新規試験法提案書

エストロゲン受容体（ER）結合親和性化学物質の検出のための、
ヒト組み換えエストロゲン受容体（hrER）in vitro 試験法に関する性能準拠試験法ガイドライン（TG493）

令和 2 年12月

国立医薬品食品衛生研究所

新規試験法提案書
令和 2 年 12 月 2 日
No.

2020-03

エストロゲン受容体（ER））結合親和性化学物質の検出のための、ヒト組み換えエストロゲン
受容体（hrER））in vitro 試験法に関する性能準拠試験法ガイドライン（TG493））に関する提案
令和 2 年 6 月 25 日に国立医薬品食品衛生研究所にて開催された新規試験法評価会議（通称：JaCVAM 評価会議）において以下の提案がなされた。
提案内容： 本試験法は、ERへの結合活性を有する化学物質を同定するための簡便で迅速なin vitroのスクリーニング方法として活用することができると考える。
この提案書は、受容体結合試験資料編纂委員会によりまとめられた文書を用いて、JaCVAM 評
価会議が評価および検討した結果、その有用性が確認されたことから作成された。
以上の理由により、行政当局の安全性評価方法としてエストロゲン受容体（ER）結合親和性化
学物質の検出のための、ヒト組み換えエストロゲン受容体（hrER）in vitro 試験法に関する性能準拠試験法ガイドラインの使用を提案するものである。

大野泰雄
JaCVAM 評価会議

平林容子
議長

JaCVAM 運営委員会

委員長

i

JaCVAM 評価会議
大 野 泰 雄 （公益財団法人 木原記念横浜生命科学振興財団）：座長
五十嵐良明 （国立医薬品食品衛生研究所）
池 田 孝 則 （日本製薬工業協会）**
石 井 雄 二 （国立医薬品食品衛生研究所

安全性生物試験研究センター）

稲 若 邦 文 （日本化学工業協会）
井 上 智 彰 （日本免疫毒性学会）*
今 井 教 安 （日本動物実験代替法学会）
岩瀬裕美子 （日本製薬工業協会）*
久 保 文 宏 （独立行政法人 医薬品医療機器総合機構）*
中村るりこ （独立行政法人 製品評価技術基盤機構）
西 川 秋 佳 （国立医薬品食品衛生研究所

病理部／済生会宇都宮病院）

西 村 次 平 （独立行政法人 医薬品医療機器総合機構）
沼澤聡 （日本毒性学会）

平 林 容 子 （国立医薬品食品衛生研究所 安全性生物試験研究センター）
廣 田 衞 彦 （日本化粧品工業連合会）
増 村 健 一 （日本環境変異原学会）
山 本 恵 子 （独立行政法人 医薬品医療機器総合機構）**
横 関 博 雄 （日本皮膚免疫アレルギー学会）
任期：平成 30 年 4 月 1 日～令和 2 年 9 月 30 日*：平成 30 年 4 月 1 日～令和 2 年 3 月 31 日**：令和 2 年 4 月 1 日～令和 2 年 9 月 30 日

ii

JaCVAM 運営委員会
平 林 容 子 （国立医薬品食品衛生研究所

安全性生物試験研究センター）：委員長

石 井 孝 司 （国立感染症研究所）
小川久美子 （国立医薬品食品衛生研究所

安全性生物試験研究センター病理部）

諫 田 泰 成 （国立医薬品食品衛生研究所

安全性生物試験研究センター

薬理部）

北嶋

安全性生物試験研究センター

毒性部）

聡 （国立医薬品食品衛生研究所

合 田 幸 広 （国立医薬品食品衛生研究所）
佐々木正広 （厚生労働省

医薬・生活衛生局

医薬品審査管理課

化学物質安全対策室）

杉 山 圭 一 （国立医薬品食品衛生研究所

安全性生物試験研究センター

変異遺伝部）

髙 橋 祐 次 （国立医薬品食品衛生研究所

安全性生物試験研究センター

毒性部

高 畑 正 浩 （厚生労働省

医薬・生活衛生局

医薬品審査管理課）

束 野 正 明 （厚生労働省

医薬・生活衛生局

医薬品審査管理課

広 瀬 明 彦 （国立医薬品食品衛生研究所
笛 木

医薬品医療機器総合機構）

横 田 雅 彦 （独立行政法人

医薬品医療機器総合機構）

足利太可雄 （国立医薬品食品衛生研究所

化学物質安全対策室）

安全性生物試験研究センター

修 （独立行政法人

動物管理室）

安全性予測評価部）

安全性生物試験研究センター

安全性予測評価部

安全性生物試験研究センター

安全性予測評価部第二室）：事務局
小島肇 （国立医薬品食品衛生研究所第二室）：事務局

iii

JaCVAM Statement on
the Performance-Based Test Guideline (TG493) for
Human Recombinant Estrogen Receptor (hrER) In Vitro Assays
to Detect Chemicals with ER Binding Affinity

At a meeting held on 25 June 2020 at the National Institute of Health Sciences (NIHS) in
Kanagawa, Japan, the Japanese Center for the Validation of Alternative Methods (JaCVAM)
Regulatory Acceptance Board unanimously endorsed the following statement:
Proposal: We consider the Human Recombinant Estrogen Receptor (hrER) In Vitro Assay to
be possibly used as an in vitro, simple, and rapid screening method for detecting
chemicals with ER binding affinity.
This statement was prepared, following the review prepared by the Receptor Binding Assay
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 Performance-Based Test Guideline (TG493) for Human
Recombinant Estrogen Receptor (hrER) In Vitro Assays as useful means for estimating ER
Binding Affinity of chemicals by regulatory agencies.

Yasuo Ohno
Chairperson
JaCVAM Regulatory Acceptance Board

Yoko Hirabayashi
Chairperson
JaCVAM Steering Committee

2 December 2020v

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 (Kihara Memorial Yokohama Foundation for the Advancement of Life
Sciences): Chairperson
Ms. Yoko Hirabayashi (Center for Biological Safety and Research: CBSR, National
Institute of Health Sciences: NIHS)
Mr. Morihiko Hirota (Japan Cosmetic Industry Association)
Mr. Yoshiaki Ikarashi (NIHS)
Mr. Takanori Ikeda (Japan Pharmaceutical Manufacturers Association)**Mr. Noriyasu Imai (Japanese Society for Alternatives to Animal Experiments)
Mr. Kunifumi Inawaka (Japan Chemical Industry Association)
Mr. Tomoaki Inoue (Japanese Society of Immunotoxicology) *Mr. Yuji Ishii (CBSR, NIHS)
Ms. Yumiko Iwase (Japan Pharmaceutical Manufacturers Association)*
Mr. Fumihiro Kubo (Pharmaceuticals and Medical Devices Agency)*
Mr. Kenichi Masumura (Japanese Environmental Mutagen Society)
Ms. Ruriko Nakamura (National Institute of Technology and Evaluation)
Mr. Akiyoshi Nishikawa (CBSR, NIHS/ Saiseikai Utsunomiya Hospital)
Mr. Jihei Nishimura (Pharmaceuticals and Medical Devices Agency)
Mr. Satoshi Numazawa (Japanese Society of Toxicology)
Ms. Keiko Yamamoto (Pharmaceuticals and Medical Devices Agency)**
Mr. Hiroo Yokozeki (Japanese Society for Cutaneous Immunology and Allergy)
Term: From 1st April 2018 to 30th September 2020*: From 1st April 2018 to 31st March 2020**: From 1st April 2020 to 30th September 2020

vi

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

Ms. Yoko Hirabayashi (CBSR, NIHS): Chairperson
Mr. Osamu Fueki (Pharmaceuticals and Medical Devices Agency)
Mr. Yukihiro Goda (NIHS)
Mr. Akihiko Hirose (Division of Risk Assessment, CBSR, NIHS)
Mr. Koji Ishii (National Institute of Infectious Diseases)
Mr. Yasunari Kanda (Division of Pharmacology, CBSR, NIHS)
Mr. Satoshi Kitajima (Division of Toxicology, CBSR, NIHS)
Ms. Kumiko Ogawa (Division of Pathology, CBSR, NIHS)
Mr. Masahiro Sasaki (Ministry of Health, Labour and Welfare)
Mr. Keiichi Sugiyama (Division of Genetics and Mutagenesis, CBSR, NIHS)
Mr. Masahiro Takahata (Ministry of Health, Labour and Welfare)
Mr. Yuhji Taquahashi (Animal Management Section of the Division of Toxicology,
CBSR, NIHS)
Mr. Masaaki Tsukano (Ministry of Health, Labour and Welfare)
Mr. Masahiko Yokota (Pharmaceuticals and Medical Devices Agency)
Mr. Takao Ashikaga (Division of Risk Assessment, CBSR, NIHS): Secretary
Mr. Hajime Kojima (Division of Risk Assessment, CBSR, NIHS): Secretaryvii

エストロゲン受容体（ER））結合親 和性化学物質の検出のための、ヒト組み換え
エストロゲン受容体（hrER））in vitro 試験法に関する性能準拠試験法ガイドライン（TG493））目

次

評 価 会 議 報 告 書 -- ---------- ------- --------- ------- --------- ------- --------- ------- --------- ------- - 1
評 価 報 告 書 --- ------- --------- ------- --------- ------- --------- ------- --------- ------- --------- ------- 7
化学品承認委員会（Chemical Approval Board, CAB）報告書の概要---------------35

OECD KEY EVENT BASED TEST GUIDELINE 442E,
IN VITRO SKIN SENSITISATION ASSAYS ADDRESSING THE KEY EVENTON ACTIVATION OF DENDRITIC CELLS ON THE ADVERSE OUTCOMEPATHWAY FOR SKIN SENSITISATION------------------------------------------------------339

評価会議報告書
エストロゲン受容体（ER））結合親和性化学物質の検出のための、
ヒト組み換えエストロゲン受容体（hrER））in vitro 試験法に関する）性能準拠試験法ガイドライン（TG493）

JaCVAM 評価会議

令和 2 年（2020 年）6 月 25 日

11

JaCVAM 評価会議
大 野 泰 雄 （公益財団法人 木原記念横浜生命科学振興財団）：座長
五十嵐良明 （国立医薬品食品衛生研究所）
池 田 孝 則 （日本製薬工業協会）**
石 井 雄 二 （国立医薬品食品衛生研究所

安全性生物試験研究センター）

稲 若 邦 文 （日本化学工業協会）
井 上 智 彰 （日本免疫毒性学会）*
今 井 教 安 （日本動物実験代替法学会）
岩瀬裕美子 （日本製薬工業協会）*
久 保 文 宏 （独立行政法人 医薬品医療機器総合機構）*
中村るりこ （独立行政法人 製品評価技術基盤機構）
西 川 秋 佳 （国立医薬品食品衛生研究所

病理部／済生会宇都宮病院）

西 村 次 平 （独立行政法人 医薬品医療機器総合機構）
沼澤聡 （日本毒性学会）

平 林 容 子 （国立医薬品食品衛生研究所 安全性生物試験研究センター）
廣 田 衞 彦 （日本化粧品工業連合会）
増 村 健 一 （日本環境変異原学会）
山 本 恵 子 （独立行政法人 医薬品医療機器総合機構）**
横 関 博 雄 （日本皮膚免疫アレルギー学会）
任期：平成 30 年 4 月 1 日～令和 2 年 9 月 30 日*：平成 30 年 4 月 1 日～令和 2 年 3 月 31 日**：令和 2 年 4 月 1 日～令和 2 年 9 月 30 日

22

経済協力開発機構 （Organization for Economic Co-operation and Development: OECD） 試
験法ガイドライン （Test Guideline: TG） では、エストロゲン活性に関する試験法として、
in vitro エストロゲン受容体転写活性化試験法 （OECD TG455） や in vivo 試験法として子宮肥大試験 （OECD TG440） が確立されているが、エストロゲン受容体 (Estrogen Receptor:ER) を介した生体反応の起点となる化学物質と受容体・リガンド結合部位への特異的相互作用の簡便で迅速なスクリーニング法として組み換えタンパク質を用いた in vitro
結合試験法の開発が進められてきた。本試験法は、ER結合親和性化学物質の検出のた
めの、ヒト組み換えエストロゲン受容体 （Human Recombinant Estrogen Receptor Alpha:
hrERα） in vitro 試験法に関する性能準拠試験法ガイドライン (Performance Based Test
Guideline: PBTG) であり、in vitroで化学物質 （競合物質） 存在下での放射標識リガンド（[3H]-17β-エストラジオール、[3H]-E2） のERへの結合能を化学物質の濃度を上昇させながら測定するスクリーニング試験法を含むガイドラインである。本PBTGには、以下の2種類の試験法が参照試験法として掲載されている。
・完全長ヒト組み換えERαを用いたFreyberger-Wilson （FW） の in vitro hrERα 結合試験
（以下、FWアッセイ）1)・ヒト組み換えリガンド結合ドメイン (Ligand Binding Domain: LBD) タンパク質を用いた化学物質評価研究機構 （Chemicals Evaluation and Research Institute: CERI） の in vitro
hrERα-LBD 結合試験 （以下、CERI アッセイ）1)
本試験法については、PBTG に掲載されている参照試験法について、日米欧 5 ～ 6 施
設でバリデーション研究が実施され2)、施設内・施設間再現性が評価された。OECD 専
門家会議でその正確性および信頼性が評価され、2015年に内分泌かく乱物質のスクリーニング評価に関する試験法として、OECD TG493が承認された1)。
Japanese Center for the Validation of Alternative Methods（JaCVAM）評価会議は、JaCVAM
受容体結合試験資料編纂委員会により作成された、「エストロゲン受容体 （ER） 結合親和性化学物質の検出のための、ヒト組換えエストロゲン受容体 （hrER） in vitro 試験法
に関する性能準拠試験法ガイドライン （TG493） の評価報告書」 （令和2年2月18日） を用いて、本試験法の妥当性について検討した。
1. 試験法の定義
名称：

完全長ヒト組み換え ERα を用いた Freyberger-Wilson（FW）の in vitro hrERα 結合試験



ヒト組み換えリガンド結合ドメイン(LBD)タンパク質を用いた化学物質評価研究機
構（CERI）の in vitro hrERα-LBD 結合試験

代替する対象毒性試験：なし。本試験法はスクリーニングとして用いるものである。
試験法の概略：FW および CERI アッセイは、ER 結合親和性化学物質を検出することを目的として開発された試験法である。これら試験は、①受容体－リガンド相互作用パ
ラメーターを特徴付ける飽和結合試験および②化学物質と標識リガンドとの間の競合
33

によって結合親和性を推定する競合結合試験の 2 つの主な要素から構成される。①飽
和結合試験では、FW および CERI アッセイプロトコルで規定される過剰濃度の非標識
リガンド（17β-estradiol）の存在下および非存在下で、放射標識リガンドの全結合量および非特異結合量が測定され、その差から特異的結合量が算出される。②競合結合試験では、飽和結合試験で得られた特異的結合量を踏まえ、FW アッセイでは 1 nM の放射標
識リガンドが 20±5%、CERI アッセイでは 0.5 nM の放射標識リガンドが 40±10%の特異的結合を示す量の hrERα を用いて、化学物質存在下での放射標識リガンドの結合量が測定される。
2. 評価に用いた資料および評価内容の科学的妥当性
本試験法については、以下の①日米欧 5 ～ 6 施設による国際バリデーション研究 2)
および ② その一環として実施された試験方法の正確性・信頼性を確認するためのバリ
デーション試験 2) の結果を基に、OECD において評価された。その結果、OECD Task
Force on Endocrine Disrupters Testing and Assessment で提案された OECD Conceptual
Framework for Testing and Assessment of Endocrine Disrupters のレベル 2 に該当する内分泌かく乱物質のスクリーニング評価に有用な試験法として妥当性が認められ、OECD
TG493 が承認された。
JaCVAM 受容体結合資料編纂委員会はこれらの資料を用いて評価し、報告書としてまとめたものを評価資料とした。
本試験法は、化学物質が hrERα に対し、アゴニスト作用を及ぼすかアンタゴニスト
作用を及ぼすかの判定はできないものの、hrERα への結合に対し、極めて良好な特異性を有する試験法であると考えられることから、本試験法は hrERα への結合作用を有す
る化学物質をスクリーニングする方法として、科学的な妥当性があると考えられる。
① 国際バリデーション研究：
FW アッセイについては、Freyberger と Wilson により作成されたプロトコルに基づいて、CERI アッセイについては CERI により作成されたプロトコルに基づいて、米国 EPAの主導により、FW アッセイについては日米欧 6 施設、CERI アッセイについては日米
欧 5 施設の参加による国際バリデーション研究が実施された。バリデーション研究は、飽和結合試験および競合結合試験が実施され、競合結合試験では 29 物質 （陽性 21 物
質、陰性 8 物質） について検討された。
② 試験方法の正確性・信頼性を確認するための検証試験:
ラット子宮のサイトゾルを用いた ER 結合試験 （Estrogen Receptor Binding Assay
Using Rat Uterine Cytosol: ER-RUC アッセイ）3) の結果が入手可能かつ ER 反応性が明確な 22 物質 （陽性 17、陰性 5） を用いて、検証試験が実施された。その結果、陽性
17 物質について、「判定不能」 を除く一致率は、ER-RUC アッセイで 94.1% （16/17）、FWおよび CERI アッセイでいずれも 100% （16/16） であった。さらに、陰性 5 物質について、ER-RUC アッセイで陰性 1、偽陽性 1、評価不能 3 であったのに対し、FW および
4
4

CERI アッセイでは全て陰性であった。以上の結果、「判定不能」を除く各々の試験法の正確度について、ER-RUC アッセイは 94.4% （17/18）、 FW および CERI アッセイは共に100% （21/21）であった。
以上より、FW および CERI アッセイ共に、米国 Environmental Protection Agency（EPA）の現行の TG OPPTS 890.1250 と同等以上の性能標準を満たすことが示された。
3. 本試験法の有用性と適用限界
本試験法は、hrERα へ結合活性を良好な特異性をもって検出できる点が優れている。
バリデーション研究に用いた物質の内、ER-RUC アッセイとの比較に用いた物質において、ER との結合性が適切に評価できた化合物 （equivocal と判定された 1 物質を除く 21 物質） では感度と特異度は良好な値を示しているが、例数を増やして正確な数値を求める必要がある。また、ER-RUC アッセイと同等以上の性能標準を満たすことも示されている。しかしながら、本試験法で評価できるのはあくまでも化学物質の hrERα への結合活性であり、化学物質のアゴニスト・アンタゴニスト活性を区別することは出来ず、本試験法で陽性と判定される化学物質が生体内でどのような影響を示すかについては評価することができない。なお、本試験法を行う上で、以下の点に留意する必要がある。


本試験法は放射性物質を取り扱う試験である。



未知の化学物質を試験する際に試験で用いる濃度は 1 mM を超えてはならない。

4. 目的とする物質又は製品の毒性を評価する試験法としての、社会的受け入れ性および行政上の利用の可能性
社会的受け入れ性：
本試験法は、放射標識リガンドを使用することから、放射性同位体の取り扱い可能な施設およびそれについての十分な知識を有した熟練した作業者による作業が必要であると考えるものの、①生きた動物を用いないという点で 3Rs の精神に合致していること、②内因性エストロゲンと競合して ER に結合する可能性のある化学物質を迅速に同定するための、有用かつ単純なツールであると考えられることから、本試験法の社会的受け入れ性は高いと考える。
行政上の利用性：
本試験法は、ER への結合活性を有する化学物質を同定するための簡便で迅速な invitro のスクリーニング法として活用することができると考える。

55

参考文献（最終アクセス日：2020 年 2 月 25 日）
1) OECD(2015) Performance-Based Test Guideline for Human Recombinant Estrogen
Receptor (hrER) In Vitro Assays to Detect Chemicals with ER Binding Affinity. Available
at:https://www.oecd-ilibrary.org/docserver/9789264242623-en.pdf?expires=1577340271&
id=id&accname=guest&checksum=AA652957D886AA1F986DE09814282317
2) OECD (2015), Integrated Summary Report: Validation of Two Binding Assays Using
Human Recombinant Estrogen Receptor Alpha (hrERα), Health and Safety Publications,
Series on Testing and Assessment (No. 226), Organisation for Economic Cooperation andDevelopment, Paris. Available at: http://www.oecd.org/env/ehs/testing/MONO(2015)35.pdf
3) Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1250: Estrogen
Receptor Binding Assay Using Rat Uterine Cytosol (ER-RUC). Available at:https://nepis.epa.gov/Exe/ZyNET.exe/P100SHEC.TXT?ZyActionD=ZyDocument&Client=EPA&Index=2006+Thru+2010&Docs=&Query=&Time=&EndTime=&SearchMethod=1&TocRestrict=n&Toc=&TocEntry=&QField=&QFieldYear=&QFieldMonth=&QFieldDay=&IntQFieldOp=0&ExtQFieldOp=0&XmlQuery=&File=D%3A%5Czyfiles%5CIndex%2
0Data%5C06thru10%5CTxt%5C00000038%5CP100SHEC.txt&User=ANONYMOUS&P
assword=anonymous&SortMethod=h%7C-&MaximumDocuments=1&FuzzyDegree=0&
ImageQuality=r75g8/r75g8/x150y150g16/i425&Display=hpfr&DefSeekPage=x&SearchB
ack=ZyActionL&Back=ZyActionS&BackDesc=Results%20page&MaximumPages=1&Zy
Entry=1&SeekPage=x&ZyPURL

