᪂つヨ㦂ἲᥦ᱌᭩             ᖹᡂ 28 ᖺ 12 ᭶ 6 ᪥                                 No. 2016-01

ER STTA ἲ㸦hERȘ-HeLa-9903 ⣽⬊ࢆ⏝࠸ࡓ࢚ࢫࢺࣟࢤࣥཷᐜయᜏᖖⓎ⌧⣔㌿෗άᛶ໬ヨ㦂ἲ㸧࡟㛵ࡍࡿᥦ᱌ ᖹᡂ 28 ᖺ 12 ᭶ 6 ᪥䛻ᮾிࠊᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ࡟࡚㛤ദࡉࢀࡓ᪂つヨ㦂ἲホ౯఍㆟㸦㏻⛠㸸JaCVAM ホ౯఍㆟㸧࡟࠾࠸࡚௨ୗࡢᥦ᱌ࡀ࡞ࡉࢀࡓࠋ  ᥦ᱌ෆᐜ㸸 ER STTA ἲ㸦hERȘ-HeLa-9903 ⣽⬊ࢆ⏝࠸ࡓ࢚ࢫࢺࣟࢤࣥཷᐜయᜏᖖⓎ⌧⣔㌿෗άᛶ໬ヨ㦂ἲ㸧ࡣᇵ㣴⣽⬊ࢆ⏝࠸ࡿ in vitro ヨ㦂ἲ࡛࠶ࡾࠊ໬Ꮫ≀㉁ࡢ࢚ࢫࢺࣟࢤࣥཷ
ᐜయ࡬ࡢస⏝ࡢ᭷↓ࢆホ౯࡛ࡁࠊㄗホ౯ࡀᑡ࡞࠸ࡇ࡜࠿ࡽࠊ㢮ఝヨ㦂ἲ࡛࠶ࡿ BG1Luc
ER TA ἲ࡜ྠࡌ⛬ᗘ࡟ࠊ⾜ᨻୖ฼⏝ࡀྍ⬟࡛࠶ࡿ࡜⪃࠼ࡿࠋࡇࡢᥦ᱌᭩ࡣࠊOrganisation for Economic Co-operation and Development (OECD) Test Guideline(TG) 455 Performance-Based Test Guideline for Stably Transfected Transactivation In Vitro Assays
to Detect Estrogen Receptor Agonists and Antagonists࠾ࡼࡧThe Validation Report of the Stablytransfected Transcriptional Activation Assay to Detect ER mediated activity, Part A (agonist assay),Part B (antagonist assay)ࢆࡶ࡜࡟ࠊෆศἪ࠿ࡃ஘ヨ㦂㈨ᩱ⦅⧩ጤဨ఍࡟ࡼࡾࡲ࡜ࡵࡽࢀࡓᩥ᭩ࢆ⏝࠸࡚ࠊJaCVAMホ౯఍㆟ࡀホ౯࠾ࡼࡧ᳨ウࡋࡓ⤖ᯝࠊࡑࡢ᭷⏝ᛶࡀ☜ㄆࡉࢀࡓࡇ࡜࠿ࡽసᡂࡉࢀ
ࡓࠋ ௨ୖࡢ⌮⏤࡟ࡼࡾࠊ⾜ᨻᙜᒁࡢᏳ඲ᛶホ౯᪉ἲ࡜ࡋ࡚ ER STTA ἲࡢ౑⏝ࢆᥦ᱌ࡍࡿࡶࡢ࡛࠶ࡿࠋ
኱㔝Ὀ㞝                      すᕝ⛅ె
JaCVAM ホ౯఍㆟ ㆟㛗               JaCVAM 㐠Ⴀጤဨ఍ ጤဨ㛗

i

JaCVAM ホ౯఍㆟኱ 㔝 Ὀ 㞝 㸦㐠Ⴀጤဨ఍᥎⸀㸧㸸ᗙ㛗㣤 ሯ ᑦ ᩥ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧஬༑ᔒⰋ᫂㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧▼ ஭ 㞝 ஧ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧ᒾ℩⿱⨾Ꮚ㸦᪥ᮏ〇⸆ᕤᴗ༠఍㸧㔠Ꮚ࿴ᘯ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∾ ᰤ஧㸦᪥ᮏච␿ẘᛶᏛ఍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఍㸧ྜྷᮧ ຌ㸦ᗙ㛗᥎⸀㸧                  ௵ᮇ㸸ᖹᡂ 26 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 28 ᖺ 3 ᭶ 31 ᪥                 JaCVAM ホ౯఍㆟኱ 㔝 Ὀ 㞝 㸦㐠Ⴀጤဨ఍᥎⸀㸧㸸ᗙ㛗㣤 ሯ ᑦ ᩥ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧஬༑ᔒⰋ᫂ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧▼ ஭ 㞝 ஧ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧஭ ୖ ᬛ ᙲ 㸦᪥ᮏච␿ẘᛶᏛ఍㸧௒ ஭ ᩍ Ᏻ 㸦᪥ᮏື≀ᐇ㦂௦᭰ἲᏛ఍㸧ᒾ℩⿱⨾Ꮚ 㸦᪥ᮏ〇⸆ᕤᴗ༠఍㸧⠛ ⏣ ࿴ ಇ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧ᮡᒣ┿⌮Ꮚ 㸦᪥ᮏ໬⢝ရᕤᴗ㐃ྜ఍㸧௰ ஭ ಇ ྖ 㸦᪥ᮏ໬Ꮫᕤᴗ༠఍㸧୰ᮧࡿࡾࡇ 㸦⊂❧⾜ᨻἲே 〇ရホ౯ᢏ⾡ᇶ┙ᶵᵓ㸧す ᕝ ⛅ ె 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧἟ ⃝  ⪽ 㸦᪥ᮏẘᛶᏛ఍㸧᳃ ⏣  ೺ 㸦᪥ᮏ⎔ቃኚ␗ཎᏛ఍㸧ᶓ 㛵 ༤ 㞝 㸦᪥ᮏ⓶⭵࢔ࣞࣝࢠ࣮࣭᥋ゐ⓶⭵⅖Ꮫ఍㸧                ௵ᮇ㸸ᖹᡂ 28 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 30 ᖺ 3 ᭶ 31 ᪥ii

JaCVAM 㐠Ⴀጤဨ఍す ᕝ ⛅ ె 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧㸸ጤဨ㛗ᕝ す  ᚭ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧ᑠᕝஂ⨾Ꮚ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ⑓⌮㒊㸧ຍ ⸨  ⠜ 㸦ᅜ❧ឤᰁ⑕◊✲ᡤ㸧᪥ୗ㒊ဴஓ 㸦ཌ⏕ປാ┬ ་⸆࣭⏕ά⾨⏕ᒁ ་⸆ရᑂᰝ⟶⌮ㄢ ໬Ꮫ≀㉁Ᏻ඲ᑐ⟇ᐊ㸧ᖹ ᯘ ᐜ Ꮚ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ẘᛶ㒊㸧⠛ ⏣ ࿴ ಇ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧㛵 㔝 ♸ Ꮚ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ⸆⌮㒊㸧㧗 ᮌ ⠜ ஓ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ẘᛶ㒊 ື≀⟶⌮ᐊ㸧᮰ 㔝 ṇ ᫂ 㸦ཌ⏕ປാ┬ ་⸆࣭⏕ά⾨⏕ᒁ ་⸆ရᑂᰝ⟶⌮ㄢ ໬Ꮫ≀㉁Ᏻ඲ᑐ⟇ᐊ㸧୰ ᮧ 㧗 ᩄ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧᪥ ⏣  ඘ 㸦ཌ⏕ປാ┬ ་⸆࣭⏕ά⾨⏕ᒁ ་⸆ရᑂᰝ⟶⌮ㄢ ໬Ꮫ≀㉁Ᏻ඲ᑐ⟇ᐊ㸧ᗈ ℩ ᫂ ᙪ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ 
Ᏻ඲ᛶண

ホ౯㒊㸧

ᮏ 㛫 ṇ ඘ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ኚ␗㑇ఏ㒊㸧୕ ⃝  㤾 㸦ཌ⏕ປാ┬ ་⸆࣭⏕ά⾨⏕ᒁ ་⸆ရᑂᰝ⟶⌮ㄢ㸧ᑠ ᓥ  ⫕ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ Ᏻ඲ᛶணホ౯㒊 ➨஧ᐊ㸧㸸஦ົᒁ

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JaCVAM statement on
the Stably transfected Transcriptional Activation Assayto Detect ER mediated activity
At a meeting held on 6 December 2016 at the National Institute of Health Sciences (NIHS) in
Tokyo, Japan, the Japanese Center for the Validation of Alternative Methods (JaCVAM)
Regulatory Acceptance Board unanimously endorsed the following statement:  
Proposal: The Stably transfected Transcriptional Activation Assay to Detect ER mediatedactivity (ER STTA) is an in vitro test method that uses cultured cells to assess theeffect of chemical substances on estrogen receptors. A high reliability with regard
to positive/negative classification means that the ER STTA is considered
equivalent to the BG1 Luc ER antagonist assay as a suitable test method for use ina regulatory context.
This statement was prepared following a review of the Organisation for Economic
Co-operation and Development (OECD) Test Guideline (TG) 455 Performance-Based Test
Guideline for Stably Transfected Transactivation In Vitro Assays to Detect Estrogen Receptor
Agonists and Antagonists as well as of a validation report on the Stably transfectedTranscriptional Activation Assay to Detect ER mediated activity, Part A (agonist assay) andPart B (antagonist assay), together with other materials prepared by the Endocrine Disruption
Testing JaCVAM Editorial Committee, to acknowledge that the results of a review and study
by the JaCVAM Regulatory Acceptance Board have confirmed the usefulness of this assay.
Based on the above, we propose the Stably transfected Transcriptional Activation Assay to
Detect ER mediated activity as a useful means for assessing ocular irritation potency duringsafety assessments by regulatory agencies.

Yasuo Ohno
Chairperson
JaCVAM Regulatory Acceptance Board

Akiyoshi Nishikawa
Chairperson
JaCVAM Steering Committee

6 December 2016v

The JaCVAM Regulatory Acceptance Board was established by the JaCVAM Steering
Committee, and is composed of nominees from the industry and academia.
This statement was endorsed by the following members of the JaCVAM Regulatory
Acceptance Board:
Mr. Yasuo Ohno (nominee by JaCVAM Steering Committee) : Chairperson
Mr. Naofumi Iizuka (Pharmaceuticals and Medical Devices Agency)
Mr. Yoshiaki Ikarashi (National Institute of Health Sciences: NIHS)
Mr. Yuji Ishii (Biological Safety Research Center: BSRC, NIHS)
Ms. Yumiko Iwase (Japan Pharmaceutical Manufacturers Association)
Mr. Kazuhiro Kaneko (Japan Chemical Industry Association)
Mr. Eiji Maki (Japanese Society of Immunotoxicology)
Mr. Takeshi Morita (Japanese Environmental Mutagen Society)
Mr. Akiyoshi Nishikawa (BSRC, NIHS)
Mr. Kazutoshi Shinoda (Pharmaceuticals and Medical Devices Agency)
Ms. Mariko Sugiyama (Japan Cosmetic Industry Association)
Ms. Koko Tanigawa (Japanese Society for Alternatives to Animal Experiments)
Mr. Takashi Yamada (National Institute of Technology and Evaluation)
Mr. Hiroo Yokozeki (Japanese Society for Dermatoallergology and Contact Dermatitis)
Mr. Takemi Yoshida (Japanese Society of Toxicology)
Mr. Isao Yoshimura (nominee by Chairperson)
Term: From 1st April 2014 to 31st March 2016

Mr. Yasuo Ohno (nominee by JaCVAM Steering Committee) : Chairperson
Mr. Naofumi Iizuka (Pharmaceuticals and Medical Devices Agency)
Mr. Yoshiaki Ikarashi (National Institute of Health Sciences: NIHS)
Mr. Noriyasu Imai (Japanese Society for Alternatives to Animal Experiments)
Mr. Tomoaki Inoue (Japanese Society of Immunotoxicology)
Mr. Yuji Ishii (BSRC, NIHS)
Ms. Yumiko Iwase (Japan Pharmaceutical Manufacturers Association)
Mr. Takeshi Morita (Japanese Environmental Mutagen Society)
Mr. Shunji Nakai (Japan Chemical Industry Association)
Ms. Ruriko Nakamura (National Institute of Technology and Evaluation)
Mr. Akiyoshi Nishikawa (BSRC, NIHS)
Mr. Satoshi Numazawa (Japanese Society of Toxicology)
Mr. Kazutoshi Shinoda (Pharmaceuticals and Medical Devices Agency)
Ms. Mariko Sugiyama (Japan Cosmetic Industry Association)
Mr. Hiroo Yokozeki (Japanese Society for Dermatoallergology and Contact Dermatitis)
Term: From 1st April 2016 to 31st March 2018vi

This statement was endorsed by the following members of the JaCVAM steering Committee
after receiving the report from JaCVAM Regulatory Acceptance Board:

Mr. Akiyoshi Nishikawa (BSRC, NIHS): Chairperson
Mr. Toru Kawanishi (NIHS)
Mr. Mitsuru Hida (Ministry of Health, Labour and Welfare)
Ms. Yoko Hirabayashi (Division of Toxicology, BSRC, NIHS)
Mr. Akihiko Hirose (Division of Risk Assessment, BSRC, NIHS)
Mr. Masamitsu Honma (Division of Genetics and Mutagenesis, BSRC, NIHS)
Mr. Atsushi Kato (National Institute of Infectious Diseases)
Mr. Tetsuya Kusakabe (Ministry of Health, Labour and Welfare)
Mr. Kaoru Misawa (Ministry of Health, Labour and Welfare)
Mr. Takatoshi Nakamura (Pharmaceutical & Medical Devices Agency)
Ms. Kumiko Ogawa (Division of Pathology, BSRC, NIHS)
Ms. Yuko Sekino (Division of Pharmacology, BSRC, NIHS)
Mr. Kazutoshi Shinoda (Pharmaceuticals and Medical Devices Agency)
Mr. Atsuya Takagi (Animal Management Section of the Division of Toxicology, BSRC,
NIHS)
Mr. Masaaki Tsukano (Ministry of Health, Labour and Welfare)Mr. Hajime Kojima (Division of Risk Assessment, BSRC, NIHS): Secretary

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ER STTA ἲ㸦hERȘ-HeLa-9903 ⣽⬊ࢆ⏝࠸ࡓ࢚ࢫࢺࣟࢤࣥཷᐜయᜏᖖⓎ⌧⣔㌿෗άᛶ໬ヨ㦂ἲ㸧┠  ḟ
ホ౯఍㆟ሗ࿌᭩ ------------------------------------------------------------------------------- 1
ホ౯ሗ࿌᭩ ------------------------------------------------------------------------------------- 7
OECD GUIDELINE FOR THE TESTING OF CHEMICALS 455-------------41ix

