ER STTA ἲ㸸hERȘ-HeLa-9903 ⣽⬊ࢆ⏝࠸ࡓ࢚ࢫࢺࣟࢤࣥཷᐜయᜏᖖⓎ⌧⣔㌿෗άᛶ໬ヨ㦂ἲࡢホ౯ሗ࿌᭩

ᖹᡂ 28 ᖺ 10 ᭶ 21 ᪥

1

7ෆศἪ࠿ࡃ஘ヨ㦂ἲ㈨ᩱ⦅⧩ጤဨ఍ጤဨྡ㸸ᑠ㔝 ᏹ㸦ጤဨ㛗㸸㸦୍㈈㸧㣗ရ⸆ရᏳ඲ࢭࣥࢱ࣮⛙㔝◊✲ᡤ㸧୸㔝ෆᲺ㸦⸨⏣ಖ೺⾨⏕኱Ꮫ㸧஭ཱྀὈἨ㸦ᶓ὾ᕷ❧኱Ꮫࠊඖ ᇶ♏⏕≀Ꮫ◊✲ᡤ㸧ᑠ㔝 ᩔ㸦ᒸᒣ኱Ꮫࠊඖ ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧

2

8

⏝ㄒ㞟
Accuracy㸸ṇ☜ᛶConcordance㸸୍⮴ᗘ17ȕ-estradiol (E2)㸸17ȕ-࢚ࢫࢺࣛࢪ࣮࢜ࣝ
ER㸸Estrogen receptor
ER Į/ȕ㸸࢚ࢫࢺࣟࢤࣥཷᐜయ Į/ȕ
ER STTA㸸hERĮ-HeLa-9903 ⣽⬊ࢆ⏝࠸ࡓ࢚ࢫࢺࣟࢤࣥཷᐜయᜏᖖⓎ⌧⣔㌿෗άᛶ໬ヨ㦂ἲ
Estrogen responsive element (ERE)㸸᰾ෆࡢ࢚ࢫࢺࣟࢤࣥᛂ⟅㓄ิEuropean Centre for Validation of Alternative Methods (ECVAM)㸸Ḣᕞ௦᭰ἲホ౯ࢭࣥࢱ࣮
False negative rate㸸ഇ㝜ᛶ⋡
False positive rate㸸ഇ㝧ᛶ⋡
GLP: Good Laboratory PracticeInteragency Committee on the Validation of Alternative Methods (ICCVAM)㸸⡿ᅜ௦᭰ἲホ౯┬ᗇ㛫㐃⤡ጤဨ఍
Inter-laboratory validation study㸸᪋タ㛫ࣂࣜࢹ࣮ࢩࣙࣥ◊✲Intra-laboratory validation study㸸᪋タෆࣂࣜࢹ࣮ࢩࣙࣥ◊✲Japanese Center for Validation of Alternative Methods (JaCVAM)㸸᪥ᮏື≀ᐇ㦂௦᭰ἲホ౯ࢭࣥࢱ࣮Performance-Based Test Guideline (PBTG)㸸ᛶ⬟‽ᣐヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥ
Performance Standards (PS)㸸ᛶ⬟ᶆ‽
Reliability㸸ಙ㢗ᛶ
Test Guideline (TG)㸸ヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥ
Transcription activation (TA)㸸㌿෗άᛶ໬
Uterotrophic assay㸸Ꮚᐑ⫧኱ヨ㦂
Organisation for Economic Co-operation and Development (OECD)㸸⤒῭༠ຊ㛤ⓎᶵᵓValidation study㸸ࣂࣜࢹ࣮ࢩࣙࣥ◊✲Validation study management team㸸ࣂࣜࢹ࣮ࢩࣙࣥ◊✲㐠Ⴀጤဨ఍

3

9㸯㸬ᮏヨ㦂ἲࡢ⛉Ꮫⓗጇᙜᛶ࡜つไヨ㦂ἲ࡜ࡋ࡚ࡢጇᙜᛶER STTA ἲ㸦ヲࡋࡃࡣࠕhERĮ-HeLa-9903 ⣽⬊ࢆ⏝࠸ࡓ࢚ࢫࢺࣟࢤࣥཷᐜయ(ER)ᜏᖖⓎ⌧⣔㌿෗άᛶ໬ヨ㦂ἲࠖ㸹The Stably Transfected Transcription Activation (STTA) assay using thehuman ERĮ-HeLa-9903 cell line㸧ࡣࠊ໬Ꮫ≀㉁ࡢ࢚ࢫࢺࣟࢤࣥ㸦࠾ࡼࡧᢠ࢚ࢫࢺࣟࢤࣥ㸧ά
ᛶࢆ໬ᏛⓎග࡟ࡼࡾ᳨ฟࡍࡿ in vitro ヨ㦂ἲࡢ୍ࡘ࡛ࠊෆศἪ࠿ࡃ஘≀㉁ᑐ⟇ࡢࡓࡵ࡟㛤Ⓨࡉࢀࡓࡶࡢ࡛࠶ࡿࠋᮏヨ㦂ἲࡣࠊᡃࡀᅜ㸦᪥ᮏ㸧ࡢఫ཭໬Ꮫᰴᘧ఍♫࡛ᵓ⠏ࡉࢀࡓhERĮ-HeLa-9903 ⣽⬊ᰴࢆ⏝࠸࡚ࠊ㸦୍㈈㸧໬Ꮫ≀㉁ホ౯◊✲ᶵᵓ㸦௨ୗࠊCERI ࡜グࡍ㸧࡟࠾࠸࡚ࣉࣟࢺࢥࣝࡀ㛤ⓎࡉࢀࡓࠋhERĮ-HeLa-9903 ⣽⬊ࡣࠊࣄࢺᏊᐑ㢁ࡀࢇ⏤᮶ࡢ HeLa ⣽⬊࡟ࣄࢺ ERĮ ࢆⓎ⌧ࡍࡿ㑇ఏᏊ㓄ิ࡜ࠊ࢔ࣇࣜ࢝ࢶ࣓࢚࢞ࣝࡢࣅࢸࣟࢪ࢙ࢽࣥ㑇ఏᏊ⏤᮶ࡢ࢚ࢫࢺࣟࢤࣥᛂ⟅㓄ิ㸦ERE㸧ࡢୗὶ࡟ mouse metallothionein MT㸧㑇ఏᏊࡢ TATA ࣎ࢵࢡࢫ㓄ิ࠾ࡼࡧ࣏࣮ࣞࢱ࣮㑇ఏᏊ࡜࡞ࡿ࣍ࢱࣝࣝࢩࣇ࢚࣮ࣛࢮ㑇ఏᏊࢆ㐃⤖ࡋࡓ㑇ఏᏊ㓄ิࢆᏳᐃⓗ࡟ᑟධࡋࡓ⣽⬊࡛࠶ࡾࠊER STTA ἲࡣᑟධࡉࢀࡓ ERĮ ࡢάᛶ໬࡟ࡼࡗ࡚㉳ࡿ࣏࣮ࣞࢱ࣮㑇ఏᏊࡢ㌿෗άᛶࡢኚ໬ࢆ໬ᏛⓎග࡟ࡼࡾᐃ㔞ⓗ࡟ᐃࡍࡿヨ㦂⣔࡛࠶ࡿࠋhERĮ-HeLa-9903 ⣽⬊ࡣࠊᅜ❧◊✲㛤Ⓨἲே་⸆ᇶ┙࣭೺ᗣ࣭ᰤ㣴◊✲ᡤ JCRB ⣽⬊ࣂࣥࢡ㸦http://cellbank.nibiohn.go.jp/㸧ࡼࡾධᡭྍ⬟࡛࠶ࡿ㸦⣽⬊Ⓩ㘓␒ྕ㸸JCRB1318㸧ࠋER STTA
ἲ࡟ࡘ࠸࡚ࡣࠊཌ⏕ປാ┬࣭⤒῭⏘ᴗ┬ඹྠ࡛ OECD ࡟ᥦ᱌ࡉࢀࠊࡑࡢᚋᐇ᪋ࡉࢀࡓࣂࣜ
ࢹ࣮ࢩࣙࣥヨ㦂ࡢ⤖ᯝࢆࡶ࡜࡟ࠊヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥ㸦TG㸧ࡢᥦ᱌࡟⮳ࡗࡓࠋෆศἪᶵ⬟࡟ᙳ㡪ࡍࡿ⏕≀άᛶࢆ᭷ࡍࡿ≀㉁ࡣࠊ࣍ࣝࣔࣥ➼ࡢኳ↛ࡢ⏕య≀㉁ࡢ࡯࠿ྜ
ᡂ໬Ꮫ≀㉁࡟ࡶከᩘ▱ࡽࢀࡿࡼ࠺࡟࡞ࡗࡓࡀࠊࡑࡢάᛶࡢ᭷↓࡜⛬ᗘ࡟ࡘ࠸࡚ᮍㄪᰝࡢ≀㉁ࡀከ࠸ࠋࡇ࠺ࡋࡓ≀㉁ࡢ࠺ࡕࠊ኱㔞ࡢ᭚㟢࡟ࡼࡗ࡚⏕యࡢෆศἪᶵ⬟࠾ࡼࡧࡇࢀ࡜㛵ಀࡋࡓ⏕ṪⓎ⏕➼࡟ᙳ㡪ࢆཬࡰࡍࡶࡢ㸦ෆศἪ࠿ࡃ஘≀㉁࡜࿧ࡤࢀࡿ㸧࡛࠶ࡿࡇ࡜ࡀᠱᛕࡉ
ࢀࡿࡶࡢࡀ࠶ࡾࠊ⌧ᅾࠊᙳ㡪ࡢ᭷↓ࢆ☜ㄆࡍࡿヨ㦂ἲࡢ㛤Ⓨࡀồࡵࡽࢀ࡚࠸ࡿࠋࡲࡓࠊࡇ
ࡢ☜ㄆࡢᑐ㇟࡜࡞ࡿ≀㉁ࡢᩘࡀ⭾኱࡞ࡶࡢ࡛࠶ࡿࡓࡵࠊຠ⋡ⓗ࡞ࢫࢡ࣮ࣜࢽࣥࢢࢆ⾜࠺ヨ㦂ἲࡢ㛤Ⓨ࡜ᐇ⏝໬ࡀồࡵࡽࢀ࡚ࡁࡓࠋࢫࢡ࣮ࣜࢽࣥࢢࡢᣦᶆ࡜ࡋ࡚᭷ᮃ࡞ᛶ㉁࡟ࡣ≀㉁ࡢෆศἪάᛶࡀ࠶ࡾࠊ≀㉁ࡢ࣍ࣝࣔࣥཷᐜయ࡜ࡢ⤖ྜᛶࡲࡓࡣෆศἪᶵ⬟ࡢάᛶ໬ࢆ

ᐃࡍࡿ᪉ἲࡀὀ┠ࡉࢀ࡚࠸ࡿࠋ࢚ࢫࢺࣟࢤࣥάᛶ࡟㛵ࡍࡿヨ㦂ἲ࡜ࡋ࡚ࡣࠊࡍ࡛࡟ in vivo ヨ㦂ἲ࡜ࡋ࡚ࠕᄿṑ㢮ࢆ⏝࠸ࡿᏊᐑ⫧኱ヨ㦂ࠖࡀ☜❧ࡉࢀࠊࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࢆ⾜ࡗࡓୖ࡛ᅜ㝿ⓗ࡞ TG㸦OECD TG440㸧࡜࡞ࡗ࡚࠸ࡿࡀࠊࡼࡾ⡆౽࡛㎿㏿࡞㠀ື≀ヨ㦂࡜ࡋ࡚ᇵ㣴⣽⬊ࢆ⏝࠸ࡿ in vitro ヨ㦂ἲࡢ㛤Ⓨࡀ㐍ࡵࡽࢀ࡚࠸ࡿࠋ≀㉁ࡢ ER ࡜ࡢ⤖ྜᛶࢆ in vitro ࡛ヨ㦂ࡍࡿ᪉ἲ࡟ࡣࠊ⏕యࡲࡓࡣᇵ㣴⣽⬊࠿ࡽᢳฟࡋࡓER ࡟ᑐࡍࡿ໬Ꮫ≀㉁ࡢ⤖ྜ཯ᛂࡢᐃἲࡀ࠶ࡾࠊࡓ࡜࠼ࡤࣛࢵࢺᏊᐑ࠿ࡽᢳฟࡋࡓ ER ࢆ⏝࠸ࡿࠕࣛࢵࢺᏊᐑ࢚ࢫࢺࣟࢤࣥཷᐜయ⤖ྜᛶヨ㦂ࠖࡀᣲࡆࡽࢀࡿࠋࡇࢀࡣ↓⣽⬊⣔ࡢ⤖410

ྜヨ㦂࡛࠶ࡾࠊヲ⣽࡞⏝㔞཯ᛂ㛵ಀࢆ☜ㄆ࡛ࡁࠊ≀㉁ࡢ ER ⤖ྜᛶࢆᶆ‽≀㉁ࠊࡓ࡜࠼ࡤ
17ȕ-estradiol (E2)࡜ẚ㍑ࡋ࡚ᐃ㔞ⓗ࡟Ỵᐃࡍࡿࡶࡢ࡛࠶ࡿࠋࡋ࠿ࡋࠊER ࡟ᑐࡋ࡚ࡣཷᐜయ่⃭≀㉁ࡤ࠿ࡾ࡛࡞ࡃᣕᢠ≀㉁ࡶ⤖ྜᛶࡀ࠶ࡿࡢ࡛ࠊ୧⪅ࡢ༊ูࡀฟ᮶࡞࠸ࠋࡑࡢ༊ูࡢࡓ
ࡵ࡟ࡣࠊ≀㉁࡜ ER ࡜ࡢ⤖ྜࡀ㉳ࡇࡍ⏕≀Ꮫⓗຠᯝࢆ☜ㄆࡍࡿᚲせࡀ࠶ࡾࠊ᭷ព⩏࡞ᣦᶆ࡜ࡋ࡚⏕య࡟ㄏⓎࡉࢀࡿ࢚ࢫࢺࣟࢤࣥຠᯝࢆ

ᐃࡍࡿ in vivo ヨ㦂㸦ࡓ࡜࠼ࡤࣛࢵࢺᏊᐑ⫧኱

ヨ㦂㸧ࡀ฼⏝ࡉࢀ࡚࠸ࡿࠋIn vitro ࡛ࡶࠊER ࡀάᛶ≀㉁࡜⤖ྜࡋࡓࡢࡕ࡟⣽⬊ෆ࡛㉳ࡇࡿᛂ⟅ࡀほᐹ࡛ࡁࢀࡤࠊ࢚ࢫࢺࣟࢤࣥຠᯝࢆ☜ㄆ࡛ࡁࡿࠋάᛶ≀㉁࡜⤖ྜࡋࡓ⣽⬊ෆ ER ࡣࠊ᰾ෆࡢ ERE ࡜ࡢ⤖ྜࢆ㏻ࡌ࡚㛵㐃≀㉁ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊ㸦DNA㸧ࡢ㌿෗ࢆ㉳ࡇࡍࠋࡇࡢࡼ
࠺࡞㑇ఏᏊࢆ௓ࡍࡿᛂ⟅࡟ᚲせ࡞ᶵᵓࢆಖᣢࡍࡿ⣽⬊࡟࠾࠸᳨࡚ᰝࡍࡿࡇ࡜ࡣព⩏ࡀ࠶ࡿࠋER STTA ἲࡣࠊ࢚ࢫࢺࣟࢤࣥάᛶ≀㉁࡟ࡼࡿ ER ⤖ྜ࡟⥆ࡃୗὶ㑇ఏᏊࡢ㌿෗άᛶ໬ࢆ࢚
ࣥࢻ࣏࢖ࣥࢺ࡜ࡋ᳨࡚ฟࡍࡿ᪉ἲ࡛࠶ࡿࠋ⣽⬊࡟ࡣ㌿෗άᛶ໬ࢆㄪ࡭ࡿࡓࡵ࡟ࠊᙜヱཷᐜయ≉␗ⓗᛂ⟅㓄ิ࡜㐃⤖ࡋࡓ఩⨨㸦ୗὶ㸧࡟࣏࣮ࣞࢱ࣮㑇ఏᏊࢆ⤌ࡳ㎸ࢇ࡛࠾ࡁࠊ㌿෗άᛶ໬࡟ࡼࡿ࣏࣮ࣞࢱ࣮ࢱࣥࣃࢡࡢⓎ⌧ࢆⓎගᇶ㉁ࡢῧຍ࡟ࡼࡾ᳨ฟࡍࡿࠋࡇࡢࡼ࠺࡞⣽⬊࡜ࡋ࡚ࡣࠊࡍ࡛࡟ࣄࢺ༸ᕢ⒴⏤᮶ᰴ⣽⬊ BG1 ࡟࣏࣮ࣞࢱ࣮㑇ఏᏊ࡜ࡋ࡚ Luciferase
responsive element ࢆྵࡴ plasmid ࢆ᰾ෆࡢ ERE ࡢୗὶ࡟Ᏻᐃⓗ࡟ᑟධࡋࡓ BG1Luc4E2 ⣽⬊ࡀ㛤Ⓨࡉࢀ࡚࠾ࡾࠊࡇࡢ⣽⬊ࢆ⏝࠸ࡓヨ㦂ἲࠕBG1Luc4E2 ⣽⬊ࢆ⏝࠸ࡿ࢚ࢫࢺࣟࢤࣥཷᐜ
య㸦ER㸧㌿෗άᛶ໬㸦TA㸧ヨ㦂㸦BG1Luc ER TA ἲ㸧ࠖࡀ OECD TG ࡟཰㍕ࡉࢀ࡚࠸ࡿ㸦OECD
TG 457, 2012㸧ࠋ
ER STTA ἲ࡜ BG1Luc ER TA ἲࡢ┦㐪ࡣࠊ(1) ᮏἲࡀ hERĮ ࢆ HeLa ⣽⬊࡟⤌ࡳ㎸ࢇࡔࡶࡢ࡛࠶ࡿࡢ࡟ᑐࡋࠊBG1Luc ER TA ἲࡣ BG1 ⣽⬊࡟ෆᅾࡍࡿ ER ࢆ฼⏝ࡍࡿࠊ(2) ᮏἲ࡛ࡣ⤌ࡳ㎸ࡲࢀࡓཷᐜయࡣࣄࢺ ERĮ ࡢࡳ࡛࠶ࡿࡀࠊBG1 ⣽⬊ࡣ ERĮ ࡜ ERȕ ࡢ཮᪉ࢆⓎ⌧ࡋ࡚࠸ࡿࠋࡓࡔࡋࠊ2 ✀ࡢ ER ࢔࢖ࢯࣇ࢛࣮࣒㸦ERĮࠊERȕ㸧࡟ᑐࡋ࡚᏶඲࡞㑅ᢥᛶࢆ♧ࡍ໬Ꮫ≀㉁ࡣ▱ࡽࢀ࡚࠾ࡽࡎࠊᐃᛶⓗホ౯࡟࠾࠸࡚୧