66

評価報告書エ ス ト ロ ゲ ン 受 容 体 （ ER））結合親和性化学物質の検出のための、
ヒト組み換えエストロゲン受容体（hrER））in vitro 試験法に関する性能準拠試験法ガイドライン（TG493））

受容体結合試験資料編纂委員会

令和 2 年（2020 年）2 月 18 日

17

受容体結合試験資料編纂委員会
小野敦 （委員長：岡山大学）

井 口 泰 泉 （横浜市立大学）
青 柳 智 子 （独立行政法人 製品評価技術基盤機構）

8北嶋聡 （国立医薬品食品衛生研究所）

児玉進 （岡山大学）

2

略語
CAB:

Chemical Advisory Board

CERI:

Chemicals Evaluation and Research InstituteCV:

Coefficient of Variation

DBP:

Di-n-Butyl Phthalate

E2:17β-estradiol

EPA:

Environmental Protection Agency

ER:

Estrogen Receptor

ERα:

Estrogen Receptor AlphaERβ:

Estrogen Receptor BetaFW:

Freyberger-Wilson

GLP:

Good Laboratory PracticehERα:

Human Estrogen Receptor Alpha

hrER:

Human Recombinant Estrogen Receptor

hrERα:

Human Recombinant Estrogen Receptor Alpha

IC50:

The half maximal effective concentration of an inhibitory test chemical

ICCVAM:

Interagency Coordinating Committee on the Validation of Alternative Methods

LBD:

Ligand Binding Domain

MMTV:

Mouse Mammary Tumor Virus

NICEATM:NTP Interagency Center for the Evaluation of Alternative Toxicological
Methods

OECD:

Organisation for Economic Co-operation and Development

OECD CF:

Conceptual Framework for Testing and Assessment of Endocrine Disrupters

OECD EDTA TF: Endocrine Disruptor Testing and Assessment Task ForceOTES:

Octyltriethoxysilane

PBTG:

Performance Based Test Guideline

PS:

Performance Standard

RLU:

Relative Light Units

SD:

Standard Deviation

STTA:

Stably Transfected Transactivation Assay

TA:

Transactivation

TG:

Test Guideline

VMG-NA:

Validation Management Group for Non-Animal testing

3

9

要旨
性能標準（PS）に基づく性能準拠試験法ガイドライン（PBTG）として経済協力開発機構（OECD）で承認された ER 結合親和性化学物質の検出のための、ヒト組み換えエストロゲ
ン受容体 （hrER） in vitro 試験法1, 2) は、in vitro で化学物質のエストロゲン受容体（ER）に対する結合活性を生体内リガンドであるエストラジオールとの競合結合活性を指標として検出するスクリーニング試験法であり、化学物質の ER への作用による生体反応の起点である ER 結合活性を評価することが出来る。本 PBTG には、国際的な多施設バリデー
ション研究により、試験法の妥当性と信頼性が証明された 2 種の試験法 （FWアッセイおよび化学物質評価研究機構 （CERI）アッセイ） が参照試験法として収載されている。また、
本 PBTG には、試験の目的である化学物質の hrER 結合親和性を検出する類似試験法の開発とバリデーション研究のための PS が設定されており、PS を満たすことが示された新規の類似試験法を追加することが可能である。本試験法に収載されている 2 つの試験法についての再現性や信頼性および試験法としての科学的妥当性と規制試験法としての妥当性については、米国環境保護庁 （米国EPA） の主導により日米欧 6 施設の参加 （CERIアッセイについてはうち 5 施設が参加）による国際バリデーション研究の結果をもとに、OECD 第三者評価により評価され、OECD 内分泌かく乱物質の試験と評価に関するタス
クフォース（OECD EDTA TF）で提案された OECD 内分泌かく乱物質の試験法と評価に関する概念枠組み（OECD CF）のレベル 2 に該当する内分泌かく乱物質のスクリーニング評
価に有用な in vitro 試験法として OECD TG493 が成立した。本試験法の課題として、本試
験法は化学物質の ER への結合活性を評価できるのみであり化学物質のアゴニスト・ア
ンタゴニスト活性を区別することは出来ず、本試験法で陽性と判定される化学物質が生体内でどのような影響を示すかについては評価できないという点があげられる。本試験
法はあくまでもスクリーニング法であり、ER を介して惹起されると想定される有害影響を確定評価する試験法と組み合わせて評価を行うことで今後の化学物質管理に大きく貢献すると考えられる。

104

1. 本試験法の科学的妥当性と規制試験法としての妥当性
ER 結合親和性化学物質の検出のための、hrER in vitro 試験法 1, 2) に関する PBTGは、invitro で化学物質の ER に対する結合親和性を検出するスクリーニング試験法である。本
試験法ガイドライン（TG）は、OECD のガイダンス文書 「Guidance Document on the Validation
and International Acceptance of New or Updated Test Methods for Hazard Assessment」3) に規定されるバリデーション研究の原則に従った新たな類似試験または改良試験の開発を促進する PBTG であり、参照試験法として国際的な多施設バリデーション研究により1) 試験法の妥当性と信頼性が証明された下記の 2 つの機序および機能的に同等の試験法が示されている。
・完全長ヒト組み換え ERα を用いた Freyberger-Wilson（FW）の in vitro hrERα 結合試験（以下、FWアッセイ）1)・ヒト組み換えリガンド結合ドメイン（LBD）タンパク質を用いた化学物質評価研究機構（CERI）の in vitro hrERα-LBD 結合試験（以下、CERIアッセイ）1)
本 PBTG には、試験の目的である化学物質のヒト組み換えヒトエストロゲンアルファ受容体（hrERα）結合親和性を検出する類似試験法の開発とバリデーション研究のためのPS 2) が設定されており、現行の PBTG を適時修正して新規類似試験法を追加することが可能である。ただし、類似試験法の追加は PS に示された参照物質 23 物質（表1-1）を用いたバリデーション研究により再現性や信頼性の基準を満たすことを示して合意された後にのみ可能である。
環境中や市場に流通する多くの化学物質が内分泌系に影響する生物活性を有することが示されており、そうした化学物質による内分泌系のかく乱による潜在的なヒト健康や環境に対する影響が指摘されている。OECD では、1998年に重点活動項目の 1 つとして、内分泌かく乱作用を有する可能性のある化学物質のスクリーニングおよび試験のためのTG の整備のため、OECD EDTA TF を設置し、化学物質の内分泌かく乱作用評価のための試験法を OECD CF として整理し、既存 TG の改訂と、新規 TG の整備を進めている。
OECD CF は、それぞれ生物学的複雑性の異なる 5 つのレベルから構成されており、本 TG
は、レベル 2 の 「機構に関する情報をもたらす in vitro 試験」 に示されている該当するホルモン受容体結合試験法である。化学物質の内分泌かく乱作用のスクリーニングに有用な指標として、OECD CF では化学物質のホルモン受容体との結合性または受容体を介した内分泌機能の活性化および阻害を測定する方法が示されている。エストロゲン活性*に関する試験法としては†、これまでに in vitro エストロゲン受容体転写活性化試験法（OECDTG455）や in vivo 試験法として子宮肥大試験（OECD TG440）が確立されているが、ER を介した生体反応の起点となる化学物質と受容体リガンド結合部位への特異的相互作用の簡便で迅速なスクリーニング法として組み換えタンパク質を用いた in vitro 結合試験法の開発が進められてきた。
*エストロゲン活性：化学物質がエストロゲン受容体結合において 17β-エストラジオールを模す能力。

hERα への結合は本 PBTG で検出することができる。

511

表 1-1hrERα結合試験(TG 493)の性能標準の参照物質 23 物質

No.

Chemical Name

CAS RN

Expected
Response #Concentration Range
Tested(M)117β‐Estradiol

50‐28‐2

Strong Binder

1x10‐11 – 1x10‐6

BinderBinderSteroid217α‐ethynyl estradiol

57‐63‐6

Strong Binder

1x10‐11 – 1x10‐6

BinderBinderSteroid3

Diethylstilbestrol (DES)

56‐53‐1

Strong Binder

1x10‐11 – 1x10‐6

BinderBinder4

Meso‐Hexestrol

84‐16‐2

Strong Binder

1x10‐11 – 1x10‐6

BinderBinder5

Zearalenone

17924‐92‐4

Strong Binder

1x10‐10 – 1x10‐3

BinderBinder6

Tamoxifen

10540‐29‐1

Strong Binder

1x10‐10 – 1x10‐3

BinderBinder7

Norethynodrel orModerate Binder

3x10‐9 – 30x10‐4

BinderBinder8(Norethindrone)
Genistein

68‐23‐5(68‐22‐4)aHydrocarbon,(Cyclic), Phenol
Hydrocarbon(cyclic), Phenol
Hydrocarbon(heterocyclic),
LactoneHydrocarbon,(Cyclic)
Steroid

446‐72‐0Moderate Binder

1x10‐10 – 1x10‐3

BinderBinder9Equol

531‐95‐3Moderate Binder

1x10‐10 – 1x10‐3

BinderBinder10Butyl paraben (n‐butyl‐4‐ 94‐26‐8hydroxybenzoate)
Nonylphenol (mixture)
84852‐15‐3

Weak Binder

1x10‐10 – 1x10‐3

BinderBinder

11
12

a

d

FW AssayClassification

CERI Assay
Classification

Weak Binder

1x10‐10 – 1x10‐3

BinderBinderAlkylphenol,

Weak Binder

1x10‐10 – 1x10‐3

BinderBinderOrganochlorine
Steroid,
Nonphenolic
Phenolo,p’‐DDT
5α‐ dihydrotestosterone

521‐18‐6

Weak Binder

1x10‐10 – 1x10‐3

BinderBinder14

Bisphenol A (BPA)

80‐05‐7

Weak Binder

1x10‐10 – 1x10‐3

BinderBinder15
16

4‐n‐heptylphenol
Kepone (Chlordecone)

1987‐50‐4
143‐50‐0

Weak Binder
Weak Binder

1x10‐10 – 1x10‐3
1x10‐10 – 1x10‐3

Equivocal
BinderBinderBinder17
18

Enterolactone* Di‐n‐butyl phthalate(DBP)
Octyltriethoxysilane

78473‐71‐9
84‐74‐2

Weak Binder
Non‐binder

1x10‐10 – 1x10‐3
1x10‐10 – 1x10‐4

BinderNon‐Binder*

BinderNon‐Binder*

20

Corticosterone

21

Benz(a)anthracene

22

Progesterone

23

AtrazineC

C

C

Hydrocarbon(heterocyclic),
Flavonoid
Phytoestrogen
Metabolite
Paraben789‐02‐6

13

19

MESH Chemical Class

2943‐75‐1

Non‐binder

1x10‐10 – 1x10‐3

Non‐Binder

Non‐Binder

Alkylphenol
Hydrocarbon,(Halogenated)
Phytoestrogen
Hydrocarbon(cyclic), Ester
Silane50‐22‐6

Non‐binder

1x10‐10 – 1x10‐3

Non‐binder

Non‐Binder

Steroid

1x10‐10 – 1x10‐3

Non‐Binder

Non‐Binder

Aromatic
Hydrocarbon
Steroid

56‐55‐3

Non‐Binder

57‐83‐0Non‐binder

1x10‐10 – 1x10‐4

Non‐Binder

Non‐Binder

1912‐24‐9

Non‐binder

1x10‐10 – 1x10‐4

Non‐Binder

Non‐Binderb

Heterocyclic
compound

Product Class
Pharmaceutical,
Veterinary Agent
Pharmaceutical,
Veterinary Agent
Pharmaceutical,
Veterinary Agent
Pharmaceutical,
Veterinary AgentNatural Product

Pharmaceutical,
Veterinary Agent
Pharmaceutical,
Veterinary AgentNatural Product

Natural ProductPreservative
Intermediate
CompoundInsecticide
Natural ProductChemicalIntermediate
Intermediate
Pesticide
Natural Product
Plasticizer,
Chemical
Surfacemodifier
Natural ProductIntermediate
Natural Product
Herbicide

Abbreviations: DES, diethylstilbestrol; HPTE, 2,2‐bis(p‐hydroxyphenyl)‐1,1,1‐trichloroethane, an intermediate by‐product of methoychlor; o,p’‐DDT, 1,1,1‐
trichloro‐2‐2‐[o‐ chlorophpenyl]‐2‐[p‐chlorophenyl]ethane#: The expected response for each chemical was based upon published data from in vitro studies and were reviewed by a Chemical Advisory Board whose
members were not directly associated with the validation study for the FW and CERI hrER Binding Assays. Chemicals were selected to represent multiple
chemical classes and cover a range of binding affinity potencies commonly associated with ER agonist activity. When tested in the FW or CERI hrER Assays, theLogIC50 for strong binders typically ranged from ‐ 9 to ‐7 (M), moderate ‐7.1 to ‐6.0 (M) and weak < ‐5.9 (M).
*: The use and classification of Di‐n‐butyl phthalate (DBP) as a non‐binder was based on testing up to 10‐4 M because the chemical was observed to be insolubleat 10‐3M (e.g. turbidity) in some laboratories during the pre‐validation studies. When DBP was tested up to 10‐3M as a coded chemical, it was classified as‘equivocal’ due to displacement of (3H)17b‐estradiol at highest in 3/5 laboratories using the CERI assay and 5/6 laboratories using the FW assay.
a: Norethindrone is provided as an alternate for the control weak binder for cases when norethynodrel is unavailable.
b: During the validation study, benz(a)anthracene was reclassified as a non‐binder (i.e., negative) based on published literature demonstrating that the in vitro
estrogenic activity reported for this chemical is primarily dependent upon its metabolic activation. Enzymatic metabolic activation of the chemical would not be
anticipated in the cell free hrER assays as used in this inter‐validation study. Thus, the correct classification for this chemical is a ‘non‐binder’ when used underthe experimental conditions for the FW and CERI assays.
c: Chemicals were observed to be insoluble at 10‐3M (e.g.,turbidity) in some laboratories during the validation study.
d: Optional where o,p’‐DDT is prohibited by regulatory authorities when replaced by chemical with comparable binding affinity.

参考文献(2) Table 1 を一部改変

12

6

本 PBTG は、ER のリガンド結合部位と被験物質との特異的結合を検出するため、被験物質（競合物質）存在下での放射標識リガンド（[3H]-17β-エストラジオール : [3H]-E2）のER への結合能を被験物質の濃度を上昇させながら測定する競合結合試験としてデザインされている。ER に対して高い親和性を有する被験物質は、受容体親和性の低い化学物
質と比べて、より低濃度で放射標識リガンドと競合する。ただし本試験法で評価できるのはあくまでも化学物質の ER への結合活性であり化学物質のアゴニスト・アンタゴニ
スト活性を区別することはできない。
参照試験法として示されている試験法のうち FW アッセイは完全長 hrERα への結合における被験物質の E2 に対する競合結合能を測定する手法であり、試験にはバキュロウイ
ルスに感染させた昆虫細胞で作製、単離した完全長 hrERα を用いる。FW アッセイに用いる hrERα は、市販（Thermo Fisher Scientific, USA, A15674）で入手可能である。一方、CERI
アッセイは hrERα の LBD を用いた in vitro hrERα-LBD 競合結合試験法であり、グルタチ
オン-S-トランスフェラーゼ融合タンパク質として E. coli で発現させた hrERα-LBD を用いる。CERI アッセイで用いる hrERα-LBD タンパク質は CERI によって作製されたものであり、入手可能である。CERI アッセイは FW アッセイと同様、hrERα へ結合する物質をスクリーニングする手法であり、hrERα-LBD への結合における被験物質の E2 に対する競合結合能を測定する。それぞれの試験プロトコルは 2 つの要素から構成されており、一つは試験に用いる組み換え受容体タンパク質と生理的リガンドである E2 との相互作用活性を明らかにする飽和結合試験であり、他の一つは、被験物質の ER への特異的結合性を評価するため放射標識リガンド（[3H]-E2）と被験物質との ER 結合への競合作用を確認する競合結合試験である。いずれのアッセイ法とも、定量的評価結果として、IC50（50%の [3H]-E2 を hrERα から解離させるのに必要な被験物質の濃度）、hrERα に関する被験物
質の E2 に対する相対結合親和性などを求めることが可能である。化学物質のスクリーニ
ングのための定性的評価結果として、結合曲線に関して設定された基準に従い被験物質を hrERα 結合物質、非結合物質、または判定不能に分類することが可能である。
これらの参照試験法として、FW アッセイについては、Freyberger と Wilson により作成されたプロトコルに基づいて、CERI アッセイについては CERI により作成されたプロト
コルに基づいて、米国 EPA の主導により日米欧 6 施設の参加 （CERI アッセイについては
うち 5 施設が参加） による国際バリデーション研究 1）が実施され、試験法の使用目的に対する妥当性と信頼性が証明されている。バリデーション研究では、サブタスク 0 から
サブタスク 4 まで、段階的に実施された。サブタスク 0 においては、飽和結合試験によりアッセイに用いるヒトエストロゲンアルファ受容体（hERα）タンパク質のリガンド結合能が試験実施性能の許容範囲を満たすことを全ての参加施設で確認した後、サブタス
ク 1 では、陽性・陰性対照の 3 物質（陽性 2、陰性 1 ）の測定、サブタスク 2 では陽性・陰性対照の 3 物質と非コード化 9 物質（陽性 8、陰性 1 ）、サブタスク 3 では陽性・陰性対照を含むコード化 14 物質（陽性 9、陰性 5 ）を被験物質として施設内および施設間再現性の評価が行われた。1 試験施設では、誤判定および不定判定、承認基準を満たせないケースが多く認められたが、施設特有の問題であると判断され、他の参加施設の測定結果の再現性は良好であった。ラット子宮のサイトゾルを使用した ER 結合試験（TG OPPTS 890.1250：
ER-RUC）結果が入手可能かつ ER 反応性が明確な 22 物質（陽性17、陰性 5 ）を用いた解析
713

の結果、CERI および FW アッセイの「判定不能」を除く分類結果は100%一致しており、各試験法での陽性物質（ER に対して結合性をもつことが予測される物質）の一致率は、CERI および FW アッセイは共に100%（16/16）、ER-RUC アッセイは94.1%（16/17）であった。一方、陰性物質（ER に対して結合性を示さないと予測される物質）については、CERIおよび FW アッセイは共に 5 物質全てを陰性と判定した。一方、ER-RUC アッセイの判定
結果の内訳は陰性 1、偽陽性 1、評価不能 3 であった。最終的に、「判定不能」を除く各々の試験法の正確度は、CERI および FW アッセイは共に100%（21/21）、一方、ER-RUC ア
ッセイは94.4%（17/18）であった。なお、比較に用いた物質の予想される結果の妥当性は、OECD VMG-NA のメンバーのうち本バリデーション研究に直接関わっていない 3 名から構成された CAB により確認された。
また、バリデーション研究開始時点のプロトコルで指定されていた弱結合陽性対照のノルエチノドレルおよび陰性対照（非結合性）のフタル酸ジ-n-ブチル（DBP）は、溶解性に問題があったため、追加のサブタスク 4 として 2 施設の参加によりコード化 7 物質を被験物質とした測定が実施され、弱結合性を示す陽性対照としてノルエチンドロンが、陰性対照としてオクチルトリエトキシシラン（OTES）が適切であることが示され、最終的なプロトコルでは、対照物質（基準エストロゲン E2、CAS 50-28-2）、弱結合物質（ノルエチ
ノドレルやノルエチンドロンなど）、非結合物質（OTES）を用いて競合結合試験を実施することとされている。
なお、いずれの試験法とも放射性リガンドを用いるため、試験施設は放射性物質取扱に関する国の認可が必要であり、放射性同位元素および有害化学物質を扱う工程についてはすべて、国の法令で定められた規則および手順に従う必要がある。
2. 試験法の妥当性
2-1. 試験法の概略
2-1-1. 目的と原理
本試験法は内分泌かく乱物質のうち hrERα に作用（結合）する化学物質を検索し、その人体および自然界に対する有害影響を避けることを目的として開発された。本法は、
hrERα に結合し、アゴニスト作用あるいはアンタゴニスト作用を示す化学物質のスクリーニング試験法であり、濃度を変えた試験物質（競合物質）の存在下において放射性同位元素［3H］で標識した内因性のリガンド（E2）と hrERα との結合能を測定し、被験物質のhrERα に対する結合親和性を推測するものである。
2-1-2. 標準測定条件
結合試験は受容体-リガンド相互作用パラメーターを特徴付ける飽和結合試験および被験物質と標識リガンドとの間の競合によって結合親和性を推定する競合結合試験の 2
つの主な要素からなる。
飽和結合試験は、競合結合試験に用いる hrERα の結合親和性（Kd）および活性受容体部
位数（Bmax）を特徴付ける目的で実施される。CERI および FW アッセイプロトコルに記載されている飽和結合実験では、それぞれ規定の過剰濃度の非標識リガンド（E2）の存在
下および非存在下で、8 濃度の標識リガンドの結合量を評価する。活性結合部位を高濃度148の標識リガンドで飽和させた全結合および非特異的結合の両方を測定し、全結合から非特異的結合を差し引くことによって特異的結合量を計算する。Kd および Bmax は、非線形回帰分析とこれに続く Scatchard プロットにより求められる。
競合結合実験では、FW アッセイでは 1 nM の標識リガンドが20±5%、CERI アッセイでは 0.5 nM の標識リガンドが40±10%の特異的結合を示す量の hrERα を用いて異なる濃度の被験物質の存在下で標識リガンドの結合量を測定する。親和性は平衡状態で標識リガ
ンドの特異的結合を50%阻害する化学物質の濃度（IC50）によって定量評価される。したがって、被験物質の結合曲線の特性が十分に示されるよう、Logistic 式における「頂点（TOP）」と「底値（BOTTOM）」を含まれるよう最終的な結果は LogIC50 の両側に十分な濃度範囲を確保する必要がある。
また、対照物質（基準エストロゲン E2）、弱結合物質（ノルエチノドレルまたはノルエ
チンドロン）、非結合物質（原則 OTES であるが、DBP を用いることもできる）の結合親和性を測定し、試験法の当該条件下での有効性および実験間での比較の指標とする。