xホ౯఍㆟ሗ࿌᭩ER STTA ἲ㸦hERȘ-HeLa-9903 ⣽⬊ࢆ⏝࠸ࡓ࢚ࢫࢺࣟࢤࣥཷᐜయᜏᖖⓎ⌧⣔㌿෗άᛶ໬ヨ㦂ἲ㸧

JaCVAM ホ౯఍㆟

ᖹᡂ 28 ᖺ㸦2016 ᖺ㸧 12 ᭶ 6 ᪥

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JaCVAM ホ౯఍㆟኱ 㔝 Ὀ 㞝 㸦㐠Ⴀጤဨ఍᥎⸀㸧㸸ᗙ㛗㣤 ሯ ᑦ ᩥ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧஬༑ᔒⰋ᫂㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧▼ ஭ 㞝 ஧ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧ᒾ℩⿱⨾Ꮚ㸦᪥ᮏ〇⸆ᕤᴗ༠఍㸧㔠Ꮚ࿴ᘯ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∾ ᰤ஧㸦᪥ᮏච␿ẘᛶᏛ఍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఍㸧ྜྷᮧ ຌ㸦ᗙ㛗᥎⸀㸧                ௵ᮇ㸸ᖹᡂ 26 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 28 ᖺ 3 ᭶ 31 ᪥኱ 㔝 Ὀ 㞝 㸦㐠Ⴀጤဨ఍᥎⸀㸧㸸ᗙ㛗㣤 ሯ ᑦ ᩥ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧஬༑ᔒⰋ᫂ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧▼ ஭ 㞝 ஧ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧஭ ୖ ᬛ ᙲ 㸦᪥ᮏච␿ẘᛶᏛ఍㸧௒ ஭ ᩍ Ᏻ 㸦᪥ᮏື≀ᐇ㦂௦᭰ἲᏛ఍㸧ᒾ℩⿱⨾Ꮚ 㸦᪥ᮏ〇⸆ᕤᴗ༠఍㸧⠛ ⏣ ࿴ ಇ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧ᮡᒣ┿⌮Ꮚ 㸦᪥ᮏ໬⢝ရᕤᴗ㐃ྜ఍㸧௰ ஭ ಇ ྖ 㸦᪥ᮏ໬Ꮫᕤᴗ༠఍㸧୰ᮧࡿࡾࡇ 㸦⊂❧⾜ᨻἲே 〇ရホ౯ᢏ⾡ᇶ┙ᶵᵓ㸧す ᕝ ⛅ ె 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧἟ ⃝  ⪽ 㸦᪥ᮏẘᛶᏛ఍㸧᳃ ⏣  ೺ 㸦᪥ᮏ⎔ቃኚ␗ཎᏛ఍㸧ᶓ 㛵 ༤ 㞝 㸦᪥ᮏ⓶⭵࢔ࣞࣝࢠ࣮࣭᥋ゐ⓶⭵⅖Ꮫ఍㸧                ௵ᮇ㸸ᖹᡂ 28 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 30 ᖺ 3 ᭶ 31 ᪥
22hERĮ-HeLa-9903 ⣽⬊ࢆ⏝࠸ࡓ࢚ࢫࢺࣟࢤࣥཷᐜయ㸦Estrogen receptor㸸ER㸧ᜏᖖⓎ⌧⣔㌿෗άᛶ໬ヨ㦂ἲ㸸The Stably Transfected TA assay using the human ERĮ-HeLa-9903 cell line㸦ER
STTA ἲࠊ௨ୗࠊᮏヨ㦂ἲ㸧ࡣࠊ໬Ꮫ≀㉁ࡢ࢚ࢫࢺࣟࢤࣥ㸦࠾ࡼࡧᢠ࢚ࢫࢺࣟࢤࣥ㸧άᛶࢆ
໬ᏛⓎග࡟ࡼࡾ᳨ฟࡍࡿ in vitro ヨ㦂ἲࡢ୍ࡘ࡛ࠊෆศἪ࠿ࡃ஘≀㉁ᑐ⟇ࡢࡓࡵ࡟㛤Ⓨࡉࢀࡓࡶࡢ࡛࠶ࡿࠋᮏヨ㦂ἲࡣ HeLa ⣽⬊࡟ᑟධࡉࢀࡓ ERĮ ࡢάᛶ໬࡟ࡼࡗ࡚㉳ࡇࡿ࣏࣮ࣞࢱ
࣮㑇ఏᏊࡢ㌿෗άᛶࡢኚ໬ࢆ໬ᏛⓎග࡟ࡼࡾᐃ㔞ⓗ࡟ᐃࡍࡿヨ㦂⣔࡛࠶ࡿࠋᮏヨ㦂ἲࡢࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡟ࡘ࠸࡚ࡣࠊERĮ ࢔ࢦࢽࢫࢺ࡜ ERĮ ࢔ࣥࢱࢦࢽࢫࢺ࡟ࡘ
࠸࡚ࠊࡑࢀࡒࢀ⊂❧ࡋࡓ᪋タ㛫ࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀᐇ᪋ࡉࢀࡓࠋ࢔ࢦࢽࢫࢺヨ㦂ࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩ 1㸧ࡣࠊOECD㸦Organisation for Economic Co-operation and Development㸧࡟ 2006ᖺ࡟ᥦฟࡉࢀࠊ࢔ࣥࢱࢦࢽࢫࢺヨ㦂ࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩ 2㸧ࡣࠊ2014 ᖺ࡟ᥦฟࡉࢀࡓࠋ๓
⪅ࡣ 2009 ᖺ࡟ヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥ㸦Test Guideline: TG㸧455 ࡜ࡋ࡚ᢎㄆࡉࢀ 3)ࠊᚋ⪅ࡣ 2015
ᖺ࡟ TG455 ࡢᨵゞ∧࡜ࡋ࡚ TG ࡜࡞ࡗࡓ 4)ࠋJaCVAM㸦Japanese Center for Validation of
Alternative Methods㸧ホ౯఍㆟ࡣࠊෆศἪ࠿ࡃ஘ヨ㦂ἲ㈨ᩱ⦅⧩ጤဨ఍࡟ࡼࡾసᡂࡉࢀࡓࠕER
STTA ἲ㸸hERȘ-HeLa-9903 ⣽⬊ࢆ⏝࠸ࡓ࢚ࢫࢺࣟࢤࣥཷᐜయᜏᖖⓎ⌧⣔㌿෗άᛶ໬ヨ㦂ἲࡢホ౯ሗ࿌᭩ࠖ
㸦ᖹᡂ 28 ᖺ 10 ᭶ 21 ᪥㸧ࢆ⏝࠸࡚ࠊᮏヨ㦂ἲࡢጇᙜᛶ࡟ࡘ࠸᳨࡚ウࡋࡓࠋ㸯㸬ヨ㦂ἲࡢᐃ⩏ྡ⛠㸸 ER STTA ἲ㸦hERȘ-HeLa-9903 ⣽⬊ࢆ⏝࠸ࡓ࢚ࢫࢺࣟࢤࣥཷᐜయᜏᖖⓎ⌧⣔㌿෗άᛶ໬ヨ㦂ἲ㸧௦᭰ࡍࡿᑐ㇟ẘᛶヨ㦂㸸 in vivo ヨ㦂ἲࡢࠕࡆࡗṑ㢮ࢆ⏝࠸ࡿᏊᐑ⫧኱ヨ㦂ࠖ㸦OECD TG440,
2007㸧5)ࠋࢆ௦᭰ࡍࡿヨ㦂ἲ࡛࠶ࡿࠋ㢮ఝࡢヨ㦂ἲ࡜ࡋ࡚ࠊin vitro ヨ㦂ἲࡢࠕࣛࢵࢺᏊᐑ࢚
ࢫࢺࣟࢤࣥཷᐜయ⤖ྜᛶヨ㦂ࠖ㸦OECD TG 493, 2015㸧6)࡜ࠕBG1Luc4E2 ⣽⬊ࢆ⏝࠸ࡿ࢚ࢫࢺ
ࣟࢤࣥཷᐜయ㌿෗άᛶ໬ヨ㦂㸸BG1Luc estrogen receptor transactivation assay㸦BG1Luc ER TA㸧ࠖ㸦OECD TG 457, 2012㸧7)ࡀ࠶ࡿࠋ
ヨ㦂ἲࡢᴫ␎㸸 ⏕యࡢ ER ࡟⤖ྜࡋࠊ࢔ࢦࢽࢫࢺస⏝ࠊ࠶ࡿ࠸ࡣ࢔ࣥࢱࢦࢽࢫࢺస⏝ࢆ♧ࡍ໬Ꮫ≀㉁ࢆࢫࢡ࣮ࣜࢽࣥࢢࡍࡿࡓࡵ࡟ࠊᮏヨ㦂ἲࡣࠊHeLa ⣽⬊࡟ࣄࢺ ERȘࢆᜏᖖⓗ࡟Ⓨ⌧ࡍࡿࣉࣛࢫ࣑ࢻ࡜࢚ࢫࢺࣟࢤࣥᛂ⟅㓄ิ㸦Estrogen responsive element㸸ERE㸧ࡢୗὶ࡟ࣝ
ࢩࣇ࢙࣮ࣛࢮ㑇ఏᏊࢆ⧅࠸ࡔ࣏࣮ࣞࢱ࣮ࣉࣛࢫ࣑ࢻࢆᑟධ࣭ᏳᐃⓎ⌧ࡉࡏࡓ⣽⬊㸦hERȘHeLa-9903 ⣽⬊㸧ࢆ⏝࠸ࡿࠋࡇࡢ⣽⬊࡟⿕㦂≀㉁ࢆ᭚㟢ࡋࡓᚋࡢࣝࢩࣇ࢙࣮ࣛࢮάᛶࡢኚ໬ࢆᐃࡍࡿࠋ㸰㸬ホ౯࡟⏝࠸ࡓ㈨ᩱ࠾ࡼࡧホ౯ෆᐜࡢ⛉Ꮫⓗጇᙜᛶ࢔ࢦࢽࢫࢺࢆホ౯ࡍࡿヨ㦂ἲ࡟㛵ࡋ࡚ࡣࠊ㸦୍㈈㸧໬Ꮫ≀㉁ホ౯◊✲ᶵᵓࡀ୰ᚰ࡜࡞ࡗ࡚ࠊ
4 ヨ㦂᪋タࡀཧຍࡋࡓࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀᐇ᪋ࡉࢀࡓࠋࡑࡢሗ࿌᭩ࡀ 2006 ᖺ࡟ OECD ࡟
33ᥦฟࡉࢀࠊ2009 ᖺ࡟ TG455 ࡜ࡋ࡚ᢎㄆࡉࢀࡓࠋࡇࡢ TG455 ࡣࠊ2012 ᖺ࡟ BG1Luc ER TA ἲࡢ࢔ࢦࢽࢫࢺヨ㦂ἲࢆໟྵࡍࡿᛶ⬟‽ᣐヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥ㸦Performance-Based Test
Guideline㸸PBTG㸧8)࡟᭦᪂ࡉࢀࠊ࠶ࢃࡏ࡚࢔ࢦࢽࢫࢺヨ㦂ࡢᛶ⬟ᇶ‽㸦Performance Standards㸸
PS㸧ࡀබ㛤ࡉࢀࡓ 9㸧ࠋ࢔ࣥࢱࢦࢽࢫࢺࢆホ౯ࡍࡿヨ㦂ἲ࡟㛵ࡋ࡚ࡣࠊJaCVAM ࡀ୰ᚰ࡜࡞
ࡗ࡚ࠊ᭱⤊ⓗ࡟ 4 ヨ㦂᪋タ࡛ࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀᐇ᪋ࡉࢀࡓࠋ࢔ࣥࢱࢦࢽࢫࢺࣂࣜࢹ࣮ࢩ
ࣙࣥሗ࿌᭩ࡣࠊඛ࡟ᥦฟࡉࢀࡓ࢔ࢦࢽࢫࢺ࡟ࡘ࠸࡚ࡢࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩ࡢ㏣ຍᩥ᭩࡜
ࡋ࡚ᥦฟࡉࢀࠊ᭱⤊ⓗ࡟࢔ࢦࢽࢫࢺホ౯ἲ㸦part A㸧࡜࢔ࣥࢱࢦࢽࢫࢺホ౯ἲ㸦part B㸧ࢆྵࡴ༢୍ࡢࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩࡜ࡋ࡚බ㛤ࡉࢀࡓ 2㸧ࠋࡇࢀࡽࡢࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩࡟ᇶ࡙
ࡁࠊ2014 ᖺ࡟࢔ࣥࢱࢦࢽࢫࢺヨ㦂ࢆໟྵࡍࡿ TG455 ᨵゞ᱌ࡀ OECD ࡟ᥦฟࡉࢀࠊᢎㄆࡉࢀ
ࡓࠋࡇࢀࡽࡢ㈨ᩱࢆ⏝࠸࡚ࠊJaCVAM ෆศἪ࠿ࡃ஘ヨ㦂ἲ㈨ᩱ⦅⧩ጤဨ఍ࡀホ౯ࡋࠊሗ࿌᭩
࡜ࡋ࡚ࡲ࡜ࡵࡓࡶࡢࢆホ౯㈨ᩱ࡜ࡋࡓࠋ
ᮏヨ㦂ἲࡣ HeLa ⣽⬊࡟ࣄࢺ ERȘࢆᑟධࡋࠊᙉไⓎ⌧ࡉࡏࡓ hER Ș-HeLa-9903 ⣽⬊ࢆ⏝࠸ࠊ࢔ࢦࢽࢫࢺ࠾ࡼࡧ࢔ࣥࢱࢦࢽࢫࢺࡢ୧⪅ࢆ᳨ฟ࡛ࡁࡿࡼ࠺࡞ in vitro ヨ㦂ἲ࡟ࡋࡓࡶ
ࡢ࡛࠶ࡿࠋỈࡸ Dimethyl sulfoxide㸦DMSO㸧➼࡟⁐ゎࡍࡿᗈ⠊࡞⿕㦂≀㉁࡟㐺⏝࡛ࡁࡿⅬ࡛౯್ࡀ㧗࠸ࠋ㢮ఝࡢ ER ㌿෗άᛶ໬ヨ㦂ἲ࡛࠶ࡿ BG1Luc ER TA ࡛⏝࠸ࡽࢀࡿ BG1Luc ⣽⬊ࡣࠊERȘ ᆺ㸭ERș ᆺ཮᪉ࢆⓎ⌧ࡋ࡚࠾ࡾࠊ୧⪅ࢆ௓ࡋࡓస⏝ࡀ᳨ฟྍ⬟࡛࠶ࡿ࡜ࡉࢀ
࡚࠸ࡿࠋࡇࢀ࡟ᑐࡋ࡚ࠊᮏヨ㦂ἲ࡛ࡣࠊ⣧⢋࡟ࣄࢺ ERȘࡢࡳࢆ௓ࡋࡓస⏝ࢆ᳨ฟྍ⬟࡛࠶
ࡿࡀࠊ2 ✀ࡢ ER ࢔࢖ࢯࣇ࢛࣮࣒㸦ERȘࠊERș㸧࡟ᑐࡋ࡚᏶඲࡞㑅ᢥᛶࢆ♧ࡍ໬Ꮫ≀㉁ࡣ
▱ࡽࢀ࡚࠾ࡽࡎࠊᐃᛶⓗホ౯࡟࠾࠸࡚୧ヨ㦂ἲࡢ⤖ᯝࡣྠ➼࡜⪃࠼ࡽࢀࠊER ࢆ௓ࡋ࡚స⏝ࡍࡿ໬Ꮫ≀㉁ࢆࢫࢡ࣮ࣜࢽࣥࢢࡍࡿ᪉ἲ࡜ࡋ࡚⛉Ꮫⓗ࡞ጇᙜᛶࡀ࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋ
㸱㸬ᮏヨ㦂ἲࡢ᭷⏝ᛶ࡜㐺⏝㝈⏺
ᮏヨ㦂ἲࡣ༢࡟࢔ࣥࢱࢦࢽࢫࢺ࡜ ER ࡜ࡢ⤖ྜ࡟࡜࡝ࡲࡽࡎ࢔ࢦࢽࢫࢺࡢάᛶࢆᢚไࡍ
ࡿຠᯝࢆ᳨ฟ࡛ࡁࡿⅬࡀඃࢀ࡚࠸ࡿࠋICCVAM㸦Interagency Coordinating Committee on the
Validation of Alternative Methods㸧ཧ↷ศ㢮ࠊER ⤖ྜヨ㦂ࠊᏊᐑ⫧኱ヨ㦂➼ࡢ⤖ᯝ࡜ࡢ୍⮴ᗘࠊᮏヨ㦂ἲࡢឤᗘࠊ≉␗ᗘࡣⰋዲ࡛࠶ࡾࠊഇ㝧ᛶࡸഇ㝜ᛶࡣᑡ࡞࠸࡜࠸࠺⤖ᯝࢆ♧ࡋ࡚࠸ࡿࠋ
BG1Luc ER TA ヨ㦂ἲ࡟ẚࡋឤᗘࡀ㧗ࡃࠊ࢔ࢦࢽࢫࢺࠊ࢔ࣥࢱࢦࢽࢫࢺࢆ 1/100 ࡢ⃰ᗘ᳨࡛ฟ࡛ࡁࡿࠋ
ᮏヨ㦂ἲ࡟ࡼࡿ໬Ꮫ≀㉁ࡢ࢔ࢦࢽࢫࢺάᛶ࣭࢔ࣥࢱࢦࢽࢫࢺάᛶࡢࢫࢡ࣮ࣜࢽࣥࢢࡢ⤖ᯝࢆ ICCVAM ཧ↷ศ㢮࡜ࡢẚ㍑ࡋࡓ⤖ᯝࠊ୍⮴⋡ࡣ 100㸣࡛࠶ࡗࡓࠋ࢚ࢫࢺࣟࢤࣥάᛶࡢ
᳨ฟ࡟⏝࠸ࡽࢀࡿ᪤Ꮡࡢ⣽⬊⣔㸦ෆᅾᛶࡢࣄࢺ ER ࢆ฼⏝ࡍࡿ BG1 ⣽⬊㸧࡞࡝࡜ẚ㍑ࡋ࡚
ࡶࠊ㝧ᛶ≀㉁ࡸ㝜ᛶ≀㉁ࡢ㆑ูᛶࡣⰋዲ࡛࠶ࡿࠋᮏヨ㦂ἲ࡛⏝࠸࡚࠸ࡿ hERȘ-HeLa-9903⣽⬊ࡣቑṪࡀ᪩࠸ࡓࡵࠊヨ㦂‽ഛࡢࡓࡵࡢ᫬㛫ࢆ▷ࡃࡍࡿࡇ࡜ࡀ࡛ࡁࠊከᩘࡢ໬Ꮫ≀㉁ࡢࢫ
ࢡ࣮ࣜࢽࣥࢢ࡟ྥ࠸࡚࠸ࡿࠋ
࣏࣮ࣞࢱ࣮࡜ࡋ࡚⏝࠸࡚࠸ࡿࣝࢩࣇ࢙࣮ࣛࢮάᛶ࡟ᙳ㡪ࢆ୚࠼ࡿ໬Ꮫ≀㉁࡛ࡣࠊER 㠀≉
44␗ⓗ࡞ஹ㐍ࡸ㜼ᐖ➼࡟ࡼࡾഇ㝧ᛶ㸦ࡶࡋࡃࡣഇ㝜ᛶ㸧཯ᛂࢆច㉳ࡍࡿྍ⬟ᛶࡀ࠶ࡿࡓࡵὀពࡀᚲせ࡛࠶ࡿࠋER ࢆ௓ࡋࡓ໬Ꮫ≀㉁ࡢ࢚ࢫࢺࣟࢤࣥάᛶࢆ୍ḟࢫࢡ࣮ࣜࢽࣥࢢࡍࡿ࡟ࡣ౽
฼࡞ヨ㦂⣔࡛࠶ࡿࡀࠊ⣽⬊ࡢ⥅௦࡟ࡼࡗ࡚ ER ࡢ཯ᛂᛶࡀኚ໬ࡋ࡞࠸ࡇ࡜ࢆ☜ㄆࡋ࡚࠾ࡃᚲ
せࡀ࠶ࡿࠋOECD TG ࡛♧ࡉࢀࡓ ERȘ࢔ࢦࢽࢫࢺ࠾ࡼࡧ ERȘ࢔ࣥࢱࢦࢽࢫࢺヨ㦂ࡑࢀࡒࢀࡢ⇍㐩ᗘ☜ㄆ≀㉁ 14 ≀㉁࡜ 10 ≀㉁ࢆ⏝࠸࡚⏝㔞-ᛂ⟅ᛶࢆㄪ࡭࡚࠾ࡃࡇ࡜ࡀᚲせ࡛࠶ࡿࠋࡲࡓࠊ⁐፹࡜ࡋ࡚Ỉࡸ Ethanol (>95%)࠶ࡿ࠸ࡣ DMSO ࡀ⏝࠸ࡽࢀ࡚࠸ࡿࡀࠊDMSO ࡟⁐ࡅ࡟ࡃ࠸≀㉁࡟ࡘ࠸࡚ࡣࠊ௚ࡢ᭷⏝࡞⁐፹ࢆ᳨ウࡋࠊࡑࢀࡀ hERȘ-HeLa-9903 ⣽⬊࡟ᙳ㡪ࡋ࡞࠸ࡇ࡜ࢆ᳨ドࡍࡿᚲせࡀ࠶ࡿࠋ⌧᫬Ⅼ࡛ࡣ᥹Ⓨᛶ≀㉁ࡢྲྀࡾᢅ࠸࡟ࡘ࠸࡚᫂☜࡞ᣦ㔪ࡀ↓࠸ࠋ௒ᚋࡢ᳨ウࡀᮇᚅࡉࢀࡿࠋ
ࡲࡓࠊ௦ㅰࡉࢀ࡚࠿ࡽ ERȘ࢔ࢦࢽࢫࢺ࠾ࡼࡧ ERȘ࢔ࣥࢱࢦࢽࢫࢺస⏝ࢆ♧ࡍ≀㉁ࡢホ౯ࡶ⾜࠼࡞࠸ࠋ
㸲㸬┠ⓗ࡜ࡍࡿ≀㉁ཪࡣ〇ရࡢẘᛶࢆホ౯ࡍࡿヨ㦂ἲ࡜ࡋ࡚ࡢࠊ♫఍ⓗཷࡅධࢀᛶ࠾ࡼࡧ
⾜ᨻୖࡢ฼⏝ࡢྍ⬟ᛶ ♫఍ⓗཷࡅධࢀᛶ㸸ᮏヨ㦂ἲࡣ㑇ఏᏊ⤌ࡳ᥮࠼࡟ࡼࡾస〇ࡉࢀࡓ hERȘ-HeLa-9903 ⣽⬊ࢆ⏝࠸ࡿヨ㦂ἲ࡛࠶ࡾࠊ⏕ࡁࡓື≀ࢆ⏝࠸࡞࠸࡜࠸࠺Ⅼ࡛ࠊ3Rs ࡢ⢭⚄࡟ྜ⮴ࡋ࡚࠸ࡿࠋࡇࡢヨ㦂࡛ᚲせ࡞ᢏ⾡ࡣࠊᇵ㣴⣽⬊ࢆ⏝࠸ࡿヨ㦂ἲ୍⯡ࡢᢏ⾡࠾ࡼࡧ⣽⬊ࡢⓎගࢆᐃࡍࡿᢏ⾡࡛࠶ࡾࠊ㐺ษ࡞カ⦎࡟ࡼࡗ࡚ᐜ᫆࡟⩦ᚓ࡛ࡁࡿࡶࡢ࡛࠶ࡿࠋࡲࡓࠊᮏヨ㦂ࡢࡓࡵ࡟ᚲせ࡞ᶵჾࡣࠊ㏻ᖖࡢ⣽⬊ᇵ㣴࡟せࡍࡿ⿦⨨ࡢ࡯࠿ࠊ⣽⬊Ⓨගࡢᐃ࡟⏝࠸ࡿගᗘィ࡛࠶ࡾࠊ㧗౯࡞ࡶࡢ࡛࡞࠸ࠋ⣽⬊ࡶබⓗ࡞⣽⬊ࣂࣥࢡ࠿ࡽධᡭྍ⬟࡛࠶ࡿࠋ௨ୖࡼࡾࠊᮏヨ㦂ἲࡢ♫఍ⓗཷࡅධࢀᛶࡣ㧗࠸࡜⪃࠼ࡿࠋ ⾜ᨻୖࡢ฼⏝ᛶ㸸 ᮏヨ㦂ἲࡣᇵ㣴⣽⬊ࢆ⏝࠸ࡿ in vitro ヨ㦂ἲ࡛࠶ࡾࠊ໬Ꮫ≀㉁ࡢ࢚ࢫࢺࣟࢤࣥཷᐜయ࡬ࡢ
స⏝ࡢ᭷↓ࢆホ౯࡛ࡁࠊㄗホ౯ࡀᑡ࡞࠸ࡇ࡜࠿ࡽࠊ㢮ఝヨ㦂ἲ࡛࠶ࡿ BG1Luc ER TA ἲ࡜ྠࡌ⛬ᗘ࡟ 10㸧ࠊ⾜ᨻୖ฼⏝ࡀྍ⬟࡛࠶ࡿ࡜⪃࠼ࡿࠋᘬ⏝ᩥ⊩1) Takeyoshi, M. (2006), Draft Report of Pre-validation and Inter-Laboratory Validation for Stably
Transfected Transcriptional Activation (TA) Assay to Detect Estrogenic Activity - The Human
Estrogen Receptor Alpha Mediated Reporter Gene Assay Using hER-HeLa-9903 Cell Line,
Chemicals Evaluation and Research Institute (CERI): Japan. p1-188.
2) OECD (2015), Report of the inter-laboratory validation for stably transfected transactivation assay
to detect estrogenic and anti-estrogenic activity, Series on Testing & Assessment No. 225.
3) OECD (2009), Test No. 455: The Stably Transfected Human Estrogen Receptor-alpha55Transcriptional Activation Assay for Detection of Estrogenic Agonist-Activity of Chemicals
4) OECD (2015), Test No. 455: Performance-Based Test Guideline for Stably TransfectedTransactivation In Vitro Assays to Detect Estrogen Receptor Agonists and Antagonists5) OECD (2007), Test No. 440: Uterotrophic Bioassay in Rodents
6) OECD (2015), Test No. 493: Performance-Based Test Guideline for Human RecombinantEstrogen Receptor (hrER) In Vitro Assays to Detect Chemicals with ER Binding Affinity
7) OECD (2012) Test No. 457: BG1Luc Estrogen Receptor Transactivation Test Method for
Identifying Estrogen Receptor Agonists and Antagonists8) OECD (2012), Test No. 455, Performance-Based Test Guideline for Stably TransfectedTransactivation In Vitro Assays to Detect Estrogen Receptor Agonists and Antagonists
9) OECD (2012) Performance standards for stably transfected transactivation in vitro assays to detect
estrogen agonists for TG 455, Series on Testing & Assessment No. 173.
10) JaCVAM ホ౯఍㆟ሗ࿌᭩㸦ᖹᡂ 25 ᖺ 6 ᭶ 11 ᪥㸧ࣄࢺ࢚ࢫࢺࣟࢤࣥཷᐜయ⤖ྜ࡟ࡼࡿά
ᛶ໬࣭ᣕᢠస⏝≀㉁ࢆ᳨ฟࡍࡿ BG1Luc ER TA ἲ

6
6

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⏝

33

39

40

OECD/OCDE

455
Adopted:28 July 2015

OECD GUIDELINE FOR THE TESTING OF CHEMICALS
Performance-Based Test Guideline for Stably Transfected Transactivation In Vitro Assays to
Detect Estrogen Receptor Agonists and Antagonists

GENERAL INTRODUCTION
Performance-Based Test Guideline
1.
This Performance-Based Test Guideline (PBTG) describes the methodology of Stably TransfectedTransactivation In Vitro Assays to detect Estrogen Receptor Agonists and Antagonists (ER TA assays).
It comprises several mechanistically and functionally similar test methods for the identification of
estrogen receptor (i.e. ERD, and/or ERE) agonists and antagonists and should facilitate thedevelopment of new similar or modified test methods in accordance with the principles for validation
set forth in the OECD Guidance Document (GD) on the Validation and International Acceptance of New
or Updated Test Methods for Hazard Assessment (1). The fully validated reference test methods (Annex
2 and Annex 3) that provide the basis for this PBTG are:x

The Stably Transfected TA (STTA) assay (2) using the (h) ERα-HeLa-9903 cell line; andx

The BG1Luc ER TA assay (3) using the BG1Luc-4E2 cell line which predominately expresses
hERα with some contribution from hERE(4) (5).