ᐃ⣔ࡢ⤖ᯝࡣྠ➼࡜⪃࠼ࡽࢀࡿࠋ(3)

BG1Luc ER TA ἲࡢ࢔ࣥࢱࢦࢽࢫࢺヨ㦂࡛ࡣࠊࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟࠾࠸࡚㧗⃰ᗘ㡿ᇦ࡛ࡢࣝࢩࣇ࢙࣮ࣛࢮάᛶࡢ㠀≉␗ⓗ㜼ᐖࡀㄆࡵࡽࢀࡓࡇ࡜࠿ࡽࠊホ౯ྍ⬟࡞⿕㦂≀㉁ࡢ᭱኱⃰ᗘࡣࠊ20ȣg/mL (⣙ 10ȣM)࡟ไ㝈ࡉࢀ࡚࠸ࡿࡢ࡟ᑐࡋ࡚ࠊᮏἲ࡛ࡣࠊ፹య࡬ࡢ⁐ゎᛶࡀⰋዲ࠿ࡘ⣽⬊ẘᛶࡀㄆࡵࡽࢀ࡞࠸ሙྜࠊ࢔ࢦࢽࢫࢺ࣭࢔ࣥࢱࢦࢽࢫࢺヨ㦂࡜ࡶ᭱኱ 1mM ࡲ࡛ホ౯ྍ⬟࡛࠶ࡿࠋER STTA ἲࡢࣂࣜࢹ࣮ࢩࣙࣥ࡟ࡘ࠸࡚ࡣࠊ࢔ࢦࢽࢫࢺヨ㦂࡜࢔ࣥࢱࢦࢽࢫࢺヨ㦂࡟ࡘ࠸ู࡚ࠎ࡟⊂❧ࡋࡓ᪋タ㛫ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡀᐇ᪋ࡉࢀࡓࠋ
࢔ࢦࢽࢫࢺヨ㦂࡟㛵ࡋ࡚ࡣࠊ2004 ᖺ࡟ CERI ࡀ୰ᚰ࡜࡞ࡗ࡚ࠊఫ཭໬Ꮫ(ᰴ)(௨ୗࠊఫ཭
511

໬Ꮫ࡜グࡍ)ࠊ኱ሯ〇⸆㸦ᰴ㸧(௨ୗࠊ኱ሯ〇⸆࡜グࡍ)ࠊ(ᰴ)࢝ࢿ࢝ࢸࢡࣀࣜࢧ࣮ࢳ(௨ୗࠊ࢝ࢿ࢝࡜グࡍ)ࡢᅜෆ 4 ᪋タࡢཧຍ࡟ࡼࡿࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡀᐇ᪋ࡉࢀࡓࠋ࢔ࢦࢽࢫࢺࣂ
ࣜࢹ࣮ࢩࣙࣥヨ㦂ࡢணヨ㦂࡛ࡣࠊ஦๓᳨ウ࡜ࡋ࡚㝧ᛶ 2 ≀㉁㸦E2, Bisphenol A㸧ࡢ 2 ᅇ⧞ࡾ㏉ࡋᐃࢆ⾜࠸ࠊཧຍヨ㦂᪋タࡢᐃᢏ⾡࠾ࡼࡧᐇ㦂ᡭἲࡢᢏ⾡⛣㌿ࡢホ౯ࡀ⾜ࢃࢀࡓࠋᮏヨ㦂࡛ࡣࠊ㝧ᛶᑐ↷ࢆྵࡴࢥ࣮ࢻ໬ࡉࢀࡓ 10 ≀㉁ࡢ 3 ᅇ⧞ࡾ㏉ࡋᐃ࡟ࡼࡾ᪋タෆࠊ᪋タ㛫෌⌧ᛶ࡟ࡘ࠸࡚ࡢ᳨ウࡀ⾜ࢃࢀࡓࠋࡲࡓࠊ࣮ࣜࢻ࡛ࣛ࣎ᐇ᪋ࡉࢀࡓ 86 ≀㉁㸦ከ᪋タࣂࣜࢹ࣮ࢩ࡛ࣙࣥࡢ

ᐃᐇ᪋ 10 ≀㉁ࢆྵࡴ㸧ࡢᐃ⤖ᯝ࠿ࡽ⤖ྜヨ㦂࡜ࡢẚ㍑ࠊᏊᐑ⫧኱ヨ㦂࡜ࡢẚ㍑ࠊICCVAM ሗ࿌࡜ࡢẚ㍑ࡀ⾜ࢃࢀࡓࠋࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡢ⤖ᯝࠊ4 ヨ㦂᪋タࡢࢹ࣮ࢱࡣ㧗࠸୍⮴⋡ࢆ♧ࡋࠊICCVAM ሗ࿌ࡢศ㢮㸦㝧ᛶ࣭㝜ᛶ㸧࡜ࡢ୍⮴⋡ࡶ㧗࠸ࡶࡢ࡛࠶ࡗࡓࠋࡇࡢ⤖ᯝ࠿ࡽࠊER STTA ἲࡣࠊ໬Ꮫ≀㉁ࡢ in vitro ER ࢔ࢦࢽࢫࢺάᛶࢆṇ☜࡟᳨ฟࡍࡿヨ㦂ἲ࡛࠶ࡿ࡜ホ౯ࡉࢀࡓࠋࡇࡢ࢔ࢦࢽࢫࢺヨ㦂ࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩ࡣࠊ2006 ᖺ࡟࢔ࢦࢽࢫࢺヨ㦂ἲࡢࡳࡢヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥ᱌࡜࡜ࡶ࡟ OECD ࡟ᥦฟࡉࢀࠊ2009 ᖺ࡟ᡂ❧ࡋࡓ᭱ึࡢ TG455 ࢆࢧ࣏࣮ࢺࡍࡿࢹ࣮ࢱ࡜࡞ࡗࡓࠋࡑࡢᚋࠊTG455 ࡣࠊ2012 ᖺ࡟ BG1Luc
ER TA ἲࡢ࢔ࢦࢽࢫࢺヨ㦂ἲࢆໟྵࡍࡿᛶ⬟‽ᣐヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥ㸦PBTG㸧࡟᭦᪂ࡉࢀࠊ
࠶ࢃࡏ࡚࢔ࢦࢽࢫࢺヨ㦂ࡢᛶ⬟ᇶ‽㸦PS㸧ࡀබ㛤ࡉࢀࡓ(1)ࠋ
࢔ࣥࢱࢦࢽࢫࢺヨ㦂࡟㛵ࡋ࡚ࡣࠊୖグࡢ࢔ࢦࢽࢫࢺヨ㦂ࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩ࡢ➨୕⪅ホ౯࡟࠾ࡅࡿせồࢆཷࡅ࡚ࠊ2008 ᖺ࡟ OECD ࡟ࣉࣟࢪ࢙ࢡࢺᥦ᱌ࡉࢀࠊ JaCVAM ࡀ୰ᚰ࡜࡞ࡗ࡚ࠊᙜึࠊᅜෆ 3 ᪋タ㸦CERIࠊ኱ሯ〇⸆ࠊ࢝ࢿ࢝㸧ࠊᾏእ 2 ᪋タ㸦㡑ᅜ KFDAࠊ࣋ࣝࢠ࣮VITO㸧ࡢཧຍ࡟ࡼࡿᅜ㝿ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡜ࡋ࡚㛤ጞࡉࢀࡓࡶࡢࡢࠊᾏእ 2 ᪋タ࠾ࡼࡧᅜෆ 1 ᪋タ㸦࢝ࢿ࢝㸧ࡣྛ᪋タࡢ஦᝟࡟ࡼࡾࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡢ㏵୰࡛᧔㏥ࡋࡓࡓࡵࠊ
ࣂࣜࢹ࣮ࢩࣙࣥ࡟ᚲせ࡞ヨ㦂ࢹ࣮ࢱࡢྲྀᚓࡢࡓࡵࠊ2010 ᖺࡼࡾ BG1Luc ER TA ἲࣂࣜࢹ࣮
ࢩࣙࣥヨ㦂࡟ཧຍ⤒㦂ࡢ࠶ࡿ(ᰴ)᪥ྜྷ(௨ୗࠊ᪥ྜྷ࡜グࡍ)ࡀ㏣ຍ࡛ཧຍࡋࡓࠋ࢔ࣥࢱࢦࢽࢫ
ࢺࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡣ 3 ẁ㝵࡟ࢃࡅ࡚⾜ࢃࢀࠊ➨ 1 ẁ㝵࡛ࡣࠊTG455 ࡛♧ࡉࢀࡓ࢔ࢦࢽ
ࢫࢺヨ㦂ࡢཧ↷≀㉁ 3 ≀㉁ࡢ 3 ᅇ⧞ࡾ㏉ࡋᐃ࡟ࡼࡾࠊཧຍヨ㦂᪋タࡢᐃᢏ⾡࠾ࡼࡧᐇ

㦂ᡭἲࡢᢏ⾡⛣㌿ࡢホ౯ࡀ⾜ࢃࢀࠊ඲࡚ࡢཧຍ᪋タࡀࠊ࢔ࢦࢽࢫࢺࡢᇶ‽ࢆ‶ࡓࡍ⤖ᯝࢆᚓࡓࠋ➨ 2 ẁ㝵࡛ࡣࠊ᪂ࡓ࡟ᵓ⠏ࡉࢀࡓ࢔ࣥࢱࢦࢽࢫࢺࡢࣉࣟࢺࢥࣝ࡟ᚑ࠸ࠊ࢔ࣥࢱࢦࢽ
ࢫࢺᐃࡢཧ↷≀㉁ೃ⿵ 4 ≀㉁ࡢ

ᐃࠊ➨ 3 ẁ㝵࡛ࡣࠊࢥ࣮ࢻ໬ࡉࢀࡓ 20 ≀㉁ࡢ

ᐃࡀ⾜ࢃࢀࡓࠋᾏእ 2 ᪋タࡣ➨ 2 ẁ㝵ࡢ㏵୰࡛ࠊᅜෆ 1 ᪋タ㸦࢝ࢿ࢝㸧ࡣ➨ 3 ẁ㝵ࡢ㏵୰࡛ࣂࣜ
ࢹ࣮ࢩࣙࣥヨ㦂ࡼࡾ᧔㏥ࡋࡓࡓࡵࠊ᭱⤊ⓗ࡞ホ౯ࡣࠊCERIࠊ኱ሯ〇⸆࠾ࡼࡧ᪥ྜྷࡢ
ᯝ࡜࢝ࢿ࢝ࡢᐃ⤖ᐃ῭ࡳ⤖ᯝࡼࡾ⾜ࢃࢀࡓࠋࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡢ⤖ᯝࠊ⿕㦂≀㉁ࡢᐃᛶⓗホ౯⤖ᯝࡣࠊ᪋タ㛫୍࡛⮴ࡍࡿࡶࡢࡢࠊ᪋タ࡟ࡼࡗ࡚ࡣཧ↷ᇶ‽ࢆ‶ࡓࡍ஦ࡀฟ᮶࡞࠿ࡗ
ࡓࠋࡇࡢཎᅉ࡜ࡋ࡚ࠊ⣽⬊ᇵ㣴ᢏ⾡࡟⏤᮶ࡍࡿ཯ᛂᛶࡢኚ໬ࡀ♧၀ࡉࢀࡓࡶࡢࡢ᏶඲࡟ࡣ
612ゎᾘฟ᮶࡞࠿ࡗࡓࡇ࡜࠿ࡽࠊ㝧ᛶ≀㉁ࡢᐃ㔞ⓗホ౯⤖ᯝ࡟ࡘ࠸࡚ಙ㢗ᛶࢆಖドฟ᮶࡞࠸ྍ
⬟ᛶࡀ♧ࡉࢀࡓࠋࡑࡢࡓࡵࠊᮏヨ㦂⣔ࡣᙜึࠊᐃ㔞ⓗホ౯⣔࡜ࡋ࡚㛤Ⓨࡉࢀࡓࡶࡢࡢᐃᛶⓗホ౯ࢆ୺║࡜ࡋࡓࣉࣟࢺࢥࣝࡢኚ᭦ࡀ⾜ࢃࢀࡓࠋ᪤࡟ヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥࡀᡂ❧ࡋ࡚࠸ࡿ BG1Luc ER TA ἲࡶᐃᛶⓗホ౯⣔࡜ࡋ࡚ヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥ໬ࡉࢀ࡚࠸ࡿࡇ࡜࠿ࡽࠊ
OECD TG 455 ᨵᐃ᱌࡟ࡘ࠸࡚ࡶᐃᛶⓗホ౯⣔࡜ࡋ࡚ᥦ᱌ࡉࢀࡓࠋ࢔ࣥࢱࢦࢽࢫࢺヨ㦂ࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩࡟ࡘ࠸࡚ࡣࠊ2014 ᖺ࡟࢔ࣥࢱࢦࢽࢫࢺヨ㦂ࢆໟྵࡍࡿ TG455 ᨵゞ᱌(2)࠾ࡼࡧ࢔ࣥࢱࢦࢽࢫࢺヨ㦂ࡢ PS(3)࡜࡜ࡶ࡟ OECD ࡟ᥦฟࡉࢀࡓࠋ࢔ࣥࢱࢦࢽࢫࢺヨ㦂ࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩ࡣࠊඛ࡟ᥦฟࡉࢀࡓ࢔ࢦࢽࢫࢺヨ㦂ࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩ࡢ㏣ຍᩥ᭩࡜
ࡋ࡚ᥦฟࡉࢀࠊ᭱⤊ⓗ࡟࢔ࢦࢽࢫࢺヨ㦂㸦part A㸧࡜࢔ࣥࢱࢦࢽࢫࢺヨ㦂㸦part B㸧ࢆྵࡴ༢୍ࡢࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩࡜ࡋ࡚බ㛤ࡉࢀࡿணᐃ࡛࠶ࡿ(4)ࠋ࡞࠾ࠊTG 455 ࡬ࡢ࢔ࣥࢱࢦࢽ
ࢫࢺヨ㦂ἲࡢ㏣ຍ࡟ࡼࡾ BG1Luc ER TA ἲࡣࠊER STTA ἲ࡜ඹ࡟ヨ㦂ἲࡢ୍ࡘ࡜ࡋ࡚ TG 455
࡟཰㍕ࡉࢀࠊBG1Luc ER TA ἲࡢࡳࡢ TG ࡛࠶ࡿ TG 457 ࡣࠊᗫṆࡉࢀࡿணᐃ࡛࠶ࡿࠋ

㸰㸬ヨ㦂ἲࡢጇᙜᛶ
2-1 ヨ㦂ἲࡢᴫ␎㸯㸧┠ⓗ࡜ཎ⌮ࡇࡇ࡛ྲྀࡾୖࡆࡿヨ㦂ἲࡣෆศἪ࠿ࡃ஘≀㉁ࡢ࠺ࡕ ER ࡟స⏝ࡍࡿ໬Ꮫ≀㉁ࢆ᳨⣴ࡋࠊࡑࡢேయࠊ⮬↛⏺࡟ᑐࡍࡿᘢᐖࢆ㑊ࡅࡿࡇ࡜ࢆ┠ⓗ࡜ࡋ࡚㛤ⓎࡉࢀࡓࠋᮏἲࠊER STTA ἲࡣࡑࡢ୍ࡘ࡛⏕యࡢ ER ࡟⤖ྜࡋࠊ࢔ࢦࢽࢫࢺస⏝ࠊ࠶ࡿ࠸ࡣ࢔ࣥࢱࢦࢽࢫࢺస⏝ࢆ♧ࡍ໬Ꮫ≀㉁ࢆࢫࢡ࣮ࣜࢽࣥࢢࡍࡿࡓࡵ࡟ HeLa ⣽⬊࡟ࣄࢺ ERĮ ࢆᜏᖖⓗ࡟Ⓨ⌧ࡍࡿࣉࣛࢫ࣑ࢻ࡜
ERE ୗὶ࡟ࣝࢩࣇ࢙࣮ࣛࢮ㑇ఏᏊࢆ⧅࠸ࡔ࣏࣮ࣞࢱ࣮ࣉࣛࢫ࣑ࢻࢆᑟධ࣭ᏳᐃⓎ⌧ࡉࡏࠊ⿕㦂≀㉁ࢆᢞ୚ࡋ࡚⣽⬊࡟཯ᛂࡉࡏࡓᚋࠊࣝࢩࣇ࢙࣮ࣛࢮάᛶࡢኚ໬ࢆࣝࢩࣇ࢙ࣜࣥࡢⓎග࡟ࡼࡾᐃ࡛ࡁࡿᵝ࡟ࡋࡓࡶࡢ࡛࠶ࡿࠋ

㸰㸧ᶆ‽

ᐃ᮲௳

ḟࡢⅬ࡟␃ពࡍࡿᚲせࡀ࠶ࡿࠋa.

