%20Report.pdf
4) Gerberick GF, Vassallo JD, Foertsch LM, Price BB, Chaney, JG, Lepoittevin JP. (2007).
Quantification of chemical peptide reactivity for screening contact allergens: A classification treemodel approach. Toxicol. Sci. 97(2), 417-427
5) JaCVAM ⓶⭵ឤసᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍㸸⓶⭵ឤసᛶヨ㦂ホ౯ሗ࿌᭩ Direct PeptideReactivity Assay (DPRA)㸦ᖹᡂ 27 ᖺ 2 ᭶ 6 ᪥㸧556