Performance standards (PS) (6) (7) are available to facilitate the development and validation ofsimilar test methods for the same hazard endpoint and allow for timely amendment of this PBTG so thatnew similar test methods can be added to an updated PBTG; however, similar test methods will only be
added after review and agreement that performance standards are met. The test methods included in thisTest Guideline can be used indiscriminately to address countries’ requirements for test results on
estrogen receptor transactivation while benefiting from the Mutual Acceptance of Data.
Background and principles of the test methods included in the PBTG
2.
The OECD initiated a high-priority activity in 1998 to revise existing, and to develop new, Test
Guidelines for the screening and testing of potential endocrine disrupting chemicals. The OECD
conceptual framework (CF) for testing and assessment of potential endocrine disrupting chemicals wasrevised in 2012. The original and revised CFs are included as Annexes in the Guidance Document
on Standardised Test Guidelines for Evaluating Chemicals for Endocrine Disruption (8). The CF
comprises five levels, each level corresponding to a different level of biological complexity. The ER
Transactivation (TA) assays described in this PBTG are level 2, which includes "in vitro assays
providing data about selected endocrine mechanism(s)/pathway(s). This PBTG is for in vitro
Transactivation (TA) test methods designed to identify estrogen receptor (ER) agonists and antagonists.
3.
The interaction of estrogens with ERs can affect transcription of estrogen-controlled genes, which
can lead to the induction or inhibition of cellular processes, including those necessary for cell proliferation,© OECD, (2015)1

You are free to use this material for personal, non-commercial purposes without seeking prior consent fromthe OECD, provided the source is duly mentioned. Any commercial use of this material is subject to written
permission from the OECD.
41

455

OECD/OCDE

normal fetal development, and reproductive function (9) (10) (11). Perturbation of normal estrogenic
systems may have the potential to trigger adverse effects on normal development (ontogenesis),
reproductive health and the integrity of the reproductive system.
4.
In vitro TA assays are based on a direct or indirect interaction of the substances with a specificreceptor that regulates the transcription of a reporter gene product. Such assays have been used
extensively to evaluate gene expression regulated by specific nuclear receptors, such as ERs (12) (13)(14) (15) (16). They have been proposed for the detection of estrogenic transactivation regulated by theER (17) (18) (19). There are at least two major subtypes of nuclear ERs, α and β, which are encoded by
distinct genes. The respective proteins have different biological functions as well as different tissue
distributions and ligand binding affinities (20) (21) (22) (23) (24) (25) (26). Nuclear ERα mediates theclassic estrogenic response (27) (28) (29) (30), and therefore most models currently being developed tomeasure ER activation or inhibition are specific to ERα. The assays are used to identify chemicals that
activate (or inhibit) the ER following ligand binding, after which the receptor-ligand complex binds
to specific DNA response elements and transactivates a reporter gene, resulting in increased cellular
expression of a marker protein. Different reporter responses can be used in these test methods. In
luciferase based systems, the luciferase enzyme transforms the luciferin substrate to a bioluminescentproduct that can be quantitatively measured with a luminometer. Other examples of common reporters arefluorescent protein and the LacZ gene, which encodes β-galactosidase, an enzyme that can transform thecolourless substrate X-gal (5- bromo-4-chloro-indolyl-galactopyranoside) into a blue product that can bequantified with a spectrophotometer. These reporters can be evaluated quickly and inexpensively withcommercially available test kits.
5.
Validation studies of the STTA and the BG1Luc TA assays have demonstrated their relevanceand reliability for their intended purpose (3) (4) (5) (30). Performance standards for luminescence-based
ER TA assays using ovarian cells lines are included in ICCVAM Test Method Evaluation Report on the
LUMI-CELL® ER (BG1Luc ER TA) Test Method: An In Vitro Assay for Identifying Human Estrogen
Receptor Agonist and Antagonist Activity of Chemicals (3). These performance standards have been
modified to be applicable to both the STTA and BG1Luc TA test methods (2).
6. Definitions and abbreviations used in this Test Guideline are described in Annex 1.
Scope and limitations related to the TA assays
7.
These test methods are being proposed for screening and prioritisation purposes, but can also
provide mechanistic information that can be used in a weight of evidence approach. They address TA
induced by chemical binding to the ERs in an in vitro system. Thus, results should not be directly
extrapolated to the complex signaling and regulation of the intact endocrine system in vivo.
8.
TA mediated by the ERs is considered one of the key mechanisms of endocrine disruption(ED), although there are other mechanisms through which ED can occur, including (i) interactions
with other receptors and enzymatic systems within the endocrine system, (ii) hormone synthesis, (iii)
metabolic activationand/or inactivation of hormones, (iv) distribution of hormones to target tissues, and(v) clearance of hormones from the body. None of the test methods under this PBTG addresses thesemodes of action.
9.
This PBTG addresses the ability of chemicals to activate (i.e. act as agonists) and also to suppress(i.e. act as antagonists) ER- dependent transcription. Chemicals that are negative in these test methods
should be evaluated in an ER binding assay before concluding that the chemical does not bind to thereceptor. In addition, the assay is only likely to inform on the activity of the parent molecule bearing in
mind the limited metabolising capacities of the in vitro cell systems. Considering that only single
substances were used during the validation, the applicability to test mixtures has not been addressed. The
test method is nevertheless theoretically applicable to the testing of multi-constituent substances and2© OECD, (2015)42

OECD/OCDE

455

mixtures. Before use of the Test Guideline on a mixture for generating data for an intended regulatory
purpose, it should be considered whether, and if so why, it may provide adequate results for that purpose.
Such considerations are not needed, when there is a regulatory requirement for testing of the mixture.
10. For informational purposes, Table 1 provides the agonist test results for the 34 substances thatwere tested in both of the fully validated test methods described in this PBTG. Of these substances,
26 are classified as definitive ER agonists and 8 negatives based upon published reports, including invitro assays for ER binding and TA, and/or the uterotrophic assay (3) (18) (31) (33) (34) (35) (36). Table
2 provides the antagonist test results for the 15 substances that were tested in both of the fully
validated test methods described in this PBTG. Of these substances, 4 are classified as
definitive/presumed ER antagonists and 10 negatives based upon published reports, including in vitro
assays for ER binding and TA (3) (18) (31) (33). In reference to the data summarised in table 1 andtable 2, there was 100% agreement between the two test methods on the classifications of all thesubstances except for one substance (Mifepristone) for antagonist assay, and each substance was correctlyclassified as an ER agonist/antagonist or negative. Supplementary information on this group ofchemicals as well as additional chemicals tested in the STTA and BG1Luc ER TA test methods
during the validation studies is provided in the Performance Standards for the ERTA (6) (7), Annex 2(Tables 1, 2 and 3).

3© OECD, (2015)

43

44

OECD/OCDE

480-16-0
68-23-5
72-43-5
57-30-750-55-550-28-2
57-91-0
57-63-6
10161-33-8
434-22-0
599-64-4
140-66-9
520-36-5
1912-24-9
80-05-7
77-40-7
85-68-7
50-22-6
479-13-0
486-66-8
56-53-1
84-74-2
120-47-8
53-16-7
446-72-0
52-86-8
520-18-3
143-50-065277-42-1
330-55-2
84-16-2
58-18-417-ß Estradiola
17-DEstradiola
17-DEthinyl estradiola
17-E-Trenbolone
19-Nortestosteronea
4-Cumylphenola
4-tert-Octylphenola
Apigenina
Atrazinea
Bisphenol Aa
Bisphenol Ba
Butylbenzyl phthalatea
Corticosteronea
Coumestrola
Daidzeina
Diethylstilbestrola
Di-n-butyl phthalate
Ethyl paraben
Estronea
Genisteina
Haloperidol
Kaempferola
Keponea
Ketoconazole
Linurona
meso-Hexestrola
Methyl testosteronea
Morin
Norethynodrelap,p’-Methoxychlora
Phenobarbitala
Reserpine© OECD, (2015)

28
29
30
31
32

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27

CASRN

Substance

ER TA
Activity
POS
POS
POS
POS
POS
POS
POS
POS
NEG
POS
POS
POS
NEG
POS
POS
POS
POS
POS
POS
POS
NEG
POS
POS
NEG
NEG
POS
POS
POS
POS
POS
NEG
NEG

STTA Assay1
PC10 Value
PC50 Valueb(M)(M)<1.00 × 10-11<1.00 × 10-11
7.24 × 10-11
6.44 u10-10-11<1.00 u10
<1.00 u10-11
1.78 × 10-8
2.73 × 10-7
9.64 u10-9
2.71 u10-7-7
1.60 × 10-6
1.49 u10-91.85 u10
7.37 u10-8-7
1.31 u10
5.71 u10-7
2.94 × 10-7
2.02 u10-8
2.36 u10-8
2.11 u10-7
1.14 u10-6
4.11 u10-6
1.23 u10-9
2.00 u10-8
1.76 u10-8
1.51 u10-7<1.00 × 10-11
2.04 × 10-11
4.09 × 10-6
5.00 x 10-6
(no PC50)
3.02 × 10-11
5.88 u10-10
2.24 × 10-92.45 × 10-8
1.36 × 10-7
1.21 u10-6
7.11 × 10-7
7.68 × 10-6
<1.00 × 10-11
2.75 × 10-11
1.73 × 10-7
4.11 u10-6-7
5.43 × 10
4.16 × 10-6
1.11 × 10-11
1.50 u10-9-6
1.23 × 10(no PC50)b-

BG1Luc ER TA Assay2
ER TA
EC50 Value b,3
Activity(M)
POS
5.63 u10-12
POS
1.40 × 10-9
POS
7.31 × 10-12
POS
4.20 × 10-8
POS
1.80 u10-6
POS
3.20 × 10-7
POS
3.19 × 10-8
POS
1.60 u10-6
NEG
POS
5.33 × 10-7
POS
1.95 u10-7
POS
1.98 × 10-6
NEG
POS
1.32 × 10-7
POS
7.95 u10-7
POS
3.34 × 10-11
POS
4.09 × 10-6
POS
2.48 x 10-5
POS
2.34 u10-10
POS
2.71 × 10-7
NEG
POS
3.99 × 10-6
POS
4.91 × 10-7
NEG
NEG
POS
1.65 × 10-11
POS
2.68 u10-6
POS
2.37 u10-6
POS
9.39 × 10-10
POS
1.92 × 10-6
NEG
NEG
-

Data Source For Classification4
ER
Uterotrophic
Other
Binding
ER TAsc
POS
POS
POS (227/227)
POS
POS
POS(11/11)
POS
POS
POS(22/22)
NT
NT
POS (2/2)
POS
POS
POS(4/4)
POS
NT
POS(5/5)
POS(21/24)
POS
POS
POS
NT
POS(26/26)
NEG (30/30)
NEG
NT
POS
POS
POS(65/65)
POS
POS
POS(6/6)
POS
NEG
POS(12/14)
NEG( 6/6 )
NEG
NT
POS
NT
POS(30/30)
POS
POS
POS(39/39)
POS(42/42)
POS
NT
POS(6/11)
POS
NEG
POS
NT
POS
POS
POS(26/28)
POS(100/102)
POS
POS
NEG (2/2)
NEG
NT
POS
NT
POS(23/23)
POS(14/18)
POS
NT
NEG (2/2)
NEG
NT
NEG (8/8 )NEG
NTPOS(4/4)
POS
NT
POS
NT
POS(5/6)

4

POS(2/2)
POS(5/5)
POS(24/27)
NEG(2/2)
NEG(4/4)

POS
POS
POS
NEG
NEG

NT
NT
POS
NT
NT

Table 1: Comparison of Results from STTA and BG1Luc ER TA Assays for Substances Tested in Both Agonist Assays and Classified as ER
Agonists (POS) or Negatives (NEG)

455

45

Spironolactonea
Testosterone

52-01-7
58-22-0

NEG
POS

2.82 × 10-8

9.78 × 10-6

NEG
POS

1.75 × 10-5

NEG(4/4)
POS(5/10)

NEG
POS

NT
NT

455

© OECD, (2015)

5Abbreviations: CASRN = Chemical Abstracts Service Registry Number; M = molar; EC50 = half maximal effective concentration of test substance; NEG = negative; POS = positive; NT= Not tested; PC10 (and PC50) = the concentration of a test substance at which the response is 10% (or 50 % for PC50) of the response induced by the positive control (E2, 1nM) in each plate.
aCommon substances tested in the STTA and BG1Luc ER TA assays that were designated as ER agonists or negatives and used to evaluate accuracy in the BG1 Luc ER TA validation
study ( ICCVAM BG1Luc ER TA Evaluation Report, Table 4-1 (3).
bMaximum concentration tested in the absence of limitations due to cytotoxicity or insolubility was 1 x 10-5 M (STTA Assay) and 1 x 10-3 M (BG1Luc ER TA Assay).
c
Number in parenthesis represents the test results classified as positive (POS) or negative (NEG) over the total number of referenced studies.
1
Values reported in Draft Report of Pre-validation and Inter-laboratory Validation For Stably Transfected Transcriptional Activation (TA) Assay to Detect Estrogenic Activity - The Human
Estrogen Receptor Alpha Mediated Reporter Gene Assay Using hER-HeLa-9903 Cell Line (31)2ICCVAM Test Method Evaluation Report on the LUMI-CELL® ER (BG1Luc ER TA) Test Method: An In Vitro Method for Identifying ER Agonists and Antagonists (3)3Mean EC50 values were calculated with values reported by the laboratories of the BG1Luc ER TA validation study (XDS, ECVAM, and Hiyoshi) (3).
4Classification as an ER agonist or negative was based upon information in the ICCVAM Background Review Documents (BRD) for ER Binding and TA test methods (32) as well as
information obtained from publications published and reviewed after the completion of the ICCVAM BRDs (3) (18) (31) (33) (34) (35) (36).

33
34

OECD/OCDE

46

OECD/OCDE

Dibenzo[a.h] anthracene

Mifepristone

Raloxifene HCl

Tamoxifen

17-b estradiol

ApigeninAtrazine

Di-n-butyl phthalate

Fenarimol

FlavoneFlutamide

Genisteinp-n-nonylphenol

Resveratrol

2

3

4

5

6

7

8

9

10

11

12

13

14

15

501-36-0

104-40-5

446-72-0

13311-84-7

525-82-6

60168-88-9

84-74-2

1912-24-9

520-36-5

50-28-2

10540-29-1

82640-04-8

84371-65-3

53-70-368047-06-3

CASRN

NEG

NEG

NEG

NEG

NEG

NEG

NEG

NEG

NEG

NEG

POS

POS

POS

POS

POS

POS
POS

7.86 × 10-10
4.91 × 10-7

-

-

-

-

-

-

-

-

-

NEG

NEG

NEG

NEG

NEG

NEG

NEG

NEG

NEG

NEG

NEG

5.61 × 10-6

-

POS

POS

3.97 × 10-9
No IC50

ER TA
Activity

IC50 Valueb
(M)

-

-

-

-

-

-

-

-

-

-

8.17 × 10-7

1.19 × 10-9-

No IC50

2.08 × 10-7

IC50 Valueb,3(M)

BG1Luc ER TA assay2

PN

not tested

PN

NEG

PN

not tested

NEG

NEG

NEG

PN

POS

moderate POS

mild POS

POS

moderate POS

ER STTAcandidateeffects4

NEG

NEG

NEG

PN

PN

PN

NEG

PN

NEG

PN

POS

POS

NEG

PP

POS

ICCVAM 5
Consensus
Classification

Flavonoid,
Heterocyclic
CompoundPhenolHydrocarbon(Cyclic)

Heterocyclic
CompoundHeterocyclic
CompoundEster,
Phthalic Acid
Heterocyclic
Compound,Pyrimidine
Flavonoid,
Heterocyclic
Compound
Amide

MeSH6
ChemicalClass
Hydrocarbon(Cyclic)
Polycyclic
CompoundSteroid
Hydrocarbon(Cyclic)
Hydrocarbon(Cyclic)
Steroid

Chemical Intermediate
Natural Product

Pharmaceutical, Veterinary
Agent
Natural Product,
Pharmaceutical

Natural Product,
Pharmaceutical

Cosmetic Ingredient, Industrial
Chemical, Plasticizer
Fungicide

Pharmaceutical, Veterinary
AgentDye, Natural Product,
Pharmaceutical Intermediate
Herbicide

Pharmaceutical

Laboratory Chemical, Natural
Product
PharmaceuticalPharmaceutical

Product Class7
Pharmaceutical© OECD, (2015)6Abbreviations: CASRN = Chemical Abstracts Service Registry Number; M = molar; IC50 = half maximal inhibitory concentration of test substance; NEG = negative; PN = presumed negative; POS= positive; PP = presumed positive.
aCommon substances tested in the STTA and BG1Luc ER TA assays that were designated as ER antagonists or negatives and used to evaluate accuracy in the BG1 Luc ER TA validation study

4-hydroxytamoxifen

1

Substancea

ER TA
Activity

ER STTA assay1

Table 2: Comparison of Results from STTA and BG1Luc ER TA assays for Substances Tested in Both Antagonist Assays and Classified as ER
Antagonists (POS) or Negatives (NEG)

455

47

455

© OECD, (2015)7(3) (31).
b
Maximum concentration tested in the absence of limitations due to cytotoxicity or insolubility was 1 x 10-3 M (STTA Assay) and 1 x 10-5 M (BG1Luc ER TA Assay).
1
The Validation Report of the Stably transfected Transcriptional Activation Assay to Detect ER mediated activity, Part B (31)2ICCVAM Test Method Evaluation Report on the LUMI-CELL ER (BG1Luc ER TA) Test Method: An In Vitro Method for Identifying ER Agonists and Antagonists (3).
3Mean IC50 values were calculated with values reported by the laboratories of the BG1Luc ER TA validation study (XDS, ECVAM, and Hiyoshi) (3).
4
ER STTA activity assumed from their reported effects known from the CERI historical data of ER receptor binding assay, the uterotrophic assay and information collated from the open literature(31)5 Classification as an ER antagonist or negative was based upon information in the ICCVAM Background Review Documents (BRD) for ER Binding and TA test methods (32) as well as
information obtained from publications published and reviewed after the completion of the ICCVAM BRDs (3) (18) (31) (33).
6
Substances were assigned to one or more chemical classes using the U.S. National Library of Medicine’s Medical Subject Headings (MeSH), an internationally recognised standardisedclassification scheme (available at http://www.nlm.nih.gov/mesh).
7
Substances were assigned to one or more product classes using the U.S. National Library of Medicine’s Hazardous Substances Data Bank (available at http://toxnet.nlm.nih.gov/cgibin/sis/htmlgen?HSDB).