ᇵ㣴⣽⬊ࡢ⥔ᣢ⟶⌮㸸ᇵ㣴⣽⬊ࡣ඲࡚↓⳦≧ែ࡛ྲྀࡾᢅ࠺ᚲせࡀ࠶ࡿࠋ‽ഛẁ㝵ࡢᇵ㣴࠿ࡽࠊ96 ࢙࣭࢘ࣝࣉ࣮ࣞࢺ୰ࡢヨ㦂࡟⮳ࡿࡲ࡛ࠊ⣽⬊ࡀࣇࣛࢫࢥࡸ࢙࢘ࣝෆ࡛
ᆒ୍࡞ᐦᗘ࡟࡞ࡿᵝ࡟⥔ᣢࡉࢀ࡞ࡅࢀࡤ࡞ࡽ࡞࠸ࠋOECD TG 455 ࡟ῧ௜ࡉࢀࡓࣉࣟ
ࢺࢥࣝ㸦ANNEX 2㸧࡛♧ࡉࢀࡓᇵ㣴᪉ἲ࠾ࡼࡧὀពⅬࢆᛅᐇ࡟Ᏺࡿࡇ࡜ࠊ⣽⬊⃰ᗘࡢㄪᩚ᪉ἲ࡞࡝ࡶྵࡵ࡚ࠊᇵ㣴⣽⬊ࡢᢅ࠸᪉ࢆ๓ࡶࡗ࡚≉ู࡟カ⦎ࢆཷࡅ࡚࠾ࡃᚲ
せࡀ࠶ࡿࠋ
7

13

b. ẚ㍑ᑐ↷ࡢタᐃ㸸⿕㦂≀㉁ࡢ

ᐃ್ࡣ඲࡚┦ᑐ್࡛ẚ㍑ࡍࡿࡢ࡛㐺ษ࡞⃰ᗘࡢ E2ࢆᖖ࡟ྠ୍ࡢ 96 ࢙࣭࢘ࣝࣉ࣮ࣞࢺୖ࡛
c.

⿕㦂≀㉁㸸⿕㦂≀㉁ྠ᫬࡟ᐃࡋࠊࡑࢀ࡟ᑐࡍࡿ┦ᑐ್࡜ࡋ࡚⾲ࡍࠋᐃ᫬࡟ࡣࠊࣉࣟࢺࢥ࡛ࣝつᐃࡉࢀࡿ㝧ᛶ࣭㝜ᛶࡢཧ↷≀㉁ࢆ

ᐃࡍࡿࡇ࡜ࡀ㔜せ࡛࠶ࡿࠋ

2-2 ጇᙜᛶࡢ᳨ウ㸯㸧⥲ྜⓗ᳨ウ㸸 ER STTA ἲࡢᇶ࡜࡞ࡿ HeLa ⣽⬊ࡣኳ↛ࡢ ER ࢆⓎ⌧ࡋ࡚࠾ࡽࡎࠊER STTA
ἲ࡛⏝࠸ࡿ HeLa-9903 ࡛ࡣᙉไⓎ⌧ࡉࢀࡓࣄࢺ ERĮ ࢆ௓ࡋࡓ࢔ࢦࢽࢫࢺ࠾ࡼࡧ࢔ࣥࢱࢦࢽ
ࢫࢺࡢ୧⪅ࢆ᳨ฟྍ⬟࡞ in vitro ⣔࡛࠶ࡿࠋ⁐፹࡟⁐ゎྍ⬟㸦DMSO ௨እࢆ⏝࠸ࡿሙྜࡶ࠶
ࡿ㸧࡞ᗈ⠊࡞⿕㦂≀㉁࡟㐺⏝࡛ࡁࡿⅬ࡛㠀ᖖ࡟౯್ࡀ㧗࠸ࠋྠᵝࡢ ER ㌿෗άᛶ໬ヨ㦂ἲ࡛࠶ࡿ BG1Luc ⣽⬊ࡣࠊERĮ ᆺ㸭ȕ ᆺ཮᪉ࢆⓎ⌧ࡋ࡚࠾ࡾࠊ୧⪅ࢆ௓ࡋࡓస⏝ࡀ᳨ฟྍ⬟࡛࠶ࡿ࡜ࡉࢀࡿࡶࡢࡢࠊ࠸ࡎࢀࡢཷᐜయ࡬ࡢస⏝࡛࠶ࡿ࠿ࢆ༊ูࡍࡿࡇ࡜ࡣฟ᮶࡞࠸ࠋࡇࢀ࡟
ᑐࡋ࡚ࠊER STTA ἲ㸦HeLa-9903 ⣽⬊㸧࡛ࡣࠊ⣧⢋࡟ࣄࢺ ERĮ ࡢࡳࢆ௓ࡋࡓస⏝ࢆ᳨ฟྍ⬟࡛࠶ࡿࠋ⌮ㄽⓗどⅬ࠿ࡽ㸸࢔ࢦࢽࢫࢺάᛶ࠾ࡼࡧ࢔ࣥࢱࢦࢽࢫࢺస⏝ࡢ࠶ࡿ໬Ꮫ≀㉁࡟ࡼࡿ ER ࡟ᑐࡍࡿ༢࡞ࡿ⤖ྜ⬟࡛ࡣ࡞ࡃ㌿෗άᛶࢆᐃ࡛ࡁࡿࠋin vitro ⣔࡛ࡣ࠶ࡿࡀࠊࣂ࢖࢜࢔ࢵࢭ࢖࡛࠶ࡿࡓࡵ཯

ᛂ⣔ࠊᇵ㣴⣽⬊ࡢ⥔ᣢ⟶⌮ࡀ⤖ᯝ࡟ᙳ㡪ࢆཬࡰࡍྍ⬟ᛶࡣྰᐃ࡛ࡁ࡞࠸ࠋࡇࡢࡇ࡜ࡣヨ㦂⤖ᯝࡢኚື࡟ࡘ࡞ࡀࡿྍ⬟ᛶࡀ㧗࠸ࡢ࡛༑ศ࡟ὀពࡀᚲせ࡛࠶ࡿࠋᐃ⣔ᵓ⠏ୖࡢどⅬ࠿ࡽ㸸a.

ᇵ㣴⣽⬊㸸ER STTA ἲ࡛ࡣ HeLa ⣽⬊࡟ࣄࢺ ERĮ ࢆᜏᖖⓗ࡟ᙉไⓎ⌧ࡉࡏࡓ
HeLa9903 ⣽⬊ࢆ⏝࠸࡚࢔ࢵࢭ࢖⣔ࢆᵓ⠏ࡋ࡚࠸ࡿࠋ୍⯡࡟ࣄࢺࡢᇵ㣴⣽⬊࡛ࡣᰁⰍయࡢᙧែ࠾ࡼࡧᩘࡢ␗ᖖࢆ㧗㢖ᗘ࡟᭷ࡍࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿࠋࡑࡢᙳ㡪ࡣᙉไ
Ⓨ⌧ࡉࡏࡓ ER ࡢⓎ⌧࡟ࡶ࠶ࡿ⛬ᗘཬࡪࡇ࡜ࡀண᝿ࡉࢀࡿࠋ⌧ᅾࡢ࡜ࡇࢁࡇ࠺ࡋࡓ
ᰁⰍయ␗ᖖࢆ㜵ࡄᡭẁࡣ▱ࡽࢀ࡚࠸࡞࠸ࠋᚑࡗ࡚ࠊヨ㦂ᑐ㇟≀㉁ࡢ
཯ᛂᛶࡢࢳ࢙ࢵࢡࢆ TG ࡛ᣦᐃࡉࢀࡓཧ↷㸦ᶆ‽㸧≀㉁ࡢ

ᐃ᫬࡟ࡣࠊERᐃ࡟ࡼࡗ࡚ᖖ࡟ホ౯ࡍࡿᚲせࡀ࠶ࡿࠋ
b. ࣏࣮ࣞࢱ࣮㑇ఏᏊ㸸ࡇࡢ࢔ࢵࢭ࢖⣔࡛ࡣ࣏࣮ࣞࢱ࣮㑇ఏᏊࢆᑟධࡋࠊER άᛶ໬࡟ࡼࡿ࣏࣮ࣞࢱ࣮㑇ఏᏊⓎ⌧࡟ࡼࡾᐃࢆ⾜࠺ࠋ୍㒊ࡢ໬Ꮫ≀㉁ࡣࠊ࣏࣮ࣞࢱ࣮࡜ࡋ

࡚⏝࠸࡚࠸ࡿࣝࢩࣇ࢙࣮ࣛࢮάᛶࡢཷᐜయ㠀≉␗ⓗ࡞ஹ㐍ࡸ㜼ᐖ➼࡟ࡼࡾ␲㝧ᛶ㸦ࡶࡋࡃࡣഇ㝜ᛶ㸧཯ᛂࢆច㉳ࡍࡿྍ⬟ᛶࡀ࠶ࡿࠋࡲࡓࠊER STTA ἲ࡛ࡣࠊ୍㒊ࡢ᳜≀࢚ࢫࢺࣟࢤ࡛ࣥ㧗⃰ᗘ࡟࠾࠸࡚ཷᐜయࢆ௓ࡋ࡞࠸ࣝࢩࣇ࢙࣮ࣛࢮࡢ㧗άᛶ໬814⌧㇟ࡀ♧ࡉࢀࡿࡇ࡜ࡀ᫂ࡽ࠿࡟ࡉࢀ࡚࠸ࡿࠋࡇࢀࡲ࡛࡟ᕤᴗ⏝໬Ꮫ≀㉁࡛ࡣࠊྠᵝࡢస⏝ࡀ᳨ฟࡉࢀࡓ౛ࡣ࡞࠸ࡀὀពࡀᚲせ࡛࠶ࡿࠋヨ㦂ἲ᧯సୖࡢどⅬ࠿ࡽ㸸a.

‽ഛẁ㝵ࢆྵࡵࡓᇵ㣴ᐜჾෆࡢ⣽⬊ᐦᗘࡢㄪᩚࠋ≉࡟⣽⬊ࢆᆒ୍࡟᧛✀࡛ࡁ࡚࠸ࡿ
࠿࡝࠺࠿ࡢࢳ࢙ࢵࢡࡀᚲせ࡛࠶ࡿࠋb.

⏕⣽⬊ࢆಽ❧㢧ᚤ㙾࡛ࢳ࢙ࢵࢡࡍࡿ㝿࡟ᐜჾ඲యࢆࢳ࢙ࢵࢡࡍࡿᚲせࡀ࠶ࡿࠋ

2-3 ER STTA ἲࡢၥ㢟Ⅼ㸯㸧࢔ࢵࢭ࢖ࢩࢫࢸ࣒

௨ୗࡢࡼ࠺࡞ၥ㢟ࡀ࠶ࡿࠋ౑⏝ᇵ㣴⣽⬊࡟ࡘ࠸࡚㸸⣽⬊ࡢ཯ᛂᛶࢆ⥔ᣢࡍࡿࡓࡵࠊࣉࣟࢺࢥ࡛ࣝࡣࠊ

ᐃ࡟౑⏝ࡍࡿ⣽⬊ࡢ⥅௦ᩘࡣ 40 ௦ࡲ࡛࡜つᐃࡉࢀ࡚࠸ࡿࠋࡋ࠿ࡋࠊ࢔ࣥࢱࢦࢽࢫࢺヨ㦂ࡢࣂࣜࢹ࣮ࢩࣙࣥ࡟࠾࠸࡚ࡣࠊ୍㒊ࡢཧຍ᪋タ࡛⥅௦ᩘࡀ 40 ௦௨ୗ࡛࠶ࡗ࡚ࡶ
⣽⬊⥅௦࡟క࠺࡜ᛮࢃࢀࡿ཯ᛂᛶࡢపୗࡸ⏝㔞཯ᛂᛶࡢኚ໬ࡀㄆࡵࡽࢀ࡚࠾ࡾὀពࡀᚲせ࡛࠶ࡿࠋࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡛ࡣࠊ⥅௦᫬ࡢࢺࣜࣉࢩࣥᾘ໬᫬㛫ࡢᘏ㛗ࡀ⣽⬊࡟ࢲ࣓࣮ࢪࢆ୚࠼ࡿྍ⬟ᛶࡀ♧၀ࡉࢀ࡚࠸ࡿࠋࡲࡓࠊ⣽⬊ᙧែ࡟␗ᖖࡀ↓ࡃ࡚ࡶࠊ཯ᛂᛶࡀኚ໬ࡍࡿࡇ࡜ࡀ♧၀ࡉࢀ࡚࠾ࡾࠊཧ↷≀㉁ࡢ

ᐃ⤖ᯝࡀࣉࣟࢺ

ࢥࣝ࡟♧ࡉࢀࡓࢡࣛ࢖ࢸࣜ࢔ࢆ㐃⥆ࡋ࡚㐓⬺ࡋࡓሙྜࡸࣉ࣮ࣞࢺ᥇⏝ᇶ‽ࢆ㐃⥆ࡋ࡚‶ࡓࡉ࡞࠿ࡗࡓሙྜࠊ⣽⬊ࢆ᪂ࡓ࡞ࣟࢵࢺ㸦ࢧࣈࣟࢵࢺ㸧࡟ษ᭰࠼ࡿ࡭ࡁ࡛
࠶ࡿࠋ㸱㸬ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟⏝࠸ࡓ≀㉁ࡢศ㢮࡜ጇᙜᛶ࢔ࢦࢽࢫࢺヨ㦂ἲࡢࣂࣜࢹ࣮ࢩ࡛ࣙࣥࡣ 86 ≀㉁㸦Table 1㸧ࡀࠊ࢔ࣥࢱࢦࢽࢫࢺヨ㦂ἲࡢࣂࣜࢹ࣮ࢩ࡛ࣙࣥࡣ 21 ≀㉁㸦Table 2㸧ࡀ⏝࠸ࡽࢀࡓ(4)ࠋࡑࢀࡽ࡟ࡣࢫࢸࣟ࢖ࢻࠊ᭷ᶵ㓟ࠊ
Ⅳ໬Ỉ⣲࡞࡝ᗈ⠊࡞✀㢮࠿ࡘᵝࠎ࡞཯ᛂᙉᗘࡢ≀㉁ࡀྵࡲࢀ࡚࠸ࡿࠋヨ㦂ἲࡢឤᗘࢆホ౯ࡍࡿࡓࡵࠊ㝜ᛶ≀㉁ࡀ࢔ࢦࢽࢫࢺヨ㦂࡛ࡣ⣙ 30%㸦27/86㸧ࠊ࢔ࣥࢱࢦࢽࢫࢺヨ㦂࡛ࡣ 60㸣
㸦12/20㸧ࢆ༨ࡵ࡚࠾ࡾࠊ㑅ࡤࢀࡓ≀㉁ࡣጇᙜ࡜ุ᩿ࡉࢀࡿࠋ

