ᣂⷙ⹜㛎ᴺឭ᩺ᦠ             ᐔᚑ 26 ᐕ 1 ᦬ 20 ᣣ
                                  No. 2013-02
ࡅ࠻ࠛࠬ࠻ࡠࠥࡦฃኈ૕⚿วߦࠃࠆᵴᕈൻ࡮ᜟ᛫૞↪‛⾰ࠍᬌ಴ߔࠆ BG1Luc ER TA ᴺߦ㑐ߔࠆឭ᩺ ᐔᚑ 25 ᐕ 6 ᦬ 11 ᣣߦ᧲੩‫ޔ‬࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲߦߡ㐿௅ߐࠇߚᣂⷙ⹜㛎ᴺ⹏ଔળ⼏㧔ㅢ
⒓㧦JaCVAM ⹏ଔળ⼏㧕ߦ߅޿ߡએਅߩឭ᩺߇ߥߐࠇߚ‫ޕ‬  ឭ᩺ౝኈ㧦ࡅ࠻ࠛࠬ࠻ࡠࠥࡦฃኈ૕⚿วߦࠃࠆᵴᕈൻ࡮ᜟ᛫૞↪‛⾰ࠍᬌ಴ߔࠆ
BG1Luc ER TA ᴺߪ‫ߩઁޔ‬㘃ૃ⹜㛎ᴺߣห⒟ᐲߦ‫ⴕޔ‬᡽਄೑↪น⢻ߢ޽ࠆ‫ޕ‬ ߎߩឭ᩺ᦠߪ‫ޔ‬OECD (Organisation for Economic Co-operation and Development) Test Guideline
OECD Test Guideline㧔TG㧕457 ߅ࠃ߮ICCVAM (Interagency Coordinating Committee on the
Validation of Alternative Methods) 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 Chemicalsࠍ߽ߣߦ‫ޔ‬ౝಽᴲ߆ߊੂ⹜㛎⹏ଔᆔຬળߦࠃࠅ߹ߣ߼ࠄࠇߚᢥᦠࠍ↪޿ߡ
JaCVAM⹏ଔળ⼏߇⹏ଔ߅ࠃ߮ᬌ⸛ߒߚ⚿ᨐ‫↪᦭ߩߘޔ‬ᕈ߇⏕⹺ߐࠇߚߎߣ߆ࠄ૞ᚑߐࠇߚ‫ޕ‬ એ਄ߩℂ↱ߦࠃࠅ‫ⴕޔ‬᡽ᒰዪߩ቟ోᕈ⹏ଔᣇᴺߣߒߡ‫ࡦࠥࡠ࠻ࠬࠛ࠻ࡅޟ‬ฃኈ૕⚿วߦࠃࠆᵴ
ᕈൻ࡮ᜟ᛫૞↪‛⾰ࠍᬌ಴ߔࠆ BG1Luc ER TA ᴺ‫ࠍ↪૶ߩޠ‬ឭ᩺ߔࠆ߽ߩߢ޽ࠆ‫ޕ‬ ศ↰ᱞ⟤                        ⷏Ꮉ⑺૫ศ↰ᱞ⟤JaCVAM ⹏ଔળ⼏ ⼏㐳               JaCVAM ㆇ༡ᆔຬળ ᆔຬ㐳

i

,C%8#/ ⹏ଔળ⼏ศ↰ᱞ⟤㧔ᣣᧄᲥᕈቇળ㧕㧦ᐳ㐳

ᵻ㊁ື⑲

㧔ᣣᧄⅣႺᄌ⇣ේቇળ㧕੖ච፲⦟᣿ 㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ↢ᵴⴡ↢ൻቇㇱ㧕৻㝩 ീ㧔ᣣᧄൻቇᎿᬺදળ㧕*ᄢ㊁ᵏ㓶㧔ᐳ㐳ផ⮈㧕*ዊ㊁ኹඳᔒ 㧔⁛┙ⴕ᡽ᴺੱ ක⮎ຠක≮ᯏེ✚วᯏ᭴㧕㤥Ỉ ദ㧔ᣣᧄേ‛ታ㛎ઍᦧᴺቇળ㧕᧖ጊ⌀ℂሶ 㧔ᣣᧄൻ♆ຠᎿᬺㅪวળ㧕⼱↰ᥓሶ㧔⁛┙ⴕ᡽ᴺੱ ක⮎ຠක≮ᯏེ✚วᯏ᭴㧕*

⷏Ꮉ⑺૫

㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ቟ోᕈ↢‛⹜㛎⎇ⓥ࠮ࡦ࠲࡯㧕’ ᩕੑ㧔ᣣᧄ఺∉Ქᕈቇળ㧕Ⴧ↰శノ㧔ᐳ㐳ផ⮈㧕ጊ↰㓉ᔒ㧔⁛┙ⴕ᡽ᴺੱ ⵾ຠ⹏ଔᛛⴚၮ⋚ᯏ᭴㧕*ᮮ㑐ඳ㓶㧔ᣣᧄ⊹⤏ࠕ࡟࡞ࠡ࡯࡮ធ⸅⊹⤏Ἳቇળ㧕ศ↰ ✛

㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ቟ోᕈ↢‛⹜㛎⎇ⓥ࠮ࡦ࠲࡯ ∛ℂㇱ㧕ศ᧛ ഞ㧔ᐳ㐳ផ⮈㧕

ᷰㇱ৻ੱ㧔ᣣᧄ⵾⮎Ꮏᬺදળ㧕                  છᦼ㧦ᐔᚑ 24 ᐕ 4 ᦬ 1 ᣣ㨪ᐔᚑ 26 ᐕ 3 ᦬ 31 ᣣ*: ᐔᚑ 25 ᐕ 4 ᦬ 1 ᣣ㨪ᐔᚑ 26 ᐕ 3 ᦬ 31 ᣣii

,C%8#/ ㆇ༡ᆔຬળ
⷏Ꮉ⑺૫

㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ቟ోᕈ↢‛⹜㛎⎇ⓥ࠮ࡦ࠲࡯㧕㧦ᆔຬ㐳Ꮉ⷏ ᔀ

㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ㧕ዊᎹਭ⟤ሶ 㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ቟ోᕈ↢‛⹜㛎⎇ⓥ࠮ࡦ࠲࡯ ∛ℂㇱ㧕⩲㊁ ⚐

㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ቟ోᕈ↢‛⹜㛎⎇ⓥ࠮ࡦ࠲࡯ Ქᕈㇱ㧕ୖᜬᙗ〝㧔ෘ↢ഭ௛⋭ ක⮎㘩ຠዪ ൻቇ‛⾰቟ోኻ╷ቶ㧕*ᢪ⮮๺ᐘ

㧔⁛┙ⴕ᡽ᴺੱ ක⮎ຠක≮ᯏེ✚วᯏ᭴㧕૒‫ᧁޘ‬ᱜᐢ 㧔ෘ↢ഭ௛⋭ ක⮎㘩ຠዪ ൻቇ‛⾰቟ోኻ╷ቶ㧕㑐㊁␭ሶ㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ቟ోᕈ↢‛⹜㛎⎇ⓥ࠮ࡦ࠲࡯ ⮎ℂㇱ㧕㜞ᧁ◊਽㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ቟ోᕈ↢‛⹜㛎⎇ⓥ࠮ࡦ࠲࡯ Ქᕈㇱ േ‛▤ℂቶ㧕ᐢἑ᣿ᒾ  㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ቟ోᕈ↢‛⹜㛎⎇ⓥ࠮ࡦ࠲࡯ ✚ว⹏ଔ⎇
ⓥቶ㧕
ᧄ㑆ᱜల

㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ቟ోᕈ↢‛⹜㛎⎇ⓥ࠮ࡦ࠲࡯ ᄌ⇣ㆮવㇱ㧕శጟବᚑ㧔ෘ↢ഭ௛⋭ ක⮎㘩ຠዪ ክᩏ▤ℂ⺖㧕ጊᧄ㗅ੑ㧔ෘ↢ഭ௛⋭ ක⮎㘩ຠዪ ൻቇ‛⾰቟ోኻ╷ቶ㧕*ዊፉ ⡸㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ቟ోᕈ↢‛⹜㛎⎇ⓥ࠮ࡦ࠲࡯ ⮎ℂㇱᣂⷙ⹜㛎⴫⹏ଔቶ㧕㧦੐ോዪ*㧦ᐔᚑ 25 ᐕ 8 ᦬ 1 ᣣࠃࠅ

iii

iv

JaCVAM statement
on BG1Luc Estrogen Receptor Transactivation Test Method for Identifying
Estrogen Receptor Agonists and Antagonists
At the meeting concerning the above method, held on 11 June 2013 at the National Instituteof Health Sciences (NIHS), Tokyo, Japan, the members of the Japanese Center for the
Validation of Alternative Methods (JaCVAM) Regulatory Acceptance Board unanimously
endorsed the following statement:
BG1Luc Estrogen Receptor Transactivation Test Method for Identifying Estrogen
Receptor Agonists and Antagonists is considered to be useful as a screening of
endocrine disrupter substances as well as similar test methods for regulatory use.
Following the review of the results of OECD (Organisation for Economic Co-operation andDevelopment) Test Guideline OECD Test Guideline (TG) 457 and ICCVAM (Interagency
Coordinating Committee on the Validation of Alternative Methods) 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, it is concluded that
BG1Luc Estrogen Receptor Transactivation Test Method for Identifying Estrogen Receptor Agonists
and Antagonists such as screening of endocrine disrupter substances are clearly beneficial.

The JaCVAM Regulatory Acceptance Board has been regularly kept informed of the
progress of the study, and this endorsement is based on an assessment of various documents,
including, in particular, the evaluation report prepared by the JaCVAM ad hoc peer review
panel for endocrine disrupter testing.

Takemi Yoshida
Chairperson
JaCVAM Regulatory Acceptance Board

Akiyoshi Nishikawa
Chairperson
JaCVAM Steering Committee

20 January, 2014v

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. Takemi Yoshida (Japanese Society of Toxicology): Chairperson
Mr. Norihide Asano (Japanese Environmental Mutagen Society)
Mr. Tsutomu Ichiki (Japan Chemical Industry Association)*
Mr. Yoshiaki Ikarashi (National Institute of Health Sciences: NIHS)
Mr. Tsutomu Miki Kurosawa (Japanese Society for Animal Experimentation)
Mr. Eiji Maki (Japanese Society of Immunotoxicology)
Mr. Mitsuteru Masuda (nominee by Chairperson)
Mr. Akiyoshi Nishikawa (NIHS)
Mr. Yasuo Ohno (nominee by Chairperson)*
Mr. Hiroshi Onodera (Pharmaceuticals and Medical Devices Agency)
Ms. Mariko Sugiyama (Japan Cosmetic Industry Association)
Ms. Tomoko Tanita (Pharmaceuticals and Medical Devices Agency)*
Mr. Takashi Yamada (National Institute of Technology and Evaluation)*
Mr. Hiroo Yokozeki (Japanese Society for Dermatoallergology and Contact
Dermatitis)
Ms. Midori Yoshida (NIHS)
Mr. Isao Yoshimura (nominee by Chairperson)
Mr. Kazuto Watanabe (Japan Pharmaceutical Manufacturers Association)
Term: From 1st April 2012 to 31st March 2014*: From 1st April 2013 to 31st March 2014vi

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. Akihiko Hirose (Division of Risk Assessment, BSRC, NIHS)
Mr. Masamitsu Honma (Division of Genetics and Mutagenesis, BSRC, NIHS)
Mr. Jun Kanno (Division of Cellular and Molecular Toxicology, BSRC, NIHS)
Mr. Toru Kawanishi (NIHS)
Mr. Kenji Kuramochi (Ministry of Health, Labour and Welfare)*
Mr. Toshinari Mitsuoka (Ministry of Health, Labour and Welfare)
Ms. Kumiko Ogawa (Division of Pathology, BSRC, NIHS)
Mr. Kazuyuki Saito (Pharmaceutical & Medical Devices Agency)
Mr. Masahiro Sasaki (Ministry of Health, Labour and Welfare)
Ms. Yuko Sekino (Division of Pharmacology, BSRC, NIHS)
Mr. Atsuya Takagi (Animal Management Section of the Division of Cellular andMolecular Toxicology, BSRC, NIHS)
Mr. Junji Yamamoto (Ministry of Health, Labour and Welfare)*Mr. Hajime Kojima (Section for the Evaluation of Novel Methods, Division of
Pharmacology, BSRC, NIHS): Secretary* Arrival at post day: 1st August 2013

vii

viii

ࡅ࠻ࠛࠬ࠻ࡠࠥࡦฃኈ૕⚿วߦࠃࠆᵴᕈൻ࡮ᜟ᛫૞↪‛⾰ࠍᬌ಴ߔࠆ
BG1Luc ER TA ᴺߩ⹏ଔળ⼏ႎ๔ᦠ⋡ ᰴ⹏ଔળ⼏ႎ๔ᦠ ------------------------------------------------------------------------------ 1⹏ଔႎ๔ᦠ ------------------------------------------------------------------------------------ 7
OECD TG457㧔2012㧕 --------------------------------------------------------------------- 33
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--------------------- 59ix

xࡅ࠻ࠛࠬ࠻ࡠࠥࡦฃኈ૕⚿วߦࠃࠆᵴᕈൻ࡮ᜟ᛫૞↪‛⾰ࠍᬌ಴ߔࠆ BG1Luc ER TA ᴺߩ⹏ଔળ⼏ႎ๔ᦠ










JaCVAM ⹏ଔળ⼏ᐔᚑ 25 ᐕ 6 ᦬ 11 ᣣ

11

JaCVAM ⹏ଔળ⼏ศ↰ᱞ⟤㧔ᣣᧄᲥᕈቇળ㧕㧦ᐳ㐳

ᵻ㊁ື⑲

㧔ᣣᧄⅣႺᄌ⇣ේቇળ㧕੖ච፲⦟᣿ 㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ↢ᵴⴡ↢ൻቇㇱ㧕৻㝩 ീ㧔ᣣᧄൻቇᎿᬺදળ㧕*ᄢፉஜᐘ㧔ᣣᧄൻቇᎿᬺදળ㧕ᄢ㊁ᵏ㓶㧔ᐳ㐳ផ⮈㧕*ዊ═ේᒄ㆏ 㧔⁛┙ⴕ᡽ᴺੱ ක⮎ຠක≮ᯏེ✚วᯏ᭴㧕
ዊ㊁ኹඳᔒ 㧔⁛┙ⴕ᡽ᴺੱ ක⮎ຠක≮ᯏེ✚วᯏ᭴㧕㤥Ỉ ദ㧔ᣣᧄേ‛ታ㛎ઍᦧᴺቇળ㧕᧖ጊ⌀ℂሶ 㧔ᣣᧄൻ♆ຠᎿᬺㅪวળ㧕⼱↰ᥓሶ㧔⁛┙ⴕ᡽ᴺੱ ක⮎ຠක≮ᯏེ✚วᯏ᭴㧕*

⷏Ꮉ⑺૫

㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ቟ోᕈ↢‛⹜㛎⎇ⓥ࠮ࡦ࠲࡯㧕㐳⼱Ꮉ㓉৻ 㧔⁛┙ⴕ᡽ᴺੱ ⵾ຠ⹏ଔᛛⴚၮ⋚ᯏ᭴㧕’ ᩕੑ㧔ᣣᧄ఺∉Ქᕈቇળ㧕Ⴧ↰శノ㧔ᐳ㐳ផ⮈㧕ጊ↰㓉ᔒ㧔⁛┙ⴕ᡽ᴺੱ ⵾ຠ⹏ଔᛛⴚၮ⋚ᯏ᭴㧕*ᮮ㑐ඳ㓶㧔ᣣᧄ⊹⤏ࠕ࡟࡞ࠡ࡯࡮ធ⸅⊹⤏Ἳቇળ㧕ศ↰ ✛

㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ቟ోᕈ↢‛⹜㛎⎇ⓥ࠮ࡦ࠲࡯ ∛ℂㇱ㧕ศ᧛ ഞ㧔ᐳ㐳ផ⮈㧕