OECD/OCDE

455

OECD/OCDEER TA TEST METHOD COMPONENTS
Essential Test Method Components
11. This PBTG applies to methods using a stably transfected or endogenous ERα receptor andstably transfected reporter gene construct under the control of one or more estrogen response
elements; however, other receptors such as ERβ may be present. These are essential test method
components.
Control substances
12. The basis for the proposed concurrent reference substances for each of agonist and antagonist assay
should be described. Concurrent controls (negative, solvent, and positive), as appropriate, serve as anindication that the test method is operative under the test conditions and provide a basis forexperiment-to-experiment comparisons; they are usually part of the acceptability criteria for a given
experiment (1).
Standard Quality Control Procedures
13.
Standard quality control procedures should be performed as described for each assay to ensure thecell line remains stable through multiple passages, remains mycoplasma-free, and retains the ability toprovide the expected ER-mediated responses over time. Cell lines should be further checked for theircorrect identity as well as for other contaminants (e.g. fungi, yeast and viruses).
Demonstration of Laboratory Proficiency
14. Prior to testing unknown chemicals with any of the test methods under this PBTG, eachlaboratory should demonstrate proficiency in using the test method by testing of the 14 proficiency
substances listed in Table 3 for agonist assay and 10 proficiency substances in Table 4 for antagonist
assay. This proficiency testing will also confirm the responsiveness of the test system. The list of
proficiency substances is a subset of the reference substances provided in the Performance Standards for
the ER TA assays (6). These substances are commercially available, represent the classes of chemicals
commonly associated with ER agonist or antagonist activity, exhibit a suitable range of potency expected
for ER agonists/antagonists (i.e. strong to weak) and negatives. Testing of these substances should be
replicated at least twice, on different days. Proficiency is demonstrated by correct classification(positive/negative) of each proficiency substance. Proficiency testing should be repeated by eachtechnician when learning the test methods.

8© OECD, (2015)

48

49

520-18-3

85-68-7

72-43-5meso-Hexestrol

4-tertOctylphenol

Genistein

Bisphenol A

Kaempferol

Butylbenzyl
phthalatep,p’Methoxychlor

Ethyl paraben

Atrazine

Spironolactone

15

11

9

6

2

3

4

1

17

20

© OECD, (2015)

57-91-017v-Estradiol

1252-01-7

1912-24-9

120-47-8

80-05-7

446-72-0

140-66-9

84-16-2

56-53-1

CASRN

Diethylstilbestrol

Substance
Name

14

N°7

NEG

NEG

POS

POS

POS

POS

POS

POS

POS

POS

POS

POS

Expected
Response1--

5.00 ×10-6

1.23 × 10-6

-

-

-

-

4.11 × 10-6

1.21 × 10-6

1.36 ×10-7

1.14 ×10-6

2.94 × 10-7

2.02 × 10-8

2.45 × 10-8

2.75 × 10-11<1.00 × 10-11

2.24 × 10-9

6.44 × 10-10

4.27 × 10-11

7.37 × 10-8

2.04 × 10-11<1.00 × 10-11

1.85 × 10-9

PC50 Value(M)2

STTA Assay
PC10 Value(M) 2

Tab le 3: List of (14) Proficiency Substances for agonist assay

8-

10-11 – 10-59-2.48 × 10-5

1.92 × 10-6

1.98 × 10-6

3.99 × 10-6

5.33 × 10-7

2.71 × 10-7

3.19 × 10-8

1.65 × 10-11

1.40 × 10-9

3.34 × 10-11

10-10 – 10-4

10-11 – 10-5

10-11 – 10-5

10-11 – 10-5

10-11 – 10-5

10-11 – 10-5

10-11 – 10-5

10-11 – 10-5

10-11 – 10-5

10-11 – 10-5

10-14 – 10-8

Test
concentration
range (M)2.40 u10-3

4.64 u10-4

6.02 u10-3

2.89 u10-3

3.20 u10-4

3.49 u10-3

4.38 u10-3

Carboxylic
Acid, Phenol
Heterocyclic
Compound
Lactone, Steroid

Hydrocarbon(Halogenated)

Flavonoid,
Heterocyclic
CompoundCarboxylic
Acid, Ester,Phthalic Acid

PhenolFlavonoid,
Heterocyclic
CompoundPharmaceutical

Herbicide

Plasticizer,
Industrial
ChemicalPesticide,
Veterinary
Agent
Pharmaceutical,
Preservative

Natural ProductChemicalIntermediate

Natural Product,
Pharmaceutical

Hydrocarbon(Cyclic), Phenol

3.70 u10-3

3.70 u10-4

Steroid3.67 u10-3

PhenolPharmaceutical,
Veterinary
Agent
Pharmaceutical,
Veterinary
Agent
Pharmaceutical,
Veterinary
Agent
ChemicalIntermediate
Hydrocarbon(Cyclic)

3.73 u10-4

4.85 u10-3

Product
Class6

MeSH
ChemicalClass5

BG1Luc ER TA Assay
HighestConcentrat
Bg1Luc
EC50 Value ion for
Range
(M)3
Finder (M)4

OECD/OCDE

455

50

Reserpine

2250-55-565277-421
NEG

NEG
-

-

OECD/OCDE---

10-11 – 10-5
10-11 – 10-5

1.64 u10-3

9.41 u10-5

Heterocyclic
CompoundHeterocyclic
Compound,IndolePharmaceutical,
Veterinary
Agent

Pharmaceutical© OECD, (2015)10

Abbreviations: CASRN = Chemical Abstracts Service Registry Number; EC50 = half maximal effective concentration of test substance; NEG = negative; POS = positive;
PC10 (and PC50) = the concentration of a test substance at which the response is 10% (or 50 % for PC50) of the response induced by the positive control (E2, 1nM) in each plate.
1
Classification as positive or negative for ER agonist activity was based upon the ICCVAM Background Review Documents (BRD) for ER Binding and TA test methods (32) (33)
as well as empirical data and other information obtained from referenced studies published and reviewed after the completion of the ICCVAM BRDs
(3) (18) (31) (32) (33) (34) (35) (36).
2
Values reported in Draft Report of Pre-validation and Inter-laboratory Validation For Stably Transfected Transcriptional Activation (TA) Assay to Detect Estrogenic Activity The Human Estrogen Receptor Alpha Mediated Reporter Gene Assay Using hER-HeLa-9903 Cell Line (30).
3Mean EC50 values were calculated with values reported by the laboratories of the BG1Luc ER TA validation study (XDS, ECVAM, and Hiyoshi) (3).
4Concentrations reported were the highest concentrations tested (range finder) during the validation of the BG1Luc ER TA Assay. If concentrations differed between thelaboratories, the highest concentration is reported. See table 4-10 of ICCVAM Test Method Evaluation Report; The LUMI-Cell®ER (BG1Luc ER TA) Test Method: An In Vitro
Assay for Identifying Human Estrogen Receptor Agonist and Antagonist Activity of Chemicals (3).
5
Substances were assigned into one or more chemical classes using the U.S. National Library of Medicine’s Medical Subject Headings (MeSH), an internationally recognised
standardised classification scheme (available at: http://www.nlm.nih.gov/mesh).
6
Substances were assigned into one or more product classes using the U.S. National Library of Medicine’s Hazardous Substances Database (available at: http://toxnet.nlm.nih.gov/cgibin/sis/htmlgen?HSDB)
7
From Table 1 (List of Reference Chemicals (22) for Evaluation of ER Agonist Accuracy) of the Performance Standards (6)8If a reference substance is no longer commercially available, a substance with the same classification and, comparable potency, mode of action and chemical class can be used.

Ketoconazole

21

455

51

NEG

NEG

84-74-2

525-826

Di-n-butyl
phthalate

Flavone67© OECD, (2015)

NEG

5

520-365

17β estradiol

4

POS

1054029-1

ApigeninTamoxifen

3

POS

8264004-8

NEG

Raloxifene HCl

2

POS

CASRN
6804706-3

50-28-24hydroxytamoxifen

1

Substancea

ER TA
Activit
y

-

-

-

-

4.91 ×10-7

7.86 ×
10-10

IC50
Value
(M)
3.97 ×10-9

10-8 – 10-3

10-8 – 10-3

NEG

NEG

NEG

NEG

10-9 – 10-4

10-9 – 10-4

POS

POS

POS

ER TA
Activit
y

-

-

-

-

8.17 ×10-7

1.19 ×10-9

IC50
Value3(M)
2.08 ×10-7

11

4.50 × 10-4

3.59 × 10-3

3.70 × 10-4

3.67 × 10-3

2.69 × 10-4

1.96 × 10-4

2.58 × 10-4

HighestConcentrat
ion for
Range
Finder(M)4

BG1Luc ER TA assay2

10-10 – 10-5

10-12 – 10-7

10-12 – 10-7

Test
concentrati
onrange(M)ER STTA assay1

Table 4: List of (10) Proficiency Substances for antagonist assay

to benegative*

NEG

PN

NEG

NEG

PN

to be
negative*

NEG

POS

POS

POS

ICCVAM 5
Consensus
Classificati
onPOS

moderate
POS

ER STTA1
candidateef
fects
moderate
POS

OECD/OCDE

Product Class7
PharmaceuticalPharmaceuticalPharmaceuticalPharmaceutical,
Veterinary Agent
Dye, Natural
Product,
Pharmaceutical
Intermediate
Cosmetic
Ingredient,Industrial
Chemical,Plasticizer
Natural Product,
Pharmaceutical

MeSH6
ChemicalClass
Hydrocarbon(Cyclic)
Hydrocarbon(Cyclic)
Hydrocarbon(Cyclic)
Steroid

Heterocyclic
CompoundEster, Phthalic
Acid

Flavonoid,
Heterocyclic
Compound455

52

p-n-nonylphenol

Resveratrol

9

10

NEG

NEG

NEG

446-720

104-405

501-360---

10-8 – 10-3

10-9 – 10-4

-4

10 – 10-9OECD/OCDE

NEG

NEG

NEG

-

-

-

4.38 × 10-4

4.54 × 10-4

3.70 × 10

-4

to be
negative*

not tested

to benegative*

NEG

NEG

NEG

Natural Product,
Pharmaceutical

ChemicalIntermediate
Natural Product

Flavonoid,
Heterocyclic
CompoundPhenolHydrocarbon(Cyclic)© OECD, (2015)12

Abbreviations: CASRN = Chemical Abstracts Service Registry Number; M = molar; IC50 = half maximal inhibitory concentration of test substance; NEG = negative; PN = presumed negative; POS =positive.
*classified negative according to literature review (31).
aCommon substances tested in the STTA and BG1Luc ER TA assays that were designated as ER antagonists or negatives and used to evaluate accuracy in the BG1 Luc ER TA validation study (3)(31).
1
The Validation Report of the Stably transfected Transcriptional Activation Assay to Detect ER mediated activity, Part B (31)2ICCVAM Test Method Evaluation Report on the LUMI-CELL ER (BG1Luc ER TA) Test Method: An In Vitro Method for Identifying ER Agonists and Antagonists (3).
3Mean IC50 values were calculated with values reported by the laboratories of the BG1Luc ER TA validation study (XDS, ECVAM, and Hiyoshi) (3).
4Concentrations reported were the highest concentrations tested (range finder) during the validation of the BG1Luc ER TA Assay. If concentrations differed between the laboratories, the highest
concentration is reported. See table 4-11 of ICCVAM Test Method Evaluation Report; The LUMI-Cell®ER (BG1Luc ER TA) Test Method: An In Vitro Assay for Identifying Human Estrogen
Receptor Agonist and Antagonist Activity of Chemicals (3).
5Classification as an ER antagonist or negative was based upon information in the ICCVAM Background Review Documents (BRD) for ER Binding and TA test methods (32) as well as information
obtained from publications published and reviewed after the completion of the ICCVAM BRDs (3) (18) ((31) (33).
6
Substances were assigned to one or more chemical classes using the U.S. National Library of Medicine’s Medical Subject Headings (MeSH), an internationally recognised standardised classification
scheme (available at http://www.nlm.nih.gov/mesh).
7
Substances were assigned to one or more product classes using the U.S. National Library of Medicine’s Hazardous Substances Data Bank (available at http://toxnet.nlm.nih.gov/cgibin/sis/htmlgen?HSDB).

Genistein

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Test Run Acceptability Criteria
15. Acceptance or rejection of a test run is based on the evaluation of results obtained for the reference
substances and controls used for each experiment. Values for the PC50(EC50) or IC50 for the reference
substances should meet the acceptability criteria as provided for the selected test method (for STTA
see Annex 2, for BG1Luc ER TA see Annex 3), and all positive/negative controls should be correctlyclassified for each accepted experiment. The ability to consistently conduct the test method should bedemonstrated by the development and maintenance of a historical database for the reference substancesand controls. Standard deviations (SD) or coefficients of variation (CV) for the means of reference
substances curve fitting parameters from multiple experiments may be used as a measure of withinlaboratory reproducibility. In addition, the following principles regarding acceptability criteria should be
met:xData should be sufficient for a quantitative assessment of ER activation (for agonist assay) or
suppression (for antagonist assay) (i.e., efficacy and potency).

x

The mean reporter activity for the reference concentration of reference estrogen should be at least
the minimum specified in the test methods relative to that of the vehicle (solvent) control to ensure
adequate sensitivity. For the STTA and BG1Luc ER TA test methods, this is four times that of themean vehicle control on each plate.

x

The concentrations tested should remain within the solubility range of the test chemicals and notdemonstrate cytotoxicity.

Analysis of data
16. The defined data interpretation procedure for each test method should be used for classifying a
positive and negative response.
17. Meeting the acceptability criteria (paragraph 15) indicates the assay system is operating properly,
but it does not ensure that any particular test will produce accurate data. Replicating the correct results ofthe first test is the best indication that accurate data were produced. If two tests give reproducible results(e.g. both test results indicate a test chemical is positive), it is not necessary to conduct a third test.
18. If two runs do not give reproducible results (e.g. a test chemical is positive in one run andnegative in the other run), or if a higher degree of certainty is required regarding the outcome of thisassay, at least three independent runs should be conducted.
General Data Interpretation Criteria
19. There is currently no universally agreed method for interpreting ER TA data. However, both
qualitative (e.g. positive/negative) and/or quantitative (e.g. EC50, PC50, IC50) assessments of ER-mediatedactivity should be based on empirical data and sound scientific judgment. Where possible, positive results
should be characterised by both the magnitude of the effect as compared to the vehicle (solvent) control orreference estrogen and the concentration at which the effect occurs (e.g. an EC50, PC50, RPCMax, IC50 , etc.).
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Test Report
20.

The test report should include the following information:

Test method:
Test method used;
Control/Reference/Test chemical− source, lot number, limit date for use, if available− stability of the test chemical itself, if known;− solubility and stability of the test chemical in solvent, if known.
− measurement of pH, osmolality and precipitate in the culture medium to which the test chemical wasadded, as appropriate.
Mono-constituent substance:− physical appearance, water solubility, and additional relevant physicochemical properties;− chemical identification, such as IUPAC or CAS name, CAS number, SMILES or InChI code,
structural formula, purity, chemical identity of impurities as appropriate and practically feasible, etc.
Multi-constituent substance, UVBCs and mixtures:− characterised as far as possible by chemical identity (see above), quantitative occurrence and relevant
physicochemical properties of the constituents.
Solvent/Vehicle:characterisation (nature, supplier and lot);justification for choice of solvent/vehicle;
solubility and stability of the test chemical in solvent/vehicle, if known;

Cells:
 type and source of cells:- Is ER endogenously expressed? If not, which receptor(s) were Transfected?
- Reporter construct(s) used (including source species);- Transfection method;- Selection method for maintenance of stable transfection (where applicable);- Is the transfection method relevant for stable lines?
 number of cell passages (from thawing);
 passage number of cells at thawing; methods for maintenance of cell cultures;
Test conditions:solubility limitations;14© OECD, (2015)

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









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description of the methods of assessing viability applied;composition of media, CO2 concentration;
concentrations of test chemical;
volume of vehicle and test chemical added;
incubation temperature and humidity;
duration of treatment;
cell density at the start of - and during treatment;
positive and negative reference substances;
reporter reagents (product name, supplier and lot);
criteria for considering tests as positive, negative or equivocal;

Acceptability check:fold inductions for each assay plate and whether they meet the minimum required by the test method
based on historical controls; actual values for acceptability criteria, e.g. log10EC50, log10PC50, logIC50 and Hillslope values, forconcurrent positive controls/reference substances;
Results:
 raw and normalised data; the maximum fold induction level;
 cytotoxicity data; if it exists, the lowest effective concentration (LEC); RPCMax, PCMax, PC50, IC50 and/or EC50 values, as appropriate; concentration-response relationship, where possible; statistical analyses, if any, together with a measure of error and confidence (e.g. SEM, SD, CV or 95%
CI) and a description of how these values were obtained;
Discussion of the results
Conclusion

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OECD/OCDELITERATURE

1)OECD. (2005). Guidance Document on the Validation and International Acceptance of New or
Updated Test Methods for Hazard Assessment. Environment, Health and Safety Publications, Series on
Testing and Assessment (No. 34.), Organisation for Economic Cooperation and Development, Paris.

2)

OECD. (2009). Test (No. 455.): The Stably Transfected Human Estrogen Receptor-alpha Transcriptional
Activation Assay for Detection of Estrogenic Agonist-Activity of Chemicals, replaced by Test Guideline.

3)

ICCVAM (2011). ICCVAM Test Method Evaluation Report on the LUMI-CELL® ER (BG1Luc ER
TA) Test Method, an In Vitro Method for Identifying ER Agonists and Antagonists, National Institute ofEnvironmental Health Sciences: Research Triangle Park, NC.

4)

Pujol P., et al. (1998). Differential Expression of Estrogen Receptor-Alpha and -Beta Messenger
RNAs as a Potential Marker of Ovarian Carcinogenesis, Cancer. Res., 58(23): p. 5367-73.

5)

Rogers J.M. and Denison M.S. (2000). Recombinant Cell Bioassays for Endocrine Disruptors:
Development of a Stably Transfected Human Ovarian Cell Line for the Detection of Estrogenic andAnti-Estrogenic Chemicals, In Vitro and Molecular Toxicology: Journal of Basic and Applied Research,
13(1): p. 67-82.

6)OECD. (2012). Performance Standards For Stably Transfected Transactivation In Vitro Assay to Detect
Estrogen Receptor Agonists (for TG 455). Environment, Health and Safety Publications, Series on
Testing and Assessment ( No.173.), Organisation for Economic Cooperation and Development, Paris.

7)

OECD. (2015). Performance Standards For Stably Transfected Transactivation In Vitro Assay to Detect
Estrogen Receptor Antagonists. Environment, Health and Safety Publications, Series on Testing andAssessment (No. 174.), Organisation for Economic Cooperation and Development, Paris.

8)OECD (2012), Guidance Document on Standardized Test Guidelines for Evaluating Chemicals for
Endocrine Disruption. Environment, Health and Safety Publications, Series on Testing and Assessment(No. 150.), Organisation for Economic Cooperation and Development, Paris.

9)

Cavailles V. (2002). Estrogens and Receptors: an Evolving Concept. Climacteric, 5 Suppl 2: p. 20- 6.

10)

Welboren W.J., et al. (2009). Genomic Actions of Estrogen Receptor Alpha: What are the Targets andhow are they Regulated? Endocr. Relat. Cancer, 16(4): p. 1073-89.

11)

Younes M. and Honma N. (2011). Estrogen Receptor Beta, Arch. Pathol. Lab. Med., 135(1): p. 63- 6.

12)

Jefferson W.N., et al. (2002). Assessing Estrogenic Activity of Phytochemicals Using Transcriptional
Activation and Immature Mouse Uterotrophic Responses, Journal of Chromatography B, 777(1-2): p.
179-189.

13)

Sonneveld E., et al. (2006). Comparison of In Vitro and In Vivo Screening Models for Androgenic andEstrogenic Activities, Toxicol. Sci., 89(1): p. 173-187.
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14)

Takeyoshi M., et al. (2002). The Efficacy of Endocrine Disruptor Screening Tests in Detecting AntiEstrogenic Effects Downstream of Receptor-Ligand Interactions, Toxicology Letters, 126(2): p. 91- 98.

15)

Combes R.D. (2000). Endocrine Disruptors: a Critical Review of In Vitro and In Vivo Testing
Strategies for Assessing their Toxic Hazard to Humans, ATLA Alternatives to Laboratory
Animals,28(1): p. 81-118.

16)

Escande A., et al. (2006). Evaluation of Ligand Selectivity Using Reporter Cell Lines Stably Expressing
Estrogen Receptor Alpha or Beta, Biochem. Pharmacol,71(10): p. 1459-69.