図2-1 競合結合試験における評価例

2-2. 生物学的妥当性
ER の内因性のリガンドである E2 の ER との結合は標的遺伝子の転写を活性化し、最終的には生理学的作用をもたらす。したがって、ER とリガンドの結合は一連の生物応答の起点であり、この試験法は生体系におけるエストロゲンシグナル伝達に必要な最初の分子的現象を反映している。また、不適切な ER シグナル伝達は、ホルモン依存性がん発症リスクの増加、生殖能力の低下および胎児の成長および発達の変化などの影響をもた
らすことも報告されている4)。
検証試験の競合結合試験から得られた結果は、既存の論文、ER 結合および試験方法に関する総説等 5-10）を基にまとめられた予想される結果と比較され、その妥当性が確認された。
ER 結合試験は、内因性エストロゲンと競合して ER に競合する可能性のある化学物質
915

を迅速に同定するための、有用かつ単純なツールであることが歴史的に証明されており、その実用性および in vivo の反応と密接に関連することが科学的資料に記載されている11, 12)。
2-3. ER 結合試験法の注意点
結合試験に共通の注意点として、以下が挙げられる。
放射標識リガンドを使用することから、放射性同位体の取り扱いに十分な知識を有した熟練した作業者によって実施される必要がある。
結合試験の結果は被験物質の in vitro 系における受容体への結合に関するものであり、invivo での内分泌システムへの影響に直接外挿することはできず、アゴニストとアンタゴ
ニストの区別もできない。また、界面活性剤やアッセイ緩衝液の pH を変化させる物質では正しい結果を得ることができない可能性がある。
3. バリデーション研究に用いた物質の分類と結果の妥当性
両アッセイともに、飽和結合試験（サブタスク 0 ）では [3H]-E2 について、競合結合試
験（サブタスク 1 ～ 3 ）では陽性対照 （ER 結合高親和性化学物質：E2、弱結合：ノルエチ
ノドレル）および陰性対照（非結合：DBP）を含むのべ 29 物質（表 3-1 および表 3-2 参照）について試験が実施された。また、適切な弱結合および非結合対照物質の探索のため、オプションとして 7 物質（表 3-1 および表 3-2 参照）が追加され競合試験（サブタスク 4 ）が実施された。
表3-1バリデーション研究に用いた物質数
競合結合試験
サブタスク1
物質数
コード化 1/非コード化2の区別対照物質 3 物質

サブタスク2

サブタスク3

サブタスク4（オプション）

対照物質 3 物質 14 物質（対照物＋ 9 物質質 3 物質含む）

7 物質

非コード化

非コード化

コード化

コード化

陽性物質3数

2 物質

10 物質

9 物質

4 物質

陰性物質4数

1 物質

2 物質

5 物質

3 物質

1

試験に際し物質の本体が何であるかわからないように、また結果を予測できないようにするためにコードを付して試験施設に配付される被験物質。コード化には、試験を実施するに当たり、意図的、非意図的に結果を偏らせることを防ぐ役割がある。

162

試験に際し物質名が明記された状態で試験施設に配付される被験物質。

3

ERに対して強い又は弱い結合性をもつことが予測される物質。

4

ERに対して結合性を示さないと予測される物質。

10

表 3-2

バリデーション研究に用いた物質名
Subtask 1(controls, uncoded)

Test chemical

CAS #dExpected binding

Expected Classification FW

aﬃnity†and CERI assays

17β-Estradiol

50-28-2

StrongBinderNorethynodrel

68-23-5Moderate

BinderDi-n-butyl phthalate (DBP)

84-74-2

Negative
Subtask 2*17β-Estradiol

50-28-2*Norethynodrel

68-23-5Moderate

Binder* Di-n-butyl phthalate (DBP) 84-74-2

Negative

Non-binder(uncoded chemicals)
Strong

Non-binder

BinderDiethylstilbestrol (DES)

56-53-1

Very strong

Binder17α-ethynyl estradiol

57-63-6

Very strong

Binder

Meso-Hexestrol

84-16-2

StrongBinder

Genistein446-72-0

Moderate

BinderEquol

531-95-3

Moderate

Binder

94-26-8

Weak

Binder84852-15-3

WeakBindero,pʼ-DDT

789-02-6

Weak

BinderCorticosterone

50-22-6

Negative

Non-binder

Butyl paraben (n-butyl-4hydroxybenzoate)
Nonylphenol (mixture)a

Subtask 3(coded chemicals)*17β-Estradiol

50-28-2*Norethynodrel

StrongBinder68-23-5Moderate

Binder* Di-n-butyl phthalate (DBP) 84-74-2

Negative

Non-binder

Zearalenone

17924-92-4

StrongBinderTamoxifen

10540-29-1

StrongBinder5α-dihydrotestosteroneb

521-18-6

Weak

BinderBisphenol A

80-05-7

Weak

Binder4-n-heptylphenol

1987-50-4

Weak

Binder

Kepone (Chlordecone)

143-50-0

Weak

BinderBenz(a)anthracene

56-55-3

Weak

Non-binder1

Enterolactone

78473-71-9

WeakBinderProgesterone

57-83-0

Negative

Non-binder

Octyltriethoxysilane

2943-75-1

Negative

Non-binder

Atrazine

1912-24-9

Negative
Subtask 4*,‡

Non-binder

Bisphenol A

80-05-7

Weak

Binder

Genistein446-72-0

Weak

BinderHPTEc2971-36-0

Weak

BinderNorethindrone

68-22-4

Weak

BinderCorticosterone

50-22-6

Negative

Non-binder(optional, coded chemicals)

Dexamethasone

50-02-2

Negative

Non-binder

Octyltriethoxysilane

2943-75-1

Negative

Non-binder

1117競合試験（サブタスク 1 ～ 3 ）で用いられた 29 物質のうち、21 物質が陽性、8 物質が陰性物質であり、オプションの競合試験（サブタスク 4 ）では、4 物質が陽性、3 物質が陰性物質である。陽性物質には強結合性を有する物質とともに弱結合物質も含まれている。
これらバリデーション研究に用いられた物質には、多様な用途の化学物質から代表的な ER 強結合性、弱結合および非結合物質が含まれていることから選択は妥当と判断される。
なお、競合試験（サブタスク 1 ～ 3 ）に用いた物質の妥当性は、OECD VMG-NA のメン
バーのうち本バリデーション研究に直接関わっていない 3 名から構成された CAB により確認された（Appendix 1 参照）。JaCVAM 受容体結合試験資料編纂委員会は、CAB の判
断はいずれも妥当であり、バリデーションに用いられた化合物セットは ER 結合アッセ
イの評価に十分なケミカルスペースを確保出来ており、バリデーションは適切に実施されたと評価した。
4. 試験法のデータと結果の有用性
バリデーションでは競合試験 （サブタスク 1 ～ 3 ）において 29 物質（陽性 21 物質、陰性 8物質）の測定が実施され、さらに、オプションとして実施されたサブタスク 4 において、陽性 4 物質、陰性 3 物質の測定が実施された。
両アッセイのバリデーション研究に用いられた化学物質は、既知見情報や入手の容易さ等をもとに可能な限り広範な用途・構造の物質が選択されており、それら陽性物質には強結合性を有する物質とともに弱結合物質も含まれており、本バリデーション研究で得られた評価結果は各アッセイ系の特性を示す有用な情報である。
また、両アッセイのバリデーション試験で評価が行われた化学物質の既知見情報から想定される結合活性および構造による分類（ステロイド類、ベンゼン環を一つ有する化合物類等）については、TG493 の習熟度確認物質リスト（表 1-1）として試験濃度域および各
アッセイでの評価結果とともに示されており、これらの情報は新たな関連試験系開発の参照物質として有用である。

1812

5. 試験方法の再現性
CERI アッセイには 5 施設（A－E）が、FW アッセイには CERI アッセイに参加した 5 施設と施設 F を含めた 6 施設が、バリデーション研究に参加した。
（A：University of Missouri-Columbia、B : Lovelace Respiratory Research Institute、C： CERI、
D：Bayer HealthCare, Bayer Pharma AG、E：CeeTox、F：University of Konstanz）
5-1. 飽和結合試験（サブタスク 0）
8 濃度段階の [3H]-E2 の hrERα に対する全結合量、非特異的結合量および特異的結合量を測定し、測定結果をもとに再現性が評価された。両アッセイの全ての測定（CERI アッ
セイ：17、FW アッセイ：20）で、ER の標識リガンドによる飽和と強い結合性を有する単一のリガンド結合部位を有することが確認された。両アッセイ共に、全施設にて妥当な
Kd 値が報告された（CERI アッセイ：各施設平均=0.244～0.758 nM、全体平均±SD=0.451±0.16
nM；FWアッセイ：各施設平均=0.230～0.707 nM、全体平均=0.423±0.20 nM）。特定の施設（FWアッセイ：施設 F）への輸送過程における受容体サンプルの分解が原因とされる場合を除き、殆どの施設が競合結合試験に必要なアッセイ条件を担保する活性受容体濃度を得た。CERI アッセイの場合、推奨される承認基準は （1） 標識リガンド0.5 nM 存在下の全特異的結合量は40±10%、（2） 標識リガンド 4.0 nM 使用時の非特異的結合量は全結合の35%未満であるのに対して、参加 5 施設の平均値±SDはそれぞれ33±5.4%、46±7.6%であった。FWアッセイの場合、推奨される承認基準は （1） 標識リガンド1.0 nM 存在下の全特異的結合量は20±10%、（2） 標識リガンド3.0 nM 使用時の非特異的結合量は全結合の35%未満であるのに対して、参加 6 施設の平均値±SDはそれぞれ20±7.7%、21±10.2%であった。
従って、両アッセイで、共に参加施設の実施性能は概ね許容範囲内であった（表 5-1､ 5-2､
5-3､ 5-4 参照）。
表 5-1

CERI アッセイ、hrERαへの標識リガンド(0.5 nM)の特異的結合の割合

Run 1
Run 2
Run 3
Run 4
Mean ± SD (n)CeeTox (%)

Lovelace (%)

Missouri (%)

Bayer (%)

CERI (%)

21
15
21
49
27 ± 15.3 (4)

27
33
27

23
43
33
50
38 ± 12.2 (4)

40
19
34

45
39
43

31 ± 10.8 (3)42 ± 3.1 (3)29 ± 3.5 (3)参考文献(1) Table 14 を引用。

表 5-2

CERI アッセイ、hrERαへの標識リガンド(4 nM)の非特異的結合の割合
CeeTox (%)

Lovelace (%)

Missouri (%)

Bayer (%)

CERI (%)

Run 1
Run 2
Run 3
Run 4

60
50
60
22

64
53
51

65
25
34
37

35
42
35

50
48
47

Mean ± SD (n)48 ± 18.0 (4)56 ± 7.6 (3)40 ± 17.3 (4)37 ± 4.0 (3)48 ±1.5 (3)参考文献(1) Table 15 を引用。

1319

表 5-3

FW アッセイ、hrERαへの標識リガンド(1.0 nM)の特異的結合の割合
CeeTox (%) Lovelace (%) Missouri (%)

Bayer (%)

CERI (%)

Konstanz (%)

Run 1
Run 2
Run 3
Run 4

27
21
23

33
30
23

22
17
16
15

23
20
24

22
21
16

6a
5a
5a
8b

Mean ±SD

24 ± 3.1

29 ± 5.1

18 ± 3.1

22 ± 2.1

20 ± 3.2

6 ±1.4a

実施施設から受容体分解の可能性が報告された（U. Konstanz）。
新しいバッチの受容体を使用して測定を実施した（U. Konstanz）。
参考文献(1) Table 18 を引用。
b

表 5-4

FW アッセイ、hrERαへの標識リガンド(3 nM)の非特異的結合の割合
CeeTox (%) Lovelace (%) Missouri (%)

Run 1
Run 2
Run 3
Run 4

12
12
14

Mean ±SD (n) 13 ± 1.2 (3)Bayer (%)

CERI (%)

Konstanz (%)

8
9
11

7
9
21
17

31
32
26

21
24
28

35a
45a
41a
16b

9 ± 1.5 (3)14 ± 6.6 (4)30 ± 3.2 (3)24 ± 3.5 (3)34 ± 12.8 (3)a

実施施設から受容体分解の可能性が報告された（U. Konstanz）。
新しいバッチの受容体を使用して測定を実施した（U. Konstanz）。
参考文献(1) Table 19 を引用。
b5-2. 競合結合試験（サブタスク 1 ～ 3 ）段階的に技術的難易度が高まる一連のサブタスクでは、8 濃度段階の被験物質の存在下、単一濃度の [3H]-E2 の hrERα に対する結合量を測定する競合アッセイの測定結果をもとに施設内および施設間での再現性の評価が行われた。
5-2-1. サブタスク 1
3 つの非コード化被験物質陽性対照（強結合性：E2、弱結合性：ノルエチノドレル）および陰性対照（非結合性：DBP）の測定結果をもとに再現性が評価された。結合競合曲線の「頂点」、「底値」および「傾き」を注視するとの助言以外のデータ分析に関する定量的な承認基準の指示は無く、各施設は、アッセイパフォーマンスの評価尺度（表 5-5 参照）に基づき「承認」し得ると判断した 3 回の測定データと共に、全測定データの提出を求められた。両アッセイ共に、承認基準を満たした場合はいずれの被験物質も正しく判定し、アッセイ間で各々の陽性対照の LogIC50 が良く一致していた。CERI アッセイについて、
E2 とノルエチノドレルおよび DBP の許容測定数の割合（許容測定数／全測定数）は、それぞれ27/28、27/28および25/28であった。一方、FW アッセイについて、E2 は20/23、ノルエチノドレルは21/22、DBP は24/24であった。全施設の全体平均値 LogIC50 （平均値±SD）と R2 値（平均値±SD）は、CERI アッセイについて、E2 は-9.03±0.20、0.97±0.06（許容測定数：27）、ノルエチノドレルは -6.14±0.28、0.98±0.02（許容測定数：27）であった（表 5-6 参照）。また、施設毎の LogIC50 の SD 値は E2 とノルエチノドレルそれぞれ、最大で0.442

20

14

と0.259であった。一方、FW アッセイについて、E2 は -8.89±0.18、0.97±0.03（許容測定数：20）、ノルエチノドレルについては -6.25±0.34、0.98±0.02（許容測定数：21）であった（表 56 参照）。さらに、施設毎の LogIC50 の SD 値は E2 とノルエチノドレルそれぞれ、最大で0.255と0.401であった。なお、両アッセイの承認基準を満たさない測定結果の多くの場合においても、競合結合曲線の「頂点」が100%予測値と著しく乖離していたが、それらは明確な標識リガンドの ER 結合に対する濃度依存的な阻害を示し、適切な LogIC50 を得ていた。従って、両アッセイで、共に施設内と施設間それぞれにおける定性的および定量的な再現性が示された。
表 5-5飽和結合試験および競合結合試験のアッセイパフォーマンスの評価尺度
Saturation Binding

1. Did the specific binding curve reach a plateau with increasing concentrations of [3H]17β-estradiol)?
2.

Was the ratio of specific binding to total radioligand used in the assay within the limits
recommended for the assay?

3. Was non-specific binding within the limits as recommended for the assay?
4.

When data were analyzed using the recommended non-linear regression model with a correction
for ligand depletion, was fit for the data linear when displayed using a Scatchard (Rosenthal) plot?
Competitive Binding

1.

Did increasing concentrations of the reference estrogen (17β-estradiol) displace [3H]17β-estradiol
from the hrERα in a manner consistent with a one-site competitive binding model?

2. Was the IC50 for the reference estrogen consistent across multiple runs?
3.

Was the ratio of specific binding to total radioligand used in the assay within the recommended
range for the assay?

4. Did the negative control (Di-n-butyl phthalate) displace less than 25% of the [3H]17β- estradiol?
5.

Did comparison of solvent and buffer controls confirm that the solvent does nothave any effect on ER binding?

参考文献(1) Table 9 を引用。

表 5-6

CERI アッセイと FW アッセイの比較、サブタスク 1（対照物質）

Reference estrogen(17-estradiol)
LogIC50
R2
Weak Binder(Norethynodrel)
Log IC50
R2
Non-binder
Di-n-butyl phthalatea
Non-binder

CERI Assay
Mean ± SD (n)FW Assay
Mean ± SD (n)-9.03 ± 0.20 (27)0.97 ± 0.06 (27)-8.88 ±0.18 (23)0.97 ± 0.03 (23)

-6.14 ± 0.28 (27)0.98 ± 0.02 (27)

-6.22 ± 0.34 (24)0.98 ± 0.02 (24)

27/29 total runsb

24/24 total runsba

対照非結合物質として、最大 10-4M まで試験した。
全測定回数に対する DBP を非結合物質と判定した測定回数を記す。
参考文献(1) Table 31 を引用。
b

15

21

5-2-2. サブタスク 2
ER 結合性が明確な 9 つの非コード化被験物質の測定結果をもとに再現性が評価された。測定データの分析は、（1） 陽性対照の曲線フィットパラメータに基づく許容判別と（2） 各試験施設の許容判定を共に満たす測定結果に限定され、測定毎に被験物質を分類判定した。なお、技術的検討を受け、「判定不能」に分類された被験物質に対して10%ルー
ル‡を適用させた後§、再分析された測定結果を含む。両アッセイにおいて、共に 9 物質の各施設における分類結果を総合すると、結合物質と非結合物質を問わず予想結果と一致しており、両アッセイで、共に施設間における非コード化被験物質の定性的な再現性が示された（表 5-7、5-8 参照）。施設 B では、両アッセイ共に分類判定に不正確な点があったが、施設特有の問題と判断された。詳細な技術的検討の結果、分類判定の不正確さの要因として、（1） 10%ルールの不適切な適用、（2） 4 パラメータロジスティック回帰分析
へのデータの不適合性、③最適数に満たない許容可能な測定結果を基にした分類の限界、などが挙げられ、これらの理由から、最終的な分類前に段階的な分析アプローチの導入が推奨された。

表 5-7

CERI アッセイ、サブタスク 2（非コード化被験物質の分類判定）

Chemical

Expected
ResultCeeTox

Lovelace

Missouri

Bayer

CERI

SimpleAverage

Butyl paraben

BinderBinderBinderBinderBinderBinderBinderCorticosterone

Non-Binder

Non-Binder

Equivocal

Non-Binder

Non-Binder

Non-Binder

Non-Bindero, p’-DDT

BinderBinderBinderBinderBinderBinderBinderDiethylstilbestrol

BinderBinderBinderBinderBinderBinderBinderEthynyl estradiol

BinderBinderEquivocal

BinderBinderBinderBinderEquol

BinderBinderEquivocal

BinderBinderBinderBinder

Genistein

BinderBinderEquivocal

BinderBinderBinderBinder

Meso-Hexestrol

BinderBinderBinderBinderBinderBinderBinderNonylphenol

BinderBinderBinderBinderBinderBinderBinder

緑色背景は、予想される結果との不一致を示す。
参考文献(1) Table 32 を引用。

‡

10%ルール：[3H]-17β-エストラジオールの特異的結合の割合について、反復試験の平均値が低濃度で認められた平均値よりも 10%以上大きい場合、解析のデータポイントから除外する選択肢。

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表 5-8FW アッセイ、サブタスク 2（非コード化被験物質の分類判定）

Chemical
Butyl paraben
Corticosterone

Expected
ResultBinder

CeeTox

Lovelace

Missouri

Bayer

CERI

Konstanz

BinderBinderBinderBinderBinderBinder

Non-Binder Non-Binder

Binder

SimpleAverage
Binder

Non-Binder Non-Binder Non- Binder Non-binder Non-Bindero, p’-DDTb

BinderBinderBinderBinderBinderBinderBinderBinderDiethylstibesterol

BinderBinderBinderBinderBinderBinderBinderBinderEthynyl estradiol

BinderBinderBinderBinderBinderBinderBinderBinderEquol

BinderBinder§BinderBinderBinderBinderBinder

Genistein

BinderBinderBinderBinderBinderBinderBinderBinder

Meso-Hexestrol

BinderBinderBinderBinderBinderBinderBinderBinderNonylphenol

BinderBinderBinderBinderBinderBinderBinderBinder

緑色背景は、予想される結果との不一致を示す。
§
許容基準を満たす測定結果の提出なし。
bo,p’-DDT: 1,1,1,-trichloro-2-[o-chlorophenyl]-2-[p-chlorophenyl]ethan
参考文献(1) Table 33 を引用。