ᷰㇱ৻ੱ㧔ᣣᧄ⵾⮎Ꮏᬺදળ㧕                  છᦼ㧦ᐔᚑ 24 ᐕ 4 ᦬ 1 ᣣ㨪ᐔᚑ 26 ᐕ 3 ᦬ 31 ᣣ*㧦ᐔᚑ 25 ᐕ 4 ᦬ 1 ᣣ㨪ᐔᚑ 26 ᐕ 3 ᦬ 31 ᣣએ਄22ౝಽᴲᡬੂ‛⾰߇ᄢ߈ߥ໧㗴ߣߥߞߡ߆ࠄ‫ࡦࠥࡠ࠻ࠬࠛߩ⾰‛ޔ‬᭽૞↪ࠍ᷹ቯߔࠆ in vitro ⹜㛎ᴺߩ㐿⊒߇␠ળ⊛ߦ᳞߼ࠄࠇߡ߈ߚ‫ޕ‬BG1LucER TA ᴺ㧔એਅ‫ޟ‬ᒰ⹥⹜㛎ᴺ‫ޠ‬㧕ߪ‫␠ߩߎޔ‬ળ⊛ⷐ⺧ߦᔕ߃ߡ㐿⊒ߐࠇߚ⹜㛎ᴺߩ৻ߟߢ‫ޔ‬ൻቇ‛⾰߇ ࡅ࠻ࠛࠬ࠻ࡠࠥࡦฃኈ૕㧔ER㧕ߦኻߒߡᵴ
ᕈ‛⾰㧔agonist㧕޽ࠆ޿ߪᜟ᛫ᕈ‛⾰㧔antagonist㧕ߢ޽ࠆߎߣࠍᬌ಴ߒߡ‫ࡦࠥࡠ࠻ࠬࠛޔ‬ᵴᕈ޽ࠆ޿ߪᜟ᛫૞↪ࠍ␜ߔౝಽᴲᡬੂᕈߩ੍᷹ߦᓎ┙ߟ߽ߩߣߒߡឭ᩺ߐࠇߚ߽ߩߢ޽ࠆ 1)‫ޕ‬ᒰ⹥⹜
㛎ᴺߦኻߒߡᓥ᧪⹜㛎ᴺߣ⸒߃ࠆ߽ߩߪ⏕┙ߒߡ޿ߥ޿‫ޔߒ߆ߒޕ‬ౝಽᴲᡬੂᕈࠍⷙቯߔࠆᲥᕈ
ߥ޿ߒ↢‛ᵴᕈߦߟ޿ߡߪ߹ߛචಽߦߪ․ቯߐࠇߡ޿ߥ޿ㇱಽ߇ᱷߐࠇߡ߅ࠅ‫ޔ‬ICCVAM ߩ̌In
vitro ER and AR binding and TA reference substances list2)̍ߢߪ‫ⶄޔ‬ᢙߩᩮ᜚ࠍ✚วߒߡౝಽᴲᡬੂᕈ
‛⾰ࠍಽ㘃ߒߡ޿ࠆ‫ޕ‬☨࿖ߢߪ 2012 ᐕߦ ICCVAM ߇ᒰ⹥⹜㛎ᴺߩ⹏ଔ⚿ᨐࠍႎ๔ߒߡ޿ࠆ 1)‫ޕ‬OECD ߦ߅޿ߡ߽
2012 ᐕ 10 ᦬ 2 ᣣߦߎࠇࠍࠟࠗ࠼࡜ࠗࡦ TG457 ߦขࠅ౉ࠇߡ޿ࠆ 3)‫ߦࠇߎޕ‬ኻߒߡᣣᧄߢߪ‫ޔ‬
JaCVAM ߩ⹏ଔᆔຬળ߇ᒰ⹥⹜㛎ᴺߩ⹏ଔࠍⴕ޿‫ޔ‬ᐔᚑ 24 ᐕ 11 ᦬ߦ⚿ᨐࠍႎ๔ߒߡ޿ࠆ 4㧕‫ޕ‬એਅߦ␜ߔߩߪ‫ߩߎޔ‬ႎ๔ߦၮߠ޿ߚ JaCVAM ⹏ଔળ⼏ߩ⹏ଔ⚿ᨐߢ޽ࠆ‫ޕ‬
1. ᒰ⹥⹜㛎ᴺߪ‫ߥ߁ࠃߩߤޔ‬ᓥ᧪⹜㛎ᴺࠍઍᦧߔࠆ߽ߩ߆‫ߥ߁ࠃߩߤޔߪߚ߹ޕ‬Ქᕈࠍ⹏ଔ޽
ࠆ޿ߪ੍᷹ߔࠆ߽ߩ߆‫ޕ‬ൻቇ‛⾰ߩࠛࠬ࠻ࡠࠥࡦᵴᕈࠍᬌ಴ߒࠃ߁ߣߔࠆ in vivo ߩᓥ᧪⹜㛎ᴺߣߒߡߪ‫ߞߍޔ‬ᱤ㘃ࠍ↪޿ߚሶች⢈ᄢ⹜㛎߇޽ࠆ‫ޕ‬ൻቇ‛⾰ߩࠛࠬ࠻ࡠࠥࡦᵴᕈࠍᬌ಴ߒࠃ߁ߣߔࠆ in vitro ߩ⹜㛎ᴺ
ߣߒߡߪ‫ࡦࠥࡠ࠻ࠬࠛޔ‬ฃኈ૕⚿วᕈ⹜㛎ߣ‫ࡦࠥࡠ࠻ࠬࠛ࠻ࡅޔ‬-Į ฃኈ૕቟ቯዉ౉⚦⢩ࠍ↪޿ࠆォ౮ᵴᕈൻ⹜㛎 (Stably Transfected Human Estrogen Receptor-Į Transcriptional Activation Assay; ER-ĮSTTA; OECD TG455㧕߇޽ࠆ‫ޕ‬ᒰ⹥⹜㛎ᴺߪߎࠇࠄߣ㘃ૃߩᓎഀ߇ᦼᓙߐࠇߡ޿ࠆ߽ߩߢ޽ࠆ‫ޕ‬
2. ᒰ⹥⹜㛎ᴺߣᓥ᧪⹜㛎ᴺߩ㑆ߦߤߩࠃ߁ߥ⑼ቇ⊛ߟߥ߇ࠅ߇޽ࠆ߆‫ޕ‬᷹ቯᜰᮡߪ⇣ߥࠆ߇‫ࡦࠥࡠ࠻ࠬࠛޔ‬ᵴᕈ߹ߚߪᜟ᛫ᕈࠍၮ␆ߣߔࠆߣ޿߁ᗧ๧ߢ‫ޔ‬ᒰ⹥⹜㛎ᴺ
ߩ⑼ቇ⊛ࡔࠞ࠾࠭ࡓߪ‫ߞߍޔ‬ᱤ㘃ሶች⢈ᄢ⹜㛎ߣ౒ㅢߢ޽ࠆ‫ޕ‬㘃ૃߩ⹜㛎ᴺߢ޽ࠆ ER-Į STTA ᴺߪ‫ޔ‬ᵴᕈ‛⾰߇ ER ߣ⚿วߔࠆߎߣߦࠃߞߡ⺃ዉߐࠇࠆDNA ォ౮ᵴᕈൻࠍ࡟ࡐ࡯࠲࡯ㆮવሶ㧔Luciferase㧕ߩᵴᕈൻߦࠃࠆ luciferin ߩ⊒శࠍᜰᮡߣߒߡ
᷹ቯߔࠆ߽ߩߢ޽ࠅ‫ޔ‬ᒰ⹥⹜㛎ᴺߩේℂߪߎࠇߣ౒ㅢߢ޽ࠆ‫ޕ‬㘃ૃߩ⹜㛎ᴺߢ޽ࠆࠛࠬ࠻ࡠࠥࡦฃኈ૕⚿วᕈ⹜㛎ߪ‫ⵍޔ‬㛎‛⾰ߣ ER ߣߩ⚿วᕈࠍ᷹ቯߔࠆ
߽ߩߢ޽ࠆ߇‫⚿ޔ‬วߒߚ‛⾰߇ ER ࠍೝỗߔࠆߩ߆࡝ࠟࡦ࠼ߦᜟ᛫ߔࠆߩ߆ߪ್೎ߢ߈ߥ޿ߣߎ
ࠈ߇ᒰ⹥⹜㛎ᴺߣ⇣ߥߞߡ޿ࠆ‫ޕ‬
3. ᒰ⹥⹜㛎ᴺߣߘߩ࠺࡯࠲ߪ‫ޔ‬ㅘ᣿ߢ⁛┙ߥ⑼ቇ⊛⹏ଔࠍฃߌߡ޿ࠆ߆‫ޕ‬EU㧔ECVAM㧕ߣᣣᧄ㧔JaCVAM㧕ࠍ૬ߖߡ ICCVAM ߇⚵❱ߒߚㆇ༡ᆔຬળߩ▤ℂߩਅߢ‫ޔ‬3⹜㛎ᣉ⸳㧔☨࿖ߩ Xenobiotic Detection Systems, Inc.‫ޔ‬ECVAM Joint Research Centre‫ޔ‬ᣣᧄߩᣣศᩣ
ᑼળ␠㧕ߦࠃࠆᬌ⸽⹜㛎߇ታᣉߐࠇߚ‫ⵍޕ‬㛎‛⾰ߪ‫ޔ‬ICCVAM ߇ࠛࠬ࠻ࡠࠥࡦ߅ࠃ߮ࠕࡦ࠼ࡠࠥ
ࡦߦ㑐ߔࠆ in vitro ⹜㛎ᴺߩ⹏ଔߦ↪޿ࠆߴ߈‛⾰ߣߒߡ 2003 ᐕߦ⊒⴫ߒߚ 78 ‛⾰ߢ޽ࠆ 2)‫ޕ‬್ቯߦᔅⷐߥ↪㊂෻ᔕᦛ✢߇ᓧࠄࠇߕ‫ࡦࠥࡠ࠻ࠬࠛޔ‬ᵴᕈ࡮ᜟ᛫ᕈߩ᦭ή߇್ቯ࿎㔍ߢ޽ߞߚ 7‛⾰߇‫ߩߎޔ‬ᬌ⸽⹜㛎ߩ⸃ᨆ࡮⹏ଔ߆ࠄ㒰ᄖߐࠇߚߎߣߪ‫ޔ‬ᒰ⹥⹜㛎ᴺߩ೑↪ߦᒰߚߞߡ⇐ᗧߔߴ߈ߎߣߢ޽ࠆ‫ޕ‬33ߎߩ⹜㛎ߦ߅ߌࠆᒰ⹥⹜㛎ᴺߩᱜ⏕ᕈߪ‫ޔ‬ER ᵴᕈ 35 ‛⾰ߦ߅޿ߡ‫ޔ‬ᗵᐲ߇ 96%㧔27/28㧕‫․ޔ‬⇣ᐲ߇ 100%㧔7/7㧕ߢ‫ ߪ₸⥌৻ޔ‬97%㧔34/35㧕ߢ޽ߞߚ‫ޕ‬ER ᜟ᛫ᕈ 25 ‛⾰ߦ߅޿ߡߪ‫ޔ‬ᗵᐲ
߇ 100%㧔3/3㧕‫⇣․ޔ‬ᐲ߽ 100%㧔22/22㧕ߢ‫ ߪ₸⥌৻ޔ‬100%㧔25/25㧕 ߢ޽ߞߚ‫ޕ‬ߎߩ⹜㛎⚿ᨐߦߟ޿ߡߩ࿖㓙⊛╙ਃ⠪⹏ଔߦ߅޿ߡ‫ޔ‬ᒰ⹥⹜㛎ᴺߪ‫ޔ‬ER ᵴᕈ߅ࠃ߮ ER ᜟ᛫ᕈࠍᬌ಴ߔࠆ in vitro ⹜㛎ᴺߣߒߡㆡᱜߥ߽ߩߢ޽ࠆߣߐࠇߡ޿ࠆ‫ޕ‬ࠃߞߡ‫ޔ‬ᒰ⹥⹜㛎ᴺߣߘߩ࠺࡯࠲ߪㅘ᣿ߢ⁛┙ߥ⑼ቇ⊛⹏ଔࠍฃߌߡ޿ࠆߣ⸒߃ࠆ‫ޕ‬
4. ᒰ⹥⹜㛎ᴺߪ‫ޔ‬ᓥ᧪⹜㛎ᴺߩઍᦧᴺߣߒߡ‫⵾ߪߚ߹⾰‛ߥ߁ࠃߩߤޔ‬ຠࠍ⹏ଔߔࠆߎߣࠍ⋡⊛ߣߒߡ޿ࠆ߆‫ޕ‬ઍᦧߔߴ߈⹜㛎ᴺߪ⏕┙ߐࠇߡ޿ߥ޿߇‫ޔ‬ᒰ⹥⹜㛎ᴺߩ⹏ଔኻ⽎ߣߥࠆߩߪ‫ޔ‬ක⮎ຠ‫ޔ‬േ‛↪
ක⮎ຠ‫ޔ‬ㄘ⮎‫↥ޔ‬ᬺൻቇ‛⾰╬ߢ޽ࠆ‫ޔߒߛߚޕ‬ਇṁᕈ‛⾰߅ࠃ߮ើ⊒ᕈ‛⾰ߦኻᔕߔࠆ⹜㛎ᚻ㗅ߪ⏕┙ߐࠇߡ޿ߥ޿‫ޕ‬
5. ᒰ⹥⹜㛎ᴺߪ‫⹏࠼࡯ࠩࡂޔ‬ଔ޽ࠆ޿ߪ࡝ࠬࠢ⹏ଔߩߤߜࠄߦ᦭↪ߢ޽ࠆ߆‫ޕ‬
ᒰ⹥⹜㛎ᴺߪ‫ᤨ⃻ޔ‬ὐߢߪࡂࠩ࡯࠼⹏ଔߦ᦭↪ߢ޽ࠆ‫ޕ‬ߥ߅‫ޔ‬ᒰ⹥⹜㛎ᴺߪ‫ⵍޔ‬㛎‛⾰ߩỚᐲߦᔕߓߚᵴᕈߩ᷹ቯ߇น⢻ߢ޽ࠆ߆ࠄ‫⹏ࠢࠬ࡝ޔ‬ଔߦ૶↪ߢ߈ࠆน⢻ᕈߪ޽ࠆ߇‫ߛ߹ޔ‬ᬌ⸽߇ⴕࠊࠇߡ޿ߥ޿ߩߢ‫↪᦭ޔ‬ᕈߪਇ᣿ߢ޽ࠆ‫ޕ‬
6. ᒰ⹥⹜㛎ᴺߪ‫⵾ߪߚ߹⾰‛ࠆߔߣ⊛⋡ޔ‬ຠߩᲥᕈࠍ⹏ଔߢ߈ࠆ߆‫ߩߘޕ‬႐ว‫ޔ‬ᒰ⹥⹜㛎ᴺߩ
ㆡ↪᧦ઙ߇᣿⏕ߦߥߞߡ޿ࠆ߆‫ޕ‬
ᒰ⹥⹜㛎ᴺߪ‫ޔ‬ൻቇ‛⾰ߩ ER ᵴᕈ߅ࠃ߮ ER ᜟ᛫ᕈࠍ⺞ߴࠆߎߣ߇ߢ߈ࠆ‫ޕ‬ᒰ⹥⹜㛎ᴺߪ‫ޔ‬ၮᧄ⊛ߥࡊࡠ࠻ࠦ࡞ߣߒߡ‫⹜ޔ‬㛎‛⾰ࠍ DMSO ߦṁ⸃ߒߡㆡ↪ߒߡ߅ࠅ‫ޔ‬DMSO ߦਇṁߩ‛⾰߅ࠃ߮ើ⊒ᕈ‛⾰ߦㆡ↪ߢ߈ࠆ߆ߤ߁߆ߪ‫ޔ‬ᬌ⸛߇ߥߐࠇߡ޿ߥ޿ߩߢਇ᣿ߢ޽ࠆ‫ޕ‬
7. ᒰ⹥⹜㛎ᴺߪࡊࡠ࠻ࠦ࡞ߩᓸ⚦ߥᄌᦝߦኻߒߡ㗎ஜߢ޽ࠆ߆‫ޕ‬ᬌ⸽⹜㛎ߦෳടߒߚ 3 ᣉ⸳ߢߩᣉ⸳ౝౣ⃻ᕈߪ 12 ⵍ㛎‛⾰ߦߟ޿ߡ 100%ߢ޽ߞߚ‫ޕ‬ER ᵴᕈ‛
⾰ߦ߅ߌࠆᣉ⸳㑆ౣ⃻ᕈߪ 67%㧔8/12㧕‫ޔ‬ER ᜟ᛫ᕈ‛⾰ߢߩᣉ⸳㑆ౣ⃻ᕈ߽ 100%㧔12/12㧕ߢ޽ߞߚߩߢ‫ࠆ޽ޔ‬⒟ᐲߩ㗎ஜᕈߪᦼᓙߢ߈ࠆ‫ޕ‬
8. ᒰ⹥⹜㛎ᴺߩᛛⴚ⠌ᓧߪ‫ޔ‬ㆡಾߥ⸠✵ࠍ⚻ߡ޿ࠆᜂᒰ⠪ߦߣߞߡኈᤃߥ߽ߩߢ޽ࠆ߆‫ޕ‬ߎߩ⹜㛎ߢᔅⷐߥᛛⴚߪ‫ޔ‬ၭ㙃⚦⢩ࠍ↪޿ࠆ⹜㛎ᴺ৻⥸ߩᛛⴚ߅ࠃ߮⚦⢩ߩ⊒శࠍ᷹ቯߔࠆᛛ
ⴚߢ޽ࠅ‫ޔ‬ㆡಾߥ⸠✵ߦࠃߞߡኈᤃߦ⠌ᓧߢ߈ࠆ߽ߩߢ޽ࠆ‫ޕ‬
9. ᒰ⹥⹜㛎ᴺߪ‫ޔ‬ᓥ᧪⹜㛎ᴺߣᲧߴߡᤨ㑆⊛⚻⾌⊛ߦఝࠇߡ޿ࠆ߆‫ޕ‬
ᒰ⹥⹜㛎ᴺߩߚ߼ߦᔅⷐߥᯏེߪ‫ޔ‬ㅢᏱߩ⚦⢩ၭ㙃ߦⷐߔࠆⵝ⟎ߩ߶߆‫⚦ޔ‬⢩⊒శߩ᷹ቯߦ↪
޿ࠆశᐲ⸘ߢ޽ࠅ‫ޔ‬㜞ଔߥ߽ߩߢߪߥ޿‫⹜ޕ‬㛎ߦⷐߔࠆᤨ㑆ߪ‫ޔ‬ಓ⚿଻ሽߐࠇߚ⚦⢩ࠍⲢ⸃ߒߡ
Ⴧᱺߔࠆߩߦ 48㨪72 ᤨ㑆‫⹜ޔ‬㛎↪ၭ࿾ߦ⚦⢩ࠍ᠞⒳ߒߡߐࠄߦ 48㨪72 ᤨ㑆‫⹜ޔ‬㛎‛⾰ߣធ⸅ߐߖߡ Luciferase ᵴᕈൻࠍ⺃ዉߔࠆߩߦ 19㨪24 ᤨ㑆ࠍⷐߔࠆ߇‫ߩߘޔ‬ᓟߪ⚦⢩ࠍⲢ⸃ߐߖߡ⊒శࠍ᷹ቯߒ‫ࠍ࠲࡯࠺ޔ‬㓸⸘⸃ᨆߔࠆߩߺߢ޽ࠆ‫ߩࠄࠇߎޕ‬૞ᬺߦⷐߔࠆᤨ㑆ߪઁߩ in vitro 㘃ૃ⹜㛎ᴺߣᄌࠊࠄߥ޿‫ޕ‬44

10. ᒰ⹥⹜㛎ᴺߪ‫ޔ‬േ‛⑔␩ߩⷰὐ෸߮⑼ቇ⊛⷗࿾߆ࠄ‫⵾ߪߚ߹⾰‛ࠆߔߣ⊛⋡ޔ‬ຠߩᲥᕈࠍ⹏
ଔߔࠆઍᦧᴺߣߒߡ‫ⴕޔ‬᡽਄೑↪ߔࠆߎߣߪน⢻߆‫ޕ‬ᒰ⹥⹜㛎ᴺߪၭ㙃⚦⢩ࠍ↪޿ࠆ in vitro ⹜㛎ᴺߢ޽ࠅ‫ߩઁޔ‬㘃ૃ⹜㛎ᴺߣหߓ⒟ᐲߦ‫ⴕޔ‬᡽਄೑↪น⢻ߢ޽ࠆ‫ޕ‬ෳ⠨ᢥ₂
1)

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. 11-7814; 2011)2)

ICCVAM Evaluation of In vitro Test Methods for Detecting Potential Endocrine
Disruptors: Estrogen Receptor and Androgen Receptor Binding and Transcriptional
Activation Assays. (NIH Publication No: 03-4503; 2003)

3)

OECD TG457 : BG1Luc Estrogen Receptor Transactivation Test Method for Identifying
Estrogen Receptor Agonists and Antagonists.

4)(2012)

BG1LucER TA (LUMI-CELL ER) ᴺ㧦in vitro ࡅ࠻ࠛࠬ࠻ࡠࠥࡦฃኈ૕ᵴᕈ‛⾰⹜㛎ᴺߩ⹏ଔႎ๔㧔ᐔᚑ 24 ᐕ 11 ᦬㧕

5
5

6

BG1LucER TA (LUMI-CELL ER) ᴺ㧦in vitro ࡅ࠻ࠛࠬ࠻ࡠࠥࡦฃኈ૕ᵴᕈ‛⾰⹜㛎ᴺߩ⹏ଔႎ๔ᦠ

ᐔᚑ 24 ᐕ 11 ᦬ 29 ᣣ17ౝಽᴲ߆ߊੂ⹜㛎ᴺ⹏ଔᆔຬળ
ᆔຬฬ㧦
ዊ㊁ ብ㧔ᆔຬ㐳㧦㧔৻⽷㧕㘩ຠ⮎ຠ቟ో࠮ࡦ࠲࡯⒌㊁⎇ⓥᚲ㧕ਣ㊁ౝ᫢㧔⮮↰଻ஜⴡ↢ᄢቇ㧕੗ญᵏᴰ㧔ၮ␆↢‛ቇ⎇ⓥᚲ㧕ਛỈᙗ৻㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ㧕

2
8

↪⺆㓸
Accuracy㧦ᱜ⏕ᐲConcordance㧦৻⥌₸
Estoregen receptor㧔ER㧕Į/ȕ㧦ࠛࠬ࠻ࡠࠥࡦฃኈ૕ Į/ȕ
Estrogen responsive element㧔ERE㧕㧦ᩭౝߩࠛࠬ࠻ࡠࠥࡦ෻ᔕㇱ૏European Centre for Validation of Alternative Methods㧔ECVAM㧕㧦᰷Ꮊઍᦧᴺᬌ⸽࠮ࡦ࠲࡯
False negative rate㧦ன㒶ᕈ₸
False positive rate㧦ன㓁ᕈ₸Federal Register㧦㧔☨࿖㧕ㅪ㇌ቭႎ
Independent International Scientific Peer Review Panel㧦࿖㓙╙ਃ⠪ኾ㐷ᛛⴚ⹏ଔᆔຬળ
Interagency Committee on the Validation of Alternative Methods㧔ICCVAM)㧦☨࿖ઍᦧᴺ⹏ଔ⋭ᐡ㑆ㅪ⛊ᆔຬળInter-laboratory validation study㧦ᣉ⸳㑆ᬌ⸽Intra-laboratory validation study㧦ᣉ⸳ౝᬌ⸽
Japanese Center for Validation of Alternative Methods㧔JaCVAM㧕㧦ᣣᧄઍᦧᴺ⹏ଔ࠮ࡦ࠲࡯
National Institute of Health㧔NIH㧕㧦☨࿖࿖┙ⴡ↢⎇ⓥᚲ
National Toxicology Program Interagency Center for the Validation of Alternative Methods㧔NICEATM㧕㧦
Ქᕈቇ࿖ኅ੐ᬺઍᦧ⹜㛎ᴺᬌ⸽⋭ᐡ㑆ㅪ⛊࠮ࡦ࠲࡯
Reliability㧦ା㗬ᕈTranscription activation㧔TA㧕㧦ォ౮ᵴᕈൻ
US Environmental Protection Agency㧔US EPA㧕㧦☨࿖ⅣႺ଻⼔ᐡ
Uterotrop(h)ic assay㧦ሶች⢈ᄢ⹜㛎
Organisation for Economic Co-operation and Development㧔OECD㧕㧦⚻ᷣදജ㐿⊒ᯏ᭴
Xenobiotic Detection Systems, Inc.㧔XDS㧕㧦XDS ␠Validation study㧦ᬌ⸽⹜㛎
Validation study management team㧦ᬌ⸽⹜㛎ㆇ༡ᆔຬળ