17)

Gray L.E. Jr. (1998). Tiered Screening and Testing Strategy for Xenoestrogens and Antiandrogens,
Toxicol. Lett, 102-103, 677-680.

18)

EDSTAC. (1998). Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) Final
Report. Available at: [http://www.epa.gov/scipoly/oscpendo/pubs/edspoverview/finalrpt.html].

19)

ICCVAM. (2003). ICCVAM Evaluation of In Vitro Test Methods for Detecting Potential
Endocrine Disruptors: Estrogen Receptor and Androgen Receptor Binding and Transcriptional
Activation Assays. Available at:[http://www.iccvam.niehs.nih.gov/docs/endo_docs/edfinalrpt0503/edfinrpt.pdf ].

20)

Gustafsson J.Ö. (1999). Estrogen Receptor ß - A New Dimension in Estrogen Mechanism of Action,
Journal of Endocrinology, 163(3): p. 379-383.

21)

Ogawa S., et al. (1998). The Complete Primary Structure of Human Estrogen Receptor ß (hERß) and its
In Vivo and In Vitro, Biochemical and Biophysical Research
Heterodimerization with ER
Communications, 243(1): p. 122-126.

22)

Enmark E., et al. (1997). Human Estrogen Receptor ß-Gene Structure, Chromosomal Localization, andExpression Pattern, Journal of Clinical Endocrinology and Metabolism,82(12): p. 4258-4265.

23)

Ball L.J., et al. (2009). Cell Type- and Estrogen Receptor-Subtype Specific Regulation of Selective
Estrogen Receptor Modulator Regulatory Elements, Molecular and Cellular Endocrinology, 299(2): p.
204-211.

24)

Barkhem T., et al. (1998). Differential Response of Estrogen Receptor Alpha and Estrogen Receptor
Beta to Partial Estrogen Agonists/Antagonists, Mol. Pharmacol, 54(1): p. 105-12.

25)

Deroo B.J. and Buensuceso A.V. (2010). Minireview: Estrogen Receptor-ß: Mechanistic Insights from
Recent Studies, Molecular Endocrinology, 24(9): p. 1703-1714.

26)

Harris D.M., et al. (2005). Phytoestrogens Induce Differential Estrogen Receptor Alpha- or BetaMediated Responses in Transfected Breast Cancer Cells, Experimental Biology and Medicine, 230(8): p.
558-568.

27)

Anderson J.N., Clark J.H. and Peck E.J.Jr. (1972). The Relationship Between Nuclear ReceptorEstrogen Binding and Uterotrophic Responses, Biochemical and Biophysical ResearchCommunications, 48(6): p. 1460-1468.

28)

Toft D. (1972). The Interaction of Uterine Estrogen Receptors with DNA, Journal of Steroid
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Biochemistry, 3(3): p. 515-522.
29)

Gorski J., et al. (1968), Hormone Receptors: Studies on the Interaction of Estrogen with the Uterus,
Recent Progress in Hormone Research, 24: p. 45-80.

30)

Jensen E.V., et al. (1967), Estrogen-Receptor Interactions in Target Tissues, Archives d'Anatomie
Microscopique et de Morphologie Experimentale, 56(3):p. 547-569.

31)

OECD. The Validation Report of the Stably Transfected Transcriptional Activation Assay to Detect ER
mediated activity, Part A (Agonist Assay), Part B (Antagonist Assay), Organisation for Economique
Cooperation and Development, Paris.

32)

ICCVAM. (2002). Background Review Document: Estrogen Receptor Transcriptional Activation (TA)Assay. Appendix D, Substances Tested in the ER TA Assay, NIH Publication Report (No. 03-4505.).
Available at: [http://www.iccvam.niehs.nih.gov/docs/endo_docs/final1002/erta_brd/ERTA034505.pdf].

33)

Kanno J., et al. (2001). The OECD Program to Validate the Rat Uterotrophic Bioassay to Screen
Compounds for In Vivo Estrogenic Responses: Phase 1, Environ. Health Persp., 109:785-94.

34)

Kanno J., et al. (2003). The OECD Program to Validate the Rat Uterotrophic Bioassay: Phase Two
Dose -Response Studies, Environ. Health Persp., 111:1530-1549.

35)

Kanno J., et al. (2003), The OECD Program to Validate the Rat Uterotrophic Bioassay: Phase Two –Coded Single-Dose Studies, Environ. Health Persp., 111:1550-1558.

36)

ICCVAM (2002). Background Review Document: Estrogen Receptor Binding, Appendix D, Substances
Tested in the ER Binding Assay, NIH Publication Report (No. 03-4504.) Available at:[http://www.iccvam.niehs.nih.gov/docs/endo_docs/final1002/erbndbrd/ERBd034504.pdf ].

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ANNEX 1
Definitions and Abbreviations
Acceptability criteria: Minimum standards for the performance of experimental controls and reference
standards. All acceptability criteria should be met for an experiment to be considered valid.
Accuracy (concordance): The closeness of agreement between test method results and an acceptedreference values. It is a measure of test method performance and one aspect of relevance. The term is often
used interchangeably with “concordance” to mean the proportion of correct outcomes of a test method (1).
Agonist: A substance that produces a response, e.g. transcription, when it binds to a specific receptor.
Antagonist: A type of receptor ligand or chemical that does not provoke a biological response itself uponbinding to a receptor, but blocks or dampens agonist-mediated responses.
Anti-estrogenic activity, the capability of a chemical to suppress the action of 17β-estradiol mediatedthrough estrogen receptors.
BG-1: An immortalised adenocarcinoma cell that endogenously express estrogen receptor.
BG-1Luc4E2: The BG-1Luc4E2 cell line was derived from BG-1 immortalised human-derived
adenocarcinoma cells that endogenously express both forms of the estrogen receptor (ERα and ERβ) andhave been stably transfected with the plasmid pGudLuc7.ERE. This plasmid contains four copies of asynthetic oligonucleotide containing the estrogen response element upstream of the mouse mammary
tumor viral (MMTV) promoter and the firefly luciferase gene.
Cell morphology: The shape and appearance of cells grown in a monolayer in a single well of a tissue
culture plate. Cells that are dying often exhibit abnormal cell morphology.
CF: The OECD Conceptual Framework for the Testing and Evaluation of Endocrine Disrupters.
Charcoal/dextran treatment: Treatment of serum used in cell culture. Treatment with charcoal/dextran(often referred to as “stripping”) removes endogenous hormones and hormone-binding proteins.
Cytotoxicity: Harmful effects to cell structure or function that can ultimately cause cell death and can bereflected by a reduction in the number of cells present in the well at the end of the exposure period or areduction of the capacity for a measure of cellular function when compared to the concurrent vehicle
control.
CV: Coefficient of variation
DCC-FBS: Dextran-coated charcoal treated fetal bovine serum.
DMEM: Dulbecco’s Modification of Eagle’s Medium
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OECD/OCDEDMSO: Dimethyl sulfoxide
E2: 17β-estradiol
EC50: The half maximal effective concentration of a test substance.
ED: Endocrine disruption
hERα: Human estrogen receptor alphahERß: Human estrogen receptor beta
EFM: Estrogen-free medium. Dulbecco’s Modification of Eagle’s Medium (DMEM) supplemented with
4.5% charcoal/dextran-treated FBS, 1.9% L-glutamine, and 0.9% Pen-Strep.
ER: Estrogen receptor
ERE: Estrogen response element
Estrogenic activity: The capability of a chemical to mimic 17β-estradiol in its ability to bind to andactivate estrogen receptors. hERα-mediated estrogenic activity can be detected with this PBTG.
FBS: Fetal bovine serum
HeLa: An immortal human cervical cell line
HeLa9903: A HeLa cell subclone into which hERD and a luciferase reporter gene have been stablytransfected
IC50: The half maximal effective concentration of an inhibitory test chemical.
ICCVAM: The Interagency Coordinating Committee on the Validation of Alternative Methods.
Inter-laboratory reproducibility: A measure of the extent to which different qualified laboratories, using
the same protocol and testing the same substances, can produce qualitatively and quantitatively similar
results. Interlaboratory reproducibility is determined during the prevalidation and validation processes, andindicates the extent to which a test method can be successfully transferred between laboratories, also
referred to as between-laboratory reproducibility (1).
Intra-laboratory reproducibility: A determination of the extent that qualified people within the samelaboratory can successfully replicate results using a specific protocol at different times. Also referred to as“within-laboratory reproducibility” (1).
LEC: Lowest effective concentration is the lowest concentration of test chemical that produces a response(i.e. the lowest test chemical concentration at which the fold induction is statistically different from theconcurrent vehicle control).
Me-too test: A colloquial expression for a test method that is structurally and functionally similar to avalidated and accepted reference test method. Interchangeably used with similar test method
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MT: Metallothionein
MMTV: Mouse Mammary Tumor Virus
OHT: 4-Hydroxytamoxifen
PBTG: Performance-Based Test Guideline
PC (Positive control): a strongly active substance, preferably 17ß-estradiol that is included in all tests to
help ensure proper functioning of the assay.
PC10: the concentration of a test chemical at which the measured activity in an agonist assay is 10% of the
maximum activity induced by the PC (E2 at 1nM for the STTA assay) in each plate.
PC50: the concentration of a test chemical at which the measured activity in an agonist assay is 50% of the
maximum activity induced by the PC (E2 at the reference concentration specified in the test method) ineach plate.
PCMax: the concentration of a test chemical inducing the RPCMax
Performance standards: Standards, based on a validated test method, that provide a basis for evaluating
the comparability of a proposed test method that is mechanistically and functionally similar. Included are(1) essential test method components; (2) a minimum list of reference chemicals selected from among thechemicals used to demonstrate the acceptable performance of the validated test method; and (3) thecomparable levels of accuracy and reliability, based on what was obtained for the validated test method,that the proposed test method should demonstrate when evaluated using the minimum list of reference
chemicals (1).
Proficiency substances: A subset of the reference substances included in the Performance Standards thatcan be used by laboratories to demonstrate technical competence with a standardised test method.
Selection criteria for these substances typically include that they represent the range of responses, arecommercially available, and have high quality reference data available.
Proficiency: The demonstrated ability to properly conduct a test method prior to testing unknown
substances.
Reference estrogen (Positive control, PC): 17β -estradiol (E2, CAS 50-28-2).
Reference standard: a reference substance used to demonstrate the adequacy of a test method. 17βestradiol is the reference standard for the STTA and BG1Luc ER TA assays.
Reference test methods: The test methods upon which this PBTG is based.
Relevance: Description of relationship of the test to the effect of interest and whether it is meaningful anduseful for a particular purpose. It is the extent to which the test correctly measures or predicts the
biological effect of interest. Relevance incorporates consideration of the accuracy (concordance) of a test
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method (1).
Reliability: Measure of the extent that a test method can be performed reproducibly within and between
laboratories over time, when performed using the same protocol. It is assessed by calculating intra- andinter-laboratory reproducibility.
RLU: Relative Light Units
RNA: Ribonucleic Acid
RPCMax: maximum level of response induced by a test chemical, expressed as a percentage of the response
induced by 1 nM E2 on the same plate
RPMI: RPMI 1640 medium supplemented with 0.9% Pen-Strep and 8.0% fetal bovine serum (FBS)
SD: Standard deviation.
Sensitivity: The proportion of all positive/active substances that are correctly classified by the test. It is ameasure of accuracy for a test method that produces categorical results, and is an important considerationin assessing the relevance of a test method (1).
Specificity: The proportion of all negative/inactive substances that are correctly classified by the test. It isa measure of accuracy for a test method that produces categorical results, and is an important considerationin assessing the relevance of a test method (1).
Stable transfection: When DNA is transfected into cultured cells in such a way that it is stably integrated
into the cells genome, resulting in the stable expression of transfected genes. Clones of stably transfected
cells are selected by stable markers (e.g. resistance to G418).
STTA Assay: Stably Transfected Transactivation Assay, the ERα transcriptional activation assay using the
HeLa 9903 Cell Line.
Substance: Used in the context of the UN GHS (1) as chemical elements and their compounds in thenatural state or obtained by any production process, including any additive necessary to preserve thestability of the product and any impurities deriving from the process used, but excluding any solvent which
may be separated without affecting the stability of the substance or changing its composition.
TA (Transactivation): The initiation of mRNA synthesis in response to a specific chemical signal, such asa binding of an estrogen to the estrogen receptor
Transcription: mRNA synthesis
Validated test method: A test method for which validation studies have been completed to determine therelevance (including accuracy) and reliability for a specific purpose. It is important to note that a validated
test method may not have sufficient performance in terms of accuracy and reliability to be foundacceptable for the proposed purpose (1).
Validation: The process by which the reliability and relevance of a particular approach, method, process or
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assessment is established for a defined purpose (1).
VC (Vehicle control): The solvent that is used to dissolve test and control chemi cal s is tested solely as
vehicle without dissolved chemical.
Weak positive control: A weakly active substance selected from the reference chemicals list that isincluded in all tests to help ensure proper functioning of the assay.

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OECD/OCDEANNEX 2

Stably Transfected Human Estrogen Receptor-α Transactivation Assay for Detection of Estrogenic
Agonist and antagonist Activity of Chemicals using the hERα-HeLa-9903 cell line

INITIAL CONSIDERATIONS AND LIMITATIONS (See also GENERAL INTRODUCTION, page1)
1.
This transactivation (TA) assay uses the hERα-HeLa-9903 cell line to detect estrogenic agonist
activity mediated through human estrogen receptor alpha (hERα). The validation study of the Stably
Transfected Transactivation (STTA) Assay by the Japanese Chemicals Evaluation and Research Institute(CERI) using the hERα-HeLa-9903 cell line to detect estrogenic agonist and antagonist activity mediatedthrough human estrogen receptor alpha (hERα) demonstrated the relevance and reliability of the assay
for its intended purpose (1).
2.
This test method is specifically designed to detect hERα-mediated TA by measuringchemiluminescence as the endpoint. However, non-receptor-mediated luminescence signals have beenreported at phytoestrogen concentrations higher than 1 μM due to the over-activation of the luciferase
reporter gene (2) (3). While the dose-response curve indicates that true activation of the ER system occurs
at lower concentrations, luciferase expression obtained at high concentrations of phytoestrogens or similar
compounds suspected of producing phytoestrogen-like over-activation of the luciferase reporter gene needs
to be examined carefully in stably transfected ER TA assay systems (Appendix 1).
3.
The “GENERAL INTRODUCTION” and “ER TA TEST METHOD COMPONENTS” (pages
1-14) should be read before using this test method for regulatory purposes. Definitions and abbreviations
used in this TG are described in Annex 1.
PRINCIPLE OF THE TEST METHOD (See also GENERAL INTRODUCTION, page 1)
4.
The assay is used to signal binding of the estrogen receptor with a ligand. Following ligand binding,
the receptor-ligand complex translocates to the nucleus where it binds specific DNA response elements andtransactivates a firefly luciferase reporter gene, resulting in increased cellular expression of luciferase
enzyme. Luciferin is a substrate that is transformed by the luciferase enzyme to a bioluminescence productthat can be quantitatively measured with a luminometer. Luciferase activity can be evaluated quickly andinexpensively with a number of commercially available test kits.
5.
The test system utilises the hERα-HeLa-9903 cell line, which is derived from a human cervicaltumor, with two stably inserted constructs: (i) the hERD expression construct (encoding the full-length
human receptor), and (ii) a firefly luciferase reporter construct bearing five tandem repeats of a vitellogenin
Estrogen-Responsive Element (ERE) driven by a mouse metallothionein (MT) promoter TATA element.
The mouse MT TATA gene construct has been shown to have the best performance, and so is commonly
used. Consequently this hERα-HeLa-9903 cell line can measure the ability of a test chemical to induce
hERα-mediated transactivation of luciferase gene expression.
6.

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level induced by a test chemical equals or exceeds an agonist response equal to 10% of that induced bya maximally inducing (1 nM) concentration of the positive control (PC) 17β estradiol (E2) (i.e. the PC10).
In case of ER antagonist assay, data interpretation is based upon whether or not the response shows at
least a 30% reduction in activity from the response induced by the spike in control (25 pM of E2) without
cytotoxicity. Data analysis and interpretation are discussed in detail in paragraphs 34 - 48.
PROCEDURE
Cell Lines
7.
The stably transfected hERα-HeLa-9903 cell line should be used for the assay. The cell line can beobtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank1, upon signing aMaterial Transfer Agreement (MTA).
8.
Only cells characterised as mycoplasma-free should be used in testing. RT-PCR (Real Time
Polymerase Chain Reaction) is the method of choice for a sensitive detection of mycoplasma infection (4)(5) (6).
Stability of the cell line
9.
To monitor the stability of the cell line, E2, 17α-estradiol, 17α-methyltestosterone and corticosterone
should be used as the reference substances for agonist assay and a complete concentration-response curve inthe test concentration range provided in Table 1 should be measured at least once each time the assay is
performed, and the results should be in agreement with the results provided in Table 1.
10. In case of antagonist assay, complete concentration curves for two reference substances, tamoxifen
and flutamide, should be measured simultaneously with each run. Correct qualitative classification as
positive or negative for the two chemicals should be monitored.
Cell Culture and Plating Conditions
11. Cells should be maintained in Eagle’s Minimum Essential Medium (EMEM) without phenol red,
supplemented with 60 mg/L of antibiotic kanamycine and 10% dextran-coated-charcoal-treated fetal bovine
serum (DCC-FBS), in a CO2 incubator (5% CO2) at 37±1˚C. Upon reaching 75 -90% confluency, cells canbe subcultured at 10 mL of 0.4 x 105 – 1 x 105 cells/mL for 100 mm cell culture dish. Cells should besuspended with 10% FBS-EMEM (which is the same as EMEM with DCC-FBS) and then plated into
wells of a microplate at a density of 1 x 104 cells/(100 μL x well). Next, the cells should be pre-incubated ina 5% CO2 incubator at 37˚±1˚C for 3 hours before the chemical exposure. The plastic-ware should be freeof estrogenic activity.
12. To maintain the integrity of the response, the cells should be grown for more than one passage fromthe frozen stock in the conditioned media and should not be cultured for more than 40 passages. For thehERα-HeLa-9903 cell line, this will be less than three months. However the performance of cells may be
reduced if they are grown in inappropriate culture conditions.
1

JCRB Cell Bank : National Institute of Biomedical Innovation, 7-6-8 Asagi Saito, Ibaraki-shi, Osaka 567-0085,
Japan Fax: +81-72-641-9812

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The DCC-FBS can be prepared as described in Appendix 2, or obtained from commercial sources.