㸲㸬ヨ㦂ἲࡢࢹ࣮ࢱ࡜⤖ᯝࡢ᭷⏝ᛶ࢔ࢦࢽࢫࢺヨ㦂ࡢࣂࣜࢹ࣮ࢩࣙࣥ࡟⏝࠸ࡽࢀࡓ 86 ≀㉁ࡢ࠺ࡕࠊཧຍ 4 ᪋タ඲࡚࡛ᐃࡀᐃࡉࢀࡓ E2 ཬࡧࢥ࣮ࢻ໬≀㉁࡜ࡋ࡚

ᐃࡀᐇ᪋ࡉࢀࡓ

9 ≀㉁ࡢィ 10 ≀㉁㸦㝧ᛶ 7 ≀㉁ࠊ㝜ᛶ 3 ≀㉁㸧࡛࠶ࡾࠊࡇࢀࡽ 10 ≀㉁ࡢ

ᐃ⤖ᯝࢆࡶ࡜࡟

⾜ࢃࢀࡓ≀㉁ࡣࠊ㝧ᛶ≀㉁࡜ࡋ࡚

915᪋タෆ࠾ࡼࡧ᪋タ㛫෌⌧ᛶࡢホ౯ࡀ⾜ࢃࢀࠊ௚ࡢ 76 ≀㉁࡟ࡘ࠸࡚ࡣࠊ࣮ࣜࢻ࡛ࣛ࣎࠶ࡿ
CERI ࡟࠾ࡅࡿ

ᐃ⤖ᯝࢆࡶ࡜࡟ࠊICCVAM ཧ↷ศ㢮ࡸ ER ⤖ྜヨ㦂ࠊᏊᐑ⫧኱ヨ㦂⤖ᯝ࡜ࡢẚ㍑ࡀ⾜ࢃࢀࡓ㸦Table 3㹼6㸧ࠋ࢔ࣥࢱࢦࢽࢫࢺヨ㦂ࡢࣂࣜࢹ࣮ࢩ࡛ࣙࣥࡣࠊཧ↷≀㉁ࢆྵࡴ 21 ≀㉁ࡢ࠺ࡕࠊࣂࣜࢹ࣮ࢩ
ࣙࣥヨ㦂➨ 3 ẁ㝵࡛ࢥ࣮ࢻ໬≀㉁࡜ࡋ࡚
≀㉁ࠊ୙᫂ 2 ≀㉁㸧ࡢᐃࡀᐇ᪋ࡉࢀࡓ 20 ≀㉁㸦㝧ᛶ 8 ≀㉁ࠊ㝜ᛶ 10ᐃ⤖ᯝࢆࡶ࡜࡟ࠊ᪋タෆ࠾ࡼࡧ᪋タ㛫෌⌧ᛶࡢホ౯ࠊ࠾ࡼࡧᩥ⊩᝟ሗ࡞࡝࠿ࡽண᝿ࡉࢀࡓ࢔ࣥࢱࢦࢽࢫࢺάᛶ࡜ࡢẚ㍑࡟ࡼࡿホ౯⢭ᗘࡢࣂࣜࢹ࣮ࢩࣙࣥࡀ
⾜ࢃࢀࡓ㸦Table 7㸧ࠋ࢔ࣥࢱࢦࢽࢫࢺヨ㦂ࡢࣂࣜࢹ࣮ࢩࣙࣥ࡟࠾࠸࡚ࡣ

ᐃ≀㉁ᩘࡀᑡ࡞࠸

ࡀࠊ࢔ࣥࢱࢦࢽࢫࢺάᛶࢆ♧ࡍࡇ࡜ࡀ᫂ࡽ࠿࡟ࡉࢀ࡚࠸ࡿ໬Ꮫ≀㉁ࡢከࡃࡣࠊ་⸆ရࡶࡋ
ࡃࡣࡑࡢ㢮ఝ≀㉁࡛࠶ࡾࠊ୍⯡ᕤᴗ໬Ꮫ≀㉁࡛᫂ࡽ࠿࡞࢔ࣥࢱࢦࢽࢫࢺάᛶࡀ▱ࡽࢀ࡚࠸ࡿ≀㉁ࡣࠊ㠀ᖖ࡟㝈ࡽࢀ࡚࠸ࡿࡇ࡜࡟ࡼࡿࠋࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡛ホ౯ࡀ⾜ࢃࢀࡓ㝧ᛶ 8≀㉁ࡣࠊ௒ᚋࡢ᪂ࡓ࡞ヨ㦂⣔ホ౯࡟࠾ࡅࡿ㔜せ࡞ཧ↷≀㉁࡜࡞ࡿ࡛࠶ࢁ࠺ࠋ
࠸ࡎࢀࡢࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟࠾࠸࡚ࡶ⏝࠸ࡽࢀࡓ໬Ꮫ≀㉁ࡣࠊICCVAM ཧ↷ศ㢮ࣜࢫࢺ➼ࢆཧ⪃࡟ࠊᗈ⠊࡞✀㢮ࡢ≀㉁ࡀ㑅ᢥࡉࢀ࡚࠸ࡿࡀࠊࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩࡟࠾࠸࡚ࡣࠊ
໬Ꮫ≀㉁ࡢᵓ㐀࡟ࡼࡿศ㢮㸦ࢫࢸࣟ࢖ࢻ㢮ࠊ࣋ࣥࢮࣥ༢⎔➼㸧ࡶ♧ࡋࡓ᪉ࡀⰋ࠸࡜⪃࠼ࡽࢀࡿࠋ㸳㸬ヨ㦂᪉ἲࡢ෌⌧ᛶ ࢔ࢦࢽࢫࢺヨ㦂࡛ࡣࠊ㝧ᛶ≀㉁࡜ࡋ࡚
≀㉁㸧ࡢཧຍ 4 ᪋タ࡟࠾ࡅࡿ 3 ᅇࡢᐃࡉࢀࡓ E2 ࢆྵࡴ 10 ≀㉁㸦㝧ᛶ 7 ≀㉁ࠊ㝜ᛶ 3ᐃ⤖ᯝࢆࡶ࡜࡟᪋タෆ࠾ࡼࡧ᪋タ㛫෌⌧ᛶࡢࣂࣜࢹ

࣮ࢩࣙࣥࡀ⾜ࢃࢀࠊ࠸ࡎࢀࡶ୍⮴⋡ࡣ 100㸣࡛࠶ࡗࡓࠋ
࢔ࣥࢱࢦࢽࢫࢺヨ㦂࡛ࡣࠊࣂࣜࢹ࣮ࢩࣙࣥヨ㦂➨ 3 ẁ㝵┠࡛᧔㏥ࡋࡓ࢝ࢿ࢝ࢆྵࡴ 4 ᪋タ࡟࠾ࡅࡿ 20 ≀㉁㸦኱ሯ〇⸆ࡣ 19 ≀㉁ࠊ࢝ࢿ࢝ࡣ 12 ≀㉁㸧ࡢ 3 ᅇ௨ୖࡢᐃ⤖ᯝࢆࡶ࡜࡟᪋タෆ෌⌧ᛶࡢゎᯒࡀ⾜ࢃࢀࡓࠋ᪋タෆ෌⌧ᛶ࡟ࡘ࠸࡚࢝ࢿ࡛࢝ࡣࠊྛ≀㉁ 5㹼6 ᅇࡢᐃ⤖ᯝࡀ඲୍࡚⮴ࡋࡓࡢࡣ 67㸣 8/12 ≀㉁ ࡛࠶ࡗࡓࡀࠊ௚ࡢ 4 ≀㉁࡟ࡘ࠸࡚ࡣࠊ
ࡀ୍⮴ࡋ࡞࠿ࡗࡓࡢࡣࠊྛ 1ᐃ⤖ᯝᐃࡢࡳ࡛࠶ࡗࡓࠋ௚ࡢ᪋タ࡛ࡣ 90㸣௨ୖࡢ෌⌧ᛶࢆ♧ࡋࡓ

㸦Table 8㸧ࠋ୍᪉ࠊ᪋タ㛫෌⌧ᛶ࡟ࡘ࠸࡚ࡶྠᵝ࡟ 20 ≀㉁࡟ࡘ࠸࡚ࡢ 2㹼4 ᪋タ࠿ࡽࡢᐃ⤖ᯝ࠿ࡽゎᯒࡀ⾜ࢃࢀࠊ2 ≀㉁㸦AtrazineࠊDibenzo[a,h]anthracene㸧࡟ࡘ࠸࡚ࡣࠊࡑࢀࡒࢀ
1/3 ᪋タ࡛௚᪋タ࡜୍⮴ࡋ࡞࠸⤖ᯝࡀ♧ࡉࢀࡓࠋࡑࡢ௚ࡢ⿕㦂≀㉁࡟ࡘ࠸࡚ࡣࠊ᪋タ㛫࡛
100㸣୍⮴ࡍࡿ⤖ᯝࢆᚓࡓࠋ⤖ᯝ࡜ࡋ࡚ᩥ⊩᝟ሗ࡞࡝࠿ࡽண᝿ࡉࢀࡓ࢔ࣥࢱࢦࢽࢫࢺάᛶ
㸦୙᫂ 2 ≀㉁ࢆ㝖ࡃ 18 ≀㉁㸦㝧ᛶ 8 ≀㉁ࠊ㝜ᛶ 10 ≀㉁㸧㸧࡜ࡢẚ㍑࡛ࡣࠊ2 ᪋タ୍࡛⮴⋡94㸣ࠊ௚ࡢ 2 ᪋タ࡛ࡣ 100㸣࡛࠶ࡗࡓ㸦Table 9㸧ࠋ1016㸴㸬ヨ㦂ἲࡢṇ☜ᛶ࣭ಙ㢗ᛶ
ER STTA ἲࡢṇ☜ᛶࢆホ౯ࡍࡿࡓࡵࠊ࢔ࢦࢽࢫࢺヨ㦂࡛ࡣࠊ35 ≀㉁㸦㝧ᛶ 25 ≀㉁ࠊ㝜ᛶ
10 ≀㉁㸧ࡢᐃ⤖ᯝ࡟ࡘ࠸࡚ ICCVAM ཧ↷ศ㢮࡜ࡢẚ㍑ࡀ⾜ࢃࢀ୍⮴⋡ࡣࠊ100㸣࡛࠶ࡗࡓ㸦Table 10㸧ࠋER ⤖ྜヨ㦂ࢹ࣮ࢱ࡜ ER STTA ἲࡢẚ㍑ࡀ 48 ≀㉁㸦⤖ྜヨ㦂㝧ᛶ 24 ≀㉁ࠊ
㝜ᛶ 24 ≀㉁㸧࡟ࡘ࠸࡚⾜ࢃࢀ୍⮴⋡ࡣ 79%㸦38/48㸧࡛࠶ࡗࡓ㸦Table 11㸧ࠋྠᵝ࡟ࠊin vivoᏊᐑ⫧኱ヨ㦂࡜ࡢẚ㍑ࡀ 48 ≀㉁㸦Ꮚᐑ⫧኱ヨ㦂㝧ᛶ 32 ≀㉁ࠊ㝜ᛶ 16 ≀㉁㸧࡟ࡘ࠸࡚⾜ࢃࢀ୍⮴⋡ࡣ 85%㸦41/48㸧࡛࠶ࡗࡓ㸦Table 12㸧ࠋ࢔ࣥࢱࢦࢽࢫࢺヨ㦂࡟ࡘ࠸࡚ࡣࠊࣂࣜࢹ࣮ࢩࣙࣥ࡟⏝࠸ࡽࢀࡓ 20 ≀㉁࡟ࡘ࠸࡚ࠊᩥ⊩᝟ሗ࡞࡝࠿ࡽண᝿ࡉࢀࡓ࢔ࣥࢱࢦࢽࢫࢺάᛶ㸦୙᫂ 2 ≀㉁ࢆ㝖ࡃ 18 ≀㉁㸦㝧ᛶ 8 ≀㉁ࠊ㝜ᛶ
10 ≀㉁㸧㸧࡜ 2㹼4 ᪋タ࡛ࡢᐃ⤖ᯝࢆ⥲ྜⓗ࡟ุ᩿ࡋࡓุᐃ⤖ᯝ࡜ࡢẚ㍑ࡀ⾜ࢃࢀࠊ୍⮴⋡ࡣࠊ100㸣࡛࠶ࡗࡓ㸦Table 9㸧ࠋ㸵㸬ࢹ࣮ࢱࡢ㉁ ࢔ࢦࢽࢫࢺࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟࠾ࡅࡿከ᪋タᐃࡣࠊ࣮ࣜࢻ࡛ࣛ࣎࠶ࡿ CERI ࡢಙ㢗ᛶಖドࢩࢫࢸ࣒ࡢࡶ࡜ࠊGLP㸦OECD principle of Good Laboratory Practice, November 26, 1997㸧࡟‽ᣐࡋ࡚ᐇ᪋ࡉࢀࡓ㸦ඛ⾜ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡣࠊ㠀 GLP ࡛ᐇ᪋ࡉࢀࡓ㸧ࠋ࢔ࣥࢱࢦࢽࢫ
ࢺࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟ࡘ࠸࡚ࡣࠊࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩࡟グ㍕ࡣ↓࠸ࡀࠊྛ᪋タ࡟࠾࠸
࡚ GLP ࡟‽ࡌ࡚ヨ㦂ࡀᐇ᪋ࡉࢀࡓࠋ

㸶㸬ヨ㦂ἲࡢ᭷⏝ᛶࠊ㝈⏺࠾ࡼࡧᥦゝ1) ࢔ࣥࢱࢦࢽࢫࢺ࡟ࡼࡿ⤖ᯝ㸸ER STTA ἲࡣཎ⌮ࡢ㡯࡛ࡶ㏙࡭ࡓᵝ࡟༢࡟࢔ࣥࢱࢦࢽࢫࢺ࡜ ER ࡜ࡢ⤖ྜ࡟Ṇࡲࡽࡎ࢔ࢦࢽࢫࢺࡢάᛶࢆᢚไࡍࡿຠᯝࢆ᳨ฟ࡛ࡁࡿⅬࡀඃࢀ࡚࠸
ࡿࠋ௚ࡢྠᵝࡢヨ㦂ἲࡢ⤖ᯝ࡜ࡢ୍⮴ᗘࠊER STTA ἲࡢឤཷᛶࠊ≉␗ᛶࡣⰋዲ࡛࠶ࡾࠊ