39㧝㧚ᧄ⹜㛎ᴺߩ⑼ቇ⊛ᅷᒰᕈߣⷙ೙⹜㛎ᴺߣߒߡߩᅷᒰᕈLUMI-Cell ER ⹜㛎ᴺߔߥࠊߜ BG1LucER TA ᴺ㧔⹦ߒߊߪ‫ޟ‬BG1Luc4E2 ⚦⢩ࠍ↪޿ࠆࠛࠬ࠻ࡠࠥ
ࡦฃኈ૕㧔ER㧕ォ౮ᵴᕈൻ㧔TA㧕⹜㛎‫ޠ‬㧕ߪ‫ࡦࠥࡠ࠻ࠬࠛߩ⾰‛ޔ‬ᵴᕈࠍ᷹ቯߔࠆ in vitro ⹜㛎ᴺߩ৻ߟߢ‫ޔ‬ౝಽᴲᡬੂ‛⾰ኻ╷ߩߚ߼ߦ㐿⊒߇᳞߼ࠄࠇߡ߈ߚ߽ߩߢ޽ࠆ‫⹜ᧄޕ‬㛎ᴺߪ‫☨ޔ‬࿖ North
Carolina Ꮊ Durham ߦ޽ࠆ Xenobiotic Detection Systems, Inc.㧔XDS㧕␠ߢ㐿⊒ߐࠇߚ߽ߩߢ‫࠻ࡅޔ‬ෆᎽ≸↱᧪ᩣ⚦⢩ BG1 ߦ࡟ࡐ࡯࠲࡯ㆮવሶߣߒߡ Luciferase responsive element ࠍ฽߻ plasmid ࠍᩭౝߩࠛ
ࠬ࠻ࡠࠥࡦ෻ᔕㇱ૏㧔ERE㧕ߩਅᵹߦ቟ቯ⊛ߦዉ౉ߒߚ⚦⢩ࠍ↪޿‫⚦ߩߎޔ‬⢩ߦౝ࿷ߔࠆ ER ߩᵴᕈൻߦࠃߞߡ⿠ࠆㆮવሶᄌൻࠍ⊒శߢᬌ಴ߒߘࠇࠍቯ㊂⊛ߦ᷹ቯߔࠆ⹜㛎ᴺߢ޽ࠆ‫ޕ‬Interagency
Committee on the Validation of Alternative Methods㧔ICCVAM㧕ߪߎߩ⹜㛎ᴺߩᬌ⸽⹜㛎㧔Validation study㧕ߦၮߠߊ⹏ଔࠍⴕ޿‫↪᦭ߩߘޔ‬ᕈࠍ⹺߼‫ޔ‬US Environmental Protection Agency㧔US EPA㧕߅ࠃ߮
Organisation for Economic Co-operation and Development㧔OECD㧕ߦ⹜㛎ᴺࠟࠗ࠼࡜ࠗࡦߣߒߡណᛯߔ
ࠆࠃ߁ឭ᩺ߒߚ‫ޕ‬ౝಽᴲᯏ⢻ߦᓇ㗀ߔࠆ↢‛ᵴᕈࠍ᦭ߔࠆ‛⾰ߪ‫ߩ╬ࡦࡕ࡞ࡎޔ‬ᄤὼߩ↢૕‛⾰ߩ߶߆วᚑൻቇ‛⾰ߦ߽ᄙᢙ⍮ࠄࠇࠆࠃ߁ߦߥߞߚ߇‫ߩߘޔ‬ᵴᕈߩ᦭ήߣ⒟ᐲߦߟ޿ߡᧂ⺞ᩏߩ‛⾰߇ᄙ޿‫‛ߚߒ߁ߎޕ‬⾰ߩ߁ߜ‫ޔ‬ᄢ㊂ߩᦑ㔺ߦࠃߞߡ↢૕ߩౝಽᴲᯏ⢻߅ࠃ߮ߎࠇߣ㑐ଥߒߚ↢ᱺ⊒↢╬ߦᓇ㗀ࠍ෸߷ߔ߽
ߩ㧔ౝಽᴲᡬੂ‛⾰ߣ๭߫ࠇࠆ㧕ߢ޽ࠆߎߣ߇ ᔨߐࠇࠆ߽ߩ߇޽ࠅ‫⃻ޔ‬࿷‫ޔ‬ᓇ㗀ߩ᦭ήࠍ⏕⹺ߔࠆ⹜㛎ᴺߩ㐿⊒߇᳞߼ࠄࠇߡ޿ࠆ‫ߩ⹺⏕ߩߎޔߚ߹ޕ‬ኻ⽎ߣߥࠆ‛⾰ߩᢙ߇⤘ᄢߥ߽ߩߢ޽ࠆߚ߼‫ޔ‬ല₸⊛ߥࠬࠢ࡝࡯࠾ࡦࠣࠍⴕ߁⹜㛎ᴺߩ㐿⊒ߣታ↪ൻ߇᳞߼ࠄࠇߡ߈ߚ‫ߩࠣࡦ࠾࡯࡝ࠢࠬޕ‬ᜰᮡߣߒߡ
᦭ᦸߥᕈ⾰ߦߪ‛⾰ߩౝಽᴲᵴᕈ߇޽ࠅ‫ࡦࡕ࡞ࡎߩ⾰‛ޔ‬ฃኈ૕ߣߩ⚿วᕈ߹ߚߪౝಽᴲᯏ⢻ߩᵴᕈൻࠍ᷹ቯߔࠆᣇᴺ߇ᵈ⋡ߐࠇߡ޿ࠆ‫ࡦࠥࡠ࠻ࠬࠛޕ‬ᵴᕈߦ㑐ߔࠆ⹜㛎ᴺߣߒߡߪ‫ ߦߢߔޔ‬in vivo ⹜
㛎ᴺߣߒߡ‫ྐྵޟ‬ᱤ㘃ࠍ↪޿ࠆሶች⢈ᄢ⹜㛎‫ޔࠇߐ┙⏕߇ޠ‬ᬌ⸽⹜㛎ࠍⴕߞߚ਄ߢ࿖㓙⊛ߥ⹜㛎ᴺࠟࠗ
࠼࡜ࠗࡦ㧔OECD TG㧕ߣߥߞߡ޿ࠆ߇‫◲ࠅࠃޔ‬ଢߢㄦㅦߥ㕖േ‛⹜㛎ߣߒߡၭ㙃⚦⢩ࠍ↪޿ࠆ in vitro⹜㛎ᴺߩ㐿⊒߇ㅴ߼ࠄࠇߡ޿ࠆ‫ޕ‬‛⾰ߩ ER ߣߩ⚿วᕈࠍ in vitro ߢ⹜㛎ߔࠆᣇᴺߦߪ‫↢ޔ‬૕߹ߚߪၭ㙃⚦⢩߆ࠄ᛽಴ߒߚ ER ߦኻߔࠆൻቇ‛⾰ߩ┹ว⚿ว෻ᔕߩ᷹ቯ߇޽ࠅ‫࠻࠶࡜߫߃ߣߚޔ‬ሶች߆ࠄ᛽಴ߒߚ ER ࠍ↪޿ࠆ‫࠻࠶࡜ޟ‬ሶችࠛࠬ࠻ࡠࠥࡦฃኈ૕⚿วᕈ⹜㛎‫߇ޠ‬᜼ߍࠄࠇࠆ‫ߪࠇߎޕ‬ή⚦⢩♽ߩ⚿ว⹜㛎ߢ޽ࠅ‫↪ߥ⚦⹦ޔ‬㊂෻ᔕ㑐ଥࠍ⏕⹺ߢ߈‫ ߩ⾰‛ޔ‬ER ⚿วᕈࠍᮡḰ‛⾰‫ߣ࡞࡯ࠝࠫ࡜࠻ࠬࠛ߫߃ߣߚޔ‬Ყセߒߡቯ㊂⊛ߦ᳿ቯߢ߈ࠆ߽ߩߢ޽ࠆ‫ޔߒ߆ߒޕ‬ER ߦኻߒߡߪฃኈ૕ೝỗ‛⾰߫߆ࠅߢߥߊᜟ᛫‛⾰߽⚿วᕈ߇޽ࠆ
ߩߢ‫ޔ‬ਔ⠪ߩ඙೎߇಴᧪ߥ޿‫ߩߘޕ‬඙೎ߩߚ߼ߦߪ‫ ߣ⾰‛ޔ‬ER ߣߩ⚿ว߇⿠ߎߔ↢‛ቇ⊛ലᨐࠍ⏕⹺ߔࠆᔅⷐ߇޽ࠅ‫᦭ޔ‬ᗧ⟵ߥᜰᮡߣߒߡ↢૕ߦ⺃⊒ߐࠇࠆࠛࠬ࠻ࡠࠥࡦലᨐࠍ᷹ቯߔࠆ in vivo ⹜㛎㧔ߚߣ߃߫࡜࠶࠻ሶች⢈ᄢ⹜㛎㧕߇೑↪ߐࠇߡ޿ࠆ‫ޕ‬In vitro ߢ߽‫ޔ‬ER ߇ᵴᕈ‛⾰ߣ⚿วߒߚߩߜߦ
⚦⢩ౝߢ⿠ߎࠆᔕ╵߇ⷰኤߢ߈ࠇ߫‫ࡦࠥࡠ࠻ࠬࠛޔ‬ലᨐࠍ⏕⹺ߢ߈ࠆ‫ޕ‬ᵴᕈ‛⾰ߣ⚿วߒߚ⚦⢩ౝER ߪ‫ޔ‬ᩭౝߩ ERE ߣߩ⚿วࠍㅢߓߡ㑐ㅪ‛⾰ࠍࠦ࡯࠼ߔࠆㆮવሶ㧔DNA㧕ߩォ౮ࠍ⿠ߎߔ‫ߩߎޕ‬ࠃ߁ߥㆮવሶࠍ੺ߔࠆᔕ╵ߦᔅⷐߥᯏ᭴ࠍ଻ᜬߔࠆ⚦⢩ߦ߅޿ߡᬌᩏߔࠆߎߣߪᗧ⟵߇޽ࠆ‫ޕ‬
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BG1LucER TA ᴺߪ‫ࡦࠥࡠ࠻ࠬࠛޔ‬ᵴᕈ‛⾰ߦࠃࠆ ER ⚿วߩᰴߩㆊ⒟ߣߒߡ㑐ㅪㆮવሶߩォ౮߇⺃ዉߐࠇࠆߎߣࠍⷰኤߔࠆᣇᴺߢ޽ࠆ‫⚦ޕ‬⢩ߦߪォ౮ᵴᕈൻࠍ⺞ߴࠆߚ߼ߦ‫ޔ‬ᒰ⹥ㆮવሶߣㅪ⚿ߒߚ૏⟎㧔ਅᵹ㧕ߦ⊒శ⺃⊒ㆮવሶࠍ⚵ߺㄟࠎߢ߅߈‫ޔ‬ォ౮߇⿠ࠇ߫⚦⢩߇⊒శߔࠆࠃ߁ߥᠲ૞ࠍട߃ߡ
޽ࠆ‫⚦ߥ߁ࠃߩߎޕ‬⢩ߣߒߡߪ‫ ߦߢߔޔ‬HeLa ⚦⢩ߦࡅ࠻ࠛࠬ࠻ࡠࠥࡦฃኈ૕ Į㧔hERĮ㧕ߣ⊒శ㉂⚛
㧔Luciferase㧕ㆮવሶࠍ⚵ߺㄟࠎߛ⚦⢩߇㐿⊒ߐࠇߡ߅ࠅ‫⚦ߩߎޔ‬⢩ࠍ↪޿ߚ⹜㛎ᴺ‫ޟ‬hERĮ-HeLa-9903⚦⢩ࠍ↪޿ࠆ቟ቯዉ౉ォ౮ᵴᕈൻ⹜㛎 Stably Transfected Human Estrogen Receptor-Į Transcriptional
Activation Assay㧔hERĮ-HeLa STTA㧕‫ޔ߇ޠ‬US EPA ߩ⹜㛎ᴺߣߒߡណᛯߐࠇ㧔OPPTS 890.1300, 2009㧕‫ޔ‬߹ߚ OECD ⹜㛎ᴺࠟࠗ࠼࡜ࠗࡦߦ෼タߐࠇߡ޿ࠆ㧔OECD TG 455, 2009㧕‫ޕ‬hERĮ-HeLa STTA ᴺߣᧄ BG1LucER TA ᴺߩ⋧㆑ߪ‫(ޔ‬1) ೨⠪߇ hERĮ ࠍ HeLa ⚦⢩ߦ⚵ߺㄟࠎߛ߽
ߩߢ޽ࠆߩߦኻߒ‫ᧄޔ‬ᴺߪ BG1 ⚦⢩ߦౝ࿷ߔࠆ ER ࠍ೑↪ߔࠆ‫(ޔ‬2) ೨⠪ߢߪ⚵ߺㄟ߹ࠇߚฃኈ૕ߪERĮ ߢ޽ࠆ߇‫ᧄޔ‬ᴺߩ BG1 ⚦⢩ߪ ERĮ ߣ ERȕ ߩ෺ᣇࠍౕ߃ߡ޿ࠆ‫(ޔ‬3) ೨⠪ߪኾࠄ ER ೝỗ‛⾰㧔ࠕࠧ࠾ࠬ࠻㧕ߩ⹜㛎ࠍⴕ߁߇‫ᧄޔ‬ᴺߢߪ‫ޔ‬ER ᜟ᛫‛⾰㧔ࠕࡦ࠲ࠧ࠾ࠬ࠻㧕ߩᵴᕈࠍ᷹ቯߔࠆࡊࡠ࠻ࠦ
࡞߽↪ᗧߐࠇߡ޿ࠆ‫ޕࠆ޽ߢߣߎޔ‬ 2004 ᐕ ICCVAM ߪ‫ޔ‬XDS ␠߆ࠄឭ᩺ߐࠇߚ BG1LucER TA ᴺࠍ⹏ଔߒ‫ޔ‬ᣉ⸳㑆ᬌ⸽⹜㛎ߦઃߒߡߘߩ᦭↪ᕈࠍᐢߊ⏕⹺ߔࠆߦ୯ߔࠆ߽ߩߢ޽ࠆߣ⹺߼ߚ‫ࠍࠇߎޕ‬ฃߌߡ National Toxicology Program
Interagency Center for the Validation of Alternative Methods㧔NICEATM㧕߇࿖㓙⊛ᣉ⸳㑆ᬌ⸽⹜㛎ࠍડ↹‫ޔ‬ታⴕߒߚ‫ޕ‬Japanese Centerre for Validation of Alternative Methods JaCVAM ߅ࠃ߮ European Centre for
Validation of Alternative Methods㧔ECVAM㧕ߪߎࠇߦදജߒߡᬌ⸽⹜㛎ㆇ༡ᆔຬળࠍ⚵❱ߒߚ‫⹜ޕ‬㛎ᣉ⸳ߣߒߡ‫☨ޔ‬࿖ߩ XDS ␠‫ޔ‬ᣣᧄߩᩣᑼળ␠ᣣศ‫߮ࠃ߅ޔ‬᰷Ꮊߩ ECVAM ߇ߎߩᬌ⸽⹜㛎ߦෳടߒ‫ޔ‬
BG1LucER TA ᴺߦࠃߞߡ‛⾰ߩ ER ࠕࠧ࠾ࠬ࠻ᵴᕈ߅ࠃ߮ ER ࠕࡦ࠲ࠧ࠾ࠬ࠻ᵴᕈߩ⹜㛎ࠍታᣉߒ‫ޔ‬ߘߩ࠺࡯࠲ߦၮߠ޿ߡᧄ⹜㛎ᴺߩ⹜㛎ᕈ⢻ߩᱜ⏕ᕈߣା㗬ᕈࠍ⹏ଔߔࠆߚ߼ߩᬌ⸽ࠍⴕߞߚ‫ޕ‬
㧔Fig.1-1㧕ᬌ⸽⹜㛎ߩ╙ 1 Ბ㓏ߢߪ‫ޔ‬ER ࠕࠧ࠾ࠬ࠻‫ޔ‬ER ࠕࡦ࠲ࠧ࠾ࠬ࠻ฦ 10 ‛⾰ߩ⹜㛎ࠍⴕ޿‫ޔ‬ෳട⹜㛎
ᣉ⸳ߩᛛ㊂ߩ⹏ଔࠍⴕߞߚ‫ ╙ޕ‬2ޯ4 Ბ㓏ߢߪ‫⹏ߦ࡞ࠦ࠻ࡠࡊޔ‬ଔߣᡷ⦟ࠍട߃ߟߟ‫ޔ‬in vitro ER ࠕࠧ࠾ࠬ࠻‫⹜࠻ࠬ࠾ࠧ࠲ࡦࠕޔ‬㛎ߩᬌ⸽ߦ↪޿ࠆߴ߈ෳᾖ‛⾰ߣߒߡ ICCVAM ߇ㆬቯߒߡ޿ߚ 78 ‛⾰ߩ⹜㛎ࠍⴕߞߚ‫ޕ‬

511ᬌ⸽⹜㛎ߩ⚿ᨐ‫ޔ‬3 ⹜㛎ᯏ㑐ߩ࠺࡯࠲ߪ㜞޿৻⥌₸ࠍ␜ߒߚ‫ޕ‬ICCVAM ෳᾖ‛⾰ߩಽ㘃㧔㓁ᕈ࡮㒶ᕈ㧕ߣ 3 ⹜㛎ᯏ㑐ߩ࠺࡯࠲ߩ৻⥌₸߽㜞޿߽ߩߢ޽ߞߚ‫ޕ‬ᓥߞߡ‫ޔ‬BG1LucER TA ᴺߪ‫ ߩ⾰‛ޔ‬in vitro
ER ࠕࠧ࠾ࠬ࠻ᵴᕈࠍᱜ⏕ߦᬌ಴ߔࠆ⹜㛎ߢ޽ࠅ‫ޔ‬ᣢሽߩ ER TA ᴺ㧔hERĮ-HeLa STTA㧕ߣห╬ߩᕈ⢻ࠍ᦭ߒ‫ ߩ⾰‛ߚ߹ޔ‬in vitro ER ࠕࡦ࠲ࠧ࠾ࠬ࠻ᵴᕈࠍᱜ⏕ߦᬌ಴ߢ߈ࠆ⹜㛎ᴺߢ޽ࠆ‫ࠇߐ⺰⚿ߣޔ‬ߚ‫⹜ޕ‬㛎ᴺߩା㗬ᕈߦߟ޿ߡߪ‫⹜ޔ‬㛎ᴺᣉ⸳ౝౣ⃻ᕈ‫ޔ‬ᣉ⸳㑆ౣ⃻ᕈߣ߽ߦ⦟ᅢߥ⚿ᨐߢ޽ࠅ‫ޔ‬ᛛⴚ
⒖ォᕈ‫⹜ޔ‬㛎ᴺߩ㗎ஜᕈߦ໧㗴ߪߥߊ‫ޔ‬ㅢᏱߩ⚦⢩ၭ㙃ታ㛎ߩᣉ⸳ߣ⊒శಽᨆᯏེ߇஻߃ࠄࠇߡ޿ࠇ
߫‫ޔ‬ᛛⴚ⊛ߦ․ߦ࿎㔍ߥㇱಽ߽ߥ޿ߩߢ‫ⴕޔ‬᡽ⷙ೙⹜㛎ߣߔࠆᅷᒰᕈߪ⹺߼ࠄࠇࠆ‫ޕ‬BG1Luc ⚦⢩ߪCalifornia ᄢቇ Davis ᩞߩ Research Technology Transfer Services ߆ࠄ‫ ߪ޿ࠆ޽ޔ‬XDS ␠߆ࠄ౉ᚻߔࠆߎ
ߣ߇಴᧪ࠆ‫ޕ‬ߎߩᬌ⸽⹜㛎ߩ⚳ੌᓟ‫ޔ‬NICEATM ߣ ICCVAM ߪ‫ޔ‬2011 ᐕ 1 ᦬‫ޔ‬Independent International ScientificPeer Review Panel㧦એਅ Panel㧕ࠍ⚵❱ߒߡ Peer Review ࠍᆔབྷߒߚ‫ޕ‬Panel ߪⶄᢙ࿁ߩ㔚⹤ળ⼏ࠍⴕߞߚᓟ‫ޔ‬2011 ᐕ 3 ᦬ 29ޯ31 ᣣ Maryland Ꮊ Bethesda ߩ National Institute of Health㧔NIH㧕ߢ㓸วળ⼏ࠍ㐿௅ߒ‫ޔ‬5 ᦬ߦႎ๔ᦠࠍឭ಴ߒߚ‫ޔߪࠇߎޕ‬2011 ᐕ 5 ᦬ 18 ᣣߩ Federal Register ߦ⊒⴫ߐࠇߚ‫ޕ‬
ߎߩ⹜㛎ᴺߪ‫ޔ‬หᐕ OECD ߦឭ᩺ߐࠇ‫ޔ‬TG455 ߦ⚵ߺ౉ࠇࠆߴߊട⋖ฦ࿖ߦᗧ⷗⡬ข࿁ⷩਛߢ޽
ࠆ‫ޕ‬೨ㅀߩࠃ߁ߦ‫ ߦߢߔޔ‬TG455 ߣߒߡ hERĮ-HeLa STTA ߇෼タߐࠇߡ޿ࠆ߇‫ޔ‬ේℂ⊛߅ࠃ߮ᯏ⢻⊛ߦห᭽ߥᕈ⢻ߦၮߠ޿ߚ⹜㛎ᴺߣߒߡ‫ޔ‬หߓ TG455 ߦ ANNEX 3 ߣߒߡട߃ࠆߎߣߣߐࠇߡ޿ࠆ‫ޕ‬㧔ERĮ-HeLa cell ᴺߪ ANNEX 2 ߣߥࠆ㧕‫ޕ‬ᧄ⹜㛎ᴺߩ੹ᓟߩ໧㗴ὐߣߒߡ‫⚦ޔ‬⢩ߦౝ࿷ߔࠆ 2 ⒳㘃ߩࠛࠬ࠻ࡠࠥࡦฃኈ૕ ERĮ ߣ ERȕ ߩ᷹ቯ612⚿ᨐߦኻߔࠆነਈߩᲧ₸ࠍ᣿ࠄ߆ߦߔࠆߎߣ߇⺖㗴ߣߒߡᱷߞߡ޿ࠆ‫ޔߪࠇߎޕ‬ౝಽᴲᡬੂᕈ߇ࠛࠬ
࠻ࡠࠥࡦᵴᕈߦ⿠࿃ߔࠆߣ޿߁⠨߃ߦ߅޿ߡ‫ޔ‬ERĮ ߣ ERȕ ߩ㑐ਈߩ⒟ᐲࠍផ㊂ߔࠆߣ޿߁㊀ⷐߥ⺖㗴ߢ޽ࠆ‫ߩߎޕ‬ಽ೎᷹ቯᴺߩ㐿⊒߇ߢ߈ࠇ߫‫ࡦࠥࡠ࠻ࠬࠛߩ⾰‛ޔ‬ᵴᕈߩ⹦⚦ߥᛠី߇น⢻ߦߥࠅ‫ޔ‬
ߎߩಽ㊁ߩ⎇ⓥߣൻቇ‛⾰▤ℂߦᄢ޿ߦ⽸₂ߔࠆߣ⠨߃ࠄࠇࠆ‫ޕ‬㧞㧚⹜㛎ᴺߩᅷᒰᕈ
2-1 ⹜㛎ᴺߩ᭎⇛㧝㧕⋡⊛ߣේℂ
ߎߎߢขࠅ਄ߍࠆ⹜㛎ᴺߪౝಽᴲᠣੂ‛⾰ߩ߁ߜࠛࠬ࠻ࡠࠥࡦߦ㑐ㅪߔࠆൻቇ‛⾰ࠍ᷹ቯߒ‫ߩߘޔ‬ੱ૕‫⥄ޔ‬ὼ⇇ߦኻߔࠆᑷኂࠍㆱߌࠆߎߣࠍ⋡⊛ߣߒߡ㐿⊒ߐࠇߚ‫ᧄޕ‬ᴺ‫ޔ‬BG1LucER TA ᴺߪߘߩ৻ߟߢ↢૕ߩ ER ߦ⚿วߒ‫࠻ࠬ࠾ࠧࠕޔ‬૞↪‫࠻ࠬ࠾ࠧ࠲ࡦࠕߪ޿ࠆ޽ޔ‬૞↪ࠍ␜ߔൻቇ‛⾰ࠍࠬࠢ࡝࡯
࠾ࡦࠣ‫ޔ‬᷹ቯߔࠆߚ߼ߦ ER ࠍ⊒⃻ߔࠆၭ㙃⚦⢩ߦ ER ᔕ╵ᕈߩ࡞ࠪࡈࠚ࡜࡯࠯ㆮવሶࠍ࡟ࡐ࡯࠲࡯
ㆮવሶߣߒߡዉ౉࡮቟ቯ⊒⃻ߐߖ‫ⵍޔ‬ᬌൻቇ‛⾰ࠍᛩਈߒߡ⚦⢩ߦ෻ᔕߐߖߚᓟ‫࠯࡯࡜ࠚࡈࠪ࡞ޔ‬ᵴᕈߩᄌൻࠍ㧔࡞ࠪࡈࠚ࡝ࡦߩ㧕⊒శߦࠃࠅ᷹ቯߢ߈ࠆ᭽ߦߒߚ߽ߩߢ޽ࠆ‫ޕ‬㧞㧕ᮡḰ᷹ቯ᧦ઙ
ᰴߩὐߦ⇐ᗧߔࠆᔅⷐ߇޽ࠆ‫ޕ‬a.