Acceptability criteria
Positive and negative reference substances for ER agonist assay
14.
Prior to and during the study, the responsiveness of the test system should be verified using theappropriate concentrations of a strong estrogen: E2, a weak estrogen (17α-estradiol), a very weak agonist(17α-methyltestosterone), and a negative substance (corticosterone). Acceptable range values derived from thevalidation study (1) are given in Table 1. These 4 concurrent reference substances should be included witheach experiment and the results should fall within the given acceptable limits. If this is not the case, the cause
for the failure to meet the acceptability criteria should be determined (e.g. cell handling, and serum andantibiotics for quality and concentration) and the assay repeated. Once the acceptability criteria have beenachieved, to ensure minimum variability of EC50, PC50 and PC10 values, consistent use of materials for cell
culturing is essential. The four concurrent reference substances, which should be included in each experiment(conducted under the same conditions including the materials, passage level of cells and technicians), canensure the sensitivity of the assay because the PC10s of the three positive reference substances should fallwithin the acceptable range, as should the PC50s and EC50s where they can be calculated (see Table 1).
Table 1. Acceptable range values of the four reference substances for the ER agonist assay
NamelogPC50

logPC10logEC50

Hill slope

Test range

17β-Estradiol (E2)CAS No: 50-28-2-11.4 ~ -10.1<-11-11.3 ~ -10.1

0.7 ~ 1.5

10-14 ~ 10-8 M

17α-Estradiol
CAS No: 57-91-0-9.6 ~ -8.1

-10.7 ~ -9.3

-9.6 ~ -8.4

0.9 ~ 2.0

10-12 ~ 10-6 M

Corticosterone
CAS No: 50-22-6

–

–

–

–

10-10 ~ 10-4M-6.0 ~ -5.1

-8.0 ~ -6.2––

10-11 ~ 10-5 M

17α-Methyltestosterone
CAS No: 58-18-4

Positive and negative reference substances for ER antagonist assay
15. Prior to and during the study, the responsiveness of the test system should be verified using theappropriate concentrations of a positive substance (Tamoxifen), and a negative substance (Flutamide).
Acceptable range values derived from the validation study (1) are given in Table 2. These two
concurrent reference substances should be included with each experiment and the results should be
judged correctly as shown in the criteria. If this is not the case, the cause for the failure to meet the criteria
should be determined (e.g. cell handling, and serum and antibiotics for quality and concentration) and theassay repeated. In addition, IC50 values for a positive substance (Tamoxifen) should be calculated and theresults should fall within the given acceptable limits. Once the acceptability criteria have been achieved, toensure minimum variability of IC50 values, consistent use of materials for cell culturing is essential. The two
concurrent reference substances, which should be included in each experiment (conducted under the same
conditions including the materials, passage level of cells and technicians), can ensure the sensitivity of
the assay (see Table 2).
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Table 2. Criteria and acceptable range values of the two reference substances for the ER antagonist assay
Name

Criteria

LogIC50

Test range

Tamoxifen
CAS No: 10540-29-1

Positive:
IC50 should be calculated-5.942㹼 -7.596

10-10㹼10-5 M

Flutamide
CAS No: 13311-84-7

Negative:
IC30 should not be calculated-

10-10 㹼10-5 M

Positive and Vehicle Controls
16. The positive control (PC) for ER agonist assay (1 nM of E2) and for ER antagonist assay (10μM
TAM) should be tested at least in triplicate in each plate. The vehicle that is used to dissolve a test
chemical should be tested as a vehicle control (VC) at least in triplicate in each plate. In addition tothis VC, if the PC uses a different vehicle than the test chemical, another VC should be tested at least in
triplicate on the same plate with the PC.
Quality criteria for ER agonist assay
17. The mean luciferase activity of the positive control (1 nM E2) should be at least 4-fold that of themean VC on each plate. This criterion is established based on the reliability of the endpoint values fromthe validation study (historically between four- and 30-fold).
18. With respect to the quality control of the assay, the fold-induction corresponding to the PC10 value ofthe concurrent PC (1 nM E2) should be greater than 1+2SD of the fold-induction value (=1) of the
concurrent VC. For prioritisation purposes, the PC10 value can be useful to simplify the data analysis
required compared to a statistical analysis. Although a statistical analysis provides information on
significance, such an analysis is not a quantitative parameter with respect to concentration-based potential,
and so is less useful for prioritisation purposes.
Quality criteria for ER antagonist assay
19. The mean luciferase activity of the spike in control (25 pM E2) should be at least 4-fold that of themean VC on each plate. This criterion is established based on the reliability of the endpoint values fromthe validation study.
20. With respect to the quality control of the assay, relative transcriptional activation (RTA) of 1 nM E2should be greater than 100%, RTA of 1μM 4-Hydroxytamoxifen (OHT) should be less than 40.6% and
RTA of 100 μM Digitonin (Dig) should be less than 0%.
Substances to Demonstrate Laboratory Proficiency (see paragraph 14 and Table 2 in « ER TA TESTMETHOD COMPONENTS» of this Test Guideline (pages 6-14)).
Vehicle21.

Dimethyl sulfoxide (DMSO), or appropriate solvent, at the same concentration used for the different
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positive and negative controls and the test chemicals should be used as the concurrent VC. Test chemicals
should be dissolved in a solvent that solubilises that test chemical and is miscible with the cell medium.
Water, ethanol (95% to 100% purity) and DMSO are suitable vehicles. If DMSO is used, the level should notexceed 0.1% (v/v). For any vehicle, it should be demonstrated that the maximum volume used is not
cytotoxic and does not interfere with assay performance.
Preparation of Test Chemicals
22. Generally, the test chemicals should be dissolved in DMSO or other suitable solvent, and seriallydiluted with the same solvent at a common ratio of 1:10 in order to prepare solutions for dilution withmedia.
Solubility and Cytotoxicity: Considerations for Range Finding.
23. A preliminary test should be carried out to determine the appropriate concentration range ofchemical to be tested, and to ascertain whether the test chemical may have any solubility and cytotoxicity
problems. Initially, chemicals are tested up to the maximum concentration of 1 μL/mL, 1 mg/mL, or 1
mM, whichever is the lowest. Based on the extent of cytotoxicity or lack of solubility observed in the
preliminary test, the first definite run should test the chemical at log-serial dilutions starting at the
maximum acceptable concentration (e.g. 1 mM, 100μM, 10μM, etc.) and the presence of cloudiness or
precipitate or cytotoxicity noted. Concentrations in the second, and if necessary third run should be
adjusted as appropriate to better characterise the concentration-response curve and to avoid concentrations
which are found to be insoluble or to induce excessive cytotoxicity.
24. For ER agonists and antagonists, the presence of increasing levels of cytotoxicity can significantly
alter or eliminate the typical sigmoidal response and should be considered when interpreting the data.
Cytotoxicity testing methods that can provide information regarding 80% cell viability should be used,utilising an appropriate assay based upon laboratory experience.
25. Should the results of the cytotoxicity test show that the concentration of the test chemical has
reduced the cell number by 20% or more, this concentration should be regarded as cytotoxic, and theconcentrations at or above the cytotoxic concentration should be excluded from the evaluation.
Chemical Exposure and Assay Plate Organisation
26. The procedure for chemical dilutions (Steps-1 and 2) and exposure to cells (Step-3) can be
conducted as follows:
Step-1: Each test chemical should be serially diluted in DMSO, or appropriate solvent, and added to thewells of a microtitre plate to achieve final serial concentrations as determined by the
preliminary range finding test (typically in a series of, for example 1 mM, 100 μM, 10 μM,
1μM, 100 nM, 10 nM, 1 nM, 100 pM, and 10 pM (10-3-10-11 M)) for triplicate testing.
Step-2: Chemical dilution: First dilute 1.5 μL of the test chemical in the solvent to a concentration of
500 μL of media.
Step-3: Chemical exposure of the cells: Add 50 μL of dilution with media (prepared in Step-2) to anassay well containing 104 cells/100 μL/well.
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The recommended final volume of media required for each well is 150 μL. Test samples and reference
substances can be assigned as shown in Table 3 and Table 4.
Table 3: Example of plate concentration assignment of the reference substances in the assay plate in ER
agonist assay
Row

17α-Methyltestosterone

Corticosterone

1
2
3
4
5
6
A
conc 1 (10 μM)
100 μM→ →→→B
conc 2 (1 μM)
10 μM
→ →
→
→
conc 3 (100 nM) → →
1 μM
C
→
→
D
conc 4 (10 nM)
100 nM→ →→→conc 5 (1 nM)
10 nM
E
→ →
→
→
F
conc 6 (100 pM) → →
1 nM
→
→
G
conc 7 (10 pM)
100 pM
→ →
→
→
VC
H
→ →
→
→
→
VC: Vehicle control (0.1% DMSO); PC: Positive control (1 nM E2)

17α-Estradiol
7
1 μM
100 nM
10 nM
1 nM
100 pM
10 pM
1 pM
PC

8
→
→
→
→
→
→
→
→

E2
9
→
→
→
→
→
→
→
→

10
10 nM
1 nM
100 pM
10 pM
1 pM
0.1 pM
0.01 pM
→

11
→
→
→
→
→
→
→
→

12
→
→
→
→
→
→
→
→

27.
The reference substances (E2, 17α-Estradiol, 17D-methyl testosterone and corticosterone) should betested in every run (Table 3). PC wells treated with 1 nM of E2 that can produce maximum induction of E2
and VC wells treated with DMSO (or appropriate solvent) alone should be included in each test assay plate(Table 4). If cells from different sources (e.g. different passage number, different lot, etc.) are used in thesame experiment, the reference substances should be tested for each cell source.
Table 4: Example of plate concentration assignment of test and plate control chemicals in the assay plate in
ER agonist assay
Test Chemical 1

Test Chemical 2

Test Chemical 3

41 mM
100 μM
10 μM
1 μM
100 nM
10 nM
1 nM
→

7
1 μM
100 nM
10 nM
1 nM
100 pM
10 pM
1 pM
PC

Test Chemical 4

Row
A
B
C
D
E
F
G
H

1
conc 1 (10 μM)
conc 2 (1 μM)
conc 3 (100 nM)
conc 4 (10 nM)
conc 5 (1 nM)
conc 6 (100 pM)
conc 7 (10 pM)
VC

2
→
→
→
→
→
→
→
→

3
→
→
→
→
→
→
→
→

5
→
→
→
→
→
→
→
→

6
→
→
→
→
→
→
→
→

8
→
→
→
→
→
→
→
→

9
→
→
→
→
→
→
→
→

10
10 nM
1 nM
100 pM
10 pM
1 pM
0.1 pM
0.01 pM
→

11
→
→
→
→
→
→
→
→

12
→
→
→
→
→
→
→
→

VC: Vehicle control (0.1% DMSO); PC: Positive control (1 nM E2)

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Table 5: Example of plate concentration assignment of the reference substances in the assay plate in ER
antagonist assay
Row

Tamoxifen
1

Flutamide
3

4

5

6

7

Test Chemical 2
10

11

12

A

conc 1 (10 μM)

→

→

10 μM

→

→

10 μM

→

→

10 μM

→

→

B

conc 2 (1 μM)

→

→

1 μM

→

→

1 μM

→

→

1 μM

→

→

C

conc 3 (100 nM)→→

100 nM→→

100 nM→→

100 nM→→Dconc 4 (10 nM)

→

→

10 nM

→

→

10 nM

→

→

10 nM

→

→

E

conc 5 (1 nM)

→

→

1 nM

→

→

1 nM

→

→

1 nM

→

→

F

conc 6 (100 pM)

→

→

→

→

100 pM→→

100 pM→→

0.1% DMSO→→

100 pM→→→

1 μM OHT

→

→

→

VC

→

→

→

→

→

PC

→

→

100 μM Dig
→

→
→

→

㻌

㻌

㻌
㻌

G
H

2

Test Chemical 1
8

9

VC: Vehicle control (0.1% DMSO), PC: Positive control (1 nM E2), OHT :4-Hydroxytamoxifen, Dig: Digitonin.
㻌: Spiked with 25pM E2
㻌
㻌
㻌
㻌
㻌
㻌
㻌

28.
To evaluate the antagonist activity of chemicals, assay wells located in rows from A to G should be
spiked with 25pM E2. The reference substances (Tamoxifen and Flutamide) should be tested in every run.
PC wells treated with 1 nM of E2 that can be control quality of hERα-HeLa-9903 cell line, VC wells treatedwith DMSO (or appropriate solvent), 0.1% DMSO wells treated with DMSO addition to the spiked E2
corresponding to “Spike-in-control”, wells treated with final concentration 1 μM OHT and wells treatedwith 100 μM Dig should be included in each test assay plate (Table 5). Subsequent assay plate should
follow the same plate layout without reference substances wells (Table 6). If cells from different sources(e.g. different passage number, different lot, etc.) are used in the same experiment, the reference substances
should be tested for each cell source.
Table 6: Example of plate concentration assignment of test and plate control chemicals in the assay plate in
ER antagonist assay
Row

Test Chemical 1
1

Test Chemical 2

2

3

4

Test Chemical 3

5

6

7

Test Chemical 4

8

9

10

11

12

A

conc 1 (10 μM)

→

→

10 μM

→

→

10 μM

→

→

10 μM

→

→

B

conc 2 (1 μM)

→

→

1 μM

→

→

1 μM

→

→

1 μM

→

→

C

conc 3 (100 nM)→→

100 nM→→

100 nM→→

100 nM→→Dconc 4 (10 nM)

→

→

10 nM

→

→

10 nM

→

→

10 nM

→

→

E

conc 5 (1 nM)

→

→

1 nM

→

→

1 nM

→

→

1 nM

→

→

F

conc 6 (100 pM)

→

→

→

100 pM→→

100 pM→→G
H

0.1% DMSO→→

100 pM→→→→

1 μM OHT

→

→

→

→

→

→

→

→

PC

→

→

100 μM Dig
→

→

VC

→

→

㻌

㻌
㻌

VC: Vehicle control (0.1% DMSO), PC: Positive control (1 nM E2), OHT: 4-Hydroxytamoxifen, Dig: Digitonin.
㻌: Spiked with 25pM E2
㻌
㻌
㻌
㻌
㻌
㻌
㻌

㻌

29. The lack of edge effects should be confirmed, as appropriate, and if edge effects are suspected, theplate layout should be altered to avoid such effects. For example, a plate layout excluding the edge wells
can be employed.
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30. After adding the chemicals, the assay plates should be incubated in a 5% CO2 incubator at 37±1ºC
for 20-24 hours to induce the reporter gene products.
31. Special considerations will need to be applied to those compounds that are highly volatile. In suchcases, nearby control wells may generate false positives and this should be considered in light of expected
and historical control values. In the few cases where volatility may be of concern, the use of “plate sealers”
may help to effectively isolate individual wells during testing, and is therefore recommended in such cases.
32. Repeat definitive tests for the same chemical should be conducted on different days, to ensure
independence.
Luciferase assay
33. A commercial luciferase assay reagent [e.g. Steady-Glo® Luciferase Assay System (Promega, E2510,
or equivalents)] or a standard luciferase assay system (Promega, E1500, or equivalents) can be used forthe assay, as long as the acceptability criteria are met. The assay reagents should be selected based on thesensitivity of the luminometer to be used. When using the standard luciferase assay system, Cell Culture
Lysis Reagent (Promega, E1531, or equivalents) should be used before adding the substrate. The
luciferase reagent should be applied following the manufacturers’ instructions.
ANALYSIS OF DATA
ER agonist assay
34. In case of ER agonist assay, to obtain the relative transcriptional activity to PC (1 nM of E2), theluminescence signals from the same plate can be analysed according to the following steps (otherequivalent mathematical processes are also acceptable):
Step 1. Calculate the mean value for the VC.
Step 2. Subtract the mean value of the VC from each well value to normalise the data.
Step 3. Calculate the mean for the normalised PC.
Step 4. Divide the normalised value of each well in the plate by the mean value of the normalised PC(PC=100%).
The final value of each well is the relative transcriptional activity for that well compared to the PC
response.
Step 5. Calculate the mean value of the relative transcriptional activity for each concentration group of thetest chemical. There are two dimensions to the response: the averaged transcriptional activity (response)
and the concentration at which the response occurs (see following section).
EC50, PC50 and PC10 induction considerations
35.

The full concentration-response curve is required for the calculation of the EC50, but this may not
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always be achievable or practical due to limitations of the test concentration range (for example due tocytotoxicity or solubility problems). However, as the EC50 and maximum induction level (corresponding to
the top value of the Hill-equation) are informative parameters, these parameters should be reported where
possible. For the calculation of EC50 and maximum induction level, appropriate statistical software should
be used (e.g. Graphpad Prism statistical software).
36. If the Hill’s logistic equation is applicable to the concentration response data, the EC50 should be
calculated by the following equation (7):
Y=Bottom + (Top-Bottom) / (1+10 exp ((log EC50 -X) x Hill slope))
Where:
X is the logarithm of concentration; and,Y is the response and Y starts at the Bottom and goes to the Top in a sigmoid curve.
Bottom is fixed at zero in the Hill’s logistic equation.
37.

For each test chemical, the following should be provided:(i) The RPCMax which is the maximum level of response induced by a test chemical, expressed as apercentage of the response induced by 1 nM E2 on the same plate, as well as the PCMax (concentration
associated with the RPCMax); and(ii) For positive chemicals, the concentrations that induce the PC10 and, if appropriate, the PC50.
38.
The PCx value can be calculated by interpolating between 2 points on the X-Y coordinate, one
immediately above and one immediately below a PCx value. Where the data points lying immediately
above and below the PCx value have the coordinates (a,b) and (c,d) respectively, then the PCx value may
be calculated using the following equation:log[PCx] = log[c]+(x-d)/(d-b)
39.

Descriptions of PC values are provided in Figure 1 below.

Figure 1: Example of how to derive PC-values. The PC (1 nM of E2) is included on each assay plate

6

5

1 nM E2

Fold induction(against VC)

fold induction of PC (1 nM E2)

fold induction corresponding to PC10
mean (=1) + 2SD of fold induction of VC

3

50㸣
2

10㸣
0㸣

1

mean (=1) of fold induction of VC

0

10 pM 100 pM 1 nM 10 nM 100 nM 1 μM 10 μM

PC10 PC50

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ER antagonist assay
40. In case of ER antagonist assay, to obtain the relative transcriptional activity (RTA) to spike incontrol (25 pM of E2), the luminescence signals from the same plate can be analysed according to thefollowing steps (other equivalent mathematical processes are also acceptable):
Step 1. Calculate the mean value for the VC.
Step 2. Subtract the mean value of the VC from each well value to normalise the data.
Step 3. Calculate the mean for the normalised spike in control.
Step 4. Divide the normalised value of each well in the plate by the mean value of the normalised
spike in control (spike in control=100%).
The final value of each well is the relative transcriptional activity for that well compared to the spikein control response.
Step 5. Calculate the mean value of the relative transcriptional activity for each treatment.
IC30 and IC50 induction considerations
41.

For positive chemicals, the concentrations that induce the IC30 and, if appropriate, the IC50 should be
provided.

42. The ICx value can be calculated by interpolating between 2 points on the X-Y coordinate, one
immediately above and one immediately below a ICx value. Where the data points lying immediately
above and below the ICx value have the coordinates (c,d) and (a,b) respectively, then the ICx value may
be calculated using the following equation:
lin ICx = a-(b-(100-x)) (a-c) /(b-d)
Figure 2: Example of how to derive IC-values. The spike in control (25 pM of E2) is included on eachassay plate

RTA: relative transcriptional activity

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43. The results should be based on two (or three) independent runs. If two runs give comparable
and therefore reproducible results, it is not necessary to conduct a third run. To be acceptable, theresults should:
xMeet the acceptability criteria (see Acceptability criteria para 14-20),x

Be reproducible.

Data Interpretation Criteria
Table 7: Positive and negative decision criteria in ER agonist assay
Positive
Negative

If the RPCMax is obtained that is equal to or exceeds 10% of the
response of the positive control in at least two of two or two of threeruns.
If the RPCMax fails to achieve at least 10% of the response of thepositive control in two of two or two of three runs.

Table 8: Positive and negative decision criteria in ER antagonist assay
Positive

If the IC30 is calculated in at least two of two or two of three runs.

Negative

If the IC30 fails to calculate in two of two or two of three runs.

44.
Data interpretation criteria are shown in Tables 7 and 8. Positive results will be characterised by boththe magnitude of the effect and the concentration at which the effect occurs. Expressing results as aconcentration at which a 50% (PC50) or 10% (PC10) of PC values are reached for the agonist assay, and 50%(IC50) or 30% (IC30) of the spike-in control value is inhibited for the antagonist assay, accomplishes both ofthese goals. However, a test chemical is determined to be positive, if the maximum response induced by thetest chemical (RPCMax) is equal to or exceeds 10% of the response of the PC in at least two of two or twoof three runs, while a test chemical is considered negative if the RPCMax fails to achieve at least 10% of
the response of the positive control in two of two or two of three runs.
45.
The calculations of PC10, PC50 and PCMax in ER agonist assay and IC30 and IC50 in ER antagonist
assay can be made by using a spreadsheet available with the Test Guideline on the OECD public website2.
46.
It should be sufficient to obtain PC10 or PC50 and IC30 or IC50 values at least twice. However,should the resulting base-line for data in the same concentration range show variability with anunacceptably high coefficient of variation (CV; %) the data may not be considered reliable and the sourceof the high variability should be identified. The CV of the raw data triplicates (i.e. luminescence intensity
data) of the data points that are used for the calculation of PC10 should be less than 20%.
47.
2

Meeting the acceptability criteria indicates the assay system is operating properly, but it does not

http://www.oecd.org/env/testguidelines

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ensure that any particular run will produce accurate data. Duplicating the results of the first run is the best
insurance that accurate data were produced.
48.
In case of ER agonist assay, where more information is required in addition to the screening andprioritisation purposes of this TG for positive test chemicals, particularly for PC10-PC49 chemicals, aswell as chemicals suspected to over-stimulate luciferase, it can be confirmed that the observed luciferaseactivity is solely an ERα-specific response, using an ERα antagonist (see Appendix 1).
TEST REPORT49.
See paragraph 20 of “ER TA TEST METHOD COMPONENTS” (Pages 6-14 of this Test
Guideline).