ഇ㝧ᛶࡸഇ㝜ᛶࡣᑡ࡞࠸࡜࠸࠺⤖ᯝࢆ♧ࡋࡓࠋ2) ヨ㦂᪉ἲ࠿ࡽぢࡓ⤖ᯝ㸸௚ࡢヨ㦂ἲ࡟ẚࡋ࢔ࢦࢽࢫࢺࠊ࢔ࣥࢱࢦࢽࢫࢺࡢ⃰ᗘࡀ 1/100⃰ᗘ᳨࡛ฟ࡛ࡁࡿࡢ࡛ࡼࡾឤᗘࡀ㧗ࡃࠊഇ㝧ᛶ࣭ഇ㝜ᛶࡢ⏕ࡌࡿྍ⬟ᛶࡣᑡ࡞࠸࡜ᛮࢃࢀࡿࠋ3) ER STTA ἲ࡟ࡼࡿ໬Ꮫ≀㉁ࡢ࢔ࢦࢽࢫࢺάᛶ࣭࢔ࣥࢱࢦࢽࢫࢺάᛶࡢࢫࢡ࣮ࣜࢽࣥࢢ⤖ᯝࡣࠊ࢚ࢫࢺࣟࢤࣥάᛶࡢヨ㦂࡟⏝࠸ࡽࢀࡿ᪤Ꮡࡢ⣽⬊⣔㸦ෆᅾᛶࡢࣄࢺ ER ࢆ฼⏝ࡍ
ࡿ MCF-7 ⣽⬊ࠊ BG1 ⣽⬊ ࡞࡝࡜ẚ㍑ࡋ࡚ࡶࠊ㝧ᛶࡸ㝜ᛶ≀㉁ࡢ㆑ูࡣⰋዲ࡛࠶ࡿࠋ
ࡲࡓࠊ໬Ꮫ≀㉁ࡢ࢚ࢫࢺࣟࢤࣥάᛶࢆࣝࢩࣇ࢙ࣜࣥⓎග࡟ࡼࡾᐃ㔞໬ࡍࡿࡀࠊMCF-7 ⣽1117⬊࡟ẚ࡭࡚ቑṪࡀ᪩࠸ࡓࡵࠊヨ㦂᫬㛫ࡀ▷⦰ࡉࢀࠊከᩘࡢ໬Ꮫ≀㉁ࡢࢫࢡ࣮ࣜࢽࣥࢢ࡟
ྥ࠸࡚࠸ࡿࠋ
4) ࣏࣮ࣞࢱ࣮࡜ࡋ࡚⏝࠸࡚࠸ࡿࣝࢩࣇ࢙࣮ࣛࢮάᛶ࡟ᙳ㡪ࢆ୚࠼ࡿ໬Ꮫ≀㉁࡛ࡣࠊER 㠀≉␗ⓗ࡞ஹ㐍ࡸ㜼ᐖ➼࡟ࡼࡾ␲㝧ᛶ㸦ࡶࡋࡃࡣഇ㝜ᛶ㸧཯ᛂࢆច㉳ࡍࡿྍ⬟ᛶࡀ࠶ࡿࡓࡵ
ὀពࡀᚲせ࡛࠶ࡿࠋ
5) ER ࢆ௓ࡋࡓ໬Ꮫ≀㉁ࡢ࢚ࢫࢺࣟࢤࣥάᛶࢆ୍ḟࢫࢡ࣮ࣜࢽࣥࢢࡍࡿ࡟ࡣ౽฼࡞ヨ㦂⣔࡛࠶ࡿࠋ⣽⬊ࡢ⥅௦࡟ࡼࡗ࡚ ER ࡢ཯ᛂᛶࡀኚ໬ࡋ࡞࠸ࡇ࡜ࢆ☜ㄆࡋ࡚࠾ࡃࡇ࡜ࡀᚲせ࡛࠶ࡿࠋTG ࡛♧ࡉࢀࡓ ER ࢔ࢦࢽࢫࢺヨ㦂ࠊ࢔ࣥࢱࢦࢽࢫࢺヨ㦂ࡑࢀࡒࢀࡢཧ↷≀㉁㸦㝧
ᛶ࣭㝜ᛶ㸧ࢆ⏝࠸࡚⏝㔞-ᛂ⟅ᛶࢆㄪ࡭࡚࠾ࡃࡇ࡜ࡀᚲせ࡛࠶ࡿࠋ6) ⁐፹࡜ࡋ࡚Ỉࡸ Ethanol (>95%)࠶ࡿ࠸ࡣ DMSO ࡀ⏝࠸ࡽࢀ࡚࠸ࡿࡀࠊDMSO ࡟⁐ࡅ࡟ࡃ࠸≀㉁࡟ࡘ࠸࡚ࡣࠊ௚ࡢ᭷⏝࡞⁐፹ࢆ᳨ウࡋࠊࡑࢀࡀ HeLa-9903 ⣽⬊࡟ᙳ㡪ࡋ࡞࠸ࡇ࡜
ࢆ☜ㄆࡋ࡚࠾ࡃᚲせࡀ࠶ࡿࠋ7) ⌧᫬Ⅼ࡛ࡣ᥹Ⓨᛶ≀㉁ࡢྲྀࡾᢅ࠸࡟ࡘ࠸࡚᫂☜࡞ᣦ㔪ࡀ↓࠸࡜ᛮࢃࢀࡿࠋ௒ᚋࡢ᳨ウࡀᮇᚅࡉࢀࡿࠋ
8) ௦ㅰࡉࢀ࡚࠿ࡽ࢚ࢫࢺࣟࢤࣥస⏝ࢆ♧ࡍ≀㉁ࡢホ౯࡟ࡘ࠸࡚ࡶࠊER STTA ἲࢆ⏝࠸࡚࡝ࡢࡼ࠺࡟ホ౯ࡍࡿ࠿ࡢ᳨ウࡀᚲせ࡛࠶ࡿࠋ9) ER STTA ἲࡣࠊ໬Ꮫ≀㉁ࡢෆศἪ࠿ࡃ஘ᛶࢆࢫࢡ࣮ࣜࢽࣥࢢࡍࡿࡓࡵࡢヨ㦂ἲ࡛࠶ࡾࠊ
໬Ꮫ≀㉁ࡢᏳ඲ᛶホ౯࡟⏝࠸ࡿ㝿࡟ࡣࠊ௚ࡢ in vivo ホ౯⣔➼࡜ࡢ⤖ᯝ࡜ేࡏ࡚ᐇ㝿ࡢ⏕యᙳ㡪࡟ࡘ࠸࡚⥲ྜⓗ࡟ุ᩿ࢆ⾜࠺࡭ࡁ࡛࠶ࡿࠋ
10) ᮏヨ㦂ἲࡢᐇ⏝ୖࡢᠱᛕ࡜ࡋ࡚ࠊヨ㦂⣔ࡢᏳᐃᛶࡢၥ㢟ࡀᣦ᦬ࡉࢀࡼ࠺ࠋ୍⯡࡟㑇ఏᏊࢆᑟධࡋࡓ⣽⬊ࡣࠊᏳᐃⓗᑟධ࡜ࡣゝ࠺ࡶࡢࡢࠊ⥅௦ࡈ࡜࡟㑇ఏᏊⓎ⌧࡟ࡣከᑡࡢኚ໬
ࡀ㉳ࡿࡓࡵࠊ㛗ᮇ࡟ࢃࡓࡗ࡚ྠࡌ཯ᛂᛶࢆಖࡘࡇ࡜ࡣᮇᚅ࡛ࡁ࡞࠸ࠋHeLa-9903 ⣽⬊࡟ࡘ࠸࡚ࡶࠊ⥅௦᪩ᮇࡢ཯ᛂᛶࡢ᫂ࡽ࠿࡞⣽⬊ࢆከ㔞࡟ศ๭෾⤖ಖᏑࡋ࡚ࠊ㏲ḟ฼⏝ࡍࡿࡼ࠺࡞ᥐ⨨ࡀᮃࡲࡋࡃࠊ⣽⬊ࢆྵࡴヨ㦂ᮦᩱࡢ౪⤥࡟㛵ࡍࡿ㓄៖ࡀᚲせ࡛࠶ࡿࠋ౑⏝ࡍࡿヨ㦂⣔ࡢཧ↷㸦ᶆ‽㸧≀㉁࡟ᑐࡍࡿ཯ᛂࢆヨ㦂ࡢᗘࡈ࡜࡟☜ㄆࡍࡿࡇ࡜ࡀᚲせ࡛࠶ࡿࠋ11) ᇶ♏ⓗ࡞ヨ㦂᧯సࡢṇ☜ᛶࢆಖドࡍࡿࡇ࡜ࡀᚲせ࡛ࠊヨ㦂᪋タࡀࡇࡢ⣽⬊⣔ࡢ౑⏝࡟⩦⇍ࡍࡿࡇ࡜ࡀࠊᙜ↛࡞ࡀࡽồࡵࡽࢀࡿࠋヨ㦂‽ഛࡢ㝿࡟ࠊᇵ㣴ᾮ୰ࡢ⣽⬊ࡢ⃰ᗘࢆ☜ㄆࡋࠊ࢙࢘ࣝࡈ࡜ࡢ⣽⬊ᩘࡀᆒ➼࡛࠶ࡿࡇ࡜ࢆಖドࡍࡿࡼ࠺࡞グ㘓ࠊࡓ࡜࠼ࡤ㢧ᚤ㙾෗┿࡛ࡢ☜ㄆࢆບ⾜ࡍࡿࡼ࠺࡞㓄៖ࡀᮃࡲࡋ࠸ࠋ

㸷. ࡑࡢ௚ࡢヨ㦂᪉ἲࡢ⛉Ꮫⓗ࡞ሗ࿌ෆศἪ࠿ࡃ஘స⏝࡟ࡘ࡞ࡀࡿ࡜⪃࠼ࡽࢀࡿ ER ࡟㛵ࡋ࡚ࡢヨ㦂ἲ࡜ࡋ࡚ࡣࠊၥ㢟࡜ࡍࡿ≀
12

18

㉁ࡢཷᐜయ࡬ࡢ⤖ྜᐇ㦂ࡀࡲࡎ᝿㉳ࡉࢀࡿࠋ⤖ྜᐇ㦂࡛ࡣ໬Ꮫ≀㉁࡜ࡢ┦஫స⏝ࡀᐜ᫆࡜࡞ࡿࡼ࠺ࠊཷᐜయࡀỈ┦࡟㟢ฟࡋ࡚࠸ࡿࡇ࡜ࡀᮃࡲࡋ࠸ࡀࠊࡇࡢ┠ⓗ࡟ࡣ⣽⬊ࢆ◚ቯࡋࡓ㠀⣽⬊⣔㸦cell-free㸧ࡀ᭷฼࡛࠶ࡾࠊᐇ㝿ࠊࡇࡢ⣔࡛ࡢ⤖ྜᐇ㦂ࡣ௨๓ࡼࡾ⾜ࢃࢀ࡚࠸ࡿࠋ
୍᪉ࠊཷᐜయ࡜┦஫స⏝ࢆ♧ࡍ≀㉁࡜ࡋ࡚ࡣࠊ࢔ࢦࢽࢫࢺ࡜࢔ࣥࢱࢦࢽࢫࢺࡀ࠶ࡾࠊ༢⣧࡞⤖ྜᐇ㦂࡛ࡣ୧⪅ࡢ༊ูࡀᅔ㞴࡛࠶ࡿࠋࡉࡽ࡟ࠊ㠀⣽⬊⣔࡛ࡢ⤖ᯝࡣ⣽⬊⣔࡜୍⮴ࡋ࡞࠸࡜࠸࠺ሗ࿌ࡶከࡃࠊ⣽⬊ෆ࡟≉᭷ࡢ⤖ྜࢆไᚚࡍࡿᅉᏊࡢᏑᅾࡀ♧၀ࡉࢀ࡚࠸ࡿࠋࡼࡗ
࡚ࠊ⤖ྜࢆྵࡵࡓཷᐜయ࡜ࡢ┦஫స⏝࡟ࡘ࠸࡚ࡣ⣽⬊⣔ࡢ฼⏝ࡀᮃࡲࡋ࠸ࠋ
⣽⬊⣔࡛ࡢ≀㉁ࡢ ER ࡬ࡢస⏝ࡣࠊෆᅾⓗ࡞㌿෗άᛶࡢቑຍࢆ┠Ᏻ࡜ࡍࡿࡇ࡜࡟ࡼࡾࠊ
࢔ࢦࢽࢫࢺస⏝ࡢ᭷↓ࢆุᐃ࡛ࡁࡿࠋࡇࡢࡼ࠺࡞࣏࣮ࣞࢱ࣮࣭࢔ࢵࢭ࢖ࡣࠊᑟධࡢ୍㐣ᛶᆺ㸭Ᏻᐃᆺࢆၥࢃࡎࠊ๓㏙ࡢ BG1Luc ⣽⬊ࢆጞࡵࠊ㓝ẕࠊࢮࣈࣛࣇ࢕ࢵࢩࣗ⫢⣽⬊ᰴࠊHepG2ࠊ
CV-1 ࡞࡝✀ࠎࡢ⣽⬊᳨࡛ウࡉࢀ࡚࠸ࡿࠋ
ୖ㏙ࡢヨ㦂ࡢ࠺ࡕࠊBG1Luc ⣽⬊࡛ࣝࢩࣇ࢙࣮ࣛࢮࢆ࣏࣮ࣞࢱ࣮࡜ࡋࡓ ER άᛶ໬࢔ࢵࢭ
࢖ࡣࠊBG1Luc ER TA ἲ࡜ࡋ࡚ OECD TG ࡟ࡶ཰㍕ࡉࢀ࡚࠸ࡿࡀࠊࡇࢀ࡟ᑐࡍࡿ ER STTA ἲࡢඃ఩ᛶ࡟ࡘ࠸࡚ࡣࠊ1 ࡢ“ᮏヨ㦂ἲࡢ⛉Ꮫⓗጇᙜᛶ࡜つไヨ㦂ἲ࡜ࡋ࡚ࡢጇᙜᛶ”࡟グࡉࢀ࡚࠸ࡿࠋ௚ࡢヨ㦂ἲ࡟ࡘ࠸࡚ࡣࠊࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡣ⾜ࢃࢀ࡚࠸࡞࠸ࠋ㸯㸮㸬⤖ㄽER STTA ἲࡣࠊ໬Ꮫ≀㉁ࡢ in vitro ࡛ࡢ࢔ࢦࢽࢫࢺάᛶ࠾ࡼࡧ࢔ࣥࢱࢦࢽࢫࢺάᛶࢆ᳨ฟ
ࡍࡿࢫࢡ࣮ࣜࢽࣥࢢヨ㦂ἲ࡛࠶ࡾࠊࡑࡢ᳨ᰝᛶ⬟ࡢ᭷⏝ᛶࢆ☜ㄆࡍࡿࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡣ࢔ࢦࢽࢫࢺ࡟ࡘ࠸࡚ࡣ 4 ヨ㦂᪋タࡢ
ࡣཧຍ 6 ヨ㦂᪋タࡢ࠺ࡕ 4 ᪋タࡢᐃ⤖ᯝࢆࡶ࡜࡟ࠊࡲࡓࠊ࢔ࣥࢱࢦࢽࢫࢺ࡟ࡘ࠸࡚

ᐃ⤖ᯝࢆࡶ࡜࡟ࡑࡢ෌⌧ᛶ࣭ಙ㢗ᛶࡀ☜ㄆࡉࢀ࡚࠸ࡿࠋ

ᮏヨ㦂ἲࡣࠊ໬Ꮫ≀㉁ࡢ ER ࡟ᑐࡍࡿ⤖ྜᛶࢆぢࡿヨ㦂ἲ࡜ࡣ␗࡞ࡾࠊࡑࡢ⤖ྜࡢ⤖ᯝ࡟ࡼࡿ RNA ㌿෗άᛶ໬ࡲ᳨࡛ฟࡍࡿࡇ࡜࡟ࡼࡾ࢔ࢦࢽࢫࢺ࡜࢔ࣥࢱࢦࢽࢫࢺࡢάᛶࢆศࡅ᳨࡚
ᰝࡍࡿࡇ࡜ࡀฟ᮶ࡿࠋࡑࡢヨ㦂ἲ࡜ࡋ࡚ࡢ⛉Ꮫⓗጇᙜᛶ࡜つไヨ㦂ἲ࡜ࡋ࡚ࡢጇᙜᛶ࡟ࡘ
࠸࡚ࡣࠊICCVAM ࡢ㑅ᐃࡋࡓཧ↷≀㉁ࠊCERI ࡟࠾ࡅࡿ⫼ᬒࢹ࣮ࢱࡸᩥ⊩᝟ሗࢆࡶ࡜࡟㑅ᐃࡉࢀࡓ໬Ꮫ≀㉁㸦࢔ࢦࢽࢫࢺヨ㦂࡛ࡣ 86 ≀㉁ࠊ࢔ࣥࢱࢦࢽࢫࢺヨ㦂࡛ࡣ 20 ≀㉁㸧ࢆ⏝࠸࡚ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡀ⾜ࢃࢀࡓࠋࣂࣜࢹ࣮ࢩࣙࣥࡢ⤖ᯝࢆࡶ࡜࡟ࠊOECD ࡢ➨୕⪅ホ౯࡛ࡣࡇࡢヨ㦂ἲࡢṇ☜ᛶ࡜ಙ㢗ᛶࡀ㧗࠸࡜ホ౯ࡉࢀ࡚࠾ࡾࠊOECD-EDTA ࡛ᥦ᱌ࡉࢀࡓ໬Ꮫ≀㉁ࡢෆศἪ࠿ࡃ஘ᛶホ౯ࡢࢥࣥࢭࣉࢳࣗ࢔ࣝࣇ࣮࣒࣮ࣞ࣡ࢡࡢࣞ࣋ࣝ 2 ࡟ヱᙜࡍࡿヨ㦂ἲ࡜ࡋ࡚ෆศἪ࠿ࡃ஘≀㉁ࡢࢫࢡ࣮ࣜࢽࣥࢢホ౯࡟᭷⏝࡛࠶ࡿ࡜ㄆࡵ࡚࠸ࡿࠋᮏヨ㦂ἲࡣᐃ㔞ⓗホ౯ἲ࡜ࡋ࡚㛤Ⓨࡉࢀࠊ࢔ࢦࢽࢫࢺヨ㦂ἲ࡟ࡘ࠸࡚ࡣࠊࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟࠾࠸࡚ᐃ㔞ⓗホ౯್ࡢಙ㢗ᛶࡀホ౯ࡉࢀࡓࡀࠊ࢔ࣥࢱࢦࢽࢫࢺヨ㦂ἲ࡛ࡣࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟1319࠾࠸࡚ヨ㦂᪋タ㛫࡛ᐃ㔞್ࡢࡤࡽࡘࡁࡀㄆࡵࡽࢀࡓࡇ࡜࠿ࡽᐃᛶⓗホ౯ἲ࡜ࡋ࡚ OECD ࢞࢖ࢻࣛ࢖ࣥ໬ࡉࢀࡓࠋ
ᮏヨ㦂ἲࡢ௒ᚋࡢၥ㢟Ⅼ࡜ࡋ࡚ࠊෆศἪ࠿ࡃ஘ᛶࡀ࢚ࢫࢺࣟࢤࣥ㸦ᢠ࢚ࢫࢺࣟࢤࣥ㸧άᛶ࡟㉳ᅉࡍࡿ࡜࠸࠺⪃࠼࡟࠾࠸࡚ࠊᮏヨ㦂ἲ࡟࠾ࡅࡿ㝧ᛶ཯ᛂࡀᐇ㝿ࡢ⏕యᙳ㡪ࢆ࡝ࡢ⛬ᗘ཯ᫎࡍࡿࡶࡢ࡛࠶ࡿ࠿࡜࠸࠺ㄢ㢟ࡀ࠶ࡆࡽࢀࡿࠋᮏヨ㦂ἲࡣ࠶ࡃࡲ࡛ࡶࢫࢡ࣮ࣜࢽࣥࢢ
ἲ࡛࠶ࡾࠊ᝿ᐃࡉࢀࡿ᭷ᐖᙳ㡪ࢆ☜ᐃホ౯ࡍࡿヨ㦂ἲ࡜⤌ࡳྜࢃࡏ࡚ホ౯ࢆ⾜࠺ࡇ࡜࡛௒
ᚋࡢ໬Ꮫ≀㉁⟶⌮࡟኱ࡁࡃ㈉⊩ࡍࡿ࡜⪃࠼ࡽࢀࡿࠋཧ⪃ᩥ⊩(1) OECD (2012), Performance standards for stably transfected transactivation in vitro assays todetect estrogen agonists for TG 455, ENV/JM/MONO(2012)18 (01-Aug-2012), Series on Testingand Assessment No. 173.
(2) OECD (2015), Draft updated TG 455 for stably transfected transactivation in vitro assays todetect estrogen receptor agonists and antagonists, ENV/JM/TG(2015)27 (02-Mar-2015) for 27th
Meeting of the Working Group of National Co-ordinators of the Test Guidelines Programme(WNT)(3) OECD (2015), Draft updated performance standards for stably transfected transactivation invitro assays to detect estrogen receptor antagonists, ENV/JM/TG(2015)28 (03-Mar-2015) for27th Meeting of the Working Group of National Co-ordinators of the Test Guidelines Programme(WNT)(4) OECD  (2015), Draft report of the inter-laboratory validation for stably transfectedtransactivation assay to detect estrogenic and anti-estrogenic activity, ENV/JM/TG(2015)29(03-Mar-2015) for 27th Meeting of the Working Group of National Co-ordinators of the Test
Guidelines Programme (WNT).