ၭ㙃⚦⢩ߩ⛽ᜬ▤ℂ㧦ၭ㙃⚦⢩ߪోߡή⩶⁁ᘒߢขࠅᛒ߁ᔅⷐ߇޽ࠆ‫ޕ‬Ḱ஻Ბ㓏ߩၭ㙃߆ࠄ‫ޔ‬96 ࠙ࠚ࡞࡮ࡊ࡟࡯࠻ਛߩ⹜㛎ߦ⥋ࠆ߹ߢ‫⚦ޔ‬⢩߇ࡈ࡜ࠬࠦ߿࠙ࠚ࡞ౝߢဋ৻ߥኒᐲߦߥࠆ᭽
ߦ⛽ᜬߐࠇߥߌࠇ߫ߥࠄߥ޿‫ޕ‬ICCVAM ႎ๔ᦠߦᷝઃߐࠇߚࡊࡠ࠻ࠦ࡞㧔Appendix B㧕ߦ޽ࠆၭ㙃ಣᣇࠍᔘታߦ቞ࠆߎߣ‫⚦ޕ‬⢩Ớᐲߩ⺞⵾ᣇᴺߥߤ߽฽߼ߡ‫ޔ‬ၭ㙃⚦⢩ߩᛒ޿ᣇࠍ೨߽
ߞߡ․೎ߦ⸠✵ࠍฃߌߡ߅ߊᔅⷐ߇޽ࠆ‫ޕ‬b. ᮡḰ᧦ઙߩ⸳ቯ㧦᷹ቯ୯ߪోߡ⋧ኻ୯ߢᲧセߔࠆߩߢ 17ȕ-ࠛࠬ࠻࡜ࠫࠝ࡯࡞㧔E2 ߩㆡಾߥỚᐲࠍᏱߦห৻ߩ 96 ࠙ࠚ࡞࡮ࡊ࡟࡯࠻਄ߢᮡḰߣߒߡ᷹ቯߒ‫ߦࠇߘޔ‬ኻߔࠆ⋧ኻ୯ߣߒߡ⴫
ߔ‫ޕ‬c.

ⵍ㛎ൻว‛㧦෻ᔕ㓁ᕈߩൻቇ‛⾰ߣหᤨߦ㒶ᕈߩൻቇ‛⾰߽⹜㛎ߔࠆߎߣ‫ ߦࠄߐޔ‬ER ࠕࡦ࠲ࠧ࠾ࠬ࠻૞↪ߩ޽ࠆൻቇ‛⾰ߩലᨐ߽หᤨߦ⹜㛎ߔࠆߎߣ߇㊀ⷐߢ޽ࠆ‫ޕ‬2-2 ᅷᒰᕈߩᬌ⸛
㧝㧕✚ว⊛ᬌ⸛㧦BG1LucER TA ᴺߩၮߣߥࠆ⚦⢩‫ޔ‬BG1 ߪᄤὼߩ ER ࠍ⊒⃻ߒߡ߅ࠅ‫ޔ‬ER ࠕࠧ࠾ࠬ࠻߅ࠃ߮ ER ࠕࡦ࠲ࠧ࠾ࠬ࠻ߩਔ⠪߇᷹ቯน⢻ߥ in vitro ♽ߢ޽ࠆ‫ޕ‬ṁᇦߦṁ⸃น⢻㧔DMSO ࠍ↪޿ࠆ႐ว߽޽ࠆ㧕ߥᐢ▸ߥⵍ㛎ൻቇ‛⾰ߦㆡ↪ߢ߈ࠆὐߢ㕖Ᏹߦଔ୯߇㜞޿‫ ߒߛߚޕ‬ERĮ ဳ㧛ȕ ဳߩൻቇ‛⾰ߣߩ⚿ว߿ߘࠇߙࠇߩ↢ℂ⊛૞↪ലᨐ߅ࠃ߮ߘߩ⼂೎߇චಽߦ⸃᣿ߐࠇߡ޿ߥ޿ߩߢߘߩ
ὐߪᣧᦼߦ⼂೎น⢻ߥ♽ߣߩᲧセᬌ⸛߇ᦸ߹ࠇࠆ‫ޕ‬ℂ⺰⊛ⷞὐ߆ࠄ㧦ER ࠕࠧ࠾ࠬ࠻ᵴᕈ߅ࠃ߮ ER ࠕࡦ࠲ࠧ࠾ࠬ࠻૞↪ߩ޽ࠆൻቇ‛⾰ߦࠃࠆ ERĮ ဳ㧛ȕ ဳߦኻߔࠆන
713ߥࠆ⚿ว⢻ߢߪߥߊᵴᕈࠍ᷹ቯߢ߈ࠆ‫ޕ‬in vitro ♽ߢߪ޽ࠆ߇‫߼ߚࠆ޽ߢࠗ࠮࠶ࠕࠝࠗࡃޔ‬෻ᔕ♽‫ޔ‬ၭ㙃⚦⢩ߩ⛽ᜬ▤ℂ߇⚿ᨐߦᓇ㗀ࠍ෸߷ߔน⢻ᕈߪุቯߢ߈ߥ޿‫⴫ߪߣߎߩߎޕ‬㕙਄ߦߪ⃻ࠇ㔍޿⹜㛎⚿ᨐߩᄌേߦߟߥ߇ࠆน⢻ᕈ߇㜞޿ߩߢචಽߦᵈᗧ߇ᔅⷐߢ޽ࠆ‫ޕ‬᷹ቯ♽᭴▽਄ߩⷞὐ߆ࠄ㧦a.

ၭ㙃⚦⢩㧦BG1LucER TA ᴺߢߪࡅ࠻ෆᎽ≸↱᧪ᩣ⚦⢩‫ޔ‬BG1 ⚦⢩߇ ER ࠍ⊒⃻ߒߡ޿ࠆߎ
ߣࠍၮߦࠕ࠶࠮ࠗ♽ࠍ᭴▽ߒߡ޿ࠆ‫ߩ࠻ࡅߦ⥸৻ޕ‬ၭ㙃⚦⢩ߢߪᨴ⦡૕ߩᒻᘒ߅ࠃ߮ᢙߩ⇣Ᏹࠍ㜞㗫ᐲߦ᦭ߔࠆߎߣ߇⍮ࠄࠇߡ޿ࠆ‫ߩߘޕ‬ᓇ㗀ߪ ER ߩ⊒⃻ߦ߽޽ࠆ⒟ᐲ෸߱ߎߣ߇੍
ᗐߐࠇࠆ‫⃻ޕ‬࿷ߩߣߎࠈߎ߁ߒߚᨴ⦡૕⇣Ᏹࠍ㒐ߋᚻᲑߪ⍮ࠄࠇߡ޿ߥ޿‫ޕ‬ᓥߞߡ‫ޔ‬ER ߩ⊒⃻ߩ࠴ࠚ࠶ࠢࠍᮡḰෳᾖ‛⾰ߩ૶↪ߦࠃߞߡᏱߦታⴕߔࠆᔅⷐ߇޽ࠆ‫ޕ‬b. ࡟ࡐ࡯࠲࡯ㆮવሶ㧦ߎߩࠕ࠶࠮ࠗ♽ߢߪ࡟ࡐ࡯࠲࡯ㆮવሶࠍዉ౉ߒ‫ޔ‬ER ߦࠃࠅ࡟ࡐ࡯࠲࡯
ࠍ⊒⃻ߐߖࠆ‫ ߩ࠻ࡅޕ‬ER ߦߪ Į ဳߣ ȕ ဳ߇ሽ࿷ߒ‫ߩߘޔ‬ਔ⠪߳ߩࠕࠧ࠾ࠬ࠻㧛ࠕࡦ࠲ࠧ࠾
ࠬ࠻⚿ว߿ߘߩᵴᕈൻߦࠃࠆ↢ℂ⊛૞↪ߩ㆑޿ߪචಽߦߪ⸃᣿ߐࠇߡ޿ߥ޿‫࠻ࡅߒ߆ߒޕ‬ERDߣ EREߩ cDNA ߪᣢߦන㔌ߐࠇߡ޿ࠆߩߢߘߩᯏ⢻⸃᣿ߪㄭ޿዁᧪᣿ࠄ߆ߣߥࠆߎߣ
߇ᦼᓙߐࠇࠆ‫ ߩࠄࠇߎޕ‬cDNA ࠍห৻߹ߚߪ೎‫⚦ߩޘ‬⢩ߦዉ౉ߒߚ࡟ࡐ࡯࠲࡯࡮ࠕ࠶࠮ࠗ♽㧔HeLa-9903㧕߽ᣢߦႎ๔ߐࠇߡ޿ࠆ‫ᧄޕ‬ᴺߢ߽ߘߩ᭽ߥ⚦⢩ࠍ↪޿ߚ♽ߩ᷹ቯ⚿ᨐߣߩᲧセᬌ⸛ࠍߔߴ߈ߢ޽ࠆ‫ޕ‬c.

ౝಽᴲᠣੂ‛⾰ߩᓇ㗀ߪࡅ࠻ߦኻߔࠆࠛࠬ࠻ࡠࠥࡦ᭽‛⾰ߦ㒢ߞߚߎߣߢߪߥߊ‫ޔ‬⒳‫ࡎߩޘ‬࡞ࡕࡦ᭽‛⾰߇ࡅ࠻ߩߺߥࠄߕ࿾⃿਄ߩ↢ᘒ♽ߦ߽ᐢ▸ߥᓇ㗀ࠍ෸߷ߒߡ޿ࠆߎߣߪࠃߊ
⍮ࠄࠇߡ޿ࠆ‫ⷰߩߘޕ‬ὐ߆ࠄࡅ࠻ ER ࠕ࠶࠮ࠗ♽ߩᄖߦࡅ࠻ࠕࡦ࠼ࡠࠥࡦฃኈ૕㧔AR㧕ࠍ↪޿ߚࠕࡦ࠼ࡠࠥࡦ᭽‛⾰߿㑐ㅪߔࠆࡎ࡞ࡕࡦ᭽‛⾰ߩࠬࠢ࡝࡯࠾ࡦࠣ߅ࠃ߮ઁߩ↢‛⒳ߦኻߔࠆห᭽ߩ᷹ቯᴺߩ㐿⊒߿ߘࠇࠄߣᲧセᬌ⸛ࠍⷞ㊁ߦ౉ࠇࠆᔅⷐ߇޽ࠆ‫ޕ‬

⹜㛎ᴺᠲ૞਄ߩⷞὐ߆ࠄ㧦a.

Ḱ஻Ბ㓏ࠍ฽߼ߚၭ㙃ኈེౝߩ⚦⢩ኒᐲߩ⺞ᢛ‫⚦ߦ․ޕ‬⢩ࠍဋ৻ߦ᠞⒳ߢ߈ߡ޿ࠆ߆ߤ߁߆
ߩ࠴ࠚ࠶ࠢ߇ᔅⷐߢ޽ࠆ‫ޕ‬b.

↢⚦⢩ࠍୟ┙㗼ᓸ㏜ߢ࠴ࠚ࠶ࠢߔࠆ㓙ߦኈེో૕ࠍ࠴ࠚ࠶ࠢߔࠆᔅⷐ߇޽ࠆ‫ޕ‬2-3 BG1LucER TA ᴺߩ໧㗴ὐ㧝㧕ࠕ࠶࠮ࠗࠪࠬ࠹ࡓ

એਅߩࠃ߁ߥ໧㗴߇޽ࠆ‫ޕ‬C ૶↪ၭ㙃⚦⢩ߦߟ޿ߡ㧦BG1 ⚦⢩ߪࡅ࠻ ERĮ ߣ ERȕ ࠍ⊒⃻ߒߡ޿ࠆ‫⃻⊒ߩߘߒ߆ߒޕ‬ᒝᐲ
߇ߤߩ⒟ᐲߢ޽ࠆ߆‫ߚ߹ޔ‬ᒝᐲᲧࠍ৻ቯߦ⛽ᜬߔࠆ▤ℂᣇᴺߩ⸥タ߇ή޿‫ ࠻ࡅޕ‬ERDߣ ERE
ߩ cDNA ߪᣢߦ౉ᚻน⢻ߥᲑ㓏ߦ޽ࠆ‫⹜ߩઁޕ‬㛎⚿ᨐߦࠃࠆߣࠕࠧ࠾ࠬ࠻‫ࠬ࠾ࠧ࠲ࡦࠕޔ‬࠻ߩ⒳㘃ߦࠃߞߡ‫ޔ‬ERĮ ߣ ERȕ ߣߢ෻ᔕᕈߩ⇣ߥࠆൻቇ‛⾰߇ⶄᢙ⍮ࠄࠇߡ޿ࠆ‫ޕ‬ㄭ޿዁᧪ ERĮ ߣ ERȕ ߩ↢ℂᯏ⢻߿⊒⃻⚦⢩ߩ㆑޿ߥߤ߇᣿ࠄ߆ߦߥࠆน⢻ᕈ߇ᄢ߈޿‫ޕ‬ᓥߞߡᣧ8
14ᦼߦਔฃኈ૕ࠍ੺ߔࠆ⚦⢩ᔕ╵ࠍ೎‫ߦޘ‬᷹ቯߢ߈ࠆ♽ࠍ⏕┙ߔߴ߈ߢ޽ࠆ‫ޕ‬D ᧄᴺߢߪࠕ࠶࠮ࠗၭ㙃ߦ౉ࠆ೨ߦ⚦⢩ߪࡈࠚࡁ࡯࡞࡟࠶࠼ਇ฽ၭ࿾ߢၭ㙃ߐࠇ‫ࠪ࡞߽߆ߒޔ‬ࡈࠚ࡝ࡦ෻ᔕߦኻߔࠆᓇ㗀ࠍ㒰ᄖߔࠆߚ߼‫ޔ‬ၭ࿾ߩ⒳㘃߽ࠕ࠶࠮ࠗ೨ߦ੤឵ߐࠇࠆ‫ߒ߁ߎޕ‬ߚᾘ㔀ߐࠍ✭๺ߔࠆߚ߼‫੹ޔ‬ᣣߢߪหߓ⮎ຠળ␠߆ࠄࡈࠚࡁ࡯࡞࡟࠶࠼߿ߘߩઁߩ⮎ຠߦࠃࠆᓇ㗀ߩ಴ߥ޿ࡈ࠶ൻ࡞ࠪࡈࠚ࡝ࡦ߇⊒ᄁߐࠇߡ޿ࠆߩߢ‫ࠍ♽ࠗ࠮࠶ࠕ࡮࠯࡯࡜ࠚࡈࠪ࡞ޔ‬࡞ࠪࡈࠚ࡝ࡦ߆ࠄߎࠇߦᄌ߃ࠆߩ߇ࠃ޿ߣᕁࠊࠇࠆ‫ޕ‬ၭ࿾ߪ DMEM ߦ⛔৻ߔߴ߈ߢ޽ࠆ‫ޕ‬E ⵍ㛎ൻว‛ߩ߁ߜើ⊒ᕈ޽ࠆ޿ߪ DMSO ਇṁᕈߩ‛⾰ߦߟ޿ߡߪㆡಾߥṁ⸃⵬ഥ೷㧔นṁ⸃೷㧕ࠍ૶↪ߔࠆ߆‫ޔ‬೎ߩ⹜㛎ᴺࠍ੹ᓟᬌ⸛ߔߴ߈ߢ޽ࠆ‫ޕ‬F 96 ࠙ࠚ࡞࡮ࡊ࡟࡯࠻ߩ࠙ࠚ࡞ਛߩ⚦⢩ߩ㗼ᓸ㏜౮⌀ࠍ࠺࡯࠲ߦᷝઃߔࠆᣇ߇⦟޿‫ޕ‬᷹ቯߩା㗬ᐲߩ࠴ࠚ࠶ࠢߦ೑↪ߢ߈ࠆ‫ޕ‬㧟㧚ᬌ⸽⹜㛎ߦ↪޿ߚ‛⾰ߩಽ㘃ߣᅷᒰᕈ Figure 1-1 ߦ␜ߔࠃ߁ߦ‫ޔ‬ᬌ⸽⹜㛎ߩ 2㨪4 Ბ㓏ߢ‫ ࠇߙࠇߘޔ‬12‫ޔ‬41‫ޔ‬25 ‛⾰‫ޔ‬ว⸘ 78 ‛⾰߇⹜㛎ߦଏߐࠇߚ‫ ࠍⷩ৻ߩࠄࠇߘޕ‬Table 1 ߦ␜ߔ‫ ߪࠄࠇߎޕ‬ER ࠕࠧ࠾ࠬ࠻‫ߩ࠻ࠬ࠾ࠧ࠲ࡦࠕޔ‬ਔ⠪ߩ⹏ଔߦ↪޿ࠄࠇߚ‫᦭ޔ࠼ࠗࡠ࠹ࠬޕ‬ᯏ㉄‫ޔ‬὇ൻ᳓⚛ߥߤᐢ▸ߥ⒳㘃߆ߟ᭽‫ߥޘ‬෻ᔕᒝᐲߩ‛⾰߇೑↪ߐࠇߡ޿ࠆ‫⹜ޕ‬㛎ᴺߩᗵᐲࠍ⹏ଔߔࠆߚ߼‫ޔ‬㒶ᕈ‛⾰ߪ⚂ 25%ࠍභ߼ߡ߅ࠅ‫ޔ‬ㆬ߫ࠇߚ‛⾰ߪᅷᒰߢ޽ࠆߣ್ᢿߐࠇߡ޿ࠆ‫ޕ‬㧠㧚⹜㛎ᴺߩ࠺࡯࠲ߣ⚿ᨐߩ᦭↪ᕈ ICCVAM ߦࠃࠅㆬ߫ࠇߚ 78 ‛⾰ߩ⚿ᨐ߆ࠄ‫ޔ‬㓁ᕈ‫ޔ‬㒶ᕈߣ⏕ታߦಽ㘃ߐࠇࠆ‛⾰ߩߺ߇ᱜ⏕ᕈߩ⹏ଔߦ૶ࠊࠇߚ‫ޕ‬ER ࠕࠧ࠾ࠬ࠻ߩ⹏ଔߢߪ‫ޔ‬ਅ⸥ Table 2 ߦ␜ߔ 42 ‛⾰㧔㓁ᕈ 33 ‛⾰‫ޔ‬㒶ᕈ 9 ‛⾰㧕‫ޔ‬ER ࠕࡦ࠲ࠧ࠾ࠬ࠻ߩ⹏ଔߢߪ Table 3 ߦ␜ߔ 25 ‛⾰㧔㓁ᕈ 3 ‛⾰‫ޔ‬㒶ᕈ 22 ‛⾰㧕ߢ޽ࠆ‫ޔߦࠄߐޕ‬ER ࠕࠧ࠾ࠬ࠻ߦ㑐ߒߡߪ 42 ‛⾰ߩ߁ߜਅߦ␜ߔ 7 ‛⾰߇ࠕ࠶࠮ࠗߩᱜ⏕ᕈࠍ᳞߼ࠆ⹏ଔ߆ࠄ㒰ᄖߐࠇߚ‫ߩߎޕ‬ℂ↱ߪ‫߇࠲࡯࠺ޔ‬ᱜ⏕ߥ್ᢿߦߪਇචಽߢ޽ߞߚߚ߼‫ޔ‬㓁ᕈ߆㒶ᕈ߆ߩ್ᢿߦߪਇㆡಾߣ
ߐࠇߚߎߣߦࠃࠆ‫⹏ޔߒߛߚޕ‬ଔߦ↪޿ࠄࠇߚ‛⾰ࠍ฽߼‫ޔ‬in vivo ⹜㛎㧔ሶች⢈ᄢ⹜㛎㧕ߩ⚿ᨐ߇ᓧࠄࠇߡ޿ࠆ‛⾰ߪዋߥ޿‫ޕ‬࡮Clomiphene citrate
࡮p,p’-DDE
࡮5Į-Dihydrotestosterone
࡮Flutamide
࡮Procymidone
࡮Resveratrol࡮Tamoxifen
Table 2 ߢߪ‫ޔ‬㓁ᕈ 33/42‫ޔ‬㒶ᕈ 9/42 ߢ޽ࠆ߇‫⚿ޔ‬ᨐࠍ⹦ߒߊ⷗ࠆߣ‫ޔ‬3 ᯏ㑐ߩ⚿ᨐ߇ߘࠈߞߡ㓁ᕈ
ߥߩߪ 22/33 ߢ‫ޔ‬11/33 ߪ 1㨪2 ߩᯏ㑐ߢ㓁ᕈߢߪߥ޿ߣႎ๔ߐࠇߡ޿ࠆ‫ߦࠄߐޕ‬㒶ᕈߩ߁ߜ 2/9 ߪ 3ᯏ㑐ߩ⚿ᨐ߇ߘࠈߞߡ޿ߥ޿‫ߩ↪ࠣࡦ࠾࡯࡝ࠢࠬߪ࠲࡯࠺ߩߎޕ‬ෳ⠨ߣߒߡ⋧ᔕߒ޿ߣߪ⸒߃ߥ޿‫ޕ‬
915