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OECD/OCDELITERATURE (2)1. CERI. (2006). Draft Validation Report of TA Assay Using HeLa-hER-9903 to Detect Estrogenic
Activity. Available at:[http://www.oecd.org/document/62/0,3343,en_2649_34377_2348606_1_1_1_1,00.html].
2. Escande A., et al. (2006). “Evaluation of Ligand Selectivity Using Reporter Cell Lines Stably Expressing
Estrogen Receptor Alpha or Beta”, Biochem. Pharmacol., 71, 1459-1469.
3. Kuiper G.G., et al. (1998). “Interaction of Estrogenic Chemicals and Phytoestrogens with Estrogen
Receptor Beta”, Endocrinol., 139, 4252-4263.
4. Spaepen M., et al. (1992). “Detection of Bacterial and Mycoplasma Contamination in Cell Cultures by
Polymerase Chain Reaction”, FEMS Microbiol. Lett., 78(1), 89-94.
5. Kobayashi H., et al. (1995). “Rapid Detection of Mycoplasma Contamination in Cell Cultures by
Enzymatic Detection of Polymerase Chain Reaction (PCR) Products”, J. Vet. Med. Sci., 57(4), 769- 71.
6. Dussurget O. and Roulland-Dussoix D. (1994). “Rapid, Sensitive PCR-Based Detection of
Mycoplasmas in Simulated Samples of Animal Sera”, Appl. Environ. Microbiol., 60(3), 953-9.
7. De Lean A., Munson P.J. and Rodbard D. (1978). Simultaneous Analysis of Families of Sigmoidal
Curves: Application to Bioassay, Radioligand Assay, and Physiological Dose-Response Curves, Am. J.
Physiol., 235, E97-El02.

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Appendix 1
False positives: Assessment of non-receptor mediated luminescence signals
1.
False positives in the ER agonist assay might be generated by non-ER-mediated activation of theluciferase gene, or direct activation of the gene product or unrelated fluorescence. Such effects areindicated by an incomplete or unusual dose-response curve. If such effects are suspected, the effect of an
ER antagonist (e.g. 4- hydroxytamoxifen (OHT) at non-toxic concentration) on the response should be
examined. The pure antagonist ICI 182780 may not be suitable for this purpose as a sufficientconcentration of ICI 182780 may decrease the VC value, and this will affect the data analysis.
2.
x
x
x
x
x
3.

To ensure validity of this approach, the following needs to be tested in the same plate:
Agonistic activity of the unknown chemical with / without 10 μM of OHT
VC (in triplicate)
OHT (in triplicate)
1 nM of E2 (in triplicate) as agonist PC
1 nM of E2 + OHT (in triplicate)
Data interpretation criteria

Note: All wells should be treated with the same concentration of the vehicle.
x
If the agonistic activity of the unknown chemical is NOT affected by the treatment with ER
antagonist, it is classified as “Negative”.
x
If the agonistic activity of the unknown chemical is completely inhibited, apply the decision criteria.
x
If the agonistic activity at the lowest concentration is equal to, or is exceeding, PC10 response theunknown chemical is inhibited equal to or exceeding PC10 response. The difference in the responses
between the non-treated and treated wells with the ER antagonist is calculated and this difference
should be considered as the true response and should be used for the calculation of the appropriate
parameters to enable a classification decision to be made.
4.

Data analysis

Check the performance standard.
Check the CV between wells treated under the same conditions.
1. Calculate the mean of the VC
2. Subtract the mean of VC from each well value not treated with OHT
3. Calculate the mean of OHT
4. Subtract the mean of the VC from each well value treated with OHT
5. Calculate the mean of the PC
6. Calculate the relative transcriptional activity of all other wells relative to the PC.

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Preparation of Serum treated with Dextran Coated Charcoal (DCC)

1.
The treatment of serum with dextran-coated charcoal (DCC) is a general method for removal of
estrogenic compounds from serum that is added to cell medium, in order to exclude the biased response
associated with residual estrogens in serum. 500 mL of fetal bovine serum (FBS) can be treated by thisprocedure.
Components
2.

The following materials and equipment will be required:
Materials
Activated charcoal
DextranMagnesium chloride hexahydrate (MgCl2·6H2O)
Sucrose1 M HEPES buffer solution (pH 7.4)
Ultrapure water produced from a filter system
Equipment
Autoclaved glass container (size should be adjusted as appropriate)
General Laboratory Centrifuge (that can set temperature at 4°C)

Procedure3.

The following procedure is adjusted for the use of 50 mL centrifuge tubes:[Day-1] Prepare dextran-coated charcoal suspension with 1 L of ultrapure watercontaining 1.5 mM of MgCl2, 0.25 M sucrose, 2.5 g of charcoal, 0.25 g dextran and 5
mM of HEPES and stir it at 4°C, overnight.
[Day-2] Dispense the suspension in 50 mL centrifuge tubes and centrifuge at 10000
rpm at 4°C for 10 minutes. Remove the supernatant and store half of the charcoal
sediment at 4°C for the use on Day-3. Suspend the other half of the charcoal with FBS
that has been gently thawed to avoid precipitation, and heat-inactivated at 56°C for 30
minutes, then transfer into an autoclaved glass container such as an Erlenmeyer flask.
Stir this suspension gently at 4°C, overnight.
[Day-3] Dispense the suspension with FBS into centrifuge tubes for centrifugation at
10000 rpm at 4°C for 10 minutes. Collect FBS and transfer into the new charcoal
sediment prepared and stored on Day-2. Suspend the charcoal sediment and stir thissuspension gently in an autoclaved glass container at 4°C, overnight.
[Day-4] Dispense the suspension for centrifugation at 10000 rpm at 4°C for 10 minutes
and sterilise the supernatant by filtration through 0.2 μm sterile filter. This DCC treated
FBS should be stored at -20°C and can be used for up a year.
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ANNEX 3
BG1Luc Estrogen Receptor Transactivation Test Method for Identifying Estrogen Receptor Agonists
and Antagonists

INITIAL CONSIDERATIONS AND LIMITATIONS (See also GENERAL INTRODUCTION, page 1)
1.
This assay uses the BG1Luc4E2 cell line. It has been validated by the National Toxicology Program
Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM), and theInteragency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) (1). The BG1Luc
cell lines predominantly express endogenous ERα and a minor amount of endogenous ERβ (2) (3) (4).
2.
This assay is applicable to a wide range of substances, provided they can be dissolved in dimethyl
sulfoxide (DMSO; CASRN 67-68-5), do not react with DMSO or the cell culture medium, and are notcytotoxic at the concentrations being tested. If use of DMSO is not possible, another vehicle such as ethanol
or water may be used (see paragraph 12). The demonstrated performance of the BG1Luc ER TA (ant)agonist
test method suggests that data generated with this test method may inform upon ER mediated mechanisms ofaction and could be considered for prioritisation of substances for further testing.
3.
This test method is specifically designed to detect hERD and hERß-mediated TA by measuringchemiluminescence as the endpoint. Chemiluminescence use in bioassays is widespread because
luminescence has a high signal-to-background ratio (10). However, the activity of firefly luciferase in cellbased assays can be confounded by substances that inhibit the luciferase enzyme, causing both apparent
inhibition or increased luminescence due to protein stabilisation (10). In addition, in some luciferase-based
ER reporter gene assays, non-receptor-mediated luminescence signals have been reported at phytoestrogenconcentrations higher than 1 μM due to the over-activation of the luciferase reporter gene (9) (11). While thedose-response curve indicates that true activation of the ER system occurs at lower concentrations, luciferase
expression obtained at high concentrations of phytoestrogens or similar compounds suspected of producing
phytoestrogen-like over-activation of the luciferase reporter gene needs to be examined carefully in stablytransfected ER TA assay systems (see Annex 2).
4.
The “GENERAL INTRODUCTION” and “ER TA TEST METHOD COMPONENTS” (pages
1-15) should be read before using this test method for regulatory purposes. Definitions and abbreviations usedin this TG are described in Annex 1.
PRINCIPLE OF THE TEST METHOD (See also GENERAL INTRODUCTION, page 1)
5.
The assay is used to indicate ER ligand binding, followed by translocation of the receptor-ligand
complex to the nucleus. In the nucleus, the receptor-ligand complex binds to specific DNA response elements
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and transactivates the reporter gene (luc), resulting in the production of luciferase and the subsequentemission of light, which can be quantified using a luminometer. Luciferase activity can be quickly andinexpensively evaluated with a number of commercially available kits. The BG1Luc ER TA utilises an ER
responsive human ovarian adenocarcinoma cell line, BG-1, which has been stably transfected with a firefly
luc reporter construct under control of four estrogen response elements placed upstream of the mouse
mammary tumour virus promoter (MMTV), to detect substances with in vitro ER agonist or antagonist
activity. This MMTV promoter exhibits only minor cross-reactivity with other steroid and non-steroid
hormones (8). Criteria for data interpretation are described in detail in paragraph 41. Briefly, a positive
response is identified by a concentration-response curve containing at least three points with non-overlapping
error bars (mean ± SD), as well as a change in amplitude (normalised relative light unit [RLU]) of at least
20% of the maximal value for the reference substance (17E-estradiol [E2; CASRN 50-28-2] for the agonist
assay, raloxifene HCl [Ral; CASRN 84449-90-1]/E2 for the antagonist assay).
PROCEDURE
Cell Line6.
The stably transfected BG1Luc4E2 cell line should be used for the assay. The cell line is currently
only available with a technical licensing agreement from the University of California, Davis, California,
USA3, and from Xenobiotic Detection Systems Inc., Durham, North Carolina, USA4.
Stability of the Cell Line
7.
To maintain the stability and integrity of the cell line, the cells should be grown for more than one
passage from the frozen stock in cell maintenance media (see paragraph 9). Cells should not be cultured formore than 30 passages. For the BG1Luc4E2 cell line, 30 passages will be approximately three months.
Cell Culture and Plating Conditions
8.
Procedures specified in the Guidance on Good Cell Culture Practice (5) (6) should be followed toassure the quality of all materials and methods in order to maintain the integrity, validity, and reproducibilityof any work conducted.
9.
BG1Luc4E2 cells are maintained in RPMI 1640 medium supplemented with 0.9% Pen-Strep and
8.0% fetal bovine serum (FBS) in a dedicated tissue culture incubator at 37ºC ± 1ºC, 90% ± 5% humidity,
and 5.0% ± 1% CO2/air.
10.
Upon reaching ~80% confluence, BG1Luc4E2 cells are subcultured and conditioned to an estrogenfree environment for 48 hours prior to plating the cells in 96-well plates for exposure to test chemicals andanalysis of estrogen dependent induction of luciferase activity. The estrogen-free medium (EFM) contains
Dulbecco’s Modification of Eagle’s Medium (DMEM) without phenol red, supplemented with 4.5%
3

Michael S. Denison, Ph.D. Professor, Dept. of Environmental Toxicology, 4241 Meyer Hall, One Shields Ave,
University of California, Davis, CA 95616, E: msdenison@ucdavis.edu, (530) 754-8649

4

Xenobiotic Detection Systems Inc. 1601 East Geer Street, Suite S, Durham NC, 27704 USA, email:info@dioxins.com, Telephone: 919-688-4804, Fax: 919-688-4404

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charcoal/dextran-treated FBS, 1.9% L-glutamine, and 0.9% Pen-Strep. All plasticware should be free of
estrogenic activity [see detailed protocol (7)].
Acceptability Criteria
11.
Acceptance or rejection of a test is based on the evaluation of reference standard and control resultsfrom each experiment conducted on a 96-well plate. Each reference standard is tested in multiple
concentrations and there are multiple samples of each reference and control concentration. Results arecompared to quality controls (QC) for these parameters that were derived from the agonist and antagonist
historical databases generated by each laboratory during the demonstration of proficiency. The historical
databases are updated with reference standard and control values on a continuous basis. Changes inequipment or laboratory conditions may necessitate generation of updated historical databases.
Agonist Test
Range Finder Testxxx

Induction: Plate induction should be measured by dividing the average highest E2 reference standard
relative light unit (RLU) value by the average DMSO control RLU value. Five-fold induction is usuallyachieved, but for purpose of acceptance, induction should be greater than or equal to four-fold.
DMSO control results: Solvent control RLU values should be within 2.5 times the standard deviation of
the historical solvent control mean RLU value.
An experiment that fails either acceptance criterion should be discarded and repeated.

Comprehensive Test
It includes acceptability criteria from the agonist range finder test and the following:x Reference standard results: The E2 reference standard concentration-response curve should be sigmoidal
in shape and have at least three values within the linear portion of the concentration-response curve.
x Positive control results: Methoxychlor control RLU values should be greater than the DMSO mean plus
three times the standard deviation from the DMSO mean.
x An experiment that fails any single acceptance criterion should be discarded and repeated.
Antagonist Test
Range Finder Test
x

x
x
x

Reduction: Plate reduction is measured by dividing the average highest Ral/E2 reference standard
RLU value by the average DMSO control RLU value. Five-fold reduction is usually achieved,
but for the purposes of acceptance, reduction should be greater than or equal to three-fold.
E2 control results: E2 control RLU values should be within 2.5 times the standard deviation of
the historical E2 control mean RLU value.
DMSO control results: DMSO control RLU values should be within 2.5 times the standard
deviation of the historical solvent control mean RLU value.
An experiment that fails any single acceptance criterion will be discarded and repeated.

Comprehensive Test
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Reference standard results: The Ral/E2 reference standard concentration-response curve should
be sigmoidal in shape and have at least three values within the linear portion of theconcentration-response curve.
Positive control results: Tamoxifen/E2 control RLU values should be less than the E2 control
mean minus three times the standard deviation from the E2 control mean.
An experiment that fails any single acceptance criterion will be discarded and repeated.

Reference Standards, Positive, and Vehicle Controls
Vehicle Control (Agonist and Antagonist Assays)
12.
The vehicle that is used to dissolve the test chemicals should be tested as a vehicle control. The
vehicle used during the validation of the BG1Luc ER TA assay was 1% (v/v) dimethylsulfoxide (DMSO,CASRN 67-68-5) (see paragraph 24). If a vehicle other than DMSO is used, all reference standards, controls,
and test chemicals should be tested in the same vehicle, if appropriate.
Reference Standard (Agonist Range Finder)
13.
The reference standard is E2 (CASRN 50-28-2). For range finder testing, the reference standard iscomprised of a serial dilution of four concentrations of E2 (1.84 x 10 -10, 4.59 x 10-11, 1.15 x 10-11 and 2.87 x
10-12 M), with each concentration tested in duplicate wells.
Reference Standard (Agonist Comprehensive)
14.
E2 for comprehensive testing is comprised of a 1:2 serial dilution consisting of 11 concentrations(ranging from 3.67 x 10-10 to 3.59 x 10-13 M) of E2 in duplicate wells.
Reference Standard (Antagonist Range Finder)
15.
The reference standard is a combination of Ral (CASRN 84449-90-1) and E2 (CASRN 50-28-2).
Ral/E2 for range finder testing is comprised of a serial dilution of three concentrations of Ral (3.06 u 10-9,
7.67 u 10-10, and 1.92 u 10-10M) plus a fixed concentration (9.18 × 10-11 M) of E2 in duplicate wells.
Reference Standard (Antagonist Comprehensive)
16.
Ral/E2 for comprehensive testing is comprised of a 1:2 serial dilution of Ral (ranging from 2.45u 10-8
to 9.57 u 10-11M) plus a fixed concentration (9.18 × 10-11 M) of E2 consisting of nine concentrations of
Ral/E2 in duplicate wells.
Weak Positive Control (Agonist)
17.
The weak positive control is 9.06 u 10-6 M p,p'-methoxychlor (methoxychlor; CASRN 72-43-5) in
EFM.
Weak Positive Control (Antagonist)
18.
The weak positive control consists of tamoxifen (CASRN 10540-29-1) 3.36 u 10-6 M with
9.18 × 10-11 M E2 in EFM.
E2 Control (Antagonist Assay Only)
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The E2 control is 9.18 × 10-11 M E2 in EFM and used as a base line negative control.

Fold-Induction (Agonist)
20.
The induction of luciferase activity of the reference standard (E2) is measured by dividing the
average highest E2 reference standard RLU value by the average DMSO control RLU value, and the result
should be greater than four-fold.
Fold-Reduction (Antagonist)
21.
The mean luciferase activity of the reference standard (Ral/E2) is measured by dividing the average
highest Ral/E2 reference standard RLU value by the average DMSO control RLU value and should be greaterthan three-fold.
Demonstration of Laboratory Proficiency
22.
To demonstrate proficiency with the BG1Luc ER TA test method, a laboratory should compile
agonist and antagonist historical databases with reference standard and control data generated from at least 10
independent agonist and 10 independent antagonist experiments, conducted on different days. Theseexperiments are the foundation for reference standards and the historical controls. Future acceptable results
should be added to enlarge the database. A successful demonstration of proficiency will be achieved byproducing values that are no more than 2.5 standard deviations of the historical controls (see paragraph 11).
23.
Once the historical databases are compiled, the agonist and antagonist proficiency substances,respectively listed in Tables 3 and 4 of “ER TA TEST METHOD COMPONENTS” (Page 9-12 of this Test
Guideline), should be tested.
Vehicle24.
Test chemicals should be dissolved in a solvent that solubilises the test chemical and is miscible withthe cell medium. Water, ethanol (95% to 100% purity) and DMSO are suitable vehicles. If DMSO is used,
the level should not exceed 1% (v/v). For any vehicle, it should be demonstrated that the maximum volume
used is not cytotoxic and does not interfere with the assay performance. Reference standards and controls are
dissolved in 100% solvent and then diluted down to appropriate concentrations in EFM.
Preparation of Test chemicals
25.
The test chemicals are dissolved in 100% DMSO (or appropriate solvent), and then diluted down toappropriate concentrations in EFM. All test chemicals should be allowed to equilibrate to room temperature
before being dissolved and diluted. Test chemical solutions should be prepared fresh for each experiment.
Solutions should not have noticeable precipitate or cloudiness. Reference standard and control stocks may be
prepared in bulk; however, final reference standard, control dilutions and test chemicals should be freshly
prepared for each experiment and used within 24 hours of preparation.
Solubility and Cytotoxicity: Considerations for Range Finding

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26.
Range finder testing consists of seven point - 1:10 serial dilutions run in duplicate. Initially, test
chemicals are tested up to the maximum concentration of 1 mg/mL (~1 mM) for agonist testing and 20
μg/mL (~10 PM) for antagonist testing. Range finder experiments are used to determine the following:xx

Test chemical starting concentrations to be used during comprehensive testing
Test chemical dilutions (1:2 or 1:5) to be used during comprehensive testing27.
An assessment of cell viability/cytotoxicity is included in the agonist and antagonist test method
protocols (7) and is incorporated into range finder and comprehensive testing. The cytotoxicity method thatwas used to assess cell viability during the validation of the BG1Luc ER TA (1) was a scaled qualitativevisual observation method; however, a quantitative method for the determination of cytotoxicity can be used(see protocol (7)). Data from test chemical concentrations that cause more than 20% reduction in viability
cannot be used.
Test chemical Exposure and Assay Plate Organisation
28.
Cells are counted and plated into 96-well tissue culture plates (2 x 105 cells per well) in EFM andincubated for 24 hours to allow the cells to attach to the plate. The EFM is removed and replaced with test
and reference chemicals in EFM and incubated for 19-24 hours. Special considerations will need to be
applied to those substances that are highly volatile since nearby control wells may generate false positive
results. In such cases, “plate sealers” may help to effectively isolate individual wells during testing, andare therefore recommended.

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Range Finder Tests
29.
Range finder testing uses all wells of the 96-well plate to test up to six test chemicals as seven point
1:10 serial dilutions in duplicate (see Figures 1 and 2).
x Agonist range finder testing uses four concentrations of E2 in duplicate as the reference standard andfour replicate wells for the DMSO control.
x Antagonist range finder testing uses three concentrations of Ral/E2 with 9.18 × 10-11 M E2 induplicate as the reference standard, with three replicate wells for the E2 and DMSO controls.
Figure 1: Agonist Range Finder Test 96-well Plate Layout
1

2

3

4

5

6

7

8

9

10

11

12

A

TC1-1

TC1-1

TC2-1

TC2-1

TC3-1

TC3-1

TC4-1

TC4-1

TC5-1

TC5-1

TC6-1

TC6-1

B

TC1-2

TC1-2

TC2-2

TC2-2

TC3-2

TC3-2

TC4-2

TC4-2

TC5-2

TC5-2

TC6-2

TC6-2

C

TC1-3

TC1-3

TC2-3

TC2-3

TC3-3

TC3-3

TC4-3

TC4-3

TC5-3

TC5-3

TC6-3

TC6-3

D

TC1-4

TC1-4

TC2-4

TC2-4

TC3-4

TC3-4

TC4-4

TC4-4

TC5-4

TC5-4

TC6-4

TC6-4

E

TC1-5

TC1-5

TC2-5

TC2-5

TC3-5

TC3-5

TC4-5

TC4-5

TC5-5

TC5-5

TC6-5

TC6-5

F

TC1-6

TC1-6

TC2-6

TC2-6

TC3-6

TC3-6

TC4-6

TC4-6

TC5-6

TC5-6

TC6-6

TC6-6

G

TC1-7

TC1-7

TC2-7

TC2-7

TC3-7

TC3-7

TC4-7

TC4-7

TC5-7

TC5-7

TC6-7

TC6-7

H

E2-1

E2-2

E2-3

E2-4

VC

VC

VC

VC

E2-1

E2-2

E2-3

E2-4

Abbreviations: E2-1 to E2-4 = concentrations of the E2 reference standard (from high to low); TC1-1 to TC1-7 = concentrations(from high to low) of test chemical 1 (TC1); TC2-1 to TC2-7 = concentrations (from high to low) of test chemical 2 (TC2); TC3-1 toTC3-7 = concentrations (from high to low) of test chemical 3 (TC3); TC4-1 to TC4-7 = concentrations (from high to low) of test
chemical 4 (TC4); TC5-1 to TC5-7 = concentrations (from high to low) of test chemical 5 (TC5); TC6-1 to TC6-7 = concentrations(from high to low) of test chemical 6 (TC6); VC = vehicle control (DMSO [1% v/v EFM.]).