5-2-3. サブタスク3
ER 結合性が明確な14のコード化被験物質の測定結果をもとに施設内および施設間再現性の評価が行われた。データ分析と分類判定はサブタスク 2 と同様に実施した。全体
として、両アッセイにおいて、共に14 物質の各施設における分類結果の単純平均は、結合物質と非結合物質を問わず予想結果と良く一致しており（CERI アッセイ：12 物質で一致、FW アッセイ：12 物質で一致）、両アッセイで、共に施設間におけるコード化被験物質の良好な定性的な再現性が示された（表 5-9、5-10 参照）。なお、これらの単純平均は、測定回数を考慮した加重平均と一致していた。特記すべき点として、両アッセイにおいてコー
ド化被験物質として評価した場合、DBP（陰性）は 「判定不能 : equivocal」 と分類され、各施設で出された分類判定の総合結果は予測結果と一致しなかった。この要因として、（1） 反
応液中の溶解度限界と （2） 最大溶解濃度での ER に対する結合性が、依然として不確定であることが挙げられた。ヘプチルフェノール（弱結合性）は CERI アッセイにおいて分類判定の総合結果は 「判定不能」 と分類されたが、非常に弱い陽性物質では、十分な高濃度域で評価ができず、完全な競合結合曲線が得られないため正しく分類できない場合が指摘された。なお、本物質は FW アッセイでは当初､各施設における分類結果の単純平均に基づいて 「結合物質 : Binder」 と判定されていたが､ CERI アッセイと同様に十分な高濃度域で評価ができず、完全な競合結合曲線が得られないため､ 「判定不能 : equivocal」 に変更された。また、E2 の様に非常に強い陽性物質では、完全な結合曲線を得るためにより低濃度の結果が含まれるべきであると指摘された。これらの結果を受けて、分類判定の不正確さについてサブタスク 2 と同様の要因が挙げられた他、最終的な分類前に段階的な分析アプローチの導入が強く推奨された。また、施設 B では誤判定および不定判定、承認基準を満たせないケースが多く認められたが、施設特有の問題であると判断された。

1723

表 5-9

CERI アッセイ、サブタスク 3（コード化被験物質の分類判定）
ChemicalAtrazine

Expected
ResultNon-Binder

Bisphenol A

CeeTox

Lovelace

Missouri

Bayer

CERI

Non-Binder

Equivocal

Non-Binder

Non-Binder

Non-Binder

SimpleAverage
Non-Binder

BinderBinderBinderBinderBinderBinderBinderBenz(a)anthracene

Non-Binder

Non-Binder

Non-binder

Non-Binder

Non-Binder

Non-Binder

Non-Binder

Dibutyl phthalate-TC

Non-Binder

BinderEquivocal

Non-Binder

Binder

Non-Binder

Equivocal

Dihydrotestosterone

BinderBinderBinderEquivocal

BinderBinderBinder17B-estradiol-TC

BinderBinderBinderBinderBinderBinderBinderEnterolactone

BinderBinderBinderEquivocal

BinderEquivocal

BinderHeptylphenol

BinderBinderEquivocal

Equivocal

BinderEquivocal

Equivocal

KeponeBinderBinderBinderBinderBinderBinderBinderNorethynodrel-TC

BinderBinderBinderBinderBinderBinderBinder

Octyltriethoxysilane

Non-Binder

Non-Binder

Non-Binder

Non-Binder

Non-Binder

Non-Binder

Non-Binder

Progesterone

Non-Binder

Non-Binder

Non-Binder

Non-Binder

Non-Binder

Non-Binder

Non-Binder

Tamoxifen

BinderBinderBinderBinderBinderBinderBinderZearalenone

BinderBinderBinderBinderBinderBinderBinderaa

緑色背景は、予想される結果と不一致を示す。
a
対照物質をコード化被験物質として試験した。
参考文献(1) Table 34 を引用。

表 5-10FW アッセイ、サブタスク 3（コード化被験物質の分類判定）

Chemical

Expected
ResultAtrazine

Non-Binder Non-Binder

Bisphenol A
Benz(a)anthracene_DMSO
Benz(a)anthracene_EtOH
Di-n-butylphthalate†Binder
Non-Binderb

CeeTox

Lovelace§

Missouri

Bayer

CERI

Non-Binder Non-Binder Non-Binder

BinderBinderBinderEquivocal§

Non-Binder

BinderBinder

SimpleAverage

Equivocal

Non-Binder

BinderBinder

Non-Binder

Non- Binder Non- Binder

Non-Binderb

Konstanz

Non-Binder
Non- Binder Non- Binder

Non-Binder Non-Binder Non-Binder Non-Binder Non-Binder Non-Binder Non-Binder Non-Binder

Di-n-butyl phthalate Non-Binder
TCa

Equivocal§

Equivocal

Equivocal

Equivocal

Equivocal

Equivocal

Dihydrotestosterone

BinderBinderBinderBinderBinderBinderBinderBinder17-β-estradiol-TCa

BinderBinderBinderBinderBinderBinderBinderBinderEnterolactone

BinderBinder§BinderBinderBinderBinderBinderHeptylphenol

BinderEquivocal

Binder

Non-Binder

BinderBinderBinderEquivocal

KeponeBinderBinderBinderBinderBinderBinderBinderBinderNorethynodrel-TCa

BinderBinderBinderBinderBinderBinderBinderBinder

Octyltriethoxysilane

Non-Binder

Equivocal

Progesterone

Non-Binder Non-Binder§

Non-Binder Non-Binder Non-Binder Non-Binder Non-Binder

Binder

Non-Binder Non-Binder Non-Binder Non-Binder Non-Binder

Tamoxifen

BinderBinderBinderBinderBinderBinderBinderBinderZearalenone

BinderBinderBinderBinderBinderBinderBinderBinder

緑色背景は、予想される結果と不一致を示す。
a
対照物質をコード化被験物質として、通常、溶解限界濃度または最大 10-3M まで試験した。
§
許容基準を満たす測定結果の提出なし。
†
DBP は陰性対照（非結合物質）として、最大 10-4M まで試験した。
参考文献(1) Table 35 を引用。

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5-2-4. R2値に基づいた評価
より包括的な施設内および施設間における再現性の評価を目的として、陽性対照のデー
タ許容の有無を問わず、実施施設が各プロトコルの承認基準に基づき除外したものを除く全ての測定データを適用させて、測定データのモデルへの適合の指標となる R2 値を用いた評価が行われた。データ分析において、各測定の R2 値が0.9（または90%）以上の場合を「良好」と判定した。
CERI アッセイについて、施設毎の全測定結果の R2 値の分布を比較すると、施設 B を除く 4 施設では、8 割超が「良好」と判定され、平均値が各々95%以上、変動係数（CV）値
は6.5%以下であった（図 5-1 参照）。施設 B では、「良好」は 6 割弱にとどまり、平均値とCV 値はそれぞれ91.8%と11.9%であった。施設毎の予測陽性物質の正判定率（R2>0.9以上）の比較では、施設 A では80%以上、施設 C－E では90%超であったが、施設 B では～70%と他 4 施設と比べて偽陰性率が有意に高かった。また、施設 A および C では偽陽性判定は無く、他 3 施設の偽陽性率は約5－12%の範囲であった。特筆すべき点として、DBP を被験物質とした場合（10-3 Mまで試験）が 「偽陽性」 の約半分を占めた。FW アッセイでは、4 施設において 「良好」 判定が高い割合を占めたが、施設 B と F では 7 割程度にとどまった（A：96.6%、B：70.3%、C：94.8%、D：88.6%、E：92.8%、F：71.0%）（図 5-2 参照）。
施設毎の平均 R2 値は、施設 B を除く 4 施設で95%以上、施設 B は93%であった。また、それらの施設毎の CV 値は、最低値0.8%（施設 A）、最大値9.4%（施設 B）、残り 4 施設では
5.5～8.4%の範囲に収まった。施設毎の予測陽性物質の正判定率（R2>0.9以上）を比較すると、4 施設では90%を超えた（偽陰性率、2－7%）一方、施設 B と施設 F では、それぞれ85%（偽陰性率、15%）、75%（偽陰性率、25%）と有意に低かった。また、施設 A および F では
偽陽性判定は無く、他 4 施設の偽陽性も2.3－7.6%の範囲に収まった。従って、R2 値に基づく分析の結果、一部の施設で技術的問題が見受けられたが、全体として、両アッセイ
では、共にコード化を問わず、良好な定性的な再現性が示された。

参考文献(1) Figure 14(A)を抜粋引用。

図 5-1

CERI アッセイ、施設毎における全測定結果の R2 値分布比較
1925

参考文献(1) Figure 16(A)を抜粋引用。

図 5-2

FW アッセイ、施設毎における全測定結果の R2 値分布比較

5-2-5. サブタスク4
両アッセイの実施に適用可能な弱結合性および非結合性対照物質の探索を目的として、
2 施設（施設 A および E）において 7 物質（ 6 物質に各々 2 つのコード、および 1 物質に 4つのコードを付した）が合計 16 の独立したコード化被験物質として提供され、それらの競合結合試験が実施された。両施設は共に、どちらのアッセイを使用した場合においても、各々コード化被験物質を正しく分類判定した。なお、幾つかの被験物質で溶解度の問題が認められたものの、弱結合性物質の分類判定には影響が無かった。サブタスク 4の結果から、アッセイに最適な弱結合性物質としてノルエチンドロンが選択され、DBPの代替えとなる最も有望な陰性対照として OTES が同定された。これらの結果を受けて、両アッセイを使用する場合のより適切なデータの分析と解釈には、（1）被験物質の溶解限界の決定とその分類判定への影響の確認と（2）10%ルールが適用されるケースの適切な判別が重要であることが示された。

26

20

6.試験方法の正確性・信頼性
CERI および FW アッセイによる評価結果の信頼性の検証のため、各々の施設で実施された全被験物質の hrERα への結合能の評価を基にした最終分類結果を用いて、米国 EPAの現行 Tier I スクリーニング試験群に含まれるラット子宮のサイトゾルを使用した ER
結合試験（TG OPPTS 890.1250：ER-RUC）の結果が比較された（表 6-1 参照）。なお、比較には、3 種の試験法全てにおいて結合試験データが入手可能であった ER 結合性が明確な22 物質（予測陽性：17、予測陰性：5）を用いた。「判定不能」を除き、3 アッセイの分類判
定結果を比較したところ、それぞれ CERI および FW アッセイの結果は、予想された ERに対する結合親和性の強弱を問わず、全ての被験物質の分類において100%一致していた。
各々の試験法の陽性物質の一致率は、CERI および FW アッセイは共に100%（16/16）、ERRUC アッセイは94.1%（16/17）であった。陰性物質については、CERI および FW アッセイ
は共に 5 物質全てを陰性と判定した一方、ER-RUCアッセイの判定結果の内訳は陰性 1、
偽陽性 1、評価不能 3 であった。また、「判定不能」を除く各々の試験法の正確度は、CERIおよび FW アッセイは共に100%（21/21）、ER-RUC アッセイは94.4%（17/18）であった。従って、CERI および FW アッセイ共に、米国 EPA の現行の TG OPPTS 890.1250と同等以上の性能標準を満たすことが示された。

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表 6-1FW アッセイ、CERI アッセイおよびテストガイドライン OPPTS 890. 1250 を用いた
化学物質分類判定の性能比較

Chemical Name

CAS RN

Expected
FW Assay
Response

CERI
Assay

TG
OPPTS
890.125
0

MESH
ChemicalClass

17β-Estradiol

50-28-2

BinderBinderBinderBinderSteroid

Norethynodrel

68-23-5

BinderBinderBinderBinderSteroid*Di-n-butyl phthalate

84-74-2

Non-binder

Binder*†Binder*†Not Tested

Ester,
Phthalic Acid

DES

56-53-1

BinderBinderBinderBinderHydrocarbon,(Cyclic)

17α-ethynylestradiol

57-63-6

BinderBinderBinderBinderSteroidMeso-Hexestrol

84-16-2

BinderBinderBinderBinderHydrocarbon,(Cyclic)

Genistein446-72-0

BinderBinderBinderBinderEquol

531-95-3

BinderBinderBinderBinderButyl paraben(n butyl-4hydroxybenzoate)

94-26-8

BinderBinderBinderBinderParabenBinderBinderBinderBinderAlkylphenol,
Intermediate
Compund

BinderBinderNonylphenol (mixture) 84852-15-3o,p’-DDT

789-02-6

Corticosterone

50-22-6

Zearalenone

17924-92-4

BinderTamoxifen

10540-29-1

5a-dihydrotestosterone

Non-

Non-

Flavonoid,
Heterocyclic
CompoundPhytoestrogen
Metabolite

BinderBinderOrganochlorine

Non-Binder

BinderBinderBinderBinderSteroid
Resorcyclic
Acid
Lactone,Mycotoxin

BinderBinderBinderBinderAntiestrogen521-18-6

BinderBinderBinderEquivocal

Steroid,
Nonphenolic

Bisphenol A

80-05-7

BinderBinderBinderBinderPhenol4-n-heptylphenol
Kepone(Chlordecone)

1987-50-4

BinderEquivocal

Equivocal

BinderBenz(a)anthracene

56-55-3

143-50-0

Non-binder* Non-binder

BinderBinderBinderBinderEquivocal

BinderBinderBinderPhytoestrogen

Non-binder* Non-Binder

Non-Binder

Equivocal

SteroidEnterolactone

78473-71-9

Progesterone

57-83-0Octyltriethoxysilane

2943-75-1

Non-binder Non-Binder

Non-Binder Non-Binder

SilaneAtrazine

1912-24-9

Non-binder* Non-Binder

Non-Binder

Triazine

Equivocal

Pharmaceutical,
Veterinary
Agent
Pharmaceutical,
Veterinary
Agent
Plasticizer,
ChemicalIntermediate
Pharmaceutical,
Veterinary
Agent
Pharmaceutical,
Veterinary
Agent
Pharmaceutical,
Veterinary
Agent
NaturalProduct

Natural Product

Pharmaceutical,
Veterinary
Agent

ChemicalIntermediate

Alkylphenol
Hydrocarbon,(Halogenated)
Aromatic
Hydrocarbon

Non-Binder* Non-Binder Non-Binder
BinderProduct Class

Pesticide

Natural Product
Herbicide

濃いグレーの背景は、予想される結果と不一致を示す。
*†
10-4M まで試験した場合、非結合物質に分類された。10-3M まで試験した場合、最大濃度で標識
リガンドとの競合が検出されたことから「判定不能」に分類された（CERI アッセイ—5 施設中 3 施設、FW アッセイ—6 施設中 5 施設、表 5-8 および 5-9 参照）。
参考文献(1) Table 40 を改編引用。

28

22

7.データの質
本 試 験 法 の バ リ デ ー シ ョ ン 研 究 に お け る 多 施 設 間 試 験 は 、 GLP（ Good Laboratory
Practice）に準拠した精神で試験責任者による科学的指導および管理下で実施されたものである。また、すべてのデータは取り纏めを行った米国 EPA に提出される前に、それぞれの試験施設において独立した品質保証担当者による査察が実施されており、データの質および信頼性は確保されていると考えられる。
8. 試験法の有用性、限界および提言
1) CERI および FW アッセイは原理の項でも述べた様に、放射性標識リガンド（[3H]-E2）と被験物質との hrERα 結合への競合作用を確認することにより、化学物質と hrERα との直接的な結合を検出できる。しかし、被験物質がアゴニスト作用を及ぼすかアンタ
ゴニスト作用を及ぼすかの判定はできない。特異性は極めて良好である。混合物への応用については検討されていない。
2) 本試験法では放射性リガンドを用いるため、試験施設は放射性物質取り扱いに関する認可を要する。放射性同位元素および有害化学物質を扱う工程についてはすべて、国の法令で定められた規則および手順に従う必要がある。
3) 標準的な品質管理を実施し、受容体の活性、化学物質濃度の正しさ、信頼限界が複数の反復試験を通じて安定な状態に保たれ、かつ試験期間中、期待される hrERα 結合反応を示す性能が保持されていることを確認することが必要である。
4) 新たに試験を実施する試験施設は、予め hrERα 標品の特異性と活性を確認する飽和試
験および E2 と TG に示された習熟度確認物質（弱結合物質および非結合物質）を用いた競合結合試験を実施し、当該試験法を使用する習熟度のあることを証明する必要がある。さらに、異なる日に実施した 3 ～ 5 試験より得られた基準エストロゲン（E2）および対照物質の結果を含む履歴データベースを作成する必要がある。
5) 未知の化学物質を試験するには、予備試験を実施し、各被験物質の溶解限界を確認するとともに、試験実施に用いる適切な濃度範囲を特定する必要がある。各被験物質の溶解度はまず溶媒で決定し、さらに試験条件下で混濁や沈殿の有無を確認する。試験で用いる最高濃度は 1 mM を超えてはならない。
6) 望ましい溶媒として FW アッセイではエタノールが、CERI アッセイでは DMSO がそれぞれ示されているが、両アッセイともエタノール及び DMSO が使用可能である。各
溶媒の試験ウエル内の最終濃度は、FW アッセイでは1.5%（最大 2 %を超えてはならない）、CERI アッセイでは、2.05%（最大2.5%を超えてはならない）とされている。
7) hrERα 結合試験（CERI および FW アッセイ）は、スクリーニングのための試験法であり、化学物質の安全性評価に用いる際には、他の in vitro および in vivo 評価系等との結果と併せて実際の生体影響について総合的に判断を行うべきである。
9.その他の試験方法の科学的な報告
内分泌かく乱に係ると考えられる ER に関わる試験法としては、被験物質の受容体への結合実験がまず考えられる。実際、本 PBTG（OECD TG493）で取り上げられている、
hrER 結合親和性化学物質の検出のための、hrER in vitro 試験法は、二つの機序および機23

29

能的に同等と評価された hrERα 結合物質確認試験法から成るが、いずれも非細胞系(cellfree)において、RI 標識リガンドに対する被験物質の競合的な結合阻害により、hrERα との結合能を調べる、というものである。結合実験では、化学物質との相互作用が容易となるように、受容体が水相に露出していることが望ましいが、この目的には細胞を破壊した cell-free が有利であり、実際、この系を用いた結合実験が本 TG にて提案されている。
一方、受容体と相互作用を示す化学物質には、受容体に結合して本来結合すべき生体内物質と同様の細胞内情報伝達系を作動する（活性化する）「アゴニスト」と、受容体に結合はするが本来結合すべき生体物質と異なり生体反応を起こさず、その結合によって本来結合すべき生体内物質と受容体の結合を阻害することで生体応答反応を起こさない「アンタゴニスト」があり、上述した結合実験のみでは、アゴニスト・アンタゴニスト両者の区別が困難である。さらに、非細胞系における結果は、細胞系の場合と一致しないという報告も多く、この理由として、細胞内への移行性による差異や細胞内に特有の結合を制御する因子の存在が示唆され、したがって、受容体との相互作用の検討には、細胞系を利用して調べることが望ましい。
この点、hERα の転写活性を指標とするレポーターアッセイでは、アゴニスト・アンタ
ゴニスト作用の有無を判定できる。現時点で hERα のレポーターアッセイに関する OECD
TG として OECD TG455 が成立している。OECD TG455 もまた PBTG であり、参照試験法
として ER STTA 法（hERα-HeLa-9903 細胞を用いた ER 恒常発現系転写活性化試験法）とVM7LucER TA 法（VM7Luc4E2 細胞を用いる ER 転写活性化（TA）試験）の 2 種の方法が示されている。
両方法の相違は、（1）ER STTA 法が hERα を HeLa 細胞に組み込んだものであるのに対し、VM7LucER TA 法では VM7 細胞に内在する hER を利用する、（2）ER STTA 法では、組み込まれた受容体は hERα のみであるが、VM7 細胞では ERα と ERβ の双方を発現している。ただし、2 種の ER アイソフォーム（hERα、hERβ）に対して完全な選択性を示す化
学物質は、知られておらず、定性的評価において両測定系の結果は、同等と考えられる。
（3）VM7LucER TA 法のアンタゴニスト試験では、バリデーション研究において高濃度領域でのルシフェラーゼ活性の非特異的阻害が認められたことから、評価可能な被験物質の最大濃度は、20μg/mL（約10μM）に制限されているのに対して、ER STTA 法では、媒体への溶解性が良好かつ細胞毒性が認められない場合、アゴニスト・アンタゴニスト試験
とも最大 1 mM まで評価可能である。
ER STTA 法については、CERI が、安定的形質移入による転写活性化試験の妥当性確認
研究を、ヒトの子宮頚癌に由来する hERα-HeLa-9903 細胞株を用いて実施し、hERα を介してエストロゲンアゴニスト活性を検出する試みを行い、実施目的に関する試験の適合性および信頼性を証明している。この細胞株には、次の 2 種類を安定的に挿入されている : （i）hERα 発現ベクターおよび （ii） マウスのメタロチオネイン（MT）遺伝子 のプロモーター部位である TATA エレメントによって駆動される、アフリカツメガエルのビテロ
ゲニン由来のエストロゲン応答エレメント（ERE）のタンデムリピートを 5 個含むホタ
ル・ルシフェラーゼのレポーター遺伝子。
もう一方の VM7LucER TA 法は、NICEATM および ICCVAM によって妥当性の確認がなされてきた。ヒト卵巣腺癌細胞株 VM7 において安定的に形質移入した hER 応答ルシ
30