ᰴߦ Table 3 ߦߟ޿ߡ⷗ࠆߣ‫ޔ‬ER ࠕࡦ࠲ࠧ࠾ࠬ࠻૞↪ߩ޽ࠆ߽ߩ㧔㓁ᕈ㧧POS 3/25 ߩ߁ߜ 1 ߟߪ
3 ᯏ㑐ߩ⚿ᨐ߇ߘࠈߞߡ޿ߥ޿‫ޕ‬㒶ᕈ㧔NEG 19/25 ߩ߁ߜߩ 1 ߟߢߪ㓁ᕈ㧔POS㧕ߩ⚿ᨐ߇ᓧࠄࠇߡ޿ࠆ‫ ߪࠄࠇߎޕ‬ER ࠕࡦ࠲ࠧ࠾ࠬ࠻૞↪ࠍ᣿ࠄ߆ߦߔࠆ⹜㛎ᴺߣߒߡචಽߢߪߥ޿ߎߣࠍ␜ߒߡ޿ࠆ‫ޕ‬ൻቇ‛⾰ߩ᭴ㅧߦࠃࠆಽ㘃㧔ࠬ࠹ࡠࠗ࠼㘃‫ࡦ࠯ࡦࡌޔ‬නⅣ╬㧕߽␜ߒߚᣇ߇⦟޿ߣ⠨߃ࠄࠇࠆ‫߹ޕ‬ߚ‫ߡߒߣࡦ࡚ࠪ࡯࠺࡝ࡃޔ‬ታᣉߔߴ߈ൻቇ‛⾰ߣߒߡ‫ޔ‬ER ࠕࡦ࠲ࠧ࠾ࠬ࠻ࠍჇ߿ߔߴ߈ߢ޽ࠆ‫ޕ‬ᜰᮡߣߔࠆൻቇ‛⾰ߩࡅ࠻ ERĮ ߣ ERȕ ߩ෻ᔕᕈ߽␜ߒߚᣇ߇⦟޿ߢ޽ࠈ߁‫ޕ‬㧡㧚⹜㛎ᴺߩᱜ⏕ᕈ
BG1LucER TA ᴺߩᱜ⏕ᕈࠍ⹏ଔߔࠆߚ߼‫ޔ‬ICCVAM ෳᾖಽ㘃ߣᲧセߒߚ‫ޕ‬ER ࠕࠧ࠾ࠬ࠻ߩ⹏ଔߪ
35 ‛⾰ࠍ↪޿ߡታᣉߐࠇߚ‫ޕ‬Table 4 ߦ␜ߔࠃ߁ߦ‫ޔ‬3 ᣉ⸳ߩᱜ⏕ᕈߩ৻⥌₸ߪ 97%㧔34/35)ߢ޽ࠅ‫ޔ‬ߘߩ߁ߜᗵᐲߪ 96%㧔27/28㧕‫⇣․ޔ‬ᐲߪ 100%㧔7/7 ߢ޽ߞߚ‫ޕ‬ன㓁ᕈߪߥߊ‫ޔ‬ன㒶ᕈ₸ߪ 4%㧔1/28㧕
ߢ޽ߞߚ‫⚿ߩߎޕ‬ᨐߪ‫ޔ‬ฦᣉ⸳ߩ⚿ᨐ߆ࠄ୘೎ߦ⸘▚ߒߚ႐วߢ߽ห᭽ߢ޽ߞߚ‫ޕ‬ER ࠕࡦ࠲ࠧ࠾ࠬ࠻ߩ⹏ଔߦߟ޿ߡߪ‫ޔ‬25 ‛⾰ࠍ↪޿ߡታᣉߐࠇߚ‫ޕ‬Table 5 ߦ␜ߔࠃ߁ߦ‫ޔ‬3 ᣉ⸳ߩᱜ⏕ᕈߩ৻⥌₸ߪ 100%㧔25/25㧕ߢ޽ࠅ‫ߜ߁ߩߘޔ‬ᗵᐲߪ 100%㧔3/3㧕‫⇣․ޔ‬ᐲߪ 100%㧔22/22㧕
ߢ޽ߞߚ‫ޕ‬ன㓁ᕈ߽ன㒶ᕈ߽ߥ߆ߞߚ‫⚿ߩߎޕ‬ᨐߪ‫ޔ‬ฦᣉ⸳ߏߣߩ⚿ᨐߢ߽ห᭽ߢ޽ߞߚ‫ޕ‬
㧔33/34㧕ߢ޽ࠅ‫ޔ‬medroxyprogesteroneER ⚿ว⹜㛎࠺࡯࠲ߣ BG1LucER TA ᴺߩᲧセߢߪ‫ ߪ₸⥌৻ޔ‬97%
acetate ߇ BG1LucER TA ߢߪ㓁ᕈߣߥࠅ‫ޕߚߞ߆ߥߒ⥌৻ޔ‬ห᭽ߦ‫ޔ‬in vivo ሶች⢈ᄢ⹜㛎ߣߩᲧセߢߪ‫ ߪ₸⥌৻ޔ‬92%㧔12/13㧕ߢ޽ࠅ‫ޔ‬butylbenzyl phthalate ߇ BG1LucER TA ᴺߢߪ㓁ᕈߣߥࠅ‫ߒ⥌৻ޔ‬ߥ߆ߞߚ‫ޕ‬㧢㧚⹜㛎ᣇᴺߩା㗬ᕈ ᣉ⸳ౝౣ⃻ᕈߪ‫ޔ‬ᬌ⸽⹜㛎ߢߩฦᣉ⸳ߩߔߴߡߩࡊ࡟࡯࠻ߩᮡḰ‛⾰߅ࠃ߮ኻᾖታ㛎ߩ⚿ᨐ‫ࠄߥޔ‬߮ߦ‫ޔ‬12 ‛⾰ߩ 3 ᣉ⸳ߦ߅ߌࠆ 3 ࿁ߩታ㛎ߢ᳞߼ࠄࠇߚ╙ 2 Ბ㓏ߩ⚿ᨐ߆ࠄ᳞߼ߚ‫ޕ‬Table 6 ߦ␜ߔࠃ߁ߦ ER ࠕࠧ࠾ࠬ࠻⹜㛎ߢߪ‫ޔ‬ᮡḰ‛⾰ߢ޽ࠆ 17ȕ-estradiol㧔E2㧕ߩ EC50 ୯ߪ 8㨪1110-12M ߢ޽ߞߚ‫ޕ‬ฦᣉ⸳ߩ 3 ࿁ߩ⚿ᨐߪ 100%৻⥌ߒߚ߇‫ޔ‬12 ‛⾰ߩ߁ߜ޿ߊߟ߆ߩ⒳㘃ߪᣉ⸳㑆ߢ৻⥌ߒߥ߆ߞߚ‫ޕ‬ Table 7 ߦ␜ߔࠃ߁ߦ ER ࠕࡦ࠲ࠧ࠾ࠬ࠻⹜㛎ߢߪ‫ޔ‬ᮡḰ‛⾰ߢ޽ࠆ raloxifene ߩ IC50 ୯ߪ 1.1㨪1.3
10-9M ߢ޽ߞߚ‫ޕ‬ฦᣉ⸳ߩ 3 ࿁ߩ⚿ᨐߪ 100%৻⥌ߒߚ߇‫ޔ‬12 ‛⾰ߩ߁ߜ޿ߊߟ߆ߩ⒳㘃ߪᣉ⸳㑆ߢ৻⥌ߒߥ߆ߞߚ‫ޕ‬ ᣉ⸳㑆ౣ⃻ᕈߦߟ޿ߡߪ‫ޔ‬ᣉ⸳ౝౣ⃻ᕈߣห᭽ߦ 12 ‛⾰ߩ 3 ᣉ⸳ߩ߅ߌࠆ 3 ࿁ߩታ㛎ߢ᳞߼ࠄࠇ
ߚ╙ 2 Ბ㓏ߩ⚿ᨐ߆ࠄ᳞߼ߚ‫ޕ‬Table 8 ߦ␜ߔࠃ߁ߦ‫ޔ‬ER ࠕࠧ࠾ࠬ࠻߇㓁ᕈߣ␜ߐࠇߚߩߪ 67%㧔8/12
ߢ޽ࠅ‫ޔ‬ER ࠕࡦ࠲ࠧ࠾ࠬ࠻ߢߪ 100%㧔12/12 ߢ޽ߞߚ‫ޕ‬Table 9 ߦ␜ߔࠃ߁ߦ‫ޔ‬41 ‛⾰ࠍ↪޿ߚ╙
3 Ბ㓏ߩ⚿ᨐߢߪ‫ ߩߜ߁ߩߎޔ‬5 ‛⾰ߩ ER ࠕࠧ࠾ࠬ࠻ߪਇㆡಾߥ࠺࡯࠲ߢ⹏ଔߢ߈ߥ߆ߞߚ‫ޕ‬ዋߥ
ߊߣ߽ 2 ᣉ⸳ߦ߅޿ߡ‫ޔ‬ᱷࠅ 36 ‛⾰ߩ⚿ᨐߪ 100%৻⥌ߒߚ‫ޕ‬ER ࠕࡦ࠲ࠧ࠾ࠬ࠻ᵴᕈߦߟ޿ߡߪ‫ޔ‬93%㧔38/41 ߇৻⥌ߒߚ‫ޕ‬
10
16㧣㧚࠺࡯࠲ߩ⾰ ⹏ଔႎ๔ᦠߢߪ⸥タ߇ߥ޿߇‫ޔ‬⢛᥊⹏ଔႎ๔ᦠߦࠃࠆߣ‫ޔ‬XDS ␠ߣ ECVAM ߪ GLP ࠟࠗ࠼࡜ࠗࡦ
ߦḰ᜚ߒߡታ㛎ࠍታᣉߒߚ‫ޕ‬ᩣᑼળ␠ᣣศߦߪᬌ⸽⹜㛎ߦవ┙ߜ‫ޔ‬OECD ߩ GLP ၮḰߦೣߞߚࠟࠗ
࠳ࡦࠬᢥᦠࠍᷰߒߚ‫ޕ‬ᩣᑼળ␠ᣣศߩ QC㧔quality control㧕ߣ଻⸽ᣇᴺߪ ISO9000 ෸߮ ISO2000 ߦ߽Ḱߓߚ‫ޕ‬QA㧔quality assurance㧕ߪߔߴߡߩᣉ⸳ߢㆡಾߦታᣉߐࠇߚ‫ޕ‬㧤㧚⹜㛎ᴺߩ᦭↪ᕈ‫ޔ‬㒢⇇߅ࠃ߮ឭ⸒
1) ER ࠕࡦ࠲ࠧ࠾ࠬ࠻ߦࠃࠆ⚿ᨐ㧦BG1LucER TA ᴺߪේℂߩ㗄ߢ߽ㅀߴߚ᭽ߦනߦࠕࡦ࠲ࠧ࠾ࠬ࠻ߣ ER ߣߩ⚿วߦᱛ߹ࠄߕࠕࠧ࠾ࠬ࠻ߩᵴᕈᛥ೙ലᨐࠍᬌ಴ߢ߈ࠆὐ߇ఝࠇߡ޿ࠆ‫ߩઁޕ‬ห᭽ߩ⹜㛎ᴺߩ⚿ᨐߣߩ৻⥌ᐲ‫ޔ‬BG1LucER TA ᴺߩᗵฃᕈ‫⇣․ޔ‬ᕈ‫ޔ‬ன㓁ᕈ࡮ன㒶ᕈߩ⼂೎ߪ⦟ᅢߥ⚿ᨐࠍ␜ߒߚ‫ޕ‬2) ⹜㛎ᣇᴺ߆ࠄ⷗ߚ⚿ᨐ㧦ઁߩ⹜㛎ᴺߦᲧߒࠕࠧ࠾ࠬ࠻‫ߩ࠻ࠬ࠾ࠧ࠲ࡦࠕޔ‬Ớᐲ߇ 1/100 ߢᬌ಴ߢ߈ࠆߩߢࠃࠅᱜ⏕ߢ޽ࠅ‫ޔ‬ன㓁ᕈ࡮ன㒶ᕈߩ↢ߓࠆน⢻ᕈߪዋߥ޿‫ޕ‬3) BG1LucER TA ᴺߦࠃࠆൻቇ‛⾰ߩ ER ࠕࠧ࠾ࠬ࠻ᵴᕈ࡮ER ࠕࡦ࠲ࠧ࠾ࠬ࠻ᵴᕈߩࠬࠢ࡝࡯࠾ࡦ
ࠣ⚿ᨐߪ‫ࡦࠥࡠ࠻ࠬࠛޔ‬ᵴᕈߩ⹜㛎ߦ↪޿ࠄࠇࠆᣢሽߩ⚦⢩♽㧔ౝ࿷ᕈߩࡅ࠻ ER ࠍ೑↪ߔࠆ
MCF-7 ⚦⢩‫ ࠻ࡅ ޔ‬ERǂ ࠍ቟ቯ⊛ߦ⚵ߺㄟࠎߛ HeLa ⚦⢩ ߥߤߣᲧセߒߡ߽‫ޔ‬ன㓁ᕈ߿ன㒶ᕈߩ⼂೎ߪ⦟ᅢߢ޽ࠆ‫ޔߚ߹ޕ‬ൻቇ‛⾰ߩࠛࠬ࠻ࡠࠥࡦᵴᕈࠍ࡞ࠪࡈࠚ࡝ࡦ⊒శߦࠃࠅቯ㊂ൻߔࠆ߇‫ޔ‬MCF-7 ⚦⢩ߦᲧߴߡჇᱺ߇ᣧ޿ߚ߼‫⹜ޔ‬㛎ᤨ㑆߇⍴❗ߐࠇ‫ޔ‬ᄙᢙߩൻቇ‛⾰ߩࠬࠢ࡝࡯࠾ࡦ
ࠣߦะ޿ߡ޿ࠆ‫ޕ‬
4㧕ERĮ ߣ ERȕ ߩਔᣇࠍౝ࿷⊛ߦ⊒⃻ߒߡ޿ࠆߚ߼ൻቇ‛⾰ߩࠛࠬ࠻ࡠࠥࡦ૞↪ࠍ✚૕ߣߒߡᬌ಴น
⢻ߢ޽ࠆ‫ޕ‬ER ࠍ੺ߒߚൻቇ‛⾰ߩࠛࠬ࠻ࡠࠥࡦᵴᕈࠍ৻ᰴࠬࠢ࡝࡯࠾ࡦࠣߔࠆߦߪଢ೑ߥ⹜㛎♽ߢ޽ࠆ‫৻ޕ‬ᣇ‫ޔ‬ㆬᛯߒߚ‛⾰߇ ERĮ ޽ࠆ޿ߪ ERȕ ߦ․⇣⊛ߦ⚿วߒߡ૞↪ߔࠆߩ߆ࠍ⺞ߴࠆߚ߼ߦߪ‫ ߩ߆ࠄߜߤޔ‬ER ࠨࡉ࠲ࠗࡊࠍ⊒⃻ߐߖߚ⚦⢩♽ࠍ↪޿ߚੑᰴ⊛ߥࠬࠢ࡝࡯࠾ࡦࠣ߇ᔅⷐߣߥࠆ‫ޕ‬5) BG1 ⚦⢩ߩౝ࿷⊛ߥ ERĮ ߣ ERȕ ߩሽ࿷Ყ߇⚦⢩ߩ⛮ઍߦࠃߞߡᄌൻߒߥ޿ߎߣࠍ଻⸽ߒߡ߅ߊߎ
ߣ߇ᔅⷐߢ޽ࠆ‫ ߩࠇߙࠇߘޕ‬ER ߦኻߔࠆࠕࠧ࠾ࠬ࠻‫ߪ࠻ࠬ࠾ࠧ࠲ࡦࠕޔ‬Ꮢ⽼ߐࠇߡ޿ࠆߩߢ‫ޔ‬ߎࠇࠄߩ‛⾰ࠍ↪޿ߚᔕ╵ᕈࠍ⺞ߴߡ߅ߊߎߣ߽ᔅⷐߢ޽ࠈ߁‫ޕ‬6) ṁ⸃⵬ഥ೷㧔นṁൻ೷㧕ߣߒߡ DMSO ߇↪޿ࠄࠇߡ޿ࠆ߇‫ޔ‬DMSO ߦṁߌߦߊ޿‛⾰ߦߟ޿ߡߪ‫ޔ‬ઁߩ᦭↪ߥ⵬ഥ೷㧔นṁൻ೷㧕ࠍᬌ⸛ߒ‫ ߇ࠇߘޔ‬BG1 ⚦⢩ߦᓇ㗀ߒߥ޿ߎߣࠍᬌ⸽ߔࠆᔅⷐ߇޽ࠆ‫ޕ‬7) ൻቇ‛⾰ߩⶄวᓇ㗀ߩน⢻ᕈ߇ᜰ៰ߐࠇߡ߈ߡ޿ࠆ߇‫ޔ‬BG1LucER TA ᴺࠍ↪޿ߡⶄᢙߩ‛⾰ࠍหᤨߦᦑ㔺ߒߚ႐วߩⶄว૞↪ߩᬌ⸛ߪⴕࠊࠇߡ޿ߥ޿‫੹ޕ‬ᓟ‫ⶄޔ‬วᦑ㔺ߩᬌ⸛߇ᔅⷐߢ޽ࠆ‫ޕ‬8) ⃻ᤨὐߢߪើ⊒ᕈ‛⾰ߩขࠅᛒ޿ߦߟ޿ߡ᣿⏕ߥᜰ㊎߇ή޿ߣᕁࠊࠇࠆ‫੹ޕ‬ᓟߩᬌ⸛߇ᦼᓙߐࠇࠆ‫ޕ‬9) ઍ⻢ߐࠇߡ߆ࠄࠛࠬ࠻ࡠࠥࡦ૞↪ࠍ␜ߔ‛⾰ߩ⹏ଔߦߟ޿ߡ߽‫ޔ‬BG1LucER TA ᴺࠍ↪޿ߡߤߩࠃ