14

20

Table 1 ER-STTA ࢔ࢦࢽࢫࢺࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡛㻌㻌Nᐃࡀ⾜ࢃࢀࡓ 86 ≀㉁ࡢ

ᐃ⤖ᯝ

ER STTA agonist assay

㻌

PC10

PC50

based

based

Chemical

CASRN

PC10 (M)

PC50 (M)

class

class

1

Atrazine

1912-24-9

-

-

N

N

2

Corticosterone

50-22-6

-

-

N

N

3

Haloperidol

52-86-8

-

-

N

N

4

Ketoconazole

65277-42-1

-

-

N

N

5

Linuron330-55-2

-

-

N

N

6

Phenobarbital(Na)57-30-7

-

-

N

N

7

Reserpine

50-55-5

-

-

N

N

8

Spironolactone

52-01-7

-

-

N

N

9

Flutamide

13311-84-7

-

-

N

N

10

Procymidone

32809-16-8

-

-

N

N

P

N

P

N

P

N

P

N

6.44E-10PP<1.00E-11

PP

1.78E-08

2.73E-07

PP

7.24E-11

6.44E-10PP<1.00E-11

P

P

o

11

Ethyl paraben120-47-8

5.00E-06

12p,p'-Methoxychlor

72-43-5

1.23E-06

13

Tamoxifen

10540-29-1

1.49E-07

14

Clomiphene citrate

50-41-9

3.68E-08

15

Zearalenone

17924-92-4

2.44E-11

16

17ȕ-Estradiol

50-28-2

17

Trenbolone

10161-33-8

18

17Į-Estradiol

57-91-0<1.00E-1
1<1.00E-1

PC10(noPC50)
PC10(noPC50)
PC10(noPC50)
PC10(noPC50)19

17Į-Ethinyl estradiol

57-63-6

20

4-Cumylphenol

599-64-4

1.49E-07

1.60E-06

PP

21

4-tert-Octylphenol

140-66-9

1.85E-09

7.37E-08

PP

22

Apigenin520-36-5

1.31E-07

5.71E-07

PP

23

Bisphenol A

80-05-7

2.02E-08

2.94E-07

PP

24

Bisphenol B

77-40-7

2.36E-08

2.11E-07

PP

25

Butylbenzyl phthalate

85-68-7

1.14E-06

4.11E-06

P

P

26

Coumestrol479-13-0

1.23E-09

2.00E-08

PP

27

Daidzein486-66-8

1.76E-08

1.51E-07

PP

2.04E-11PP

1<1.00E-1

28

Diethylstilbestrol

56-53-1

29

Estrone53-16-7

3.02E-11

5.88E-10PP

30

Genistein446-72-0

2.24E-09

2.45E-08

P

P

31

Kaempferol

520-18-3

1.36E-07

1.21E-06

PP

32

Chlordecone (Kepone)

143-50-0

7.11E-07

7.68E-06

PP

33

Methyl testosterone

58-18-4

1.73E-07

4.11E-06

P

P

34

Morin

480-16-0

5.43E-07

4.16E-06

P

P

1

15

21

35

Norethynodrel

68-23-5

1.11E-10

1.50E-09

PP

36

Phenolphthalin

81-90-3

-

-

N

N

37

Progesterone

57-83-0

-

-

N

N

38

Cyproterone acetate427-51-0

-

-

N

N

39

Mifepristone

84371-65-3

-

-

N

N

40

Diethylhexyl phthalate

117-81-7

-

-

N

N

P

N

P

N

41

L-Thyroxine

51-48-9

1.32E-06

42

4-Androstenedione

63-05-8

2.56E-07

43

Testosterone

58-22-0

2.82E-08

9.78E-06

PP

44

Vinclozolin

50471-44-8

1.33E-07

7.65E-06

PP

45

Dibutyl phthalate

84-74-2

46

Nonylphenol

25154-52-3

47

Phenobarbital

50-06-648

Medroxyprogesterone

520-85-4

49

Dihydrotestosterone (DHT)521-18-6(noPC50)
PC10(noPC50)

-

-

N

N

1.37E-08

1.58E-07

P

P

-

-

N

N

-

-

N

N

1.04E-07

5.28E-07

P

P

50

Testosterone propionate

57-85-2

2.03E-09

2.91E-07

PP

51

Fenarimol

60168-88-9

-

-

N

N

52

p,p'-DDE

72-55-9

-

-

N

N

53

Hexestrol

84-16-2

2.75E-11PP

54(2,4,5-Trichlorophenoxy)acetic acid

93-76-5

-

-

N

N

55

para-sec-butylphenol

99-71-8

1.38E-06

PC10(noPC50)

P

N

56

Norgestrel

797-63-7

1.05E-07

PC10(noPC50)

P

N

57

Norethindrone

68-22-4

1.01E-09

4.95E-08

PP

7.54E-11

P

P

P

N

P

N

<1.00E-1
1<1.00E-1

58

Equilin474-86-2

59

Dicyclohexylphthalate

84-61-7

2.53E-06

60

Diethyl phthalate

84-66-2

4.46E-06

61

di(2-ethylhexyl)adipate

103-23-1

-

-

N

N

62

p-dodecyl-phenol

104-43-8

2.36E-08

4.10E-07PP

63

4-n-octylphenol

1806-26-4

1.26E-06

PN

64

4-n-amylphenol

14938-35-3

1.78E-07

4.62E-06

PP

65

Testosterone enanthate

315-37-7

1.71E-08

2.71E-07PP

66

p-(tert-pentyl)phenol

80-46-6

4.02E-07

3.46E-06

PP

67

4-cyclohexylphenol

1131-60-8

6.43E-08

1.51E-06

PP

68

4-(1-adamantyl)phenol

29799-07-3

1.25E-09

1.86E-08PP

69

4-(phenylmethyl)phenol

101-53-1

1.20E-06

4.07E-06

PP

70

2,2-bis(4-hydroxyphenyl)-4-methyl-n-pentane

6807-17-6

1.89E-09

1.99E-08PP

71

4,4'-(Hexafluoroisopropylidene)diphenol

1478-61-1

6.91E-09

8.02E-08

PP

72

4,4'-(octahydro-4,7-methano-5H-inden-5-lidene)

1943-97-1

3.72E-08

PC10

P

N

16

22

PC10

1

PC10
(noPC50)
PC10(noPC50)

PC10(noPC50)

bisphenol(noPC50)73

4,4'-dimethoxytriphenylmethane

7500-76-7

-

-

N

N

74

Benzophenone

119-61-9

-

-

N

N

75

4-hydroxybenzophenone

1137-42-4

1.10E-06

2.60E-06

PP

76

4,4'-dihydroxybenzophenone

611-99-4

1.24E-07

1.65E-06

PP

77

2,4,4'-trihydroxybenzophenone

1470-79-7

4.38E-08

3.75E-07PP

78

4,4'-dimethoxybenzophenone

90-96-0

2.50E-06

PN

79

2,2',4,4'-tetrahydroxybenzophenone

131-55-5

1.06E-07

3.28E-07PP

80

4-hydroxyazobenzene

1689-82-3

1.64E-07

1.08E-06

PP

77-08-7

1.43E-07

3.16E-06

PP

2.14E-07

P

P

P

N

N

N

81

3,3,3',3'-tetramethyl-1,1'-spirobisindane-5,5',6,6'tetrol

82

4,4'-thiobis-phenol

2664-63-3

2.01E-08

83

Diphenyl-p-phenylenediamine

74-31-7

2.30E-06

84

Octachlorostyrene

29082-74-4-

PC10(noPC50)

PC10(noPC50)-

85

Hematoxylin517-28-2

-

-

N

N

86

Tributyltin chloride

1461-22-9

-

-

N

N

Abbreviations: CASRN = Chemical Abstracts Service Registry Number, PC10 (PC50) = the concentration of a test
chemical at which the response is 10% (or 50% for PC50) of the response induced by the positive control (E2, 1nM)
in each plate, P = Positive, N = Negative ͤ ཧ⪃ᩥ⊩(1)ࡢ ANNEX2, Table 2 ࢆࡶ࡜࡟సᡂ

17

23

Table 2 ER-STTA ࢔ࣥࢱࢦࢽࢫࢺࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡛㻌 No.

Chemical117ȕ-estradiol

24,4'-(Hexafluoroisopropylidene)diphenol

3ᐃࡀ⾜ࢃࢀࡓ 21 ≀㉁ࡢ

CASRN

4,4'-[1-[4-[1-(4-Hydroxyphenyl)-1-methylethyl]phenyl]ethylidene]bis[phenol]ᐃ⤖ᯝ

ER STTA 1

ER STTA 1

antagonist

Mean

Classification

IC50(M)

50-28-2

NEG-

1478-61-1

NEG-

110726-28-8

POS

2.51 × 10-6

843-55-0NEG-68047-06-3

POS

3.97 × 10-9

44,4'-Cyclohexylidenebisphenol

54-Hydroxytamoxifen

6

Apigenin520-36-5

NEG

-

7

Atrazine

1912-24-9

NEG

-

8

Clomiphene citrate (cis and trans mixture)

50-41-9

POS

4.26 × 10-7

9Dibenzo[a,h]anthracene

53-70-3POS

No IC50

10

Dibutyl phthalate

84-74-2

NEG

-

11

Fenarimol

60168-88-9

NEG

-

12

Flavone525-82-6

NEG

-

13

Flutamide

13311-84-7

NEG

-

14

Genistein446-72-0

NEG

-

15

ICI 182,780

129453-61-8

POS

2.67 × 10-10

16

Methylpiperidinylpyrazole dihydrochloride

289726-02-9

POS

3.11 × 10-8

17

Mifepristone (Mifeprex)䠙RU-486

84371-65-3

POS

5.61 × 10-618

p-n-nonylphenol

104-40-5

NEG

-

19

Raloxifene HCl

82640-04-8

POS

7.86 × 10-10

20

Resveratrol

501-36-0

NEG

-

21

Tamoxifen

10540-29-1

POS

4.91 × 10-7

Abbreviations: CASRN = Chemical Abstracts Service Registry Number; M = molar; IC50 = half maximal
inhibitory concentration of test chemical; NEG = negative; POS = positive.
1

The Validation Report of the Stably transfected Transcriptional Activation Assay to Detect ER

mediated activity, Part B (9) ͤ ཧ⪃ᩥ⊩(3)ࡢ ANNEX2, Table 2 ࢆᘬ⏝

18

24

Table 3 ࢔ࢦࢽࢫࢺࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟࠾࠸࡚᪋タ㛫ẚ㍑࡟⏝࠸ࡽࢀࡓ 10 ≀㉁
Chemical

CAS No.

Manufacturer

Lot. No.

Purity17ȕ-Estradiol

50-28-2

Wako

ACK575499%

17Į-Estradiol

57-91-0

Wako

ELJ1532

97% ,HPLC ,for Biochem.

Genistein446-72-0

Wako

VIR171198%

4-tert-Octylphenol

140-66-9

Wako

YWE9213

97% ,cGC ,for Environment Anal.

Bisphenol A

80-05-7

TCI

GF01

>99%

p-tert-Pentylphenol

80-46-6

Wako

KSQ2664

97% ,GC

17Į-Methyltestosterone

58-18-4

Wako

TPE6748

97% ,HPLC ,for Biochem.

Hematoxylin517-28-2

Wako

LDK7723N.S.

Diethylhexyl phthalate

117-81-7

Wako

ELE179997% ,GC

Benzophenone

119-61-9

Wako

RLH911499% ,cGC ,for Environment Anal.

TCI : Tokyo Kasei Kogyo Co., Ltd.
Wako : Wako Pure Chemical Industries, Ltd.
N.S. : not specifiedͤ ཧ⪃ᩥ⊩(4), Table 9 ࢆᘬ⏝

1925

Table 4 ࢔ࢦࢽࢫࢺࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟࠾࠸࡚ ICCVAM ཧ↷ศ㢮࡜ࡢẚ㍑࡟⏝࠸ࡽࢀࡓ46 ≀㉁Chemical

CAS No.

Manufacturer

Lot. No.

Purity17Į-Ethinyl estradiol

57-63-6

Wako

KSN3933

98%

Diethylstilbestrol

56-53-1

Wako

7488C

N.S.

17Į-Estradiol

57-91-0

Wako

ELJ1532

97%

17ȕ-Estradiol

50-28-2

Wako

ACL1188>97%

Zearalenone

17924-92-4

Sigma

50K4014

N.S.

Estrone53-16-7

Wako

TPN4558

98%

Methyl testosterone

58-18-4

Wako

TPE6748

97%

Coumestrol479-13-0

Fluka

400248<95%

Genistein446-72-0

Wako

NNP171298%

p- n-Nonylphenol

104-40-5

Wako

N.S.

Bisphenol B

77-40-7

TCI

FIC01

N.S.

Daidzein486-66-8

Wako

HC-1408

97%

4-Cumylphenol

599-64-4

Wako

PAK114498%

Bisphenol A

80-05-7

TCI

GF01

>99%

p,p’ -Methoxychlor

72-43-5Wako

YWL9207>97%

Apigenin520-36-5

Aldrich00902BU

N.S.

Tamoxifen

10540-29-1

ICN

4636C

N.S.

Kepone (Chlordecone)

143-50-0

Wako

Butylbenzyl phthalate

85-68-7

Wako

KSJ840899%

Kaempferol

520-18-3

Wako

ELK6128

95%

99%

4-tert- Octylphenol

140-66-9

Wako

YWE9213

97%

Atrazine

1912-24-9

Wako

MSF9593>98%

Progesterone

57-83-0

Sigma

98H0893

99%

Testosterone

58-22-0

Wako

ACG5233>97%

Corticosterone

50-22-6

Sigma

128H0744

95%

Phenobarbital

57-30-7

Wako

ACE1373

98%

Vinclozolin

50471-44-8

Wako

HCQ9724

99%

Cyproterone acetate427-51-0

Sigma

65H0687

N.S.

Flutamide

13311-84-7

Sigma

87H151198%

Linuron330-55-2

Dr.Ehrensofter

70226

100%

Mifepristone

84371-65-3

Sigma

19H082898%

Procymidone

32809-16-8

Wako

HCH9638

99%

Clomiphene citrate

50-41-9

Sigma

28

0308

N.S.