24

フェラーゼ・レポーター遺伝子を用いて in vitro hER アゴニスト活性またはアンタゴニス
ト活性を有する物質に関する濃度反応データを提供する。具体的には、マウス乳癌ウイ
ルス（MMTV）プロモーターの上流に位置する 4 つの ERE の制御の下でルシフェラーゼ・
レポーター遺伝子を安定的に形質移入した ER 応答ヒト卵巣腺癌細胞株 VM7 を利用して、in vitro の ER アゴニストまたはアンタゴニスト活性を有する物質を検出する。アッセイには安定的に形質移入した VM7 細胞株を使用する。さらに PS を満たす類似（me too）
試 験 法 と し て 、 ERα CALUX ア ッ セ イ （ Stably Transfected Human Estrogen Receptor-αTransactivation Assay for Detection of Estrogenic Agonist and Antagonist Activity of Chemicals
using the ERα CALUX cell line）が収載されている。このアッセイは、BioDetection Systems
BV（オランダ）によって妥当性の確認がなされてきた。CALUX とは、Chemical-Activated
Luciferase Expression の略であり、ヒト骨肉腫細胞株 U-2 OS において安定的に形質移入した ERα CALUX 細胞株を使用し、3 つの ERE 制御による、hER 応答ルシフェラーゼ・
レポーター遺伝子を用いて、in vitro hER アゴニスト活性またはアンタゴニスト活性を、
ルシフェラーゼの活性（発光量）を測定することにより検出する。内因性のレポーター活
性はほとんど検出されないと報告されている。
内分泌かく乱作用としては他にも、リガンド結合ポケット以外の部位での ERαとの相互作用や、エストロゲンシグナル伝達に関連する他の受容体である ERβ、Gタンパク質
共役エストロゲン受容体およびその他の受容体あるいは、内分泌系内の酵素系との相互作用なども考慮する必要があるが、現時点でこうした点を考慮したバリデーション研究が終了している試験系はない。
なお、2012年に改訂された OECD CF は 5 つのレベル（level 1 〜 5 ）から構成され、それぞれの異なったレベルの生物学的複雑性に対応している。上記のレポーターアッセイは、
レベル 2 の「選択された内分泌機構/経路に関する情報をもたらす in vitro アッセイ（非哺乳類の方法）」に相当する。
10. 結論ER 結合親和性化学物質の検出のための、hrER in vitro 試験法に関する PBTG は、in vitro
で化学物質の ER に対する結合親和性を生体内リガンドであるエストラジオールとの競合結合により検出するスクリーニング試験法である。現在、本 PBTG には、2 種の試験法（CERI および FW アッセイ）が参照試験法として掲載されている。また、本 PBTG には、類似試験法の開発とバリデーション研究のための PS が設定されており、PS を満たすことが示された新規の試験法を追加することが可能である。
現在、本 PBTG に参照試験法として掲載されている 2 種の試験法についての科学的妥
当性と規制試験法としての妥当性については、日米欧 6 施設の参加によるバリデーショ
ン研究において陽性 21 物質、陰性 8 物質を用いた評価が行われ、施設内・施設間再現性とも良好な結果が示されており、OECD の第三者評価では、それらバリデーション研究の結果をもとに本試験法の正確性・信頼性が評価され、OECD EDTA TF で提案されたOECD CF のレベル 2 に該当する内分泌かく乱物質のスクリーニング評価に有用な試験法として OECD TG493 が成立した。
本試験法による評価の課題として、本試験法で評価できるのはあくまでも化学物質の25

31

ER への結合活性であり化学物質のアゴニスト・アンタゴニスト活性を区別することは出来ず、本試験法で陽性と判定される化学物質が生体内でどのような影響を示すかについては評価出来ないというという点があげられる。本試験法はあくまでもスクリーニング法であり、ER を介して惹起されると想定される有害影響を確定評価する試験法と組み合わせて評価を行うことで今後の化学物質管理に大きく貢献すると考えられる。
11. 利益相反（COI）について利益相反の観点から、CERI アッセイが適切に評価されたことを受容体結合資料編纂委
員会は確認した。
謝辞
本評価報告書を編纂するに当たり、武吉正博博士（一般財団法人

化学物質評価研究機構）より CERI アッセイに関する具体的な方法などを詳細に解説いただきました｡ ここに深く感謝申し上げます。
1) OECD (2015), Integrated Summary Report: Validation of Two Binding Assays Using Human
Recombinant Estrogen Receptor Alpha (hrERα), Health and Safety Publications, Series on
Testing and Assessment (No. 226), Organisation for Economic Cooperation and Development,
Paris.
2) OECD (2015), Performance Standards for Binding Assays Using Human RecombinantEstrogen Receptor Alpha (hrERα), Health and Safety Publications, Series on Testing andAssessment (No. 222), Organisation for Economic Cooperation and Development, Paris.
3) OECD (2005), Guidance Document on the Validation and International Acceptance of New or
Updated Test Methods for Hazard Assessment. Environmental, Health and Safety Publications,
Series on Testing and Assessment (No. 34), Organisation for Economic Cooperation andDevelopment, Paris.
4) Diamanti-Kandarakis, E., Bourguignon, J-P., Giudice, LC., Hauser, R., Prins, GS., Soto，AM.,
Zoeller, RT., Gore, AC. (2009). Endocrine-disrupting chemicals: An Endocrine Society ent.
Endocr. Rev., 30: 293–342.
5) ICCVAM (2003), ICCVAM Evaluation of In Vitro Test Methods for Detecting Potential
Endocrine Disruptors: Estrogen Receptor and Androgen Receptor Binding and Transcriptional
Activation Assays.
6) ICCVAM (2006), ICCVAM Evaluation of In Vitro Test Methods for Detecting Potential
Endocrine Disruptors: Estrogen Receptor and Androgen Receptor Binding and Transcriptional
Activation Assays.
7) Akahori, Y., Nakai, M., Yamasaki, K., Takatsuki, M., Shimohigashi, Y., Ohtaki, M. (2008),
Relationship between the results of in vitro receptor binding assay to human estrogen receptoralpha and in vivo uterotrophic assay: Comparative study with 65 selected chemicals, Toxicol.
In Vitro, 22(1): 225-231.
8) OECD (2007). Additional Data Supporting the Test Guideline on the Uterotrophic Bioassay inRodents, Environment, Health and Safety Publications, Series on Testing and Assessment (No.
67.), Organisation for Economic Cooperation and Development, Paris.
9) Takeyoshi, M. (2006), Draft Report of Pre-validation and Inter-laboratory Validation For

32

26

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. p. 1-188.
10) USEPA (2009). Endocrine Disruptor Screening Program Test Guidelines. OPPTS
11) 890.1250: Estrogen receptor binding assay using rat uterine cytosol (ER-RUC). Available at:
http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0576-0005.
12) Yamasaki, K., Noda, S., Imatanaka, N., Yakabe, Y. (2004), Comparative study of theuterotrophic potency of 14 chemicals in a uterotrophic assay and their receptor-binding affinity,
Toxicol. Lett., 146: 111-120.

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Appendix１

2006 年 8 月 1 日付
化学品承認委員会（Chemical Approval Board, CAB）報告書の概要

35

CAB 委員名簿
Dr. Taisen Iguchi, Okazaki Institute for Integrative Bioscience, NIBB
Dr. William Kelce, Pozen Pharmaceuticals
Dr. Weida Tong, NCTR, US FDA

1996 年 、 US Environmental Protection Agency (EPA) は 、 食 品 品 質 保 護 法 (Food Quality
Protection Act: FQPA)により、内分泌かく乱物質スクリーニングプログラム(Endcrine Disruptor
Screening Program: EDSP)の開発を義務付けられた。EDSP プログラムの一環として、EPA は
エストロゲン受容体（Estrogen Receptor: ER）と相互作用する化学物質のスクリーニングに
ER 結合アッセイを使用することを提案した。EPA は2000年に、代替法の妥当性確認に関す
る ICCVAM に対し、ER 結合アッセイに関する文献をレビューし、公表された文献のデータに基づいてアッセイの妥当性を確認するよう依頼した。Interagency Coordinating Committee
on the Validation of Alternative Methods (ICCVAM) は、多くの ER 結合アッセイが文献で報告されているが、ER 供給源（組換え、組織、細胞株）は共通のものが使用されておらず、共通のプロトコールも使用されていないことから、どの ER 結合アッセイも検証するための情報が不十分であるとの結論に至った。ICCVAM は、2003年に「ラットまたはヒト組み換え ER（a および b サブタイプ）結合アッセイが、試験方法の標準化、事前検証、および検証のための最優先事項である」と勧告した（ICCVAM, 2003）。
これを受けて、2003 年 3 月に、米国 EPA は、Organisation for Economic Co-operation andDevelopment (OECD)の非動物試験用バリデーション管理グループ（Validation Management
Group for Non-Animal Tests: VMG-NA）が、組換え ER 結合アッセイの開発を主導し、調整することに合意した。この目的のために、2 種類のヒト組み換え ERαタンパク質 (humanrecombinant (hr) ERa) と、2 つの異なる結合試験のプロトコルが検証に用いられた。2 種類の
hrERa タンパク質は、Pan Vera hrERa（PV-ER；バキュロウイルス発現全長 ERa 受容体）とCERI タンパク質（CERI-ER；大腸菌産生 ER リガンド結合ドメインに Glutathion S-Transferase(GST)融合タンパク質を連結したもの）である。2 つのプロトコルは、CERI アッセイ（日本CERI が開発）と Freyberger-Wilson Assay（FW）アッセイ（Alexius Freyberger 氏が開発し、Vickie Wilson と Alexius Freyberger の両者によって最適化）である。FW アッセイは PV-ER
を使用し、CERI プロトコールは CERI-ER を使用した。
これらの hrER 結合アッセイの使用を検証するための国際的な取り組みが、OECD を通じて行われた。バリデーションのステップには、標準化されたプロトコルの開発、他の検査機
関研究室へのプロトコルの技術移転性の検証、プロトコルが施設内および施設間で、同一化
学物質について再現性のある結果を出すことの証明、および化学物質のアッセイの信頼性の検証が含まれる。プロトコールの標準化に次いで、プロトコルの移転性と適用性を検証す36

るために、施設間の検証で使用する一連の化学物質のセットを決めることが必要である。
OECD の hrER 結合アッセイ検証グループが、結合アッセイにおける過去の使用実績、結合強度、化学構造の多様性に基づいて一連の化学物質を提案した。ER に対する親和性が知られている化学物質を、構造的に多様であるが ER 結合試験履歴のない化学物質よりも優先的に選択した。既知および報告されている結合親和性値と一致する結合活性に基づいている。
OECD の hrER バリデーショングループは、化学品承認委員会（CAB）に対して、タスク I、
II、III にノミネートされた化学物質をレビューし、修正を提案することを任務としている。
施設間試験は、両方のプロトコルとそれぞれの hrER タンパク質を用いて、3 段階のタスクで実施されることになった。施設間バリデーションに参加する施設には、FW アッセイとCERI アッセイの両方での経験がある 2 つの施設（ヨーロッパ 1、日本 1）と、結合アッセイの経験はあるが FW アッセイや CERI アッセイを特別に実行したことがない 3 つの施設（ヨーロッパ 1、米国 2）が含まれている。3 つのタスクについては、下記の通り。
タスク I： 試験実施施設は、標準化学物質と陰性化学物質を使用してアッセイを実施する能力を実証する。タスクⅠの化学物質は、高結合親和性の標準物質（17b-Estradiol）、中程度結合親和性の標準物質（Norethynodrel）、および陰性物質（Dibutyl phthalate）である。これら 3 物質は、参加施設の試験の試験能力が基準を満たすことを検証するために、すべての試
験参加研究室で評価する。17 b -Estradiol は内因性リガンドであり、Norethynodrel は、中等度のエストロゲン作用物質であり、Dibutyl phthalate は、2 つのアッセイプロトコルの両方で陰性である。これらの 3 つの基準物質は、内部コントロールとして、タスク II および III のすべての実験で試験した（すなわち、それぞれの実験で、またはアッセイ実施日毎に）。
タスク II：タスク 2 は、以前に FW アッセイと CERI アッセイで試験され、 EU Reference
Laboratory for alternatives to animal testing (EURL ECVAM)でレビューした 12 の物質を用いて、施設内および施設間で再現性のある結果を得るための参加施設の能力を試験する。これらの化学物質は、化学物質をコード化することなく試験した。このタスクでの試験物質の総数
は、9 種類の化学物質（超強結合物質 DES, 17 b -Ethynylestradiol； 強結合物質 Meso-Hexestrol；
中結合物質 Genistein, Equal； 弱結合物質 Butyl paraben, n-butyl 4-hydrobezoate, Nonylphenol,o.p’-DDT；非結合物質 Corticosterone）に 3 つの標準物質(17 b -Estradiol, Norethynodrel, Dibutyl
phthalate)を加えた 12 種類である。
タスク III：タスク 3 は、過去に他の結合アッセイプロトコル（ラット子宮サイトゾール
または hrER のいずれか）で試験したことがあるが、まだ FW アッセイまたは CERI アッセ
イでは試験の経験がない施設の、9 物質を用いた試験で、施設内および施設間で再現性の高い実験結果を得るための参加施設の能力を調べるため、試験物質をコード化した。既知の結合親和性に対応した結合結果が得られると期待された。これらの化学物質は、期待される結37

合親和性とその構造的多様性に基づいて選択された。リストには多数の弱結合物質や非結合の陰性の物質が含まれており、施設間での再現が最も困難である。
この提案に対して、CAB は、タスク I および II に指定された化学物質が適切であることに合意した。タスク I および II の化学物質はそれぞれのタスクで示した。
タスク III については、CAB は、特異度、感度、正確度の点でアッセイの性能をより良く評価するために、結合活性が弱い、または全くない化学物質に重きを置くことを提案した。
具体的には、CAB は Estrone（強結合剤）を外し、Enterolactone と Benz(a)anthracene（弱結合剤）と Atrazine（非結合剤）を追加することを提案した。Enterolactone の追加は、化学物質
リストの構造の多様性を高め、食品の腸内代謝物であるリグナンを追加することになる。
Benz(a)anthracene は、既知の弱結合物質の追加であり、Atrazine は、ER を介さないエストロ
ゲン活性化合物の追加となる。このことは、ER 結合がエストロゲン活性化合物の作用モードを区別するためのメカニズムツールとして使用できることを示すことになる。結果として、タスク III の化学物質は、標準の 3 物質に加えて、強結合物質（Zearalenone, Tamoxifen）、
弱 結 合 物 質 (5a-Dihydrotestosterone, Bisphenol A, 4-n-Heptylphenol, Kepone (Chlordecone),Benz(a)anthracene, Enterolactone)、非結合（陰性）物質(Progesterone,

Octyltriethoxysilane,

Atrazine)の 11 物質を試験物質として承認した。
CAB は、Equol（Apin 社より提供）がラセミ混合物であることを認め、承認した。天然のEquol は S(-)エナンチオマーであるが、両方のエナンチオマーは hrER に対して類似の結合親和性を有しており、そのため CAB はこれが被験物質選択上の障害になるとは考えていない。

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OECD GUIDELINE FOR THE TESTING OF CHEMICALS
Performance-Based Test Guideline for Human Recombinant Estrogen Receptor(hrER) In Vitro Assays to Detect Chemicals with ER Binding Affinity

GENERAL INTRODUCTION
Performance-Based Test Guideline
1.
This Performance-Based Test Guideline (PBTG) describes the methodology for humanrecombinant in vitro assays to detect substances with estrogen receptor binding affinity (hrER binding
assays). It comprises two mechanistically and functionally similar test methods for the identification of
estrogen receptor (i.e. ERα) binders and should facilitate the development 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 thisPBTG are:• The Freyberger-Wilson (FW) In Vitro Estrogen Receptor (ER) Binding Assay Using a Full
Length Human Recombinant ERα (2), and• The Chemical Evaluation and Research Institute (CERI) In Vitro Estrogen Receptor Binding
Assay Using a Human Recombinant Ligand Binding Domain Protein (2).
Performance standards (PS) (3) are available to facilitate the development and validation of similar test
methods for the same hazard endpoint and allow for timely amendment of this PBTG so that new 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 this Test
Guideline can be used indiscriminately to address countries’ requirements for test results on estrogen
receptor binding while benefiting from the Mutual Acceptance of Data.

1© OECD, (2015)
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.

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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 (4). The CF comprises
five levels, each level corresponding to a different level of biological complexity. The ER binding 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 receptor binding test methods designed to
identify ligands for the human estrogen receptor alpha (ER).
3.
The relevance of the in vitro ER binding assay to biological functions has been clearly
demonstrated. ER binding assays are designed to identify chemicals that have the potential to disrupt the
estrogen hormone pathway, and have been used extensively during the past two decades to characterise
ER tissue distribution as well as to identify ER agonists/antagonists. These assays reflect the ligandreceptor interaction which is the initial step of the estrogen signaling pathway and essential for
reproduction function in all vertebrates.
4.
The interaction of estrogens with ERs can affect transcription of estrogen-controlled genes andinduce non-genomic effects, which can lead to the induction or inhibition of cellular processes, including
those necessary for cell proliferation, normal fetal development, and reproductive function (5) (6) (7).
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.
Inappropriate ER signaling can lead to effects such as increased risk of hormone dependent cancer,
impaired fertility, and alterations in fetal growth and development (8).
5.
In vitro binding assays are based on a direct interaction of a substance with a specific receptor
ligand binding site that regulates the gene transcription. The key component of the human recombinantestrogen receptor alpha (hrERα) binding assay measures the ability of a radiolabeled ligand ([3H]17βestradiol) to bind with the ER in the presence of increasing concentrations of a test chemical (i.e.
competitor). Test chemicals that possess a high affinity for the ER compete with the radiolabeled ligand at
a lower concentration as compared with those chemicals with lower affinity for the receptor. This assay
consists of two major components: a saturation binding experiment to characterise receptor-ligand
interaction parameters and document ER specificity, followed by a competitive binding experiment thatcharacterises the competition between a test chemical and a radiolabeled ligand for binding to the ER.
6.
Validation studies of the CERI and the FW binding assays have demonstrated their relevance andreliability for their intended purpose (2).
7.

Definitions and abbreviations used in this Test Guideline are described in Annex 1.

Scope and limitations related to the receptor binding assays
8.
These test methods are being proposed for screening and prioritisation purposes, but can also
provide information for a molecular initiation event (MIE) that can be used in a weight of evidence
approach. They address chemical binding to the ERα ligand binding domain in an in vitro system. Thus,
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results should not be directly extrapolated to the complex signaling and regulation of the intact endocrine
system in vivo.
9.
Binding of the natural ligand, 17β-estradiol, is the initial step of a series of molecular events that
activates the transcription of target genes and ultimately, culminates with a physiological change (9). Thusbinding to the ERα ligand binding domain is considered one of the key mechanisms of ER mediatedendocrine disruption (ED), although there are other mechanisms through which ED can occur, including(i) interactions with sites of ER other than the ligand binding pocket, (ii) interactions with otherreceptors relevant for estrogen signaling, ER and G-protein coupled estrogen receptor, other receptors
and enzymatic systems within the endocrine system, (iii) hormone synthesis, (iv) metabolic activation
and/or inactivation of hormones, (v) distribution of hormones to target tissues, and (vi) clearance ofhormones from the body. None of the test methods under this PBTG address these modes of action.
10.
This PBTG addresses the ability of substances to bind to human ERα and does not distinguishbetween ERα agonists or antagonists. This assay does not address either further downstream events suchas gene transcription or physiological changes. Considering that only single substances were used during
the validation, the applicability to test mixtures has not been addressed. The test method is neverthelesstheoretically applicable to the testing of multi-constituent substances and 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 notneeded, when there is a regulatory requirement for testing of the mixture.
11.
The cell free receptor systems have no intrinsic metabolic capability and they were not validated
in combination with metabolic enzyme systems. However, it might be possible to incorporate metabolic
activity in a study design but this would require further validation efforts.
12.
Chemicals that may denature the protein (i.e. receptor protein), such as surfactant or chemicals
that can change the pH of the assay buffer, may not be tested or may only be tested at concentrations
devoid of such interactions. Otherwise, the concentration range that can be tested in the assays for a test
chemical is limited by its solubility in the assay buffer.
13.
For informational purposes, Table 1 provides the test results for the 24 substances that were
tested in both of the fully validated test methods described in this PBTG. Of these substances, 17 areclassified as ER binders and 6 as non-binders based upon published reports, including in vitro assays for
ER transcriptional activation and/or the uterotrophic assay (9) (10) (11) (12) (13) (14) (15). In reference tothe data summarised in Table 1, there was almost 100% agreement between the two test methods on theclassifications of all the substances up to 10-4M, and each substance was correctly classified as an ER
binder or non-binder. Supplementary information on this group of substances as well as additional
substances tested in the ER binding test methods during the validation studies is provided in the
Performance Standards for the hrER binding assay (3), Annex 2 (Tables 1, 2 and 3).