1117߁ߦࠬࠢ࡝࡯࠾ࡦࠣߔࠆ߆ߩᬌ⸛߇ᔅⷐߢ޽ࠆ‫ޕ‬10) BG1LucER TA ᴺࠍ࿖ౝߩൻቇ‛⾰⹏ଔߦߤߩࠃ߁ߦ೑↪ߔࠆ߆ߦߟ޿ߡߩᬌ⸛߽੹ᓟߩ⺖㗴ߢ޽ࠆ‫ޕ‬11) ᧄᴺߢߪࠕ࠶࠮ࠗၭ㙃ߦ౉ࠆ೨ߦ⚦⢩ߪࡈࠚࡁ࡯࡞࡟࠶࠼ਇ฽ၭ࿾ߢၭ㙃ߐࠇ‫߽߆ߒޔ‬ၭ࿾ߩ⒳㘃߽ࠕ࠶࠮ࠗ೨ߦ੤឵ߐࠇࠆ‫੹ޕ‬ᣣߢߪหߓ⮎ຠળ␠߆ࠄࡈࠚࡁ࡯࡞࡟࠶࠼߿ߘߩઁߩ⮎ຠߦࠃࠆᓇ㗀ߩ಴ߥ޿⹜⮎㧔ࡈ࠶ൻ࡞ࠪࡈࠚ࡝ࡦ㧕߇⊒ᄁߐࠇߡ޿ࠆߚ߼ߎࠇࠍ೑↪ߔࠇ߫ࡈࠚࡁ࡯࡞
࡟࠶࠼฽᦭ߩ᦭ήߪ໧㗴ߦߥࠄߥ޿‫ޔߒ߆ߒޕ‬ၭ࿾ߪ DMEM ߦ⛔৻ߔߴ߈ߢ޽ࠆ‫ޕ‬12) ᧄ⹜㛎ᴺߩታ↪਄ߩ ᔨߣߒߡ‫⹜ޔ‬㛎♽ߩ቟ቯᕈߩ໧㗴߇ᜰ៰ߐࠇࠃ߁‫ߦ⥸৻ޕ‬ㆮવሶࠍዉ౉ߒ
ߚ⚦⢩ߪ‫ޔ‬቟ቯ⊛ዉ౉ߣߪ⸒߁߽ߩߩ‫⛮ޔ‬ઍߏߣߦㆮવሶ⊒⃻ߦߪᄙዋߩᄌൻ߇⿠ࠆߚ߼‫ޔ‬㐳ᦼߦࠊߚߞߡหߓ෻ᔕᕈࠍ଻ߟߎߣߪᦼᓙߢ߈ߥ޿‫ޕ‬BG1 ⚦⢩ߦߟ޿ߡ߽‫⛮ޔ‬ઍᣧᦼߩ෻ᔕᕈߩ᣿
ࠄ߆ߥ⚦⢩ࠍᄙ㊂ߦಽഀಓ⚿଻ሽߒߡ‫ޔ‬ㅙᰴ೑↪ߔࠆࠃ߁ߥភ⟎߇ᦸ߹ߒߊ‫⚦ޔ‬⢩ࠍ฽߻⹜㛎᧚ᢱߩଏ⛎ߦ㑐ߔࠆ㈩ᘦ߇ᔅⷐߢ޽ࠆ‫ޕ‬ዋߥߊߣ߽‫⹜ࠆߔ↪૶ޔ‬㛎♽ߩᮡḰኻᾖ‛⾰ߦኻߔࠆ෻ᔕ
ࠍ⹜㛎ߩᐲߏߣߦ⏕⹺ߔࠆߎߣ߇ᔅⷐߢ޽ࠆ‫ޕ‬13) ၮ␆⊛ߥ⹜㛎ᠲ૞ߩᱜ⏕ᕈࠍ଻⸽ߔࠆߎߣ߇ᔅⷐߢ‫⹜ޔ‬㛎ᣉ⸳߇ߎߩ⚦⢩♽ߩ૶↪ߦ⠌ᾫߔࠆߎߣ߇‫ޔ‬ᒰὼߥ߇ࠄ᳞߼ࠄࠇࠆ‫⹜ޕ‬㛎Ḱ஻ߩ㓙ߦ‫ޔ‬ၭ㙃ᶧਛߩ⚦⢩ߩỚᐲࠍ⏕⹺ߒ‫ߩߣߏ࡞ࠚ࠙ޔ‬⚦⢩ᢙ߇ဋ╬ߢ޽ࠆߎߣࠍ଻⸽ߔࠆࠃ߁ߥ⸥㍳‫߫߃ߣߚޔ‬㗼ᓸ㏜ߢߩ⏕⹺ࠍബⴕߔࠆࠃ߁ߥ㈩ᘦ
߇ᦸ߹ߒ޿‫ޕ‬㧥. ߘߩઁߩ⹜㛎ᣇᴺߩ⑼ቇ⊛ߥႎ๔ౝಽᴲᠣੂ૞↪ߦߟߥ߇ࠆߣ⠨߃ࠄࠇࠆ ER ߦ㑐ߒߡߩ⹜㛎ᴺߣߒߡߪ‫ޔ‬໧㗴ߣߔࠆ‛⾰ߩฃኈ૕߳ߩ⚿วታ㛎߇߹ߕᗐ⿠ߐࠇࠆ‫⚿ޕ‬วታ㛎ߢߪൻቇ‛⾰ߣߩ⋧੕૞↪߇ኈᤃߣߥࠆࠃ߁‫ޔ‬ฃኈ૕߇᳓
⋧ߦ㔺಴ߒߡ޿ࠆߎߣ߇ᦸ߹ߒ޿߇‫⚦ߪߦ⊛⋡ߩߎޔ‬⢩ࠍ⎕უߒߚ㕖⚦⢩♽㧔cell-free㧕߇᦭೑ߢ޽
ࠅ‫ޔ‬ታ㓙‫⚿ߩߢ♽ߩߎޔ‬วታ㛎ߪએ೨ࠃࠅⴕࠊࠇߡ޿ࠆ‫ޕ‬ฃኈ૕ߣ⋧੕૞↪ࠍ␜ߔ‛⾰ߣߒߡߪ‫ࠕޔ‬ࠧ࠾ࠬ࠻ߣࠕࡦ࠲ࠧ࠾ࠬ࠻߇޽ࠅ‫ޔ‬න⚐ߥ⚿วታ㛎ߢߪਔ⠪ߩ඙೎߇࿎㔍ߢ޽ࠆ‫ޔߦࠄߐޕ‬㕖⚦⢩♽ߢߩ⚿ᨐߪ⚦⢩♽ߣ৻⥌ߒߥ޿ߣ޿߁ႎ๔߽ᄙߊ‫⚦ޔ‬⢩ౝߦ․᦭ߩ⚿วࠍ೙ᓮߔࠆ࿃ሶߩሽ࿷߇␜ໂ
ߐࠇߡ޿ࠆ‫⚿ޔߡߞࠃޕ‬วࠍ฽߼ߚฃኈ૕ߣߩ⋧੕૞↪ߦߟ޿ߡߪ⚦⢩♽ߩ೑↪߇ᦸ߹ߒ޿‫ޕ‬⚦⢩♽ߢߩ‛⾰ߩ ER ߳ߩ⚿วߪ‫ޔ‬ౝ࿷⊛ߥォ౮ᵴᕈߩჇടࠍ⋡቟ߣߔࠆߎߣߦࠃࠅ‫ࠬ࠾ࠧࠕޔ‬࠻૞↪ߩ᦭ήࠍ್ቯߢ߈ࠆ‫ޔߪࠗ࠮࠶ࠕ࡮࡯࠲࡯ࡐ࡟ߥ߁ࠃߩߎޕ‬ዉ౉ߩ৻ㆊဳ㧛቟ቯဳࠍ໧ࠊߕ‫ޔ‬㉂Უ‫ࡘࠪ࠶ࠖࡈ࡜ࡉ࠯ޔ‬⢄⚦⢩ᩣ‫ޔ‬HepG2‫ޔ‬HeLa‫ޔ‬CV-1 ߥߤ⒳‫⚦ߩޘ‬⢩ߢⴕࠊࠇߡ޿ࠆ‫ޕ‬ એ਄ߩߎߣߪਥߣߒߡవߦ⊒⷗ߐࠇߚ Į ဳߩฃኈ૕㧔ERĮ㧕ߦߟ޿ߡߩ⸥ㅀߢ޽ࠆ߇‫ޔ‬ER ߦߪߎࠇߣߪ೎ߦ ȕ ဳ㧔ERȕ㧕߇ሽ࿷ߔࠆ‫ޕ‬ERĮ‫ޔ‬ERȕ ߩਔ⠪ߪႮၮ㈩೉ߦ⋧หᕈ߇޽ࠆ߇‫ࠆߔ࠼࡯ࠦޔ‬ㆮવሶߪ⇣ߥߞߡ߅ࠅ‫↢ޔ‬૕ౝߢߩ⊒⃻ߩ᭽⋧ߦ߽Ꮕ⇣߇޽ࠆ‫ޔߚ߹ޕ‬ౝಽᴲᠣੂൻቇ‛⾰߇ ER ࠍ੺ߒߡᓇ㗀ࠍ෸߷ߔߣߒߡ߽ߘߩోߡ߇ ERE ਅᵹㆮવሶߩォ౮ߩୃ㘼ߦ⋥ធ⿠࿃ߔࠆߣᢿߓࠆߦߪ໧
㗴߇޽ࠅ‫ࠢ࠶ࠖ࠹ࡀࠚࠫޔ‬㧔genetic㧧ㆮવሶ⊛㧕ߥォ౮ߩୃ㘼ߢߪߥߊ‫ޔ‬ᓇ㗀ߩࠛࡇࠫࠚࡀ࠹ࠖ࠶ࠢ
㧔epigenetic㧧ᓟᚑ⊛㧕ߥ஥㕙ߦߟ޿ߡ߽⠨ኤ߇ട߃ࠄࠇߡ޿ࠆ‫ޔ߽ߡߒߦࠇߕ޿ޕ‬ERĮ‫ޔ‬ERȕ ߩਔฃ1218ኈ૕߇⊒⃻ߒߡ߅ࠅ‫ߥࠢ࠶ࠖ࠹ࡀࠚࠫࡇࠛޔ‬ᓇ㗀߽ⷰኤߒ߁ࠆ⚦⢩♽߇೑↪ߦㆡߒߡ޿ࠆ‫ޕ‬ERĮ‫ޔ‬ERȕ ߪߣ߽ߦ⚿᥏ൻߐࠇ X ✢ߦࠃࠅ᭴ㅧ߇⸃ᨆߐࠇߡ޿ࠆ‫⚿ߩߎޕ‬ᨐࠃࠅ‫ޔ‬ERĮ‫ޔ‬ERȕ ߩࠛࠬ࠻ࡠࠥࡦ߅ࠃ߮㑐ㅪ‛⾰ߩ⚿วߦߟ޿ߡߩᬌ⸛߿⠨ኤ߇ߥߐࠇߡ޿ࠆ‫ߦࠇߎޕ‬ട߃‫ޔ‬ൻቇ‛⾰ߩ
ER ߦኻߔࠆቯ㊂⊛᭴ㅧ-ᵴᕈ⋧㑐㧔QSAR㧕߽⹜ߺࠄࠇߡ޿ࠆ߇‫⃻ޔ‬࿷ߩߣߎࠈ⚿ᨐߩ㓸Ⓧߣߘߩ⸃ᨆߩᲑ㓏ߦᱛ߹ߞߡ߅ࠅ‫ޔ‬േ‛ታ㛎ߩઍᦧߦ㆐ߒߡߪ޿ߥ޿‫ޕ‬਄ㅀߩ⹜㛎ߩ߁ߜ‫ޔ‬HeLa ⚦⢩ߢ࡞ࠪࡈࠚ࡜࡯࠯ࠍ࡟ࡐ࡯࠲࡯ߣߒߚ ER ᵴᕈൻࠕ࠶࠮ࠗߪ‫ޔ‬hERĮ-HeLa STTA ᴺߣߒߡ OECD ߩ⹜㛎ᴺࠟࠗ࠼࡜ࠗࡦߦ߽෼タߐࠇߡ޿ࠆ߇‫ߦࠇߎޔ‬ኻߔࠆ
BG1LucER TA ᴺߩఝ૏ᕈߦߟ޿ߡߪ‫ޔ‬1 ߩ“ᧄ⹜㛎ᴺߩ⑼ቇ⊛ᅷᒰᕈߣⷙ೙⹜㛎ᴺߣߒߡߩᅷᒰᕈ”ߦ⸥ߐࠇߡ޿ࠆ‫⹜ߩઁޕ‬㛎ᴺߦߟ޿ߡߪ‫ޔ‬ઍᦧᴺߣߒߡߩቯ⟵‫ޔߪ޿ࠆ޽ޔ‬᥉ㆉᕈ╬ߩⷐઙࠍలߚߔ
ߣߪ⸒߃ߕ‫ޔ‬ᒰὼᬌ⸽⹜㛎ߪⴕࠊࠇߡ޿ߥ޿‫ޔߡߞࠃޕ‬ERĮ‫ޔ‬ERȕ ߩਔฃኈ૕ࠍ੺ߔࠆ↢૕߳ߩᓇ㗀ߣ޿߁ὐߢ‫ޔ‬BG1LucER TA ⹜㛎ᴺߩ੹ᓟߩ⎇ⓥߩ⊒ዷ߇ᦸ߹ࠇࠆ‫ޕ‬㧝㧜㧚⚿⺰
BG1LucER TA ᴺߪ‫ޔ‬ൻቇ‛⾰ߩ in vitro ߢߩ ER ࠕࠧ࠾ࠬ࠻ᵴᕈ߅ࠃ߮ ER ࠕࡦ࠲ࠧ࠾ࠬ࠻ᵴᕈࠍ
ᬌ಴ߔࠆࠬࠢ࡝࡯࠾ࡦࠣ⹜㛎ᴺߢ޽ࠅ‫ߩߘޔ‬ᬌᩏᕈ⢻ߩ᦭↪ᕈࠍ⏕⹺ߔࠆᬌ⸽⹜㛎ߪ࿖㓙⊛ߦ 3 ⹜㛎ᣉ⸳౒ห⹜㛎ߦࠃߞߡቢੌߒߡ޿ࠆ‫⹜ᧄޕ‬㛎ᴺߪ‫ޔ‬ൻቇ‛⾰ߩ ER ߦኻߔࠆ⚿วᕈࠍ⷗ࠆ⹜㛎ᴺߣߪ
⇣ߥࠅ‫⚿ߩߘޔ‬วߩ⚿ᨐߦࠃࠆ DNA ߩォ౮ᵴᕈൻ߹ߢ⍮ࠆߎߣ߇಴᧪‫ޔߦࠄߐޔ‬ER ࠕࠧ࠾ࠬ࠻ߣ
ࠕࡦ࠲ࠧ࠾ࠬ࠻ߩᵴᕈࠍಽߌߡᬌᩏߔࠆߎߣ߇಴᧪ࠆ‫⹜ߩߘޕ‬㛎ᴺߣߒߡߩ⑼ቇ⊛ᅷᒰᕈߣⷙ೙⹜㛎ᴺߣߒߡߩᅷᒰᕈߦߟ޿ߡ‫ޔ‬ICCVAM ߩㆬቯߒߚ 78 ⒳ߩෳᾖ‛⾰ࠍ↪޿ߡᬌ⸽⹜㛎߇ⴕࠊࠇߚ‫ޕ‬ᬌ⸽ߩ⚿ᨐ‫ޔ‬ICCVAM ߩ⹏ଔႎ๔ᦠߢߪߎߩ⹜㛎ᴺߩᱜ⏕ᕈߣା㗬ᕈࠍ㜞ߊ⹏ଔߒߡ߅ࠅ‫ⴕޔ‬᡽ⷙ೙
ߩ⹜㛎ᴺߣߒߡ߽‫ޔ‬ᣢሽߩ ER TA ᴺ㧔hERĮ-HeLa STTA㧦OECD TG455㧕ߣห╬ߢ޽ࠆߣ⹺߼ߡ޿ࠆ‫ޕ‬ߚߛߒ‫⹜ᧄޔ‬㛎ᴺߢߪᬌ⸽⹜㛎ߦ߅޿ߡ‫⹜ޔ‬㛎ᣉ⸳㑆ߦ޽ࠆ⒟ᐲߩ⚿ᨐߩ⋧㆑߇⷗ࠄࠇߡ޿ࠆ‫ߘޕ‬ߩࠃ߁ߥ႐ว‫ޔ‬ౣ⹜㛎ࠍⴕ߁ߥߤߒߡ‫⹜ޔ‬㛎ᣉ⸳ߦ߅ߌࠆ⹜㛎ᠲ૞਄ߩ໧㗴ߩ᦭ήߦߟ޿ߡ᣿ࠄ߆ߦ
ߔߴ߈ߢ޽ߞߚ‫ޔߚ߹ޕ‬ෳᾖ‛⾰ߣߒߡో 78 ‛⾰߇ⷙቯߐࠇߡ޿ߚ߇‫ోߡ޿ߟߦߡోߩߘޔ‬ෳട⹜㛎ᣉ⸳ߩ࠺࡯࠲߇Ყセᬌ⸛ߐࠇߚࠊߌߢߪߥ߆ߞߚ‫৻ޕ‬ㇱߩ‛⾰ߪ‫ޔ‬෻ᔕ߇ਇ᣿⏕ߢ޽ࠆߣߒߡ⹏ଔ
߆ࠄ㒰ᄖߐࠇߡ޿ࠆ‫ߩߘޕ‬ℂ↱ߦߟ޿ߡᬌ⸛ߔߴ߈ߢ޽ߞߚ‫ޕ‬
ᧄ⹜㛎ᴺߪ‫࠻ࡅޔ‬ෆᎽ≸↱᧪⚦⢩ᩣ BG1 ⚦⢩ࠍ⿠Ḯߣߒߡ߅ࠅ‫ޔ‬ER ߪ⚦⢩ߦౝ࿷ߔࠆ߽ߩࠍ೑↪
ߒߡ޿ࠆߩߢ‫ ࠻ࡅޔ‬ER ߩ෻ᔕ߇⚦⢩ౝߦ޽ࠆ߇߹߹ߢⷰኤߢ߈ࠆߎߣ߇೑ὐߣߐࠇߡ޿ࠆ‫ޕ‬ߒ߆ߒ‫ޔ‬
ߎߩ⚦⢩ߦߪ ERĮ ߣ ERȕ ߩ෺ᣇ߇ሽ࿷ߒ‫⹜߇ࠄࠇߘޔ‬㛎ߦࠃࠆ TA ෻ᔕߩߤߩ⒟ᐲߕߟࠍᜂߞߡ޿
ࠆ߆߇ਇ᣿ߢ޽ࠆ‫ޔߪࠇߎޕ‬ICCVAM ႎ๔ᦠߦ߽ㅀߴߡ޽ࠆߣ߅ࠅ‫੹ޔ‬ᓟߩ‫⥋ߒ߆ߒޔ‬ᕆߩ⎇ⓥ⺖㗴ߢ޽ࠆ‫ޕ‬13
19