Ethyl paraben120-47-8

Wako

ELH6061

99%

Norethynodrel

68-23-5

Sigma

88F0192

N.S.

4-Androstenedione

63-05-8

Sigma

116H0463

98%

2-sec- Butylphenol

89-72-5

Wako

KSR1873

98%

Diethylhexyl phthalate

117-81-7

Wako

ELE1799

97%

Morin

480-16-0

Fluka404144

N.S.

Phenolphthalin

81-90-3

Wako

ELP7131

N.S.

Haloperidol

52-86-8

ICN

85689

90%

Ketoconazole

65277-42-1

Wako

78353

N.S.

20

26

Reserpine

50-55-5

RBI

SNV-494A

97.5%

Spironolactone

52-01-7

Sigma

41K1534

97%

L-Thyroxine

51-48-9

TCI

GF01

98%

17ȕ-Trenbolone

10161-33-8

Sigma aldrich

024K0877>98%

Aldrich : Aldrich Chemical Co., Inc. (Sigma-Aldrich corp.)
Fluka : Fluka Chemie AG (Sigma-Aldrich corp.) ICN : ICN Biomedicals, Inc.
Kanto : Kanto Chemical Co,. Inc.
Sigma : Sigma Chemical Co. (Sigma-Aldrich corp.) RBI : SIGMA-RBITCI : Tokyo Kasei

Kogyo Co., Ltd.

Wako : Wako Pure Chemical Industries, Ltd.
N.S. : not specifiedͤ ཧ⪃ᩥ⊩(4), Table 6 ࢆᘬ⏝

21

27

Table 5 ࢔ࢦࢽࢫࢺࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟࠾࠸࡚ ER ⤖ྜヨ㦂⤖ᯝ࡜ࡢẚ㍑࡟⏝࠸ࡽࢀࡓ 48≀㉁
Chemical

CAS No.

Manufacturer

Lot. No.

PurityEthynyl estradiol

57-63-6

Wako

KSN393398%

17ȕ-Estradiol

50-28-2

Wako

ACL1188>97%

Hexestrol

84-16-2

Wako

LDQ2218

N.S.

Estrone53-16-7

Wako

TPN455898%

17Į-Estradiol

57-91-0

Wako

ELJ1532

97%

Norethynodrel

68-23-5

Sigma

88F0192

N.S.

Coumestrol479-13-0

Fluka

400248<95%

Genistein446-72-0

Wako

NNP171298%

4-tert- Octylphenol

140-66-9

Wako

YWE921397%

Daidzein486-66-8

Wako

HC-1408

97%

Nonylphenol (mixture)25154-52-3

Aldrich00504CU

N.S.

Bisphenol B

77-40-7

TCI

FIC01

N.S.

Testosterone propionate

57-85-2

Sigma

98H0566

N.S.

Bisphenol A

80-05-7

TCI

GF01

>99%

5D-Dihydrotestosterone

521-18-6

Wako

TPJ4827

95%

Kaempferol

520-18-3

Wako

ELK6128

95%

4-alpha-Cumylphenol

599-64-4

Wako

PAK114498%

17Į-Methyltestosterone

58-18-4

Wako

TPE6748

97%

Morin

480-16-0

Fluka404144N.S.

Vinclozolin

50471-44-8

Wako

HCQ9724

99%

Testosterone

58-22-0

Wako

ACG5233>97%

Tamoxifen

10540-29-1

ICN

4636C

N.S.

Clomiphene citrate

50-41-9

Sigma

28

N.S.

di(2-Ethylhexyl)phthalate

117-81-7

Wako

ELE1799

97%

RU-48684371-65-3

Sigma

19H0828

98%

Methoxychlor

72-43-5Wako

YWL9207>97%

Fenarimol

60168-88-9

Kanto

707S7109

97%

para-sec -butylphenol

99-71-8

TCI

FHF01

>98%

Dibutyl phthalate

84-74-2

Wako

0308

for Anal. of Phthalic
Acid Esters

Phenolphthalin

81-90-3

Wako

ELP7131

N.S.

Cyproterone acetate427-51-0

Sigma

65H0687

N.S.

Ethyl p -Hydroxybenzoate

120-47-8

Wako

ELH6061

99%

2,4,5-Trichlorophenoxyacetic acid

93-76-5

Wako

HCL9884

98.7%

p,p' -DDE

72-55-9

Wako

YWG9700

99%

Ketoconazol

65277-42-1

Wako

78353

N.S.

Androstenedione

63-05-8

Sigma

116H0463

98%

Progesterone

57-83-0

Sigma

98H0893

99%

Haloperidol

52-86-8

ICN

85689

90%

Medroxyprogesterone

520-85-4

Sigma

59H0579

N.S.

Spironolactone

52-01-7

Sigma

41K1534

97%

L-thyroxine

51-48-9

TCI

GF01

98%

22

28

Reserpine

50-55-5

RBI

SNV-494A

97.5%

Corticosterone

50-22-6

Sigma

128H0744

95%

Phenobarbital

50-06-6

Maruishi

8603

N.S.

Linuron = Lorox

330-55-2

Dr.Ehrensofter

70226

100%

Procymidon

32809-16-8

Wako

HCH9638

99%

Atrazine

1912-24-9

Wako

MSF9593>98%

Flutamide

13311-84-7

Sigma

87H151198%

Aldrich : Aldrich Chemical Co., Inc. (Sigma-Aldrich corp.)
Fluka : Fluka Chemie AG (Sigma-Aldrich corp.) ICN : ICN Biomedicals, Inc.
Kanto : Kanto Chemical Co., Inc.
N.S. : not specified
Maruishi : Maruishi Pharmaceutical. Co., Ltd.
Sigma : Sigma Chemical Co. (Sigma-Aldrich corp.) RBI : SIGMA-RBITCI : Tokyo Kasei

Kogyo Co., Ltd.

Wako : Wako Pure Chemical Industries, Ltd.
ͤ ཧ⪃ᩥ⊩(4), Table 7 ࢆᘬ⏝

23

29

Table 6 ࢔ࢦࢽࢫࢺࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟࠾࠸࡚Ꮚᐑ⫧኱ヨ㦂⤖ᯝ࡜ࡢẚ㍑࡟⏝࠸ࡽࢀࡓ 48≀㉁
ChemicalCas No.

Manufacture

Lot No.

PurityEthynyl Estradiol

57-63-6

Wako

KSN3933>97%

Equilin474-86-2

Sigma

97H1529

100%

Estrone53-16-7

Wako

TPN4558

98%

17D-Estradiol

57-91-0

Wako

ACL1188>97%

Zearalenone

17924-92-4

Sigma

50K4014N.S.

4-(1-Adamantyl)phenol

29799-07-3

Aldrich11608MR97%

2,2-bis(4-Hydroxyphenyl)-4-methyl-n-pentane

6807-17-6

Wako

PTM1337

100%

Genistein446-72-0

Wako

NNP171298%

Norethrindrone

68-22-4

Wako

DWM4647

100%

4-tert -Octylphenol

140-66-9

Wako09802JQ

99%

4,4'-(Hexafluoroisopropylidene)diphenol

1478-61-1

Aldrich05328PI

97%

Daidzein486-66-8

Wako

HC-1408

97%

Nonylphenol (mixture)25154-52-3

Kanto

10928197%

Bisphenol B

77-40-7

TCI

FIC01

100%

4,4'-Thiobisphenol

2664-63-3

TCI

JC01

100%

Testosterone enanthate

315-37-7

Wako

KSL4869

100%

Bisphenol A

80-05-7

TCI

GF01

>99%

2,2',4,4'-Tetrahydroxybenzophenone

131-55-5

Wako

ELN6605

98%

2,4,4'-Trihydroxybenzophenone

1470-79-7

Aldrich04417JN

95%

p -Dodecyl-phenol

104-43-8

Kanto

209D2209

N.S.

5D-Dihydrotestosterone

521-18-6

Wako

TPJ4827

95%

4-Hydroxyazobenzene

1689-82-3

Wako

LDM7343

96%

4-Cyclohexylphenol

1131-60-8

TCI

FIJ01

100%

4-D-Cumylphenol

599-64-4

Wako

PAK114498%

4,4'-Dihydroxybenzophenone

611-99-4

Wako

LDR1808

99%

4-Hydroxybenzophenone

1137-42-4

Aldrich04419CO98%

3,3,3',3'-Tetramethyl-1,1'-spirobisindane-5,5',6,6'-tetrol

77-08-7

TCI

GG01

99%

p -(tert- Pentyl)phenol

80-46-6

Wako

ELF1567100%

4-(Phenylmethyl)phenol

101-53-1

TCI

FHG01

100%

17D-Methyltestosterone

58-18-4

Wako

ELG7538

100%

4-n -Amylphenol

14938-35-3

TCI

FIF01

99%

4,4'-(Octahydro-4,7-methano-5H-inden-5-ylidene)

1943-97-1

ACROS

A008394601

100%

Levonorogestrel

797-63-7

Sigma

30K0711

99%

Methoxychlor

72-43-5Wako

YWL9207>97%

bisphenol

4-n -Octylphenol

1806-26-4

Wako

JSL994499%

Diphenyl-p -Phenylenediamine

74-31-7

Wako

ELH7269

97%

4,4'-Dimethoxybenzophenone

90-96-0

TCI

FIH01

100%

Dicyclohexyl phthalate

84-61-7

Wako

RIG9061

100%

Diethyl phthalate

84-66-2

Wako

ELH6895

99%

di-n -Butyl phthalate

84-74-2

Wako

ACE7193N.S.

di(2-Ethylhexyl)adipate

103-23-1

Wako

LDR4958

100%

24

30

p-n -Nonylphenol

104-40-5

TCI

10425

99%

di(2-Ethylhexyl)phthalate

117-81-7

Wako

ELH6895

99%

Benzophenone

119-61-9

Wako

HCM9879

100%

Tributyltin chloride

1461-22-9

Wako

LDN550898%

Octachlorostyrene

29082-74-4

Kanto

106121100%

Hematoxylin517-28-2

Sigma

99H3645N.S.

4,4'-Dimethoxytriphenylmethane

7500-76-7

ERC

1040701100%

Aldrich : Aldrich Chemical Co., Inc. (Sigma-Aldrich corp.)
Fluka : Fluka Chemie AG (Sigma-Aldrich corp.) ICN : ICN Biomedicals, Inc.
Kanto : Kanto Chemical Co,. Inc.
N.S. : not specified
Sigma : Sigma Chemical Co. (Sigma-Aldrich corp.)
RBI : SIGMA-RBITCI : Tokyo Kasei Kogyo Co., Ltd.
Wako : Wako Pure Chemical Industries, Ltd.
ͤ ཧ⪃ᩥ⊩(4), Table 8 ࢆᘬ⏝

25

31

Table 7 ࢔ࣥࢱࢦࢽࢫࢺࣂࣜࢹ࣮ࢩࣙࣥヨ㦂 Task3 ࡟࠾࠸࡚᪋タෆ෌⌧ᛶࠊ᪋タ㛫෌⌧ᛶࡢホ౯ࡢ⏝࠸ࡽࢀࡓ 20 ≀㉁
CodeChemical name

CASRN

Manufacturer

Catalog ID.

Lot No.

ATG001

ICI 182,780

129453-61-8

Sigma

I4409

068K4711

ATG002

Mifepristone (Mifeprex, RU-486)

84371-65-3

Sigma

M8046

125K1071

4,4'-(Hexafluoroisopropylidene)

1478-61-1

TCI

B0945

MMSFBTQ

289726-02-9

Sigma-Aldrich

M7068

076K47051

ATG003diphenol
ATG004

Methylpiperdinylpyrazole
dihydrochloride

ATG0054-Hydroxytamoxifen

68047-06-3

Sigma

H7904

018K4132

ATG006

Raloxifene HCl

82640-04-8

Wako (LKT

513-22901(R023923004

Labs, Inc)

243)

ATG007

Clomiphene citrate(cis and trans50-41-9

Sigma

C6272

126K1525

mixture)
ATG008

Dibutyl phthalate

84-74-2

Wako

047-16521

ALQ8564

ATG009Atrazine

1912-24-9

Wako

010-15631

TSG9681ATG010Flutamide

13311-84-7

Wako

069-04851

WKR0694

㻌

㻌

㻌

Wako

069-04851

PEF2340

ATG0114,4'-Cyclohexylidenebisphenol

843-55-0

Wako

028-11071

DPR2259

ATG0124,4'-[1-[4-[1-(4-Hydroxyphenyl)-1-

110726-28-8

Wako

322-28032

EWH0280

methylethyl]phenyl]ethylidene]bis[phenol]
ATG013

Apigenin520-36-5

Wako

012-18913

PEK4270

ATG014

Genistein446-72-0

Sigma

G6649

018K1203

㻌

㻌

㻌

Sigma

G6649

098K0735

ATG015Dibenzo[a,h]anthracene

53-70-3

Wako

041-26791

HSQ8748

㻌

㻌

㻌

Wako

041-26791

EPQ4053

ATG016p-n-nonylphenol

104-40-5

Wako

146-06791

DPF8978

ATG017

Flavone525-82-6

Wako

061-02231

PEM1887

ATG018Resveratrol

501-36-0

Sigma

R5010

038K5202

ATG019Fenarimol

60168-88-9

SupelcoPS1073393-118B

㻌㻌㻌SupelcoPS1073404-67B

ATG02017ȕ-estradiol

50-28-2

Wako

056-04044

ALL2384

㻌

㻌

㻌

Wako

056-04044

ALH5038

Tamoxifen*

10540-29-1

Sigma

T5648

117K1079

Sigma

T5648

089K1381* Tamoxifen ࡣࠊཧ↷໬ྜ≀࡜ࡋ࡚ࡢࡳᐃࡉࢀࡓͤ ཧ⪃ᩥ⊩(4), Part B, Table 2-1,2-2 ࢆࡶ࡜࡟సᡂ

26

32

Table 8 ࢔ࣥࢱࢦࢽࢫࢺࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟࠾ࡅࡿ᪋タෆ෌⌧ᛶActivity per TestCERI

OTSUKAKANEKAHIYOSHI

Agreement
WithinLaboratory
Positive
candidate
Negative
candidateUnknown
DiscordanceWithinLaboratory
Positive
candidate(Results*)
Negative
candidate(Results)
Unknown(Results)

19/20
(95%)

19/19
(100%)

8/12
(67%)

18/20
(90%)

8/9

9/9

3/5

8/9

9/9

8/9

6/7

8/9

2/2
1/20
(5%)

1/1
0/19
(0%)

0/1
4/12
(33%)

2/2
2/20
(10%)

1/9
(NNP)*

0/9

1/9
(NPP)

0/9

1/9
(PNN)

2/5
(PPPPNP)(PPPNP)
1/6(NNNPN)

0/2

0/1

1/1
(PNNNNN)

0/2

1/9
(NPP)

*: Details of discordant results, (NNP) means two negatives and one positive.
ͤ ཧ⪃ᩥ⊩(3), ANNEX2, Table 5 ࢆᘬ⏝

27

33

Table 9 ࢔ࣥࢱࢦࢽࢫࢺࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟࠾ࡅࡿ᪋タ㛫෌⌧ᛶ࠾ࡼࡧᩥ⊩᝟ሗ➼࠿ࡽண᝿ࡉࢀࡓస⏝࡜ࡢẚ㍑
Code
ATG001
ATG002
ATG003
ATG004

Chemical name

Candidate effect

CERI

OTSUKAKANEKA HIYOSHI

strongPositive

Positive

Positive

Positive

Positive (4/4)

mild

Positive

Positive

Positive

Positive

Positive (4/4)

Negative

Negative

Negative

Negative

Negative (4/4)

mild

Positive

Positive㻌Positive

Positive (3/3)

ICI 182,780
Mifepristone(Mifeprex) 䠙RU-4864,4'-(Hexafluoroisopropylid

Negative in ERene)diphenol

STTA assay

Methylpiperdinylpyrazole
dihydrochloride

Total

ATG0054-Hydroxytamoxifen

moderate

Positive

Positive

Positive

Positive

Positive (4/4)

ATG006

Raloxifene HClmoderate

Positive

Positive㻌Positive

Positive (3/3)

moderate

Positive

Positive

Positive

Positive

Positive (4/4)

ATG007

Clomiphene citrate(cis andtrans mixture)

ATG008

Dibutyl phthalate

Negative

Negative

Negative

Negative

Negative

Negative (4/4)

ATG009Atrazine

Negative

Negative

Negative㻌Positive

Negative (2/3)

ATG010Flutamide

Negative

Negative

Negative

Negative

Negative

Negative (4/4)

Negative

Negative

Negative

Negative

Negative (4/4)

mild

Positive

Positive㻌Positive

Positive (3/3)

ATG0114,4'-Cyclohexylidenebisphe

Negative in ER

nol

STTA assay

4,4'-[1-[4-[1-(4-Hydroxyphe
ATG012nyl)-1-methylethyl]phenyl]ethylidene]bis[phenol]