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Table 1: Classification of substances as ER binders or non-binders when tested in the
FW and CERI hrER Binding Assays with comparison with expected response

1Classification

Product Class

1x10-11 – 1x10-6

BinderSteroid

Pharmaceutical,
Veterinary
Agent

Binder

3x10-9 – 30x10-4

BinderSteroid

Pharmaceutical,
VeterinaryAgent3x10-9 – 30x10-4

Binder

3x10-9 – 30x10-4

BinderSteroid

Pharmaceutical,
Veterinary
Agent

Nonbinder*

1x10-10 – 1x10-4

Non*†
Binder

1x10-10 – 1x10-4

Non*†
BinderHydrocarbon(cyclic), Ester

Plasticizer,
ChemicalIntermediate56-531

Binder

1x10-10 – 1x10-3

Binder

1x10-10 – 1x10-3

BinderHydrocarbon(Cyclic),
PhenolPharmaceutical,
VeterinaryAgent17α-ethynylestradiol

57-636

Binder

1x10-10 – 1x10-3

Binder

1x10-10 – 1x10-3

BinderSteroid

Pharmaceutical,
Veterinary
Agent

Meso-Hexestrol

84-162Binder

1x10-10 – 1x10-3

Binder

1x10-10 – 1x10-3

BinderHydrocarbon(Cyclic),
PhenolPharmaceutical,
Veterinary
Agent

Genistein

44672-0

Binder

1x10-10 – 1x10-3

Binder

1x10-10 – 1x10-3

BinderEquol

Binder

1x10-10 – 1x10-3

Binder

1x10-10 – 1x10-3

BinderButyl paraben (nbutyl-4hydroxybenzoate)

53195-3
94-268Binder

1x10-10 – 1x10-3

Binder

1x10-10 – 1x10-3

BinderParabenPreservative

Nonylphenol(mixture)

8485215-3

Binder

1x10-10 – 1x10-3

Binder

1x10-10 – 1x10-3

BinderAlkylphenolIntermediate
Compoundo,p’-DDT

78902-6

Binder

1x10-10 – 1x10-3

Binder

1x10-10 – 1x10-3

BinderOrganochlorine

Insecticide

Corticosterone

50-226

Nonbinder*

1x10-10 – 1x10-4

Non-binder

1x10-10 – 1x10-4

Non-Binder

Steroid

Natural Product

Zearalenone

1792492-4

Binder

1x10-10 – 1x10-3

Binder

1x10-10 – 1x10-3

BinderHydrocarbon(heterocyclic),
LactoneNatural ProductTamoxifen

1054029-1

Binder

1x10-10 – 1x10-3

Binder

1x10-10 – 1x10-3

BinderHydrocarbon,(Cyclic)

Pharmaceutical,
VeterinaryAgent5αdihydrotestosterone

52118-6

Binder

1x10-10 – 1x10-3

Binder

1x10-10 – 1x10-3

BinderSteroid,
Nonphenolic

Natural ProductBisphenol A

80-057

Binder

1x10-10 – 1x10-3

Binder

1x10-10 – 1x10-3

BinderPhenolChemicalIntermediate

4-n-heptylphenol

198750-4

Binder

1x10-10 – 1x10-3

Equivocal a

1x10-10 – 1x10-3

BinderAlkylphenolIntermediate

17β-Estradiol

50-282

Norethynodrel

Classification

Binder

1x10-11 – 1x10-6

Binder68-235Binder

3x10-9 – 30x10-4

Norethindrone

68-224

BinderDi-n-butyl phthalate

84-742

DES

5

6

7

9
10
11
12
13
14
15

16
17
18

© OECD, (2015)

42

CERI Assay
Concentration
Range (M)3

8

FW AssayConcentration
Range (M)2

4

Expected
Response

MESH
ChemicalClass

Substance NameCAS
RN

Hydrocarbon(heterocyclic),
Flavonoid
Phytoestrogen
Metabolite

Natural Product
Natural ProductOECD/OCDE
19

Kepone(Chlordecone)

14350-0

Binder

1x10-10 – 1x10-3

20

Benz(a)anthracene

56-553

NonBinder

21Enterolactone

7847371-9

Progesterone

22

23 Octyltriethoxysilane

24

Atrazine493

1x10-10 – 1x10-3

BinderHydrocarbon,(Halogenated)

Pesticide

1x10-10 – 1x10-3 Non-Binder b

1x10-10 – 1x10-3

Non-Binder b

Aromatic
Hydrocarbon

Intermediate

Binder

1x10-10 – 1x10-3

Binder

1x10-10 – 1x10-3

BinderPhytoestrogen

Natural Product57-830Nonbinder*

1x10-10 – 1x10-4

Non-Binder

1x10-10 – 1x10-4

Non-Binder

Steroid

Natural Product294375-1

Nonbinder

1x10-10 – 1x10-3

Non-Binder

1x10-10 – 1x10-3

Non-Binder

SilaneSurface
Modifier

191224-9

Nonbinder*

1x10-10 – 1x10-4

Non-Binder

1x10-10 – 1x10-4

Non-Binder

Heterocyclic
compoundHerbicide

Binder*Limit of solubility < 1x 10-4M.
*
The use and classification of di-n-butyl phthalate (DBP) as a non-binder was based on testing up to 10-4 M because the substance had
been observed to be insoluble at 10-3M (e.g. turbidity) in some laboratories during the pre-validation studies.
†
During the validation study, di-n-butyl phthalate (DBP) was tested as a coded test substance at concentrations up to 10-3M. Under
these conditions, some laboratories observed either a decrease in radioligand binding at the highest concentration (10-3M) and/or an
ambiguous curve fit. For these runs, DBP was classified as ‘equivocal’ or ‘binder’ in 3/5 laboratories using the CERI assay and 5/6
laboratories using the FW assay (see Reference (2), Sections IV.B.3a,b and VI.A).
a
Classification was not consistent with expected classification. Classification of 4-n-heptylphenol as ‘equivocal’ or ‘non-binder’ by
3/5 labs resulted in an average classification of equivocal. Closer inspection revealed that this was due to chemical solubility
limitations that prevented the production of a full binding curve.
b
During the validation study, benz(a)anthracene was reclassified as a non-binder (i.e. negative) based on published literaturedemonstrating that the in vitro estrogenic activity reported for this substance (16) is primarily dependent upon its metabolic
activation (17) (18). Enzymatic metabolic activation of the substance would not be anticipated in the cell free hrER binding assays
as used in this inter-validation study. Thus, the correct classification for this substance is a ‘non-binder’ when used under theexperimental conditions for the FW and CERI assays.

hrER BINDING TEST METHOD COMPONENTS
Essential Test Method Components
14.
This PBTG applies to methods using an ER receptor and a suitably strong ligand to the receptor thatcan be used as a marker/tracer for the assay and can be displaced with increasing concentrations of a test
chemical. Binding assays contain the following two major components: 1) saturation binding and 2)
competitive binding. The saturation binding assay is used to confirm the specificity and activity of thereceptor preparations, while the competitive binding experiment is used to evaluate the ability of a test
chemical to bind to hrER.
Control substances
15.
The basis for the proposed concurrent reference estrogen and controls should be described.
Concurrent controls (solvent (vehicle), positive (ER binder; strong and weak affinity), negative (nonbinder)), as appropriate, serve as an indication that the test method is operative under the test conditions
and provide a basis for experiment-to-experiment comparisons; they are usually part of the acceptability
criteria for a given experiment (1). Full concentration curves for the reference estrogen and controls (i.e.
weak binder and non-binder) should be used in one plate during each run. All other plates should contain: 1)
a high- (approximately full displacement of radiolabeled ligand) and medium- (approximately the IC50)
concentration each of E2 and weak binder in triplicate; 2) solvent control and non-specific binding, each intriplicate.
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Standard Quality Control Procedures

16.
Standard quality control procedures should be performed as described for each assay to ensure
active receptors, the correct chemical concentrations, tolerance bounds remain stable through multiple
replications, and retain the ability to provide the expected ER-binding responses over time.
Demonstration of Laboratory Proficiency
17.
Prior to testing unknown chemicals with any of the test methods under this PBTG, eachlaboratory should demonstrate proficiency in using the test method by performing saturation assays toconfirm specificity and activity of the ER preparation, and competitive binding assays with the reference
estrogen and controls (weak binder and non-binder). A historical database with results for the reference
estrogen and controls generated from 3-5 independent experiments conducted on different days should beestablished by the laboratory. These experiments will be the foundation for the reference estrogen andhistorical controls for the laboratory and will be used as a partial assessment of assay acceptability for
future runs.
18.
The responsiveness of the test system will also be confirmed by testing the proficiency substances
listed in Table 2. The list of proficiency substances is a subset of the reference substances provided in the
Performance Standards for the ER binding assays (3). These substances are commercially available, represent
the classes of chemicals commonly associated with ER binding activity, exhibit a suitable range of potency
expected for ER binding (i.e. strong to weak) and non-binders (i.e. negatives). For each proficiency
substance, concentrations tested should cover the range provided in Table 2. At least three experiments
should be performed for each substance and results should be in concordance with expected chemical activity.
Each experiment should be conducted independently (i.e. with fresh dilutions of receptor, chemicals, andreagent), with three replicates for each concentration. Proficiency is demonstrated by correct classification(positive/negative) of each proficiency substance. Proficiency testing should be performed by each technician
when learning the test methods.

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Tab le 2: List of controls and proficiency substances for the hrER competitive binding assays.1
No.

Substance Name

CAS RN2

Test
concentration
range (M)Expected
Response3,4

MeSH
chemical class5

Product class6

Controls (Reference estrogen, weak binder, non-binder)
50-28-2

Binder

1x10-11 – 1x10-6

Steroid

Pharmaceutical,
Veterinary agent

Norethynodrel (or)Norethindrone

68-23-5 (or)68-22-4

Binder

3x10-9 – 30x10-6

Steroid

Pharmaceutical,
Veterinary agent

Octyltriethoxysilane

2943-75-1

Non-binder

1x10-10 – 1x10-3

SilaneSurface modifier

Hydrocarbon(cyclic), Phenol

Pharmaceutical,
Veterinary agent

Steroid

Pharmaceutical,
Veterinary agent

Hydrocarbon(cyclic), Phenol

Pharmaceutical,
Veterinary agent

117-estradiol

2
3Proficiency substances645Diethylstilbestrol

56-53-1

Binder

1x10-11 – 1x10-6

1x10-11 – 1x10-6

Binder17α-ethynylestradiol

57-63-6

6

meso-Hexestrol

84-16-2

Binder7

Tamoxifen

10540-29-1

Binder

1x10-11 – 1x10-6

Hydrocarbon(cyclic)

Pharmaceutical,
Veterinary agent

8

Genistein446-72-0

Binder

1x10-10 – 1x10-3

Heterocyclic
compound,Flavonoid,

Natural product9Bisphenol A

80-05-7

Binder

1x10-10 – 1x10-3

Phenol17924-92-4

Binder10

Zearalonone

1x10-11 – 1x10-6

1x10-11 – 1x10-3

Heterocyclic
compound, Lactone

Chemicalintermediate
Natural Product11

Butyl paraben

94-26-8

Binder

1x10-11 – 1x10-3

Carboxylic acid,
Phenol12

Atrazine

1912-24-9

Non-binder

1x10-11 – 1x10-6

Heterocyclic
compoundHerbicide

13

Di-n-butylphthalate(DBP)784-74-2

Non-binder8

1x10-10 – 1x10-4

Hydrocarbon (cyclic),
Ester

Plasticizer,
Chemical intermediate

14

Corticosterone

50-22-6

Non-binder

Steroid

Natural product1x10-11 – 1x10-4

Preservative1
If a proficiency substance is no longer commercially available, a substance with the same ER binding classification, comparable
potency, and chemical class can be used.
2
Abbreviations: CAS RN = Chemical Abstracts Service Registry Number.

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3

Classification as an ERBinder or Non-binder during the validation study for the CERI and FW hrER Binding Assays (2).
ER binding activity was based upon the ICCVAM Background Review Documents (BRD) for ER Binding and TA test methods(9) as well as empirical data and other information obtained from referenced studies published and reviewed (10) (11) (12) (13)(14) (15).
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 recognized standardized 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/cgi-bin/sis/htmlgen?HSDB)
7
DPB can be used as an alternate control non-binder tested with maximum concentration of 10-4 M.
8Limit of solubility for this substance is 10-4 M. The use and classification of di-n-butyl phthalate (DBP) as a non-binder has been
based on testing up to 10-4 M because the substance had been observed to be insoluble at 10-3M (e.g. turbidity) in some laboratories
during the pre-validation studies.
4

Solubility Testing and Concentration Range Finding for Test Chemicals
19.
A preliminary test should be conducted to determine the limit of solubility for each test chemical
and to identify the appropriate concentration range to use when conducting the test. The limit of solubility ofeach test chemical is to be initially determined in the solvent and further confirmed under assay conditions.
The final concentration tested in the assay should not exceed 1 mM. Range finder testing consists of asolvent control along with eight, log serial dilutions, starting at the maximum acceptable concentration (e.g.
1 mM or lower, based upon the limit of solubility), and the presence of cloudiness or precipitate noted.
Concentrations in the second and third experiments should be adjusted as appropriate to better characterise
the concentration-response curve.
Test Run Acceptability Criteria
20.
Acceptance or rejection of a test run is based on the evaluation of results obtained for thereference estrogen and control used for each experiment. First, for plate 1, the full concentration curves forthe reference controls from each experiment should meet the measures of performance with curve-fit
parameters (e.g. IC50 and Hillslope) based upon the results reported for the respective protocols for the
CERI and FW assays (Annex 2 and 3), and the historical control data from the laboratory conducting the test.
All controls (reference estrogen, weak binder, and non-binder) should be correctly classified for eachexperiment. Secondly, the controls on all subsequent plates need to be assessed for consistency with plate 1.
A sufficient range of concentrations of the test chemical should be used to clearly define the top of thecompetitive binding curve. Variability among replicates at each concentration of the test chemical as well as
among the three independent runs should be reasonable and scientifically defensible. The ability toconsistently conduct the test method should be demonstrated by the development and maintenance of ahistorical database for the reference estrogen and controls. Standard deviations (SD) or coefficients ofvariation (CV) for the means of reference estrogen and control weak binder curves fitting parameters frommultiple experiments may be used as a measure of within-laboratory reproducibility. Professional judgment
should be applied when reviewing the plate control results from each run as well as for each test chemical.
In addition, the following principles regarding acceptability criteria should be met:

Data should be sufficient for a quantitative assessment of ER bindingThe concentrations tested should remain within the solubility range of the test chemical.
Analysis of data

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the key principles for characterising receptor-ligand interactions. Typically, saturation binding data are
analyzed using a non-linear regression model that accounts for total and non-specific binding. A correction
for ligand depletion (e.g. Swillens, 1995 (19)) may be needed when determining Bmax and Kd. Data fromcompetitive binding assays are typically transformed (e.g. percent specific binding and concentration of test
chemical (log M)). Estimates of log (IC50) for each test chemical should be determined using an appropriate
nonlinear curve fitting software to fit a four parameter Hill equation. Following an initial analysis, the curve
fit parameters and a visual review of how well the binding data fit the generated competitive binding curveshould be conducted. In some cases, additional analysis may be needed to obtain the best curve fit (e.g.
constraining top and/or bottom of curve, use of 10% rule, see Annex 4 and Reference 2 (Section III.A.2).
22.
Meeting the acceptability criteria (paragraph 20) indicates the assay system is operating
properly, but it does not ensure that any particular test will produce accurate data. Replicating the correct
results of the first test is the best indication that accurate data were produced.
General Data Interpretation Criteria
23.
There is currently no universally agreed method for interpreting ER binding data. However, both
qualitative (e.g. binder/non-binder) and/or quantitative (e.g. log IC50, Relative Binding Affinity (RBA), etc.)
assessments of hrER-mediated activity should be based on empirical data and sound scientific judgment.
Test Report
24.
The test report
information:
Test method:− test method used;shouldincludethefollowing

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;© OECD, (2015)

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Receptors:- source of receptors (supplier, catalog No., lot, species of receptor, active receptor concentration
provided from supplier, certification from supplier)− characterization of receptors (including saturation binding results): Kd, Bmax,− storage of receptors− radiolabeled ligand:− supplier, catalog No., lot, specific activity
Test conditions:− solubility limitations under assay conditions;− composition of binding buffer;− concentration of receptor;− concentration of tracer (i.e. radiolabeled ligand);− concentrations of test chemical;− percent vehicle in final assay;− incubation temperature and time;− method of bound/free separation;− positive and negative controls/reference substances;− criteria for considering tests as positive, negative or equivocal;
Acceptability check:− actual IC50 and Hillslope values for concurrent positive controls/reference substances;
Results:− raw and bound/free data;− denaturing confirmation check, if appropriate;− if it exists, the lowest effective concentration (LEC);− RBA and/or IC50 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:− application of 10% rule
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LITERATURE

1) OECD (2005), Guidance Document on the Validation and International Acceptance of New or
Updated Test Methods for Hazard Assessment. Environmental, Health and Safety Publications, Series on
Testing and Assessment (No. 34), Organisation for Economic Cooperation and Development, Paris.
2) OECD (2015), Integrated Summary Report: Validation of Two Binding Assays Using Human RecombinantEstrogen Receptor Alpha (hrERα), Health and Safety Publications, Series on Testing and Assessment (No.
226), Organisation for Economic Cooperation and Development, Paris.
3) OECD (2015), Performance Standards for Binding Assays Using Human Recombinant Estrogen Receptor
Alpha (hrERα), Health and Safety Publications, Series on Testing and Assessment (No. 222), Organisation
for Economic Cooperation and Development, Paris.
4) OECD (2012), Guidance Document on Standardized Test Guidelines for Evaluating Chemicals for
Endocrine Disruption. Environmental, Health and Safety Publications, Series on Testing and Assessment(No. 150), Oganisation for Economic Coopeation and Development, Paris.
5) Cavailles V. (2002), Estrogens and Receptors: an Evolving Concept, Climacteric, 5 Suppl 2: p.20-6.
6) 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): p. 1073-89.
7) Younes M. and Honma N. (2011), Estrogen Receptor Beta, Arch. Pathol. Lab. Med., 135(1): p. 63-6.
8) Diamanti-Kandarakis et al. (2009), Endocrine-Disrupting Chemicals: an Endocrine Society Sci. Statement,
Endo Rev 30(4):293-342.
9) ICCVAM (2002), Background Review Document. Current Status of Test Methods for Detecting Endocrine
Disruptors: In Vitro Estrogen Receptor Binding Assays. (NIH Publication No. 03-4504). National Instituteof Environmental Health Sciences, Research Triangle Park, NC.
10) ICCVAM (2003), ICCVAM Evaluation of In Vitro Test Methods for Detecting Potential Endocrine
Disruptors: Estrogen Receptor and Androgen Receptor Binding and Transcriptional Activation Assays.
11) ICCVAM (2006), ICCVAM Evaluation of In Vitro Test Methods for Detecting Potential Endocrine
Disruptors: Estrogen Receptor and Androgen Receptor Binding and Transcriptional Activation Assays.
12) Akahori Y. et al. (2008), Relationship Between the Results of In Vitro Receptor Binding Assay to Human
Estrogen Receptor Alpha and In Vivo Uterotrophic Assay: Comparative Study with 65 Selected Chemicals,
Toxicol. In Vitro, 22(1): 225-231.
13) OECD. (2007). Additional Data Supporting the Test Guideline on the Uterotrophic Bioassay in Rodents,
Environment, Health and Safety Publications, Series on Testing and Assessment (No. 67.), Organisation
for Economic Coopeation and Development, Paris.

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14) 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. p. 1-188.
15) Yamasaki, K; Noda, S; Imatanaka, N; Yakabe, Y. (2004), Comparative Study of the Uterotrophic Potencyof 14 Chemicals in a Uterotrophic Assay and their Receptor-Binding Affinity, Toxicol. Letters, 146: 111120.
16) Kummer V; Maskova, J; Zraly, Z; Neca, J; Simeckova, P; Vondracek, J; Machala, M. (2008), Estrogenic
Activity of Environmental Polycyclic Aromatic Hydrocarbons in Uterus of Immature Wistar Rats. Toxicol.
Letters, 180: 213-221.
17) Gozgit, JM; Nestor, KM; Fasco, MJ; Pentecost, BT; Arcaro, KF. (2004), Differential Action of Polycyclic
Aromatic Hydrocarbons on Endogenous Estrogen-Responsive Genes and on a Transfected EstrogenResponsive Reporter in MCF-7 Cells. Toxicol. and Applied Pharmacol., 196: 58-67.
18) Santodonato, J. (1997), Review of the Estrogenic and Antiestrogenic Activity of Polycyclic Aromatic
Hydrocarbons: Relationship to Carcinogenicity. Chemosphere, 34: 835-848.
19) Swillens S (1995), Interpretation of Binding Curves Obtained with High Receptor Concentrations:
Practical Aid for Computer Analysis, Mol Pharmacol 47(6):1197-1203.

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ANNEX 1
Definitions and Abbreviations
10% Rule: Option to exclude from the analyses data points where the mean of the replicates for the percent[3H]17β-estradiol specific bound is 10% or more above that observed for the mean value at a lower
concentration (see annex 4).
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).
CF: The OECD Conceptual Framework for the Testing and Evaluation of Endocrine Disrupters.
CV: Coefficient of variation
E2: 17β-estradiol
ED: Endocrine disruption
hERα: Human estrogen receptor alpha
ER: Estrogen receptor
Estrogenic activity: The capability of a chemical to mimic 17β-estradiol in its ability to bind
estrogen receptors. Binding to the hERα can be detected with this PBTG.
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).
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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
PBTG: Performance-Based Test Guideline
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
that can be used by laboratories to demonstrate technical competence with a standardized 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: 17ß-estradiol (E2, CAS 50-28-2).
Reference test methods: The test methods upon which this PBTG is based.
RBA: Relative Binding Affinity. The RBA of a substance is calculated as a percent of the log (IC50) for thesubstance relative to the log (IC50) for 17β-estradiol
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
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.
SD: Standard deviation.
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 found acceptable
for the proposed purpose (1).
Validation: The process by which the reliability and relevance of a particular approach, method, process orassessment is established for a defined purpose (1).