Annex

Table 1.

20

Reference Substaces Tested for ER TA Activity

21

22

23

24

25

Table 2. 42 ICCVAM-Recommended Substances Used to Evaluate ER Agonist Accuracy

26

27

Table 3. 25 ICCVAM-Recommended Substances Used to Evaluate ER Antagonist Accuracy

28

Table 4. Accuracy of the BG1LucER TA agonist Data

Table 5. Accuracy of the BG1LucER TA antagonist Data

Table 6. Agonist E2 EC50 Control Values

29

Table 7.

Antagonist Raloxifene EC50 Control Values

Table 8. Interlaboratory Agreement for phase 2 Test Substances30

Table 9. Interlaboratory Agreement for phase 3 substances Tested Once at Each Laboratory

31

32

OECD/OCDE

457
Adopted:
2 October 2012

OECD GUIDELINE FOR THE TESTING OF CHEMICALSBG1Luc Estrogen Receptor Transactivation Test Method for Identifying Estrogen Receptor
Agonists and Antagonists
INTRODUCTION
1.
In 1998, the Organisation for Economic Co-operation and Development (OECD) initiated therevision of existing and the development of new Test Guidelines for the screening and testing ofEndocrine Disrupting Chemicals. Since that time, several potential assays have been developed into Test
Guidelines (TG), with additional assays still under development. These assays are contained within the“OECD Conceptual Framework for Testing and Assessment of Endocrine Disrupters” (CF), which 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 (1). The revised CF
comprises five levels, each level corresponding to a difference level of biological complexity (1). The
BG1Luc Estrogen Receptor Transactivation (BG1Luc ER TA) Test Method for Identifying Estrogen
Receptor Agonists and Antagonists is included in level 2 for "in vitro assays providing data about
selected endocrine mechanism(s)/pathway(s) (Mammalian and non mammalian methods)" (1).
2.
In vitro TA assays are based upon the production of a reporter gene product induced by achemical, following binding of the chemical to a specific receptor and subsequent downstream
transactivation. TA assays using activation of reporter genes are screening assays that have long beenused to evaluate the specific gene expression regulated by specific nuclear receptors, such as the estrogen
receptors (ERs) (2) (3) (4) (5). They have been proposed for detection of estrogenic transactivation
regulated by the ER (6) (7) (8).
3.
In vertebrate species, WKHUHDUHDWOHDVWWZRPDMRUVXEW\SHVRIQXFOHDU(5VĮDQGȕZKLFKDUH
encoded by distinct genes. The respective proteins have different biological functions as well as different
tissue distributions and ligand binding affinities (9) (10) (11) 1XFOHDU (5Į PHGLDWHV WKH FODVVLF
estrogenic response (12) (13) (14) (15), and therefore, most models currently being developed to measure(5DFWLYDWLRQDUHVSHFLILFWR(5Į7KH%*/XFFHOOOLQHVSUHGRPLQDQWO\H[SUHVVHQGRJHQRXV(5ĮDQGDPLQRU DPRXQW RI HQGRJHQRXV (5ȕ       7KLV PHWKRG LV EHLQJ SURSRVHG IRU VFUHHQLQJ DQGprioritisation purposes, but can also provide mechanistic information that can be used in a weight ofevidence approach.
4.
The BG1Luc ER TA test method 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) (16). It
utilizes a stably transfected ER responsive luciferase reporter gene in the human ovarian adenocarcinoma
cell line, BG-1, to provide concentration-response data for substances with in vitro ER agonist or
antagonist activity (17). Performance Standards are available to facilitate the development and validation© OECD, (2012)
You are free to use this material for personal, non-commercial purposes without seeking prior consent
from the OECD, provided the source is duly mentioned. Any commercial use of this material is subject towritten permission from the OECD.

33

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OECD/OCDEof similar test methods [(31) for the agonist part, and (32) for the antagonist part]. The Mutual
Acceptance of Data will only be guaranteed for test methods, developed according to the PerformanceStandards, if they have been reviewed and adopted by OECD.
5.

Definitions and abbreviations used in this TG are described in Appendix 1.

INITIAL CONSIDERATIONS AND LIMITATIONS
6.
The interaction of estrogens with ERs can affect transcription of estrogen-controlled genes,
which could lead to the initiation or inhibition of cellular processes, including those necessary for cellproliferation, normal fetal development, and adult homeostasis (18) (19) (20). Perturbation of normal
estrogenic systems may have the potential to trigger adverse health effects.
7.
This TG describes an assay that uses the BG1Luc4E2 cell line to evaluate TA mediated by both
ERD and ERß. 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 ofother receptor and enzymatic systems with the endocrine system, (ii) metabolic activation and/or
inactivation of hormones, (iii) distribution of hormones to tissues, and (iv) clearance of hormones fromthe body. This test method addresses TA induced by chemical binding to the ERs as indicated by the
production of luciferase in an in vitro system. Thus, results should not be directly extrapolated to thecomplex signalling and regulation of the intact endocrine system in vivo.
8.
This TG 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 20). The demonstrated performance of the BG1Luc ER TA(ant)agonist test method suggests that data generated with this test method may inform upon ER mediatedmechanisms of action, and could be considered for prioritization of substances for further testing.
9.
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 (21). However, the activity of firefly luciferase incell-based assays can be confounded by compounds that inhibit the luciferase enzyme, causing both
apparent inhibition or increased luminescence due to protein stabilization (21). In addition, in someluciferase-based ER reporter gene assays, non-receptor-mediated luminescence signals have been
UHSRUWHG DW SK\WRHVWURJHQ FRQFHQWUDWLRQV KLJKHU WKDQ  ȝ0 GXH WR WKH RYHU-activation of the luciferase
reporter gene (2) (22). While the dose-response curve indicates that true activation of the ER systemoccurs at lower concentrations, luciferase expression obtained at high concentrations of phytoestrogens orsimilar 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 (23).
PRINCIPLE OF THE TEST
10.
In vitro TA assays using a reporter gene provide mechanistic data. 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 and transactivates thereporter gene (luc), resulting in the production of luciferase and the subsequent emission of light, which
can be quantified using a luminometer. Luciferase activity can be quickly and inexpensively evaluatedwith a number of commercially available kits.
2© OECD, (2012)

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OECD/OCDE11.
The BG1Luc ER TA utilizes 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 tumor 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 (17). The protocols (agonist and antagonist)
for this TG incorporate essential test method components for in vitro ER TA assays that wererecommended by ICCVAM (8).
12.
Criteria for data interpretation are described in detail in paragraphs 51 through 53. Briefly, a
positive response is identified by a concentration-response curve containing at least three points with
nonoverlapping error bars (mean ± SD), as well as a change in amplitude (normalized 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 Line13.
The stably transfected BG1Luc4E2 cell line is used for the assay. The cell line is available witha technical licensing agreement from the University of California, Davis, California, USA 1, and fromXenobiotic Detection Systems Inc., Durham, North Carolina, USA2.
Stability of the Cell Line14.
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 (paragraph 16). Cells should not be cultured
for more than 30 passages. For the BG1Luc4E2 cell line, 30 passages will be approximately three months.
Cell Culture and Plating Conditions
15.
Procedures specified in the Guidance on Good Cell Culture Practice (24) (25) should be
followed to assure the quality of all materials and methods in order to maintain the integrity, validity, andreproducibility of any work conducted.
16.
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.
17.
Upon reaching ~80% confluence, BG1Luc4E2 cells are subcultured and conditioned to an
estrogen-free environment for 48 hours prior to plating the cells in 96-well plates for exposure to test
substances and analysis 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

1Michael 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

2

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
3© OECD, (2012)

35

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OECD/OCDE

with 4.5% charcoal/dextran-treated FBS, 1.9% L-glutamine, and 0.9% Pen-Strep. All plasticware shouldbe free of estrogenic activity.
Acceptability Criteria
18.
Acceptance or rejection of a test is based on the evaluation of reference standard and control
results from 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 is 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 usually achieved, but for the purposes of acceptance, induction should be greater than orequal 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 will be discarded and repeated.

Comprehensive Test
It includes acceptance criteria from the agonist range finder test and the following:xxx

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 theconcentration-response curve.
Positive control results: Methoxychlor control RLU values should be greater than the DMSO
mean plus three times the standard deviation from the DMSO mean.
An experiment that fails any single acceptance criterion will 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 usuallyachieved, but for the purposes of acceptance, reduction should be greater than or equal tothree-fold.
E2 control results: E2 control RLU values should be within 2.5 times the standard deviationof 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
4
© OECD, (2012)

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OECD/OCDE
It includes acceptance criteria from the antagonist range finder test and the following:xxx

Reference standard results: The Ral/E2 reference standard concentration-response curveshould 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
19.

Reference standards and controls are listed in paragraphs 20 through 29.

Vehicle Control (Agonist and Antagonist Assays)
20.
The vehicle that is used to dissolve the test substances should be tested as a vehicle control. The
vehicle used during the validation of the BG1Luc method was 1% v/v dimethyl sulfoxide (DMSO,(CASRN 67-68-5)) (see paragraph 33). If a vehicle other than DMSO is used, all reference standards,
controls, and test substances should be tested in the same vehicle, if appropriate.
Reference Standard (Agonist Range Finder)21.
The reference standard is E2 (CASRN 50-28-2). For range finder testing the reference standard
is comprised of a serial dilution of four concentrations of E2 (1.84 u 10-10, 4.59 u 10-11, 1.15 u 10-11, and2.87 u 10-12M), with each concentration tested in duplicate wells.
Reference Standard (Agonist Comprehensive)
22.
E2 for comprehensive testing is comprised of a 1:2 serial dilution consisting of
11 concentrations (ranging from 3.67 u 10-10 to 3.59 u 10-13M) of E2 in duplicate wells.
Reference Standard (Antagonist Range Finder)23.
The reference standard is a combination of Ral (CASRN 84449-90-1) and E2 (CASRN 50-282). 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 induplicate wells.
Reference Standard (Antagonist Comprehensive)24.
Ral/E2 for comprehensive testing is comprised of a 1:2 serial dilution of Ral (ranging from
2.45 u 10-8 to 9.57 u 10-11M) plus a fixed concentration (9.18 × 10-11 M) of E2 consisting of nineconcentrations of Ral/E2 in duplicate wells.
Weak Positive Control (Agonist)
25.
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)
26.
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)27.

The E2 control is 9.18 × 10-11 M E2 in EFM and used as a base line negative control.
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Fold-Induction (Agonist)
28.
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 theresult should be greater than four-fold.
Fold-Reduction (Antagonist)
29.
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 andshould be greater than three-fold.

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Demonstration of Laboratory Proficiency

30.
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 atleast 10 independent agonist and 10 independent antagonist experiments, conducted on different days.
These experiments are the foundation for reference standards and the historical controls. Futureacceptable results should be added to enlarge the database. A successful demonstration of proficiency will
be achieved by producing values that are no more than 2.5 standard deviations of the historical controls(see paragraph 18).
31.
Once the historical databases are compiled, the agonist and antagonist proficiency substances
listed in Tables 1 and 2, respectively, should be tested. EC50 and IC50 values reported in Tables 1 and 2
are provided for information. Laboratories should obtain EC50 and IC50 values approximating thosereported here.
Table 1: Agonist Substances for Demonstration of Laboratory Proficiency
Substance

Ethyl paraben

CASRN

Expected
Responsea

BG1LucER TA
Mean EC50(M)b,cMeSH
ChemicalClassdProduct Classe

120-47-8

POS

2.48 × 10-5

Carboxylic
Acid, Phenol

Pharmaceutical,
Preservative-6

Flavonoid,
Heterocyclic
CompoundNatural Product

Carboxylic
Acid, Ester,Phthalic Acid

Plasticizer,
Industrial
ChemicalHeterocyclic
CompoundDye, Natural
Product,
Pharmaceutical
Intermediate

Flavonoid,
Heterocyclic
CompoundNatural Product

Kaempferol

520-18-3

POS

3.99 × 10

Butylbenzyl
phthalate

85-68-7

POS

1.98 × 10-6

-6

Apigenin520-36-5

POS

1.60 × 10

Daidzein486-66-8

POS

7.95 × 10-7

Bisphenol A

80-05-7

POS

5.33 × 10-7

PhenolChemicalIntermediate,
Flame Retardant,
Fungicide

Genistein446-72-0

POS

2.71 × 10-7

Flavonoid,
Heterocyclic
CompoundNatural Product,
Pharmaceutical

Coumestrol479-13-0

POS

1.32 × 10-7

Heterocyclic
CompoundNatural Product7© OECD, (2012)

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Expected
Responsea

BG1LucER TA
Mean EC50(M)b,cMeSH
ChemicalClassdProduct Classe

17v-Estradiol

57-91-0POS

1.40 × 10-9

Steroid

Pharmaceutical,
Veterinary Agent

Estrone53-16-7

POS

2.34 × 10-10

Steroid

Pharmaceutical,
Veterinary Agent

Diethylstilbestrol

56-53-1

POS

3.34 × 10-11

Hydrocarbon(Cyclic)

Pharmaceutical,
Veterinary Agent

17v-Ethinyl
estradiol

57-63-6

POS

7.31 × 10-12

Steroid

Pharmaceutical,
Veterinary AgentAtrazine

1912-24-9

NEG-

Heterocyclic
CompoundHerbicide

Corticosterone

50-22-6

NEG-

Steroid

Pharmaceutical

Linuron330-55-2

NEG

-

Urea

Herbicide

Spironolactone

52-01-7NEG-

Lactone,Steroid

Pharmaceutical

Substance

Abbreviations: CASRN = Chemical Abstracts Service Registry Number; EC50 = half maximal effective
concentration of a test substance; MeSH = U.S. National Library of Medicine’s Medical Subject Headings; NEG =negative; POS = positive.
a

ICCVAM consensus data compiled and reported in Independent Scientific Peer Review Panel Report: Evaluation of
the LUMI-CELL® ER (BG1Luc ER TA) Test Method (16).

bcMean EC50 calculated from values reported by the laboratories of the BG1Luc ER TA validation study (26).

Table is sorted in the order of expected EC50 (M) of response in the BG1Luc assay.

d

Substances were assigned into one or more chemical classes using the U.S. National Library of Medicine’s Medical
Subject Headings (MeSH), an internationally recognized standardized classification scheme (available at:
http://www.nlm.nih.gov/mesh).

eSubstances 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/cgi-bin/sis/htmlgen?HSDB)

Table 2: Antagonist Substances for Demonstration of Laboratory Proficiency
Substance

Tamoxifen

CASRN

ExpectedResponsea

BG1LucER TA
Mean IC50(M)b,cMeSH
ChemicalClassdProduct Classe

10540-29-1

POS

8.17 × 10-7

Hydrocarbon(Cyclic)

Pharmaceutical

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Substance

CASRN

ExpectedResponsea

4-Hydroxytamoxifen

68047-06-3

POS

2.08 × 10-7

Hydrocarbon(Cyclic)

Pharmaceutical

Raloxifene HCl

82640-04-8

POS

1.19 × 10-9

Hydrocarbon(Cyclic)

Pharmaceutical-

Steroid

Pharmaceutical,
Veterinary
AgentDye, Natural
Product,
Pharmaceutical
Intermediate

17v- Ethinyl
estradiol

57-63-6

BG1LucER TA
Mean IC50(M)b,cMeSH
ChemicalClassdProduct Classe

NEG

Apigenin520-36-5

NEG-

Heterocyclic
CompoundChrysin

480-40-0

NEG-

Flavonoid,
Heterocyclic
CompoundNatural ProductCoumestrol479-13-0

NEG-

Heterocyclic
CompoundNatural Product

Genistein446-72-0

NEG-

Flavonoid,
Heterocyclic
CompoundNatural Product,
Pharmaceutical

Kaempferol

520-18-3

NEG-

Flavonoid,
Heterocyclic
CompoundNatural Product

Resveratrol

501-36-0

NEG-

Hydrocarbon(Cyclic)

Natural Product

Abbreviations: CASRN = Chemical Abstracts Service Registry Number; IC50 = half maximal inhibitory
concentration; MeSH = U.S. National Library of Medicine’s Medical Subject Headings; NEG = negative; POS =positive.
a

ICCVAM consensus data compiled and reported in Independent Scientific Peer Review Panel Report: Evaluation of
the LUMI-CELL® ER (BG1Luc ER TA) Test Method (16).

bMean IC50 calculated from values reported by the laboratories of the BG1Luc ER TA validation study.

cTable is sorted in the order of expected IC50 (M) of response in the BG1Luc assay.

d

Substances were assigned into one or more chemical classes using the U.S. National Library of Medicine’s Medical
Subject Headings (MeSH), an internationally recognized standardized classification scheme (available at:
http://www.nlm.nih.gov/mesh).
eSubstances 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/cgi-bin/sis/htmlgen?HSDB)32.
For each proficiency substance, starting concentrations should first be selected based on range
finder test results (paragraphs 42 and 43), and then at least two comprehensive tests conducted. Each
9
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comprehensive test should be conducted on a separate experimental day. If the results of the tests
contradict each other (e.g., one test is positive, the other negative), or if one of the tests is inadequate, athird additional test should be conducted. Proficiency is demonstrated by correct classification(positive/negative) of each proficiency substance (see Tables 1, 2, and 3). Proficiency testing should be
repeated by each technician learning the test methods.
Vehicle33.
Test substances should be dissolved in a solvent that solubilises that test substance 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 not exceed 1.0% (v/v). For any vehicle, it should be demonstrated thatthe maximum volume used is not cytotoxic and does not interfere with assay performance. Reference
standards and controls are dissolved in 100% solvent and then diluted down to appropriate concentrationsin EFM.
Preparation of Test Substances
34.
Test substances are dissolved in 100% DMSO (or appropriate solvent), and then diluted down
to appropriate concentrations in EFM. All test substances should be allowed to equilibrate to room
temperature before being dissolved and diluted. Test substance solutions should be prepared fresh foreach experiment. Solutions should not have noticeable precipitate or cloudiness. Reference standard andcontrol stocks may be prepared in bulk however, final reference standard, control dilutions and test
substances should be freshly prepared for each experiment and used within 24 hours of preparation.
Solubility and Cytotoxicity: Considerations for Range Finding
35.
Range finder testing consists of seven point, 1:10 serial dilutions run in duplicate. Initially, test
substances 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.
36.

Range finder experiments are used to determine the following:xx

Test substance starting concentrations to be used during comprehensive testing
Test substance dilutions (1:2 or 1:5) to be used during comprehensive testing37.
An assessment of cell viability/cytotoxicity is included in the agonist and antagonist test method
protocols 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 (16) was a scaled qualitativevisual observation method, however, a quantitative method for the determination of cytotoxicity can beused (see protocol (30)). Data from test substance concentrations that cause more than 20% reduction in
viability cannot be used.
Test Substance Exposure and Assay Plate Organization
38.
Cells are counted and plated into 96-well tissue culture plates (2 x 105 cells per well) in EFM
and incubated for 24 hours to allow the cells to attach to the plate. The EFM is removed and replacedwith test and reference chemicals in EFM and incubated for 19-24 hours.