ATG013

Apigenin

Negative

Negative

Negative㻌

Negative

Negative (3/3)

ATG014

Genistein

Negative*

Negative

Negative

Negative

Negative

Negative (4/4)

ATG015Dibenzo[a,h]anthracene

Positive*

Negative

Positive㻌Positive

Positive (2/3)

ATG016p-n-nonylphenol

not tested

Negative

Negative

Negative

Negative

Negative (4/4)

ATG017

FlavoneNegative*

Negative

Negative㻌

Negative

Negative (3/3)

ATG018Resveratrol

Negative*

Negative

Negative

Negative

Negative

Negative (4/4)

ATG019Fenarimol

not tested

Negative㻌㻌Negative

Negative (2/2)

ATG02017ȕ-estradiol

Negative*

Negative

Negative

Negative

Negative

Negative (4/4)

Accuracy:

94%

100%

100%

94%

100% (97%)**

Sensitivity:

88%

100%

100%

100%

100% (97%)**

Specificity :

100%

100%

100%

88%

100% (97%)**

2x2

table

analysiscandidate effects

comparedwith* classified positive/negative according to literature review**Performance compared with candidate effect excluding chemicals noted as "not tested"(n=18),
parentheses indicate the value based on the total number of tests.
ͤ ཧ⪃ᩥ⊩(4), Part B, Table19 ࢆᘬ⏝

28

34

Table 10 ࢔ࢦࢽࢫࢺࣂࣜࢹ࣮ࢩࣙࣥヨ㦂⤖ᯝ࡜ ICCVAM ཧ↷ศ㢮࡜ࡢẚ㍑⤖ᯝ
A: ಶูヨ㦂ࢹ࣮ࢱUpdatedClassificationsof Chemical

ER STTA agonist assay

No

Chemical1 Atrazine
2 Corticosterone
3 Haloperidol
4 Ketoconazole
5 Linuron
6 Phenobarbital(Na)7 Reserpine
8 Spironolactone
9 Flutamide
10 Procymidone
11 Ethyl_paraben
12 p,p'-M ethoxychlor
13 Tamoxifen
14 Clomiphene citrate
15 Zearalenone b
16 17ȕ-Estradiol
17 17ȕ-Trenbolone
18 17Į-Estradiol
19 17Į-Ethinyl estradiol
20 4-Cumylphenol
21 4-tert-Octylphenol
22 Apigenin
23 BisphenolA
24 BisphenolB
25 Butylbenzyl phthalate
26 Coumestrol
27 Daidzein
28 Diethylstilbestrol
29 Estrone
30 Genistein
31 Kaempferol
32 Kepone(Chlordecone)
33 M ethyl testosterone
34 M orin
35 Norethynodrel

CASRN

PC10 (M )PC50 (M )1912-24-9
50-22-6
52-86-865277-42-1
330-55-2
57-30-750-55-5
52-01-7
13311-84-7
32809-16-8
120-47-8
72-43-5
10540-29-1
50-41-917924-92-4
50-28-2
10161-33-8
57-91-0
57-63-6
599-64-4
140-66-9
520-36-5
80-05-7
77-40-7
85-68-7
479-13-0
486-66-8
56-53-1
53-16-7
446-72-0
520-18-3
143-50-0
58-18-4
480-16-0
68-23-5

5.00E-06
1.23E-06
1.49E-07
3.68E-08
2.44E-11<1.00E-11
1.78E-08
7.24E-11<1.00E-11
1.49E-07
1.85E-09
1.31E-07
2.02E-08
2.36E-08
1.14E-06
1.23E-09
1.76E-08<1.00E-11
3.02E-11
2.24E-09
1.36E-07
7.11E-07
1.73E-07
5.43E-07
1.11E-10

PC10(noPC50)
PC10(noPC50)
PC10(noPC50)
PC10(noPC50)
6.44E-10<1.00E-11
2.73E-07
6.44E-10<1.00E-11
1.60E-06
7.37E-08
5.71E-07
2.94E-07
2.11E-07
4.11E-06
2.00E-08
1.51E-07
2.04E-11
5.88E-10
2.45E-08
1.21E-06
7.68E-06
4.11E-06
4.16E-06
1.50E-09

PC10
based
class
N
N
N
N
N
N
N
N
N
N
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P

PC50
based
class
N
N
N
N
N
N
N
N
N
N
N
N
N
N
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P

ICCVAM
class
N
N
N
N
N
N
N
N
N
N
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P

Abbreviations: CASRN = Chemical Abstracts Service Registry Number, PC10 (PC50) = the concentration of atest chemical at which the response is 10% (or 50% for PC50) of the response induced by the positive control (E2,1nM) in each plate, P = Positive, N = Negativeͤ ཧ⪃ᩥ⊩(1)ࡢ ANNEX2, Table 2 ࢆࡶ࡜࡟సᡂ

B: 㞟ィ⾲
STTA (PC10 based)

Updatedclassificationof ICCVAM
Chemicals

Overall Accuracy

100%

35/35

Sensitivity

100%

25/25

Positive

Negative

Total

Specificity

100%

10/10

Positive

25

0

25

False positive

0%

0/10

Negative

0

10

10

False negative

0%

0/25

Total

25

10

35

Positive predictivity

100%

25/25

Negative predictivity

100%

10/10

ͤ ཧ⪃ᩥ⊩(1)ࡢ ANNEX2, Table 8 ࢆᘬ⏝

29

35

Table 11 ࢔ࢦࢽࢫࢺࣂࣜࢹ࣮ࢩࣙࣥヨ㦂⤖ᯝ࡜ ER ⤖ྜヨ㦂ࢹ࣮ࢱ࡜ࡢẚ㍑
A: ಶูヨ㦂ࢹ࣮ࢱ
ER STTA agonist assay

No

Chemical1 Atrazine
2 Corticosterone
3 Haloperidol
4 Ketoconazole
5 Linuron
7 Reserpine
8 Spironolactone
9 Flutamide
10 Procymidone
11 Ethyl paraben
12 p,p'-M ethoxychlor
13 Tamoxifen
14 Clomiphene_citrate
16 17ȕ-Estradiol
18 17Į-Estradiol
19 17Į-Ethinyl estradiol
20 4-Cumylphenol
21 4-tert-Octylphenol
23 BisphenolA
24 BisphenolB
26 Coumestrol
27 Daidzein
29 Estrone
30 Genistein
31 Kaempferol
33 M ethyl testosterone
34 M orin
35 Norethynodrel
36 Phenolphthalin
37 Progesterone
38 Cyproterone acetate
39 M ifepristone
40 Diethylhexyl phthalate
41 L-Thyroxine
42 4-Androstenedione
43 Testosterone
44 Vinclozolin
45 Dibutyl phthalate
46 Nonylphenol
47 Phenobarbital
48 M edroxyprogesterone
49 DHT
50 Testosterone propionate
51 Fenarimol
52 p,p'-DDE
53 Hexestrol
54 2,4,5-Trichlorophenoxyaceticacid
55 para-sec-butylphenol

CASRN

PC10 (M )PC50 (M )1912-24-9
50-22-6
52-86-865277-42-1
330-55-2
50-55-5
52-01-7
13311-84-7
32809-16-8
120-47-8
72-43-5
10540-29-1
50-41-950-28-2
57-91-0
57-63-6
599-64-4
140-66-9
80-05-7
77-40-7
479-13-0
486-66-8
53-16-7
446-72-0
520-18-3
58-18-4
480-16-0
68-23-5
81-90-3
57-83-0427-51-0
84371-65-3
117-81-7
51-48-9
63-05-8
58-22-050471-44-8
84-74-225154-52-3
50-06-6
520-85-4
521-18-6
57-85-2
60168-88-9
72-55-9
84-16-2
93-76-5
99-71-8

5.00E-06
1.23E-06
1.49E-07
3.68E-08<1.00E-11
7.24E-11<1.00E-11
1.49E-07
1.85E-09
2.02E-08
2.36E-08
1.23E-09
1.76E-08
3.02E-11
2.24E-09
1.36E-07
1.73E-07
5.43E-07
1.11E-10
1.32E-06
2.56E-07
2.82E-08
1.33E-07
1.37E-08
1.04E-07
2.03E-09<1.00E-11
1.38E-06

PC10(noPC50)
PC10(noPC50)
PC10(noPC50)
PC10(noPC50)<1.00E-11
6.44E-10<1.00E-11
1.60E-06
7.37E-08
2.94E-07
2.11E-07
2.00E-08
1.51E-07
5.88E-10
2.45E-08
1.21E-06
4.11E-06
4.16E-06
1.50E-09
PC10(noPC50)
PC10(noPC50)
9.78E-06
7.65E-06
1.58E-07
5.28E-07
2.91E-07
2.75E-11
PC10(noPC50)

ER Binding Assay
PC10
based
class
N
N
N
N
N
N
N
N
N
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
N
N
N
N
N
P
P
P
P
N
P
N
N
P
P
N
N
P
N
P

PC50
based
class
N
N
N
N
N
N
N
N
N
N
N
N
N
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
N
N
N
N
N
N
N
P
P
N
P
N
N
P
P
N
N
P
N
N

ER-RBA
E2=100%ER-RBA
class

N.B.
N.B.
N.B.
N.B.
N.B.
N.B.
N.B.
N.B.
N.B.
N.B.

N
N
N
N
N
N
N
N
N
N
0.00238 P
47 P
37 P
126 P
80.1 P
142 P
0.107 P
0.124 P
0.195 P
0.593 P
0.264 P
0.18 P
44.2 P
0.12 P
0.029 P
N.D.
N
0.0011 P
0.282 P
N.D.
N
N.B.
N
N.D.
N
0.0594 P
0.071 P
N.B.
N
N.B.
N
N.D.
N
N.B.
N
N.D.
N
0.143 P
N.B.
N
N.B.
N
0.0218 P
N.B.
N
0.00179 P
N.B.
N
37.6 P
N.B.
N
0.00177 P

Abbreviations: CASRN = Chemical Abstracts Service Registry Number, PC10 (PC50) = the concentration of atest chemical at which the response is 10% (or 50% for PC50) of the response induced by the positive control (E2,1nM) in each plate, P = Positive, N = Negativeͤ ཧ⪃ᩥ⊩(1)ࡢ ANNEX2, Table 2 ࢆࡶ࡜࡟సᡂ

30

36

B: 㞟ィ⾲
STTA (PC10 based)
Positive
ER binding
assay

Overall accuracy

79%

38/48

Sensitivity

88%

21/24

Negative

Total

Specificity

71%

17/24

Positive

21

3

24

False positive

29%

7/24

Negative

7

17

24

False negative

13%

3/24

Total

28

20

48

Positive predictivity

75%

21/28

Negative predictivity

85%

17/20

 ͤ ཧ⪃ᩥ⊩(1)ࡢ ANNEX2, Table 9 ࢆᘬ⏝

31

37

Table 12 ࢔ࢦࢽࢫࢺࣂࣜࢹ࣮ࢩࣙࣥヨ㦂⤖ᯝ࡜Ꮚᐑ⫧኱ヨ㦂࡜ࡢẚ㍑
A: ಶูヨ㦂ࢹ࣮ࢱ
Uterotrophic
Assay

ER STTA agonist assay

No

Chemical

CASRN

12 p,p'-M ethoxychlor
72-43-5
15 Zearalenone_b
17924-92-4
18 17Į-Estradiol
57-91-0
19 17Į-Ethinyl_estradiol
57-63-6
20 4-Cumylphenol
599-64-4
21 4-tert-Octylphenol
140-66-9
23 BisphenolA
80-05-7
24 BisphenolB
77-40-7
27 Daidzein
486-66-8
29 Estrone
53-16-730 Genistein446-72-0
33 M ethyl_testosterone
58-18-4
40 Diethylhexyl_phthalate
117-81-7
45 Dibutyl phthalate
84-74-2
46 Nonylphenol
25154-52-3
49 DHT
521-18-6
56 Norgestrel
797-63-7
57 Norethindrone
68-22-4
58 Equilin474-86-2
59 Dicyclohexyl phthalate
84-61-7
60 Diethyl phthalate
84-66-2
61 di(2-ethylhexyl)adipate
103-23-1
62 p-dodecyl-phenol
104-43-8
63 4-n-octylphenol
1806-26-4
64 4-n-amylphenol
14938-35-3
65 Testosterone_enanthate
315-37-7
66 p-(tert-pentyl)phenol
80-46-6
67 4-cyclohexylphenol
1131-60-8
68 4-(1-adamantyl)phenol
29799-07-3
69 4-(phenylmethyl)-phenol
101-53-1
2,2-bis(4-hydroxyphenyl)-4-methyl-n70
6807-17-6
pentane71 4,4'-exafluoroisopropylidene)diphenol 1478-61-1
4,4'-(octahydro-4,7-methano-5H-inden72
1943-97-1
5-lidene)bisphenol
73 4,4'-dimethoxytriphenylmethane
7500-76-7
74 Benzophenone
119-61-9
75 4-hydroxybenzophenone
1137-42-4
76 4,4'-dihydroxybenzophenone
611-99-4
77 2,4,4'-trihydroxybenzophenone
1470-79-7
78 4,4'-dimethoxybenzophenone
90-96-0
79 2,2',4,4'-tetrahydroxybenzophenone
131-55-5
80 4-hydroxyazobenzene
1689-82-3
3,3,3',3'-tetramethyl-1,1'81
77-08-7
spirobisindane-5,5',6,6'-tetrol
82 4,4'-thiobis-phenol
2664-63-3
83 Diphenyl-pphenylenediamine
74-31-7
84 Octachlorostyrene
29082-74-4
85 Hematoxylin517-28-2
86 Tributyltin-chloride
1461-22-9

PC10 (M )PC50 (M )1.23E-06
2.44E-11
7.24E-11<1.00E-11
1.49E-07
1.85E-09
2.02E-08
2.36E-08
1.76E-08
3.02E-11
2.24E-09
1.73E-07
1.37E-08
1.04E-07
1.05E-07
1.01E-09<1.00E-11
2.53E-06
4.46E-06
2.36E-08
1.26E-06
1.78E-07
1.71E-08
4.02E-07
6.43E-08
1.25E-09
1.20E-06

PC10(noPC50)
6.44E-10
6.44E-10<1.00E-11
1.60E-06
7.37E-08
2.94E-07
2.11E-07
1.51E-07
5.88E-10
2.45E-08
4.11E-06
1.58E-07
5.28E-07
PC10(noPC50)
4.95E-08
7.54E-11
PC10(noPC50)
PC10(noPC50)
4.10E-07
PC10(noPC50)
4.62E-06
2.71E-07
3.46E-06
1.51E-06
1.86E-08
4.07E-06

1.89E-09

1.99E-08

PC10
based
class
P
P
P
P
P
P
P
P
P
P
P
P
N
N
P
P
P
P
P
P
P
N
P
P
P
P
P
P
P
P

PC50
based
class
N
P
P
P
P
P
P
P
P
P
P
P
N
N
P
P
N
P
P
N
N
N
P
N
P
P
P
P
P
P

N
P
P
P
P
P
P
P
N
P
P
P
N
N
P
P
P
P
P
N
N
N
P
N
P
P
P
P
P
P

P

P

P

Utero.
Class

6.91E-09

8.02E-08

PP

P

3.72E-08

PC10(noPC50)

P

N

P

1.10E-06
1.24E-07
4.38E-08
2.50E-06
1.06E-07
1.64E-07

2.60E-06
1.65E-06
3.75E-07
PC10(noPC50)
3.28E-07
1.08E-06

N
N
P
P
P
P
P
P

N
N
P
P
P
N
P
P

N
N
P
P
P
N
P
P

1.43E-07

3.16E-06

PP

N

2.01E-08
2.30E-06-

2.14E-07
PC10(noPC50)
-

P
P
N
N
N

P
N
N
N
N

P
P
N
N
N

Abbreviations: CASRN = Chemical Abstracts Service Registry Number, PC10 (PC50) = the concentration of atest chemical at which the response is 10% (or 50% for PC50) of the response induced by the positive control (E2,1nM) in each plate, P = Positive, N = Negativeͤ ཧ⪃ᩥ⊩(1)ࡢ ANNEX2, Table 2 ࢆࡶ࡜࡟సᡂ

32

38

B: 㞟ィ⾲
STTA (PC10 based)

Uterotrophic
assay

Overall accuracy

85%

41/48

Sensitivity

100%

32/32

Positive

Negative

Total

Specificity

56%

9/16

Positive

32

0

32

False positive

44%

7/16

Negative

7916

False negative

0%

0/32

Total

39

9

48

Positive predictivity

82%

32/39

Negative predictivity

100%

9/9

ͤ ཧ⪃ᩥ⊩(1)ࡢ ANNEX2, Table 10 ࢆᘬ⏝

3339