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ANNEX 2The Freyberger-Wilson In Vitro Estrogen Receptor (ER) Saturation andCompetitive Binding Assays Using Full Length Recombinant ER
INITIAL CONSIDERATIONS AND LIMITATIONS (See also GENERAL INTRODUCTION, page1)
1.
This in vitro Estrogen Receptor (ER) saturation and competitive binding test method uses fulllength human receptor ER(hrER) that is produced in and isolated from baculovirus-infected insect cells.
The protocol, developed by Freyberger and Wilson, underwent an international multi-laboratory validation
study (2) which has demonstrated its relevance and reliability for the intended purpose of the test method.
2.
This test method is a screening procedure for identifying substances that can bind to the full length
hrERα. It is used to determine the ability of a test chemical to compete with 17β-estradiol for binding to
hrERα. Quantitative assay results may include the IC50 (a measure of the concentration of test chemical
needed to displace half of the [3H]-17β-estradiol from the hrERα) and the relative binding affinities of test
chemicals for the hrERα compared to 17β-estradiol. For chemical screening purposes, acceptable qualitative
assay results may include classifications of test chemicals as either hrERα binders, non-binders, or
equivocal based upon criteria described for the binding curves.
3.
The test method uses a radioactive ligand that requires a radioactive materials license for thelaboratory. All procedures with radioisotopes and hazardous chemicals should follow the regulations andprocedures as described by national legislation.
4.
The “GENERAL INTRODUCTION” and “hrER BINDING TEST METHODCOMPONENTS” (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.

PRINCIPLES OF THE TEST METHOD (See also GENERAL INTRODUCTION, page 1)
5. The hrERα binding assay measures the ability of a radiolabeled ligand ([ 3H]17β-estradiol) to bind with
the ER in the presence of increasing concentrations of a test chemical (i.e. competitor). Test chemicals thatpossess a high affinity for the ER compete with the radiolabeled ligand at a lower concentration ascompared with those chemicals with lower affinity for the receptor.
6. This test method consists of two major components: a saturation binding experiment to characterise
receptor-ligand interaction parameters, followed by a competitive binding experiment that characterises thecompetition between a test chemical and a radiolabeled ligand for binding to the ER.
7. The purpose of the saturation binding experiment is to characterise a particular batch of receptors forbinding affinity and number in preparation for the competitive binding experiment. The saturation binding
experiment measures, under equilibrium conditions, the affinity of a fixed concentration of the estrogen
receptor for its natural ligand (represented by the dissociation constant, Kd), and the concentration of active
receptor sites (Bmax).

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8. The competitive binding experiment measures the affinity of a substance to compete with [3H]17βestradiol for binding to the ER. The affinity is quantified by the concentration of test chemical that, atequilibrium, inhibits 50% of the specific binding of the [ 3H]17β-estradiol (termed the “inhibitory
concentration 50%” or IC50). This can also be evaluated using the relative binding affinity (RBA, relative to
the IC50 of estradiol measured separately in the same run). The competitive binding experiment measures
the binding of [3H]17β-estradiol at a fixed concentration in the presence of a wide range (eight orders ofmagnitude) of test chemical concentrations. The data are then fit, where possible, to a form of the Hillequation (Hill, 1910) that describes the displacement of the radioligand by a one-site competitive binder.
The extent of displacement of the radiolabeled estradiol at equilibrium is used to characterise the test
chemical as a binder, non-binder, or generating an equivocal response.
PROCEDURE
Demonstration of Acceptable hrERα Protein Performance9.
Prior to routinely conducting the saturation and competitive binding assays, each new batch of
hrERα should be shown to be performing correctly in the laboratory in which it will be used. A two-stepprocess should be used to demonstrate performance. These steps are the following:Conduct a saturation [3H]-17β-estradiol binding assay to demonstrate hrERα specificity andsaturation. Nonlinear regression analysis of these data (e.g. BioSoft; McPherson, 1985; Motulsky,
1995) and the subsequent Scatchard plot should document hrERα binding affinity of the [3H]-17βestradiol (Kd) and the number of receptors (Bmax) for each batch of hrERα.



Conduct a competitive binding assay using the control substances (reference estrogen (17βestradiol), a weak binder (e.g. norethynodrel or norethindrone), and a non-binder(octyltriethoxysilane, OTES). Each laboratory should establish an historical database to document
the consistency of IC50 and other relevant values for the reference estrogen and weak binder amongexperiments and different batches of hrERα. The parameters of the competitive binding curves forthe control substances should be within the limits of the 95%confidence interval (see Table 1) thatwere developed using data from laboratories that participated in the validation study for this test
method (2).

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Table 1. Performance criteria developed for the reference estrogen and weak binder, FW hrER
Binding Assay.
Substance

Parameter

17β-estradiol

NorethynodrelcNorethindrone

a

Mean

95% Confidence Intervalsb
Standard
Deviation (n)Lower Limit

Upper Limit

Top (%)

100.4410.84 (67)

97.8

103.1

Bottom (%)

0.29

1.25 (67)

-0.01

0.60

Hill Slope-1.06

0.20 (67)

-1.11

-1.02

LogIC50 (M)-8.92c0.18 (67)

-8.97

-8.88

Top (%)

99.42

8.90 (68)97.27

101.60Bottom (%)

2.02

3.42 (68)

1.19

2.84

Hill Slope-1.01

0.38 (68)

-1.10

-0.92

LogIC50 (M)-6.39

0.27 (68)

-6.46

-6.33

Top (%)

96.14

8.44 (27)

92.80

99.48

Bottom (%)

2.38

5.02 (27)

0.40

4.37

Hill Slope-1.41

0.32 (27)

-1.53

-1.28

LogIC50(M)-5.73

0.27 (27)

-5.84

-5.62

a

Mean (n) ± Standard Deviation (SD) were calculated using curve fit parameter estimates (4-parameter Hill
Equation) for control runs conducted in four laboratories during the validation study (see Annex N of Reference 2).
b
The 95% confidence intervals are provided as a guide for acceptability criteria.
cTesting of norethindrone was optional for Subtask 4 during validation study (see Reference 2, see Subtask 4). Thus,
the mean ± SD (n) were calculated using curve fit estimates (4-parameter Hill equation) for control runs conducted in
two laboratories.
The range for the IC50 will be dependent upon the Kd of the receptor preparation and concentration of radiolabeled
ligand used within each laboratory. Appropriate adjustment for the range of the IC50 based upon the conditions used
to conduct the test method will be acceptable.

Demonstration of laboratory proficiency
10.
See paragraphs 17 and 18 and Table 2 in “ hrER BINDING TEST METHODCOMPONENTS” of this Test Guideline. Each assay (saturation and competitive binding) should consist of
three independent runs (i.e. with fresh dilutions of receptor, chemicals, and reagents) on different days, andeach run should contain three replicates.
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Determination of Receptor (hrERα) Concentration
11.
The concentration of active receptor varies slightly by batch and storage conditions. For thisreason, the concentration of active receptor as received from the supplier should be determined. This will
yield the appropriate concentration of active receptor at the time of the run.
12.
Under conditions corresponding to competitive binding (i.e. 1 nM [3H]-estradiol), nominal
concentrations of 0.25, 0.5, 0.75, and 1 nM receptor should be incubated in the absence (total binding) andpresence (non-specific binding) of 1 µM unlabeled estradiol. Specific binding, calculated as the differenceof total and non-specific binding, is plotted against the nominal receptor concentration. The concentrationof receptor that gives specific binding values corresponding to 20% of added radiolabel is related to thecorresponding nominal receptor concentration, and this receptor concentration should be used for saturationand competitive binding experiments. Frequently, a final hrER concentration of 0.5 nM will comply withthis condition.
13.
If the 20% criterion repeatedly cannot be met, the experimental set up should be checked forpotential errors. Failure to achieve the 20% criterion may indicate that there is very little active receptor in
the recombinant batch, and the use of another receptor batch should then be considered.
Saturation assay
14. Eight increasing concentrations of [3H]17β-estradiol should be evaluated in triplicate, under thefollowing three conditions (see Table 2):
a. In the absence of unlabelled 17β-estradiol and presence of ER. This is the determinationof total binding by measure of the radioactivity in the wells that have only [3H]17βestradiol.
b. In the presence of a 1000- fold excess concentration of unlabelled 17β-estradiol overlabelled 17β-estradiol and presence of ER. The intent of this condition is to saturate theactive binding sites with unlabelled 17β-estradiol, and by measuring the radioactivity inthe wells, determine the non-specific binding. Any remaining hot estradiol that can bind to
the receptor is considered to be binding at a non-specific site as the cold estradiol should beat such a high concentration that it is bound to all of the available specific sites on thereceptor.
c. In the absence of unlabelled 17β-estradiol and absence of ER (determination of total
radioactivity)
Preparation of [3H]-17β-estradiol and unlabelled 17β-estradiol solutions
15.
Dilutions of [3H]-17β-estradiol should be prepared by adding assay buffer to a 12 nM stock
solution of [3H]-17β-estradiol to obtain concentrations initially ranging from 0.12nM to 12 nM. By adding
40 µL of these solutions to the respective assay wells of a 96-well microtiter plate (in a final volume of 160μL), the final assay concentrations, ranging from 0.03 to 3.0 nM, will be obtained. Preparation of assay
buffer, [3H]-17β-estradiol stock solution and dilutions and determination of the concentrations aredescribed in depth in the FW protocol (2).
16.

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achieve eight increasing concentrations initially ranging from 0.06 µM to 6 µM. By adding 80 μL of thesesolutions to the respective assay wells of a 96-well microtiter plate (in a final volume of 160 μL), the final
assay concentrations, ranging from 0.03µM to 3µM, will be obtained. The final concentration of unlabeled
17β-estradiol in the individual non-specific binding assay wells should be 1000-fold of the labeled [3H]17β- estradiol concentration. Preparation of unlabelled 17β-estradiol dilutions is described in depth in the
FW protocol (2).
17.
The nominal concentration of receptor that gives specific binding of 20±5% should be used (seeparagraphs 12-13). The hrERα solution should be prepared immediately prior to use.
18.
The 96-well microtiter plates are prepared as illustrated in Table 2, with 3 replicates perconcentration. Example of plate concentration and volume assignment of [3H]-17β-estradiol, unlabeled
17β-estradiol, buffer and receptor are provided in Appendix 2.
Table 2: Saturation Binding Assay Microtiter Plate Layout

1
A

2

3

4

5

6

3

3

0.03 nM [ H] E2 + ER

0.06 nM [ H] E2 + ER

3

3

7

8

9
3

10

11

12

0.08 nM [ H] E2 +ER

0.10 nM [3H] E2 + ER

0.30 nM [ H] E2 + ER

0.60 nM [ H] E2 + ER

1.0 nM [ 3H] E2 + ER

3.0 nM [3H] E2 + ER

0.03 nM [3H] E2 + ER
+ 0.03 µM E2
0.30 nM [3H] E2 + ERE
+ 0.30 µM E2

0.06 nM [3H] E2 +ER + 0.06 µM E2
0.60 nM [3H] E2 +ER + 0.60 µM E2

0.08 nM [3H] E2 +ER + 0.08 µM E231.0 nM [ H] E2 + ER
+ 1.0 µM E2

0.10 nM [3H] E2 + ER
+ 0.10 µM E2
3.0 nM [3H] E2 + ER +3.0 µM E2

B

Total
Binding(Solvent)

C
D

NonSpecificBindingF
G
H[3H] E2: [3H]-17β-estradiol
ER: estrogen receptor
E2: unlabelled 17β-estradiol

19.
Assay microtiter plates should be incubated at 2° to 8°C for 16 to 20 hours and placed on a rotator
during the incubation period.
Measurement of [3H]-17β-Estradiol bound to hrERα20.
[3H]-17β-Estradiol bound to hrERα should be separated from free [3H]-17β-Estradiol by adding
80 μL of cold DCC suspension to each well, shaking the microtiter plates for 10 minutes and centrifugatingfor 10 minutes at about 2500 RPM. To minimize dissociation of bound [ 3H]-17β-estradiol from the hrERαduring this process, it is extremely important that the buffers and assay wells be kept between 2 and 8°C
and that each step be conducted quickly. A shaker for microtiter plates is necessary to process plates
efficiently and quickly.
21.
50 µL of supernatant containing the hrERα-bound [3H]-17β-estradiol should then be taken withextreme care, to avoid any contamination of the wells by touching DCC, and should be placed on a second
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microtiter plate.
22.
200 μL of scintillation fluid, capable of converting the kinetic energy of nuclear emissions into
light energy, should then be added to each well (A1-B12 and D1 to E12). Wells G1-H12 (identified as totaldpms) represent serial dilutions of the [3H]-17β-estradiol (40 μL) that should be delivered directly into thescintillation fluid in the wells of the measurement plate as indicated in Table 3, i.e. these wells contain only
200 μL of scintillation fluid and the appropriate dilution of [3H]-17β-estradiol. These measures demonstrate
how much [3H]-17β-estradiol in dpms was added to each set of wells for the total binding and non-specificbinding.
Table 3: Saturation Binding Assay Microtiter Plate Layout, Radioactivity Measurement

1

2

3

4

5

6

0.03 nM [3H] E2 + ER

0.06 nM [3H] E2 + ER

0.30 nM [3H] E2 + ER

0.60 nM [3H] E2 + ER

0.03 nM [3H] E2 + ER
+ 0.03 µM E2
0.30 nM [3H] E2 + ERE
+ 0.30 µM E2

A
B

7

8

9

10

11

12

0.08 nM [3H] E2 +ER

0.10 nM [3 H] E2 + ER

1.0 nM [ 3H] E2 + ER

3.0 nM [3H] E2 + ER

0.06 nM [3H] E2 +ER + 0.06 µM E2
0.60 nM [3H] E2 +ER + 0.60 µM E2

0.08 nM [3H] E2 +ER + 0.08 µM E231.0 nM [ H] E2 + ER
+ 1.0 µM E2

0.10 nM [3H] E2 + ER
+ 0.10 µM E2
3.0 nM [3H] E2 + ER +3.0 µM E2

Total
Binding(Solvent)

C
D

NonSpecificBindingF
G

0.03 nM [3H] E2(total dpms)

0.06 nM [3H] E2

0.08 nM [3H] E2

0.10 nM [3H] E2

H0.30 nM [3H] E2

0.60 nM [3H] E2

1.0 nM [3H] E2

3.0 nM [3H] E2

Total
dpms*

[3H] E2: [3H]-17β-estradiol
ER: estrogen receptor
E2: unlabelled 17β-estradiol
dpms: disintegrations per minute*The hot serial dilutions of [3H]-labeled estradiol here should be directly added into 200 μL of scintillation fluid in wells
G1 – H12.

23.
Measurement should start with a delay of at least 2 hours and counting time should be 40 minutes
per well. A microtiter plate scintillation counter should be used for determination of dpm/well with quench
correction. Alternatively, if a scintillation counter for a microtiter plate is not available, samples may bemeasured in a conventional counter. Under these conditions, a reduction of counting time may beconsidered.

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Competitive binding assay
24.
The competitive binding assay measures the binding of a single concentration of [3H]-17βestradiol in the presence of increasing concentrations of a test chemical. Three concurrent replicates should
be used at each concentration within one run. In addition, three non-concurrent runs should be performed
for each chemical tested. The assay should be set up in one or more 96-well microtiter plates
Controls
25.
When performing the assay, concurrent solvent and controls (i.e. reference estrogen, weakbinder, and non-binder) should be included in each experiment. Full concentration curves for thereference estrogen and controls (i.e. weak binder and non-binder) should be used in one plate during eachrun. All other plates should contain (i) a high- (maximum displacement) and medium- (approximately
the IC50) concentration each of E2 and weak binder in triplicate; (ii) solvent control and non-specificbinding, each at least in triplicate. Procedures for the preparation of assay buffer, controls, [3H]-17βestradiol, hrERα and test chemical solutions are described in Reference 2 (Annex K, see FW Assay
Protocol).
- Solvent control:
26.
The solvent control indicates that the solvent does not interact with the test system and alsomeasures total binding (TB). Ethanol is the preferred solvent. Alternatively, if the highest concentration of
the test chemical is not soluble in ethanol, DMSO may be used. The concentration of ethanol or DMSO, ifused, in the final assay wells is 1.5% and may not exceed 2%.
- Buffer control:
27.
The buffer control (BC) should contain neither solvent nor test chemical, but all of the othercomponents of the assay. The results of the buffer control are compared to the solvent control to verify thatthe solvent used does not affect the assay system.
- Strong binder (reference estrogen)28.
17β-estradiol (CAS 50-28-2) is the endogenous ligand and binds with high affinity to the ER,
alpha subtype. A standard curve using unlabeled 17β-estradiol should be prepared for each hrERαcompetitive binding assay, to allow for an assessment of variability when conducting the assay over timewithin the same laboratory. Eight solutions of unlabeled 17β-estradiol should be prepared in ethanol, withconcentrations in the assay wells ranging from 100 nM – 10 pM (-7[logM] to -11[logM]), spaced asfollows: (-7[logM], -8[logM], -8.5[logM], -9[logM], - 9.5[logM], -10[logM], -11[logM]). The highest
concentration of unlabeled 17β-estradiol (1 µM) also serves as the non-specific binding indicator. This
concentration is distinguished by the label “NSB” in Table 4 even though it is also part of the standard
curve.
- Weak binder
29.
A weak binder (norethynodrel (CAS68-23-5) or norethindrone (CAS 68-22-4)) should be included
to demonstrate the sensitivity of each experiment and to allow an assessment of variability when conducting
the assay over time. Eight solutions of the weak binder should be prepared in ethanol, with concentrations in
the assay wells ranging from 3 nM to 30 μM (-8.5[logM] to -4.5[logM]), spaced as follows: -4.5[logM], 5[logM], -5.5[logM], -6[logM], -6.5[logM], -7[logM],-7.5[logM], -8.5[logM].
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OECD/OCDE- Non binder30.
Octyltriethoxysilane (OTES, CAS 2943-75-1) should be used as the negative control (nonbinder). It provides assurance that the assay as run, will detect when test chemicals do not bind to thehrERα. Eight solutions of the non-binder should be prepared in ethanol, with concentrations in the assay
wells ranging from 0.1nM to 1000 μM (-10[logM] to -3[logM]), in log increments. Di-n-butyl phtalate(DBP) can be used as an alternate control non-binder. Its maximum solubility has been shown to be 4[logM].
hrERα concentration
31.
The amount of receptor that gives specific binding of 20±5% of 1 nM radioligand should be used(see paragraphs 12-13 of Annex 2). The hrERα solution should be prepared immediately prior to use.
[3H]-17β-estradiol
32.

The concentration of [3H]-17β-estradiol in the assay wells should be of 1.0 nM.

Test Chemicals
33.
In the first instance, it is necessary to conduct a solubility test to determine the limit of solubility
for each test chemical and to identify the appropriate concentration range to use when conducting the test
protocol. The limit of solubility of each test chemical is to be initially determined in the solvent and further
confirmed under assay conditions. The final concentration tested in the assay should not exceed 1 mM.
Range finder testing consists of a solvent control along with 8 log serial dilutions, starting at the maximum
acceptable concentration (e.g. 1 mM or lower, based upon the limit of solubility), and the presence ofcloudiness or precipitate noted (see also paragraph 35). The test chemical should be tested using 8 log
concentration spaced curves as defined by the preceding range finding test. Concentrations in the second
and third experiments should be adjusted as appropriate to better characterise the concentration-response
curve.
34.
Dilutions of the test chemical should be prepared in the appropriate solvent (see paragraph 26 ofAnnex 2). If the highest concentration of the test chemical is not soluble in either ethanol or DMSO, andadding more solvent would cause the solvent concentration in the final tube to be greater than theacceptable limit, the highest concentration may be reduced to the next lower concentration. In this case, anadditional concentration may be added at the low end of the concentration series. Other concentrations inthe series should remain unchanged.
35.
The test chemical solutions should be closely monitored when added to the assay well, as thetest chemical may precipitate upon addition to the assay well. The data for all wells that containprecipitate should be excluded from curve-fitting, and the reason for exclusion of the data noted.
36.
If there is prior existing information from other sources that provide a log(IC 50) of a test chemical,
it may be appropriate to geometrically space the dilutions (i.e. 0.5 log units around the expected log(IC50).
The final result should reflect sufficient spread of concentrations on either side of the log(IC50), including
the “top” and “bottom”, such that the binding curve can be adequately characterised.
Assay plate organisation
37.
Labeled microtiter plates should be prepared considering sextuple incubations with codes for thesolvent control, the highest concentration of the reference estrogen which also serves as the non-specificbinding (NSB) indicator, and the buffer control and considering triplicate incubations with codes for each of© OECD, (2015)

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the eight concentrations of the non-binding control (octyltriethoxysilane), the 7 lower concentrations for thereference estrogen, the eight concentrations dose levels of the weak binder, and the 8 concentrations of eachtest chemical (TC). An example layout of the plate diagram for the full concentration curves for thereference estrogen and control is given below in Table 4. Additional microtiter plates are used for the test
chemicals and should include plate controls (i.e. 1) a high- (maximum displacement) and medium(approximately the IC50) concentration each of E2 and weak binder in triplicate; 2) solvent control andnon-specific binding, each in sextuple (Table 5). An example of a competitive assay microtiter plate layout
worksheet using three unknown test chemicals is provided in Appendix 3 of Annex 2. The concentrations
indicated in Tables 4 and 5 are the final concentrations of the assay. The maximum concentration for E2
should be 1×10-7 M and for the weak binder, the highest concentration used for the weak binder on plate 1should be used. The IC50 concentration has to be determined by the laboratory based on their historicalcontrol database. It is expected that this value would be similar to that observed in the validation studies(see Table 1).
Table 4: Competitive Binding Assay Microtiter Plate Layout, Full Concentration Curves for
Reference Estrogen and Controls (Plate 1).
1

2

3

4

5

6

7

8

9

10

11

12

A

TB (Solvent only)

TB (Solvent only)

NSB

NSB

B

-7
E2 (1×10 )-8E2 (1×10 )E2 (1×10CE2 (1×10-9.5)

E2 (1×10-10)E2 (1×10-11)

Blank*

D

NE (1×10-4.5)

NE (1×10-5)

NE (1×10-5.5)

NE (1×10-6)ENE (1×10-6.5)

NE (1×10-7)

NE (1×10-7.5)

NE (1×10-8.5)

F

OTES (1×10-3)

OTES (1×10-4)

OTES (1×10-5)

OTES (1×10-6)

G

OTES (1×10-7)

OTES (1×10-8)

OTES (1×10-9)

OTES (1×10-10)

H

Blank (for hot)**

Blank (for hot) **Buffer control

Buffer control

-8.5

)

-9
E2 (1×10 )In this example, the weak binder is norethinodrel (NE)*real blank, well not used**blank not used during the incubation, but used to confirm the total radioactivity added.