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39.
Special considerations will need to be applied to those compounds that are highly
volatile since nearby control wells may generate false positive results. In such cases, “plate sealers” mayhelp to effectively isolate individual wells during testing, and is therefore recommended in such cases.
Range Finder Tests
40.
Range finder testing uses all wells of the 96-well plate to test up to six substances as seven point
1:10 serial dilutions in duplicate (see Figures 1 and 2).
xx

Agonist range finder testing uses four concentrations of E2 in duplicate as the reference
standard and four replicate wells for the DMSO control.
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

A
B
C
D
E
F
G
H

1

2

3

4

5

6

7

8

9

10

11

12

TS11
TS12
TS13
TS14
TS15
TS16
TS17

TS11
TS12
TS13
TS14
TS15
TS16
TS17

TS21
TS22
TS23
TS24
TS25
TS26
TS27

TS21
TS22
TS23
TS24
TS25
TS26
TS27

TS31
TS32
TS33
TS34
TS35
TS36
TS37

TS31
TS32
TS33
TS34
TS35
TS36
TS37

TS41
TS42
TS43
TS44
TS45
TS46
TS47

TS41
TS42
TS43
TS44
TS45
TS46
TS47

TS51
TS52
TS53
TS54
TS55
TS56
TS57

TS51
TS52
TS53
TS54
TS55
TS56
TS57

TS61
TS62
TS63
TS64
TS65
TS66
TS67

TS61
TS62
TS63
TS64
TS65
TS66
TS67

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); TS1-1 to
TS1-7 = concentrations (from high to low) of test substance 1 (TS1); TS2-1 to TS2-7 = concentrations(from high to low) of test substance 2 (TS2); TS3-1 to TS3-7 = concentrations (from high to low) of test
substance 3 (TS3); TS4-1 to TS4-7 = concentrations (from high to low) of test substance 4 (TS4); TS5-1
to TS5-7 = concentrations (from high to low) of test substance 5 (TS5); TS6-1 to TS6-7 = concentrations(from high to low) of test substance 6 (TS6); VC = vehicle control (DMSO [1% v/v EFM.]).
Figure 2: Antagonist Range Finder Test 96-well Plate Layout

A
B

1

2

3

4

5

6

7

8

9

10

11

12

TS11
TS12

TS11
TS12

TS21
TS22

TS21
TS22

TS31
TS32

TS31
TS32

TS41
TS42

TS41
TS42

TS51
TS52

TS51
TS52

TS61
TS62

TS61
TS62

11
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OECD/OCDETS13
D TS14
E TS15
F TS16
G TS17
H Ral-1
C

TS13
TS14
TS15
TS16
TS17

TS23
TS24
TS25
TS26
TS27

TS23
TS24
TS25
TS26
TS27

TS33
TS34
TS35
TS36
TS37

TS33
TS34
TS35
TS36
TS37

TS43
TS44
TS45
TS46
TS47

TS43
TS44
TS45
TS46
TS47

TS53
TS54
TS55
TS56
TS57

TS53
TS54
TS55
TS56
TS57

TS63
TS64
TS65
TS66
TS67

TS63
TS64
TS65
TS66
TS67

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); TS1-1 to TS1-7 = concentrations (from high to low) of test substance 1 (TS1); TS2-1
to TS2-7 = concentrations (from high to low) of test substance 2 (TS2); TS3-1 to TS3-7 = concentrations(from high to low) of test substance 3 (TS3); TS4-1 to TS4-7 = concentrations (from high to low) of test
substance 4 (TS4); TS5-1 to TS5-7 = concentrations (from high to low) of test substance 5 (TS5); TS6-1
to TS6-7 = concentrations (from high to low) of test substance 6 (TS6); VC = vehicle control (DMSO[1% v/v EFM.]).
Note: All test compounds are tested in the presence of 9.18 × 10-11 M E2.
41.
The recommended final volume of media required for each well is 200 ȝL. Only use test plates
in which the cells in all wells give a viability of 80% and above.
42.
Determination of starting concentrations for comprehensive agonist testing is described in depth
in the agonist protocol (30). Briefly, the following criteria are used:xx

If there are no points on the test substance concentration curve that are greater than the meanplus three times the standard deviation of the DMSO control, comprehensive testing will be
conducted using an 11-point 1:2 serial dilution starting at the maximum soluble
concentration.
If there are points on the test substance concentration curve that are greater than the meanplus three times the standard deviation of the DMSO control, the starting concentration to be
used for the 11-point dilution scheme in comprehensive testing should be one log higher thanthe concentration 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 1:5 dilution should be used.

x

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

43.
Determination of starting concentrations for comprehensive antagonist testing is described in
depth in the antagonist protocol (30). Briefly, the following criteria are used:x

If there are no points on the test substance concentration curve that are less than the mean
minus three times the standard deviation of the E2, control comprehensive testing will be
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conducted using an 11-point 1:2 serial dilution starting at the maximum soluble
concentration.
If there are points on the test substance concentration curve that are less than the mean minus
three times the standard deviation of the E2 control, the starting concentration to be used forthe 11-point dilution scheme in comprehensive testing should be one of the following:x-

The concentration giving the lowest adjusted RLU value in the range finder
The maximum soluble concentration (See antagonist protocol (30), Figure 14-2)
The lowest cytotoxic concentration (See antagonist protocol (30), Figure 14-3 for a
related example).

The 11-point dilution scheme will be based on either a 1:2 or 1:5 serial or dilution according
to the following criteria:x

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
44.
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 intriplicate wells of 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 u 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
in duplicate 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.

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
each other (e.g., one test is positive, the other negative), or if one of the tests is inadequate, a third
additional test should be conducted.
Figure 3: Agonist Comprehensive Test 96-well Plate Layout
1

2

3

4

5

6

7

8

9

10

11

12

A

TS11

TS12

TS13

TS14

TS15

TS16

TS17

TS18

TS19

TS110

TS111

VC

B

TS11

TS12

TS13

TS14

TS15

TS16

TS17

TS18

TS19

TS110

TS111

VC

C

TS11

TS12

TS13

TS14

TS15

TS16

TS17

TS18

TS19

TS110

TS111

VC

D

TS21

TS22

TS23

TS24

TS25

TS26

TS27

TS28

TS29

TS210

TS211

VC

13
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OECD/OCDE

E

TS21

TS22

TS23

TS24

TS25

TS26

TS27

TS28

TS29

TS210

TS211

Meth

F

TS21

TS22

TS23

TS24

TS25

TS26

TS27

TS28

TS29

TS210

TS211

Meth

G

E2-1

E2-2

E2-3

E2-4

E2-5

E2-6

E2-7

E2-8

E2-9

E210

E211

Meth

H

E2-1

E2-2

E2-3

E2-4

E2-5

E2-6

E2-7

E2-8

E2-9

E210

E211

Meth

Abbreviations: TS11-1 to TS1-11 = concentrations (from high to low) of test substance 1; TS2-1 to TS211 = concentrations (from high to low) of test substance 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

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

2

3

4

5

6

7

8

9

10

11

12

A

TS11

TS12

TS13

TS14

TS15

TS16

TS17

TS18

TS19

TS110

TS111

VC

B

TS11

TS12

TS13

TS14

TS15

TS16

TS17

TS18

TS19

TS110

TS111

VC

C

TS11

TS12

TS13

TS14

TS15

TS16

TS17

TS18

TS19

TS110

TS111

VC

D

TS21

TS22

TS23

TS24

TS25

TS26

TS27

TS28

TS29

TS210

TS211

VC

E

TS21

TS22

TS23

TS24

TS25

TS26

TS27

TS28

TS29

TS210

TS211

Tam

F

TS21

TS22

TS23

TS24

TS25

TS26

TS27

TS28

TS29

TS210

TS211

Tam

G Ral-1

Ral-2

Ral-3

Ral-4

Ral-5

Ral-6

Ral-7

Ral-8

Ral-9

E2

E2

Tam

H Ral-1

Ral-2

Ral-3

Ral-4

Ral-5

Ral-6

Ral-7

Ral-8

Ral-9

E2

E2

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; TS1-1 to TS1-11 = concentrations (fromhigh to low) of test substance 1 (TS1); TS2-1 to TS2-11 = concentrations (from high to low) of test
substance 2 (TS2); 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)
Measure of Luminescence
45.
Luminescence is measured in the range of 300 to 650 nm, using an injecting luminometer andwith software that controls the injection volume and measurement interval (30). Light emission from eachwell is expressed as RLU per well.
ANALYSIS OF DATA
EC50/IC50 Determination
46.
The EC50 value (half maximal effective concentration of a test substance [agonists]) and the IC50
value (half maximal inhibitory concentration of a test substance [antagonists]) are determined from theconcentration-response data. For substances that are positive at one or more concentrations, theconcentration of test substance that causes a half-maximal response (IC50 or EC50) is calculated using a
Hill function analysis or an appropriate alternative. The Hill function is a four-parameter logistic
mathematical model relating the substance concentration to the response (typically following a sigmoidal
curve) using the equation below:
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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 and Bottom; 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 Prism® statistical software).
Determination of Outliers
47.
Good statistical judgment could be facilitated by including (but not limited to) the Q-test (seeagonist and antagonist protocols (30)), for determining “unusable” wells that will be excluded from thedata analysis.
48.
For E2 reference standard replicates (sample size of two), any adjusted RLU value for a
replicate at a given 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 Testing49.
Raw data from the luminometer are 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

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

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 normalize 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 substances.

Collection and Adjustment of Luminometer Data for Comprehensive Testing50.
Raw data from the luminometer are transferred to a spreadsheet template designed for the test
method. Determine whether there are outlier data points that need to be removed. (See Test AcceptanceCriteria for parameters that are determined in the analyses). The following calculations are performed:
Agonist16© OECD, (2012)48

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Step 1
Step 2
Step 3
Step 4
Step 5457

Calculate the mean value for the DMSO VC.
Subtract the mean value of the DMSO VC from each well value to normalize the data.
Calculate the mean fold induction for the reference standard (E2).
Calculate the mean EC50 value for E2 and the test substances.
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 normalize the data.
Calculate the mean fold induction for the reference standard (Ral/E2).
Calculate the mean IC50 value for Ral/E2 and the test substances.
Calculate the mean adjusted RLU value for tamoxifen.
Calculate the mean value for the E2 reference standard.

Data Interpretation Criteria
51.
The BG1Luc ER TA is intended as part of a weight of evidence approach to help prioritize
substances for ED testing in vivo. Part of this prioritization procedure will be the classification of the test
substance 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 3.
Table 3: Positive and Negative Decision Criteria
AGONIST ACTIVITY

Positive

Negative

Inadequate All test substances 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.
 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.
 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).
 If possible, an EC50 value should be calculated for each positive substance.
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 substance is positiveor negative. Substance should be retested.

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OECD/OCDEANTAGONIST ACTIVITY

Positive

Negative

Inadequate Test substance data produce a concentration-response curve consisting of abaseline, which is followed by a negative slope.
 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.
 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).
 The highest non-cytotoxic concentrations of the test substance should be lessthan or equal to 1x10-5 M.
 If possible, an IC50 value should be calculated for each positive substance.
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.
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 substance is positiveor negative. Substance should be retested.

52.
Positive results will be characterized by both the magnitude of the effect and the concentration
at which the effect occurs, where possible. Examples of positive, negative, and inadequate data are shownin Figures 5 and 6.
Figure 5: Agonist Examples of Positive, Negative and Inadequate Data

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

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

Dashed line indicates 80% of Ral/E2 response, 8000 adjusted and normalized 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 at concentrations less than 1.00 u 10-5M.
Asterixed 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.
53.
The calculations of EC50 and IC50 can be made using a four-parameter Hill Function (Seeagonist protocol and antagonist protocol (30) for more details). Meeting the acceptability criteria
indicates the assay system is operating properly, but it does not ensure that any particular run willproduce accurate data. Duplicating the results of the first run is the best assurance that accurate data
were produced.
Test Report
54.

The test report should contain the following information:

Test substance and control test substances:–––

identification data (e.g. CAS number, if available; source; purity; known impurities; lot
number);physical nature and physicochemical properties (e.g. volatility, stability, solubility);
if mixture, composition and relative percentages of components.

–
–
–
–

source of cells;
passage number of cells at thawing;number of cell passages (from thawing);
methods for maintenance of cell cultures.

Cells:

Test conditions:–––cytotoxicity data and solubility limitations;concentration of test substance;
volume of vehicle and test substance added;
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OECD/OCDE–––

incubation temperature, humidity, and CO2 concentration;
duration of treatment;
cell density during treatment.

Acceptability check (See agonist protocol and antagonist protocol (30) for more details):
For range finder tests:– DMSO control RLU values (mean, SD, CV);– fold inductions or reductions for each assay plate;– E2 control values (antagonist assay only);– did experiment pass or fail acceptance; if fail, what criteria were failed;
For comprehensive experiments:– DMSO control RLU values (mean, SD, CV);– fold inductions or reductions for each assay plate;– positive control results;– reference standard results;– E2 control results (antagonist assay only)– did experiment pass or fail acceptance; if fail, what criteria were failed;
Results:

–
–
–
–
–

raw and normalised data of luminescent signals;
dilution (1:2 or 1:5) used for each test substance;
were test substance results positive, negative, or inadequate;
IC50/EC50 values, if appropriate;statistical analyses, if any, together with a measures of error and confidence (e.g., SEM, SD,
CV or 95% CI) and a description of how these values were obtained.

Discussion of results:
Conclusion:

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LITERATURE
1.
OECD (2012), Guidance Document on Standardised Test Guidelines for Evaluating Chemicals
for Endocrine Disruption Paris, Series on Testing and Assessment No. 150, OECD, Paris.
2.
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.
3.
Jefferson, W.N. et al. (2002), "Assessing estrogenic activity of phytochemicals usingtranscriptional activation and immature mouse uterotrophic responses", J Chromatogr B.: 777(1-2):17989.
4.
Sonneveld, E. et al. (2006), "Comparison of in vitro and in vivo screening models for androgenic
and estrogenic activities", Toxicol Sci. [Comparative Study Journal Article Research Support, Non-U.S.
Gov't], 89(1):173-87.
5.
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):91-8.
6.
Gray, L.E. Jr. (1998), "Tiered screening and testing strategy for xenoestrogens andantiandrogens", Toxicol Lett:102-103:677-80.
7.
EPA (1998), Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) Final
Report, Washington, DC: U.S. Environmental Protection Agency.
8.
ICCVAM (2003), ICCVAM Evaluation of In Vitro Test Methods for Detecting Potential
Endocrine Disruptors: Estrogen Receptor and Androgen Receptor Binding and Transcriptional Activation
Assays, Research Triangle Park, NC: National Institute of Environmental Health Sciences.
9.
Gustafsson, J.-Å. (1999), "(VWURJHQ UHFHSWRU ȕ- A new dimension in estrogen mechanism ofaction", Journal of Endocrinology 163(3):379-83.
10.
Ogawa, S. et al. (1998), "7KHFRPSOHWHSULPDU\VWUXFWXUHRIKXPDQHVWURJHQUHFHSWRUȕ K(5ȕ 
DQGLWVKHWHURGLPHUL]DWLRQZLWK(5Įin vivo and in vitro", Biochem Biophys Res Commun. 243(1):122-6.
11.
Enmark, E. et al. (1997), "+XPDQHVWURJHQUHFHSWRUȕ-gene structure, chromosomal localization,and expression pattern", J. Clin. Endocrinol. Metab. 82(12):4258-65.
12.
Anderson, J.N., J.H. Clark and E.J. Peck Jr (1972), "The relationship between nuclear receptorestrogen binding and uterotrophic responses", Biochem. Biophys. Res. Commun. 48(6):1460-8.
13.
Toft, D. (1972), "The interaction of uterine estrogen receptors with DNA", Journal of Steroid
Biochemistry 3(3):515-22.
14.
Gorski, J. et al. (1968), "Hormone receptors: studies on the interaction of estrogen with theuterus", Recent Progress in Hormone Research 24:45-80.
15.
Jensen, E.V. et al. (1967), "Estrogen-receptor interactions in target tissues", Archives d'Anatomie
Microscopique et de Morphologie Experimentale 56(3):547-69.
21© OECD, (2012)

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OECD/OCDE16.
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, Research
Triangle Park, NC: National Institute of Environmental Health Sciences, NIH Publication No. 11-7850
http://iccvam.niehs.nih.gov/methods/endocrine/ERTA-TMER.htm[cid:3419914858_20826441]
17.
Rogers, J.M. and M.S. Denison (2000), "Recombinant cell bioassays for endocrine disruptors:
development of a stably transfected human ovarian cell line for the detection of estrogenic and antiestrogenic chemicals", In Vitr. Mol. Toxicol. 13(1):67-82.
18.
Cavailles, V. (2002), "Estrogens and receptors: an evolving concept", Climacteric J. 5 Suppl
2:20-6.
19.
Welboren, W.J. et al. (2009), "Genomic actions of estrogen receptor alpha: what are the targets
and how are they regulated?" Endocr. Relat. Cancer 16(4):1073-89.
20.

Younes, M. and N. Honma (2011), "Estrogen receptor beta", Arch Pathol Lab Med.135(1):63-6.

21.
Thorne, N., J. Inglese and D.S. Auld (2010), "Illuminating Insights into Firefly Luciferase andOther Bioluminescent Reporters Used in Chemical Biology", Chemistry and Biology 17(6):646-57.
22.
Kuiper, G.G. et al. (1998), "Interaction of estrogenic chemicals and phytoestrogens with estrogen
receptor beta", Endocrinology 139(10):4252-63.
23.
OECD (2009), Test No. 455: The Stably Transfected Human Estrogen Receptor-alphaTranscriptional Activation Assay for Detection of Estrogenic Agonist-Activity of Chemicals, OECD
GuidelinesfortheTestingofChemicals,
Section4,
OECD
Publishing.
doi: 10.1787/9789264076372-en
24.
Balls, M. et al. (2006), "The importance of good cell culture practice (GCCP)", ALTEX
23(Suppl):270-3.
25.
Coecke, S., et al. (2005), "Guidance on good cell culture practice: a report of the Second
ECVAM Task Force on good cell culture practice", Altern. Lab. Anim. 33:261-87.
26.
ICCVAM (2011), Independent Scientific Peer Review Panel Report: Evaluation of the LUMICELL® ER (BG1Luc ER TA) Test Method, Research Triangle Park, NC: National Institute ofEnvironmental Health Sciences.
27.
Monje, P. and R. Boland (2001), "6XEFHOOXODU GLVWULEXWLRQ RI QDWLYH HVWURJHQ UHFHSWRU Į DQG ȕisoforms in rabbit uterus and ovary", J. Cell Biochem. 82(3): 467-479.
28.
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): 5367-5373.
29.
Weihua, Z., et al. (2000), "EVWURJHQ UHFHSWRU (5  ȕ D PRGXODWRU RI (5Į LQ WKH XWHUXV",Proceedings of the National Academy of Sciences of the United States of America 97(11): 5936-5941.

22© OECD, (2012)54

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30.
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 ofChemicals, NIH Publication No. 11-7850
Appendix B1, BG1Luc ER TA – Agonist Protocol
http://iccvam.niehs.nih.gov/docs/endo_docs/ERTA-TMER/AppxB1-AgonProtocol.pdf
Appendix B2, BG1Luc ER TA – Antagonist Protocol
http://iccvam.niehs.nih.gov/docs/endo_docs/ERTA-TMER/AppxB2-AntagProtocol.pdf
31.
OECD (2012), Performance Standards For Stably Transfected Transactivation In Vitro Assay to
Detect Estrogen Receptor Agonists (for TG 455), Series on Testing and Assessment No. 173, OECD,
Paris.
32.
OECD (2012), Performance Standards for the BG1Luc ER TA Transactivation Method to Detect
Estrogen Receptor Antagonists, Series on Testing and Assessment No. 174, OECD, Paris

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OECD/OCDEAPPENDIX 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: (a) The closeness of agreement between a test method result and an accepted reference value. (b)The proportion of correct outcomes of a test method.
Agonist: A substance that produces a response, e.g., transcription, when it binds to a specific receptor.
Antagonist: A substance that inhibits a response, e.g., transcription, when it binds to a specific receptor.
BG-1: Immortalized human ovarian adenocarcinoma cells that endogenously express estrogen receptors
alpha and beta.
BG-1Luc4E2: The BG-1Luc4E2 cell line was derived from BG-1 immortalized adenocarcinoma cells thatHQGRJHQRXVO\H[SUHVVERWKIRUPVRIWKHHVWURJHQUHFHSWRU (5ĮDQG(5ȕ DQGKDYHEHHQVWDEO\WUDQVIHFWHG
with the plasmid pGudLucERE. This plasmid contains four copies of a synthetic 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 Assessment 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: The adverse effects resulting from interference with structures and/or processes essential forcell survival, proliferation, and/or function. For most substances, toxicity is a consequence of non-specificalterations in “basal cell functions” (i.e., via mitochondria, plasma membrane integrity, etc.).
DMEM: Dulbecco’s Modification of Eagle’s Medium
DMSO: Dimethyl sulfoxide
E2: ȕ-estradiol
EC50: The half maximal effective concentration of a test substance.
ED: Endocrine disruption
EE: 17Į-ethynyl estradiol

24© OECD, (2012)

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OECD/OCDE

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
FBS: Fetal bovine serumh(5Į: Human estrogen receptor alphahERß: Human estrogen receptor beta
IC50: The half maximal effective concentration of an inhibitory test substance.
ICCVAM: The Interagency Coordinating Committee on the Validation of Alternative Methods
MMTV: Mouse Mammary Tumor Virus
Proficiency: The demonstrated ability to properly conduct a test method prior to testing unknown
substances.
Proficiency Chemicals: A list of substances that can be used by laboratories to demonstrate technicalcompetence with a standardized test method. Selection criteria for these substances typically include thatthey represent the range of responses, are commercially available, and have high quality reference data
available.
Ral: raloxifene HCl
Ral/E2: The antagonist reference standard, which is a combination of raloxifene HCl (Ral) and ȕestradiol (E2).
Reference standard: D UHIHUHQFH VXEVWDQFH XVHG WR GHPRQVWUDWH WKH DGHTXDF\ RI D WHVW PHWKRG ȕestradiol is the estrogenic reference standard and Raloxifene HCl the anti-estrogenic reference standard for
the BG1Luc ER TA.
Reliability: A measure of the degree to which a test method can be performed reproducibly within andamong laboratories over time.
RLU: Relative Light Units
RNA: Ribonucleic Acid
RPMI: RPMI 1640 medium supplemented with 0.9% Pen-Strep and 8.0% fetal bovine serum (FBS)
SD: Standard deviation
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).
TA: Transactivation
TG: Test Guideline
25© OECD, (2012)

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OECD/OCDE

Transcription: mRNA synthesis
Transactivation: The initiation of mRNA synthesis in response to a specific chemical signal, such as abinding of an estrogen to the estrogen receptor.
Validation: The process by which the reliability and accuracy of a procedure are established for a specificpurpose.
VC: The vehicle (DMSO) that is used to dissolve test and control chemicals is tested solely as vehicle
without dissolved chemical.

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5HIHUHQFHVWDQGDUGDQGFRQWUROUHVXOWV
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