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Figure 2: Antagonist Range Finder Test 96-well Plate Layout
1

2

3

4

5

6

7

8

9

10

11

12

A

TC1-1

TC1-1

TC2-1

TC2-1

TC3-1

TC3-1

TC4-1

TC4-1

TC5-1

TC5-1

TC6-1

TC6-1

B

TC1-2

TC1-2

TC2-2

TC2-2

TC3-2

TC3-2

TC4-2

TC4-2

TC5-2

TC5-2

TC6-2

TC6-2

C

TC1-3

TC1-3

TC2-3

TC2-3

TC3-3

TC3-3

TC4-3

TC4-3

TC5-3

TC5-3

TC6-3

TC6-3

D

TC1-4

TC1-4

TC2-4

TC2-4

TC3-4

TC3-4

TC4-4

TC4-4

TC5-4

TC5-4

TC6-4

TC6-4

E

TC1-5

TC1-5

TC2-5

TC2-5

TC3-5

TC3-5

TC4-5

TC4-5

TC5-5

TC5-5

TC6-5

TC6-5

F

TC1-6

TC1-6

TC2-6

TC2-6

TC3-6

TC3-6

TC4-6

TC4-6

TC5-6

TC5-6

TC6-6

TC6-6

G

TC1-7

TC1-7

TC2-7

TC2-7

TC3-7

TC3-7

TC4-7

TC4-7

TC5-7

TC5-7

TC6-7

TC6-7

H

Ral-1

Ral-2

Ral-3

VC

VC

VC

E2

E2

E2

Ral-1

Ral-2

Ral-3

Abbreviations: E2 = E2 control; Ral-1 to Ral-3 = concentrations of the Raloxifene/E2 reference standard (from high to low); TC1-1 to
TC1-7 = concentrations (from high to low) of test chemical 1 (TC1); TC2-1 to TC2-7 = concentrations (from high to low) of test
chemical 2 (TC2); TC3-1 to TC3-7 = concentrations (from high to low) of test chemical 3 (TC3); TC4-1 to TC4-7 = concentrations(from high to low) of test chemical 4 (TC4); TC5-1 to TC5-7 = concentrations (from high to low) of test chemical 5 (TC5); TC6-1 to
TC6-7 = concentrations (from high to low) of test chemical 6 (TC6); VC = vehicle control (DMSO [1% v/v EFM.]).
Note: All test chemicals are tested in the presence of 9.18 × 10-11 M E2.

30.
The recommended final volume of media required for each well is 200 μL. Only use test plates inwhich the cells in all wells give a viability of 80% and above.
31.
Determination of starting concentrations for comprehensive agonist testing is described in depth in
the agonist protocol (7). Briefly, the following criteria are used:o

If there are no points on the test chemical concentration curve that are greater than the mean plus
three times the standard deviation of the DMSO control, comprehensive testing will be conductedusing an 11-point 1:2 serial dilution starting at the maximum soluble concentration.

o

If there are points on the test chemical concentration curve that are greater than the mean plus
three times the standard deviation of the DMSO control, the starting concentration to be used forthe 11-point dilution scheme in comprehensive testing should be one log higher than theconcentration giving the highest adjusted RLU value in the range finder. The 11-point dilution
scheme will be based on either 1:2 or 1:5 dilutions according to the following criteria:
An 11-point 1:2 serial dilution should be used if the resulting concentration range willencompass the full range of responses based on the concentration response curve generated in
the range finder test. Otherwise, use a 1:5 dilution.

o If a test chemical exhibits a biphasic concentration response curve in the range finder test, bothphases should also be resolved in comprehensive testing.

32.
Determination of starting concentrations for comprehensive antagonist testing is described in depth
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x

If there are no points on the test chemical concentration curve that are less than the mean minus
three times the standard deviation of the E2, control comprehensive testing will be conductedusing an 11-point 1:2 serial dilution starting at the maximum soluble concentration.

x

If there are points on the test chemical concentration curve that are less than the mean minus
three times the standard deviation of the E2 control, the starting concentration to be used for the
11-point dilution scheme in comprehensive testing should be one of the following:
The concentration giving the lowest adjusted RLU value in the range finder
The maximum soluble concentration (See antagonist protocol (7), Figure 14-2)
The lowest cytotoxic concentration (See antagonist protocol (7), Figure 14-3 for a related
example).

x

The 11-point dilution scheme will be based on either a 1:2 or 1:5 serial or dilution according to
the following criteria:
An 11-point 1:2 serial dilution should be used if the resulting concentration range willencompass the full range of responses based on the concentration response curve generated
in the range finder test. Otherwise a 1:5 dilution should be used.

Comprehensive Tests
33.
Comprehensive testing consists of 11-point serial dilutions (either 1:2 or 1:5 serial dilutions based on
the starting concentration for comprehensive testing criteria) with each concentration tested in triplicate wellsof the 96-well plate (see Figures 3 and 4).
x

Agonist comprehensive testing uses 11 concentrations of E2 in duplicate as the reference
standard. Four replicate wells for the DMSO control and four replicate wells for the
methoxychlor control (9.06 x 10-6 M) are included on each plate.

x

Antagonist comprehensive testing uses nine concentrations of Ral/E2 with 9.18 × 10-11 M E2 induplicate as the reference standard, with four replicate wells for the E2 9.18 u 10-11 M control,
four replicate wells for DMSO controls, and four replicate wells for tamoxifen 3.36 x 10-6M.

Figure 3: Agonist Comprehensive Test 96-well Plate Layout
1

2

3

4

5

6

7

8

9

10

11

12

A

TC1-1

TC1-2

TC1-3

TC1-4

TC1-5

TC1-6

TC1-7

TC1-8

TC1-9

TC1-10

TC1-11

VC

B

TC1-1

TC1-2

TC1-3

TC1-4

TC1-5

TC1-6

TC1-7

TC1-8

TC1-9

TC1-10

TC1-11

VC

C

TC1-1

TC1-2

TC1-3

TC1-4

TC1-5

TC1-6

TC1-7

TC1-8

TC1-9

TC1-10

TC1-11

VC

D

TC2-1

TC2-2

TC2-3

TC2-4

TC2-5

TC2-6

TC2-7

TC2-8

TC2-9

TC2-10

TC2-11

VC

E

TC2-1

TC2-2

TC2-3

TC2-4

TC2-5

TC2-6

TC2-7

TC2-8

TC2-9

TC2-10

TC2-11

Meth

F

TC2-1

TC2-2

TC2-3

TC2-4

TC2-5

TC2-6

TC2-7

TC2-8

TC2-9

TC2-10

TC2-11

Meth

G

E2-1

E2-2

E2-3

E2-4

E2-5

E2-6

E2-7

E2-8

E2-9

E2-10

E2-11

Meth

H

E2-1

E2-2

E2-3

E2-4

E2-5

E2-6

E2-7

E2-8

E2-9

E2-10

E2-11

Meth

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Abbreviations: TC11-1 to TC1-11 = concentrations (from high to low) of test chemical 1; TC2-1 to TC2-11 = concentrations (fromhigh to low) of test chemical 2; E2-1 to E2-11 = concentrations of the E2 reference standard (from high to low); Meth = p,p’
methoxychlor weak positive control; VC = DMSO (1% v/v) EFM vehicle control

Figure 4: Antagonist Comprehensive Test 96-well Plate Layout
1

2

3

4

5

6

7

8

9

10

11

A

TC1-1

TC1-2

TC1-3

TC1-4

TC1-5

TC1-6

TC1-7

TC1-8

TC1-9

B

TC1-1

TC1-2

TC1-3

TC1-4

TC1-5

TC1-6

TC1-7

TC1-8

TC1-9

C

TC1-1

TC1-2

TC1-3

TC1-4

TC1-5

TC1-6

TC1-7

TC1-8

TC1-9

D

TC2-1

TC2-2

TC2-3

TC2-4

TC2-5

TC2-6

TC2-7

TC2-8

TC2-9

E

TC2-1

TC2-2

TC2-3

TC2-4

TC2-5

TC2-6

TC2-7

TC2-8

TC2-9

F

TC2-1

TC2-2

TC2-3

TC2-4

TC2-5

TC2-6

TC2-7

TC2-8

TC2-9

G

Ral-1

Ral-2

Ral-3

Ral-4

Ral-5

Ral-6

Ral-7

Ral-8

H

Ral-1

Ral-2

Ral-3

Ral-4

Ral-5

Ral-6

Ral-7

Ral-8

12

TC110
TC110
TC110
TC210
TC210
TC210

TC111
TC111
TC111
TC211
TC211
TC211

Ral-9

E2

E2

Tam

Ral-9

E2

E2

Tam

VC
VC
VC
VC
Tam
Tam

Abbreviations: E2 = E2 control; Ral-1 to Ral-9 = concentrations of the Raloxifene/E2 reference standard (from high to low); Tam =Tamoxifen/E2 weak positive control; TC1-1 to TC1-11 = concentrations (from high to low) of test chemical 1 (TC1); TC2-1 to TC211 = concentrations (from high to low) of test chemical 2 (TC2); VC = vehicle control (DMSO [1% v/v EFM.]).
Note: As noted, all reference and test wells contain a fixed concentration of E2 (9.18 x 10-11M)

34.
Repeat comprehensive tests for the same chemical should be conducted on different days, to ensure
independence. At least two comprehensive tests should be conducted. If the results of the tests contradict eachother (e.g. one test is positive, the other negative), or if one of the tests is inadequate, a third additional test
should be conducted.

Measure of Luminescence
35.
Luminescence is measured in the range of 300 to 650 nm, using an injecting luminometer and with
software that controls the injection volume and measurement interval (7). Light emission from each well isexpressed as RLU per well.
ANALYSIS OF DATA
EC50 /IC50 determination
36.
The EC50 value (half maximal effective concentration of a test chemical [agonists]) and the IC50 value(half maximal inhibitory concentration of a test chemical [antagonists]) are determined from theconcentration-response data. For test chemicals that are positive at one or more concentrations, theconcentration of test chemical that causes a half-maximal response (IC50 or EC50) is calculated using a Hill
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function analysis or an appropriate alternative. The Hill function is a four-parameter logistic mathematicalmodel relating the test chemical concentration to the response (typically following a sigmoidal curve) using
the equation below:

Y = Bottom +(Top – Bottom)

1 + 10 (lgEC50-X)HillSlope
where Y = response (i.e. RLUs); X = the logarithm of concentration; Bottom = the minimum response; Top =the maximum response; lg EC50 (or lg IC50) = the logarithm of X as the response midway between Top andBottom; and Hillslope describes the steepness of the curve. The model calculates the best fit for the Top,
Bottom, Hillslope, and IC50 and EC50 parameters. For the calculation of EC50 and IC50 values, appropriate
statistical software should be used (e.g. Graphpad PrismR statistical software).
Determination of Outliers
37.
Good statistical judgment could be facilitated by including (but not limited to) the Q-test (see agonist
and antagonist protocols (7) for determining “unusable” wells that will be excluded from the data analysis.
38.
For E2 reference standard replicates (sample size of two), any adjusted RLU value for a replicate at agiven concentration of E2 is considered an outlier if its value is more than 20% above or below the adjusted
RLU value for that concentration in the historical database.
Collection and Adjustment of Luminometer Data for Range Finder Testing39.
Raw data from the luminometer should be transferred to a spreadsheet template designed for the test
method. It should be determined whether there are outlier data points that need to be removed. (See Test
Acceptance Criteria for parameters that are determined in the analyses.) The following calculations should be
performed:
AgonistStep 1
Step 2
Step 3
Step 4

Calculate the mean value for the DMSO vehicle control (VC).
Subtract the mean value of the DMSO VC from each well value to normalise the data.
Calculate the mean fold induction for the reference standard (E2).
Calculate the mean EC50 value for the test chemicals.

Antagonist
Step 1
Step 2
Step 3
Step 4
Step 5

Calculate the mean value for the DMSO VC.
Subtract the mean value of the DMSO VC from each well value to normalise the data.
Calculate the mean fold reduction for the reference standard (Ral/E2).
Calculate the mean value for the E2 reference standard.
Calculate the mean IC50 value for the test chemicals.
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Collection and Adjustment of Luminometer Data for Comprehensive Testing40.
Raw data from the luminometer should be transferred to a spreadsheet template designed for the test
method. It should be determined whether there are outlier data points that need to be removed. (See Test
Acceptance Criteria for parameters that are determined in the analyses.) The following calculations are
performed:
AgonistStep 1
Step 2
Step 3
Step 4
Step 5

Calculate the mean value for the DMSO VC.
Subtract the mean value of the DMSO VC from each well value to normalise the data.
Calculate the mean fold induction for the reference standard (E2).
Calculate the mean EC50 value for E2 and the test chemicals.
Calculate the mean adjusted RLU value for methoxychlor.

Antagonist
Step 1
Step 2
Step 3
Step 4
Step 5
Step 6

Calculate the mean value for the DMSO VC.
Subtract the mean value of the DMSO VC from each well value to normalise the data.
Calculate the mean fold induction for the reference standard (Ral/E2).
Calculate the mean IC50 value for Ral/E2 and the test chemicals.
Calculate the mean adjusted RLU value for tamoxifen.
Calculate the mean value for the E2 reference standard.

Data Interpretation Criteria
41.
The BG1Luc ER TA is intended as part of a weight of evidence approach to help prioritise
substances for ED testing in vivo. Part of this prioritisation procedure will be the classification of the test
chemical as positive or negative for either ER agonist or antagonist activity. The positive and negativedecision criteria used in the BG1Luc ER TA validation study are described in Table 1.

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Table 1:455

Positive and Negative Decision Criteria
AGONIST ACTIVITY

Positive

Negative

Inadequate

x All test chemicals classified as positive for ER agonist activity should have aconcentration–response curve consisting of a baseline, followed by a positive
slope, and concluding in a plateau or peak. In some cases, only two of thesecharacteristics (baseline–slope or slope–peak) may be defined.
x The line defining the positive slope should contain at least three points with
non-overlapping error bars (mean ± SD). Points forming the baseline areexcluded, but the linear portion of the curve may include the peak or first pointof the plateau.
x A positive classification requires a response amplitude, the difference between
baseline and peak, of at least 20% of the maximal value for the reference
substance, E2 (i.e. 2000 RLUs or more when the maximal response value of thereference substance [E2] is adjusted to 10,000 RLUs).
x If possible, an EC50 value should be calculated for each positive test chemical.
The average adjusted RLU for a given concentration is at or below the meanDMSO control RLU value plus three times the standard deviation of the DMSO
RLU.
Data that cannot be interpreted as valid for showing either the presence or absenceof activity because of major qualitative or quantitative limitations are consideredinadequate and cannot be used to determine whether the test chemical is positive or
negative. Chemicals should be retested.
ANTAGONIST ACTIVITY

Positivex Test chemical data produce a concentration-response curve consisting of abaseline, which is followed by a negative slope.
x The line defining the negative slope should contain at least three points with
non-overlapping error bars; points forming the baseline are excluded but thelinear portion of the curve may include the first point of the plateau.
x There should be at least a 20% reduction in activity from the maximal value forthe reference substance, Ral/E2 (i.e. 8000 RLU or less when the maximal
response value of the reference substance [Ral/E2] is adjusted to 10,000 RLUs).
x The highest non-cytotoxic concentrations of the test chemical should be lessthan or equal to 1x10-5 M.
x If possible, an IC50 value should be calculated for each positive test chemical.

Negative

All data points are above the ED80 value (80% of the E2 response, or 8000 RLUs),
at concentrations less than 1.0 u 10-5 M.

Inadequate

Data that cannot be interpreted as valid for showing either the presence or absenceof activity because of major qualitative or quantitative limitations are consideredinadequate and cannot be used to determine whether the test chemical is positive or
negative. Chemical should be retested.

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42.
Positive results will be characterised by both the magnitude of the effect and the concentration atwhich the effect occurs, where possible. Examples of positive, negative and inadequate data are shown in
Figures 5 and 6.
Figure 5: Agonist Examples: Positive, Negative and Inadequate Data

Dashed line indicates 20% of E2 response, 2000 adjusted and normalised RLUs.

Figure 6: Antagonist Examples: Positive, Negative, and Inadequate Data

Dashed line indicates 80% of Ral/E2 response, 8000 adjusted and normalised RLUs.
Solid line indicates 1.00 u 10-5 M. For a response to be considered positive, it should be below the 8000 RLU line, and atconcentrations less than 1.00 u 10-5M.
Asterisked concentrations in the meso-hexestrol graph indicate viability scores of "2" or greater.
The test results for meso-hexestrol are considered inadequate data because the only response that is below 8,000 RLU occurs at
1.00 u 10-5M.

43.
The calculations of EC50 and IC50 can be made using a four-parameter Hill Function (see agonist
protocol and antagonist protocol for more details (7)). Meeting the acceptability criteria indicates the systemis operating properly, but it does not ensure that any particular run will produce accurate data. Duplicating
the results of the first run is the best assurance that accurate data were produced.
TEST REPORT
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44.
See paragraph 20 of “ER TA TEST METHOD COMPONENTS” (Page 14-15 of this Test
Guideline)
LITERATURE (3)(1)

ICCVAM. (2011). ICCVAM Test Method Evaluation Report on the LUMI-CELL® ER (BG1Luc ER
TA) Test Method: An In Vitro Method for Identifying ER Agonists and Antagonists, National
Institute of Environmental Health Sciences: Research Triangle Park, NC.

(2)

Monje P., Boland R. (2001). Subcellular Distribution of Native Estrogen Receptor α and β IsoformsinRabbit Uterus and Ovary, J. Cell Biochem., 82(3): 467-479.

(3)

Pujol P., et al. (1998). Differential
Expression of Estrogen Receptor-Alpha and -Beta
Messenger
RNAs as a Potential Marker of Ovarian Carcinogenesis, Cancer Res., 58(23): 53675373.

(4)

Weihua Z., et al. (2000). Estrogen
Receptor (ER) β, a Modulator of ERα in the Uterus,
Proceedings of the National Academy of Sciences of the United States of America
97(11):
936-5941.

(5)

Balls M., et al. (2006). The Importance of Good Cell Culture Practice (GCCP), ALTEX, 23(Suppl):
p. 270-273.

(6)

Coecke S., et al. (2005). Guidance on Good Cell Culture Practice: a Report of the Second ECVAM
Task Force on Good Cell Culture Practice, Alternatives to Laboratory Animals, 33: p. 261-287.

(7)

ICCVAM. (2011). ICCVAM Test Method Evaluation Report, The LUMI-CELL® ER (BG1Luc ER
TA)
Test Method: An In Vitro Assay for Identifying Human Estrogen Receptor Agonist andAntagonist
Activity of Chemicals, NIH Publication No. 11-7850.

(8)

Rogers J.M., Denison M.S. (2000). Recombinant Cell Bioassays for Endocrine Disruptors:
Development of a Stably Transfected Human Ovarian Cell Line for the Detection of Estrogenic andAnti-Estrogenic Chemicals, In Vitro Mol. Toxicol.,13(1):67-82.

(9)

Escande A., et al. (2006). Evaluation of Ligand Selectivity Using Reporter Cell Lines Stably
Expressing
Estrogen Receptor
Alpha or Beta, Biochem. Pharmacol., 71(10):1459-69.

(10) Thorne N., Inglese J., Auld D.S. (2010). Illuminating Insights into Firefly Luciferase and Other
Bioluminescent Reporters Used in Chemical Biology, Chemistry and Biology,17(6):646-57.
(11) Kuiper G.G, et al. (1998). Interaction of Estrogenic Chemicals and Phytoestrogens with Estrogen
Receptor Beta, Endocrinology,139(10):4252-63.

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