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Table 5: Competitive Binding Assay Microtiter Plate Layout, Full Concentration Curves for Test
Chemicals and Plate Controls.
1

2

3

4

5

6

7

8

9

10

11

12

A

TB (Solvent only)

TB (Solvent only)

NSB

NSB

B

-3
TC1 (1×10 )-4
TC1 (1×10 )-5
TC1 (1×10 )-6
TC1 (1×10 )C

TC1 (1×10-7)

TC1 (1×10-8)

TC1 (1×10-9)

TC1 (1×10-10)

D

TC2 (1×10-3)

TC2 (1×10-4)

TC2 (1×10-5)

TC2 (1×10-6)E-7
TC2 (1×10 )-8
TC2 (1×10 )-9TC2 (1×10 )TC2 (1×10F-3TC3 (1×10 )-4
TC3 (1×10 )-5
TC3 (1×10 )-6
TC3 (1×10 )G

TC3 (1×10-7)

TC3 (1×10-8)

TC3 (1×10-9)

TC3 (1×10-10)

H

NE (IC50)

NE (1×10-4.5)

E2 (IC50)

E2 (1×10-7)-10)

In this example, the weak binder is norethinodrel (NE)

Completion of competitive binding assay

38.
As shown in Table 6, 80 μL of the solvent control, buffer control, reference estrogen, weakbinder, non-binder, and test chemicals prepared in assay buffer should be added to the wells. Then, 40 µl of
a 4 nM [3H]-17β-estradiol solution should be added to each well. After gentle rotation for 10 to 15 minutes
between 2° to 8°C, 40 µl of hrERα solution should be added. Assay microtiter plates should be incubated at
2° to 8°C for 16 to 20 hours, and placed on a rotator during the incubation period.
Table 6: Volume of Assay Components for hrER Competitive Binding Assay, Microtiter Plates
Volume (μL)

Constituent

80

Unlabeled 17β-estradiol, norethynodrel, OTES, test chemicals,solvent or buffer

40

4 nM [3H]-17β-estradiol solution

40

hrERα solution, concentration as determined160

Total volume in each assay well

39.
The quantification of [3H]-17β-Estradiol bound to hrERα, following separation of [3H]-17βEstradiol bound to hrERα from free [3H]-17β-Estradiol by adding 80 μL of cold DCC suspension to eachwell, should then be performed as described in paragraphs 20-23 for the saturation binding assay.
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40.
Wells H1-6 (identified as blank (for hot) in table 4) represent the dpms of the [3H]-labeledestradiol in 40 μL. The 40 μL aliquot should be delivered directly into the scintillation fluid in wells H1 –H6.
Acceptability criteria
Saturation binding assay
41.
The specific binding curve should reach a plateau as increasing concentrations of [ 3H]-17βestradiol were used, indicating saturation of hrERα with ligand.
42.
The specific binding at 1 nM of [3H]-17β-estradiol should be inside the acceptable range 15% to
25% of the average measured total radioactivity added across runs. Occasional slight excursions outside ofthis range are acceptable, but if runs are consistently outside this range or a particular run is significantly
outside this range, the protein concentration should be adjusted and the saturation assay repeated.
43.

The data should produce a linear Scatchard plot.

44.
The non-specific binding should not be excessive. The value for non-specific binding should
typically be <35% of the total binding. However, the ratio might occasionally exceed this limit whenmeasuring very low dpm for the lowest concentration of radiolabeled 17β-Estradiol tested.
Competitive binding assay
45.
Increasing concentrations of unlabeled 17β-estradiol should displace [3H]-17β- estradiol from thereceptor in a manner consistent with a one-site competitive binding.
46.
The IC50 value for the reference estrogen (i.e. 17β-estradiol) should be approximately equal tothe molar concentration of [3H]-17β-estradiol plus the Kd determined from the saturation binding assay.
47.
The total specific binding should be consistently within the acceptable range of 20 ± 5 % when
the average measured concentration of total radioactivity added to each well was 1 nM across runs.
Occasional slight excursions outside of this range are acceptable, but if runs are consistently outside thisrange or a particular run is significantly outside this range, the protein concentration should be adjusted.
48.
The solvent should not alter the sensitivity or reproducibility of the assay. The results of thesolvent control (TB wells) are compared to the buffer control to verify that the solvent used does notaffect the assay system. The results of the TB and Buffer control should be comparable if there is noeffect of the solvent on the assay.
49.
The non-binder should not displace more than 25% of the [3H]-17β-estradiol from the hrERα
when tested up to10-3 M (OTES) or 10-4 M (DBP).
50.
Performance criteria were developed for the reference estrogen and two weak binders (e.g.
norethynodrel, norethindrone) using data from the validation study of the FW hrER Binding Assay (Annex
N of Reference 2). 95% confidence intervals are provided for the mean (n) +/- SD for all control runs
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and for the log10RBA of the weak binders relative to the reference estrogen and are provided as
performance criteria for the positive controls. Table 1 provides expected ranges for the curve fit parametersthat can be used as performance criteria. In practice, the range of the IC50 may vary slightly based upon the
Kd of receptor preparation and ligand concentration.
51.
No performance criteria was developed for curve fit parameters for the test chemicals because of
the wide array of existing potential test chemicals and variation in potential affinities and outcomes (e.g.
Full curve, partial curve, no curve fit). However, professional judgment should be applied when reviewing
results from each run for a test chemical. A sufficient range of concentrations of the test chemical should be
used to clearly define the top (e.g. 90 - 100% of binding) of the competitive curve. Variability among
replicates at each concentration of test chemical as well as among the 3 non-concurrent runs should bereasonable and scientifically defensible. Controls from each run for a test chemical should approach themeasures of performance reported for this FW assay and be consistent historical control data from each
respective laboratory.
ANALYSIS OF DATA
Saturation binding assay
52.
Both total and non-specific binding are measured. From these values, specific binding ofincreasing concentrations of [3H]-17β-estradiol under equilibrium conditions is calculated by subtracting
non-specific from total. A graph of specific binding versus [3H]-17β-estradiol concentration should reach aplateau for maximum specific binding indicative of saturation of the hrERα with the [3H]-17β-estradiol. In
addition, analysis of the data should document the binding of the [3H]-17β- estradiol to a single, high-affinity
binding site. Non-specific, total, and specific binding should be displayed on a saturation binding curve.
Further analysis of these data should use a non-linear regression analysis (e.g. BioSoft; McPherson, 1985;
Motulsky, 1995) with a final display of the data as a Scatchard plot.
53.
The data analysis should determine Bmax and Kd from the total binding data alone, using theassumption that non- specific binding is linear, unless justification is given for using a different method. In
addition, robust regression should be used when determining the best fit unless justification is given. The
method chosen for robust regression should be stated. Correction for ligand depletion (e.g. using the methodof Swillens 1995) should always be used when determining Bmax and Kd from saturation binding data.
Competitive binding assay
54.
The competitive binding curve is plotted as specific [3H]-17β-estradiol binding versus theconcentration (log10 units) of the competitor. The concentration of the test chemical that inhibits 50% of
the maximum specific [3H]-17β-estradiol binding is the IC50 value.
55.
Estimates of log(IC50) values for the positive controls (e.g. reference estrogen and weak binder)
should be determined using an appropriate nonlinear curve fitting software to fit a four parameter Hillequation (e.g. BioSoft; McPherson, 1985; Motulsky, 1995). The top, bottom, slope, and log(IC50) should
generally be left unconstrained when fitting these curves. Robust regression should be used when
determining the best fit unless justification is given. Correction for ligand depletion should not be used.
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Following the initial analysis, each binding curve should be reviewed to ensure appropriate fit to the model.
The relative binding affinity (RBA) for the weak binder should be calculated as a percent of the log (IC 50)
for the weak binder relative to the log (IC50) for 17β-estradiol. Results from the positive controls and thenon-binder control should be evaluated using the measures of the test method performance in paragraphs
45-50 in this Annex 2.
56.
Data for all test chemicals should be analysed using a step-wise approach to ensure that data areappropriately analysed and that each competitive binding curve is properly classified. It is recommended
that each run for a test chemical initially undergo a standardised data analysis that is identical to that used
for the reference estrogen and weak binder controls (see paragraph 55 above). Once completed, a technicalreview of the curve fit parameters as well as a visual review of how well the data fit the generated
competitive binding curve for each run should be conducted. During this technical review, theobservations of a concentration dependent decrease in the percent [3H]-17β-estradiol specifically bound,low variability among the technical replicates at each chemical concentration, and consistency in fit
parameters among the three runs are a good indication that the assay and data analyses were conducted
appropriately.
Data interpretation
57.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be a binder forthe hrERα if a binding curve can be fit and the lowest point on the response curve within the range of thedata is less than 50% (Figure 1).
58.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be a nonbinder for the hrERα if: A binding curve can be fit and the lowest point on the fitted response curve within the range of thedata is above 75%, or A binding curve cannot be fit and the lowest unsmoothed average percent binding among theconcentration groups in the data is above 75%.
59. Test chemicals are considered equivocal if none of the above conditions are met (e.g. the lowest pointon the fitted response curve is between 76 – 51%).

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Table7. Criteria for assigning classification based upon competitive binding curve for a test
chemical.
Classification

Criteria

BinderaA binding curve can be fit.

Non-binderb

The lowest point on the response curve within the range of thedata is less than 50%.
If a binding curve can be fit,the lowest point on the fitted response curve within the range ofthe data is above 75%.
If a binding curve cannot be fit,the lowest unsmoothed average percent binding among theconcentration groups in the data is above 75%.
Any testable run that is neither a binder nor a non-binder

Equivocalc(e.g., The lowest point on the fitted response curve is between 76 – 51%).

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Figure 1. Examples of test chemical classification using competitive binding curve.

% Radioligand Bound

125
Non-Binder

100

75

Equivocal

50
Binder25

Binder

0
-12

-11

-10

-9

-8

-7

-6

-5

Concentration (log Molar)

-4

-3

60.
Multiple runs conducted within a laboratory for a test chemical are combined by assigning
numeric values to each run and averaging across the runs as shown in Table 8. Results for the combined
runs within each laboratory are compared with the expected classification for each test chemical.

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Table 8. Method for classification of test chemical using multiple runs within a laboratory
To assign value to each run:Classification

Numeric Value

Binder2

Equivocal

1

Non-binder0

To classify average of numeric value across runs:
Classification

Numeric Value

BinderAverage ≥ 1.5

Equivocal

0.5 ≤ Average < 1.5

Non-binder

Average < 0.5

TEST REPORT
61. See paragraph 24 of “hrER BINDING TEST METHOD COMPONENTS” of this Test Guideline.

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Appendix 1: List of Terms[3H]E2: 17β-Estradiol radiolabeled with tritium
DCC: Dextran-coated charcoal
E2: Unlabeled 17β-estradiol (inert)
Assay buffer: 10 mM Tris, 10 mg Bovine Serum Albumin /mL, 2 mM DTT, 10% glycerol, 0.2 mM
leupeptin, pH 7.5
hrERα: Human recombinant estrogen receptor alpha (ligand binding domain)
Replicate: One of multiple wells that contain the same contents at the same concentrations and are assayedconcurrently within a single run. In this protocol, each concentration of test chemical is tested in triplicate;that is, there are three replicates that are assayed simultaneously at each concentration of test chemical.
Run: A complete set of concurrently-run microtiter plate assay wells that provides all the informationnecessary to characterize binding of a test chemical to the hrERα (viz., total [3H]-17β-estradiol added to theassay well, maximum binding of [3H]-17β-estradiol to the hrERα, nonspecific binding, and total binding at
various concentrations of test chemical). A run could consist of as few as one assay well (i.e. replicate) per
concentration, but since this protocol requires assaying in triplicate, one run consists of three assay wells perconcentration. In addition, this protocol requires three independent (i.e. non-concurrent) runs per chemical.

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OECD/OCDEAppendix 2:
Typical [3H]-17β-Estradiol Saturation Assay with Three Replicate Wells

Position

Replicate

Well Type CodeHot E2 Initial
Concentration(nM)

Hot E2 Volume(uL)

Hot E2 Final
Concentration(nM)

Cold E2 Initial
Concentration(uM)

Cold E2 Volume(uL)

Cold E2 Final
Concentration(uM)

Buffer Volume(uL)

Receptor
Volume (uL)

Total volume inwells

Typical [3H]-17β-Estradiol Saturation Assay with Three Replicate Wells

A1
A2
A3
A4

1
2
3
1

H
H
H
H

0.12
0.12
0.12
0.24

40
40
40
40

0.03
0.03
0.03
0.06

―
―
―
―

―
―
―
―

―
―
―
―

80
80
80
80

40
40
40
40

160
160
160
160

A5
A6
A7
A8

2
3
1
2

H
H
H
H

0.24
0.24
0.32
0.32

40
40
40
40

0.06
0.06
0.08
0.08

―
―
―
―

―
―
―
―

―
―
―
―

80
80
80
80

40
40
40
40

160
160
160
160

A9
A10
A11
A12
B1
B2
B3
B4
B5
B6
B7
B8
B9
B10
B11
B12
D1
D2

3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2

H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
HC
HC

0.32
0.40
0.40
0.40
1.20
1.20
1.20
2.40
2.40
2.40
4.00
4.00
4.00
12.00
12.00
12.00
0.12
0.12

40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40

0.08
0.10
0.10
0.10
0.30
0.30
0.30
0.60
0.60
0.60
1.00
1.00
1.00
3.00
3.00
3.00
0.03
0.03

―
―
―
―
―
―
―
―
―
―
―
―
―
―
―
―

―
―
―
―
―
―
―
―
―
―
―
―
―
―
―
―
80
80

―
―
―
―
―
―
―
―
―
―
―
―
―
―
―
―
0.03
0.03

80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
―
―

40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40

160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160

D3
D4
D5
D6
D7
D8
D9
D10
D11
D12

3
1
2
3
1
2
3
1
2
3

HC
HC
HC
HC
HC
HC
HC
HC
HC
HC

0.12
0.24
0.24
0.24
0.32
0.32
0.32
0.40
0.40
0.40

40
40
40
40
40
40
40
40
40
40

0.03
0.06
0.06
0.06
0.08
0.08
0.08
0.10
0.10
0.10

80
80
80
80
80
80
80
80
80
80

0.03
0.06
0.06
0.06
0.08
0.08
0.08
0.1
0.1
0.1

―
―
―
―
―
―
―
―
―
―

40
40
40
40
40
40
40
40
40
40

160
160
160
160
160
160
160
160
160
160

© OECD, (2015)

70

0.06
0.06
0.06
0.12
0.12
0.12
0.16
0.16
0.16
0.2
0.2
0.2

32

OECD/OCDE

493

Cold E2 Volume(uL)

Cold E2 Final
Concentration(uM)

Buffer Volume(uL)

Receptor
Volume (uL)

Total volume in
wells

160
160
160
160
160
160
160
160
160
160
160
160

0.03
0.03
0.03
0.06

―
―
―
―

―
―
―
―

―
―
―
―

―
―
―
―

―
―
―
―

40
40
40
40

40
40

0.06
0.06

―
―

―
―

―
―

―
―

―
―

40
40

0.32
0.32
0.32
0.40

40
40
40
40

0.08
0.08
0.08
0.10

―
―
―
―

―
―
―
―

―
―
―
―

―
―
―
―

―
―
―
―

40
40
40
40

Hot
Hot
Hot
Hot
Hot
Hot
Hot
Hot
Hot
Hot

0.40
0.40
1.20
1.20
1.20
2.40
2.40
2.40
4.00
4.00

40
40
40
40
40
40
40
40
40
40

0.10
0.10
0.30
0.30
0.30
0.60
0.60
0.60
1.00
1.00

―
―
―
―
―
―
―
―
―
―

―
―
―
―
―
―
―
―
―
―

―
―
―
―
―
―
―
―
―
―

―
―
―
―
―
―
―
―
―
―

―
―
―
―
―
―
―
―
―
―

40
40
40
40
40
40
40
40
40
40

Hot
Hot
Hot
Hot

4.00
12.00
12.00
12.00

40
40
40
40

1.00
3.00
3.00
3.00

―
―
―
―

―
―
―
―

―
―
―
―

―
―
―
―

―
―
―
―

40
40
40
40

Hot E2 Final
Concentration
(nM)

40
40
40
40
40
40
40
40
40
40
40
40

Hot E2 Volume
(uL)

―
―
―
―
―
―
―
―
―
―
―
―

Hot E2 Initial
Concentration
(nM)

0.3
0.3
0.3
0.6
0.6
0.6
1
1
1
3
3
3

Well Type Code

6

80
80
80
80
80
80
80
80
80
80
80
80

Replicate

0.6

Position

Cold E2 Initial
Concentration(uM)

Typical [3H]-17β-Estradiol Saturation Assay with Three Replicate Wells

E1
E2
E3
E4
E5
E6
E7
E8
E9
E10
E11
E12

1
2
3
1
2
3
1
2
3
1
2
3

HC
HC
HC
HC
HC
HC
HC
HC
HC
HC
HC
HC

1.20
1.20
1.20
2.40
2.40
2.40
4.00
4.00
4.00
12.00
12.00
12.00

40
40
40
40
40
40
40
40
40
40
40
40

0.30
0.30
0.30
0.60
0.60
0.60
1.00
1.00
1.00
3.00
3.00
3.00

G1
G2
G3
G4

1
2
3
1

Hot
Hot
Hot
Hot

0.12
0.12
0.12
0.24

40
40
40
40

G5
G6

2
3

Hot
Hot

0.24
0.24

G7
G8
G9
G10

1
2
3
1

Hot
Hot
Hot
Hot

G11
G12
H1
H2
H3
H4
H5
H6
H7
H8

2
3
1
2
3
1
2
3
1
2

H9
H10
H11
H12

3
1
2
3

0.6
0.6
1.2
1.2
1.2
2
2
2
6
6

Note that the "hot" wells are empty during incubation. The 40 µl are added only for scintillation counting.

© OECD, (2015)

33

71

493

OECD/OCDE

Plate

Position

Replicate

Well type

Well Code

Concentration CodeCompetitor Initial
Concentration (M)hrER stock (uL)

Buffer Volume (uL)

Tracer (Hot E2) Volume(uL)

Volume from dilution
plate( uL)

Final Volume (ul)Competitor Final
Concentration (M)Appendix 3 :Competitive Binding Assay Well Layout

S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S

A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
B1
B2
B3
B4
B5
B6
B7
B8
B9
B10
B11
B12
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
D1
D2
D3
D4
D5
D6
D7
D8
D9
D10
D11
D12

1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
1
1
1
1
1
1
1
1
1
1
1

total binding
total binding
total binding
total binding
total binding
total binding
cold E2 (high)cold E2 (high)cold E2 (high)cold E2 (high)cold E2 (high)cold E2 (high)cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
blank
blank
blank
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel

TB
TB
TB
TB
TB
TB
NSB
NSB
NSB
NSB
NSB
NSB
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
blank
blank
blank
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE

TB1
TB2
TB3
TB4
TB5
TB6
S0
S0
S0
S0
S0
S0
S1
S1
S1
S2
S2
S2
S3
S3
S3
S4
S4
S4
S5
S5
S5
S6
S6
S6
S7
S7
S7
B1
B2
B3
WP1
WP1
WP1
WP2
WP2
WP2
WP3
WP3
WP3
WP4
WP4
WP4

2.00E-06
2.00E-06
2.00E-06
2.00E-06
2.00E-06
2.00E-06
2.00E-07
2.00E-07
2.00E-07
2.00E-08
2.00E-08
2.00E-08
6.00E-09
6.00E-09
6.00E-09
2.00E-09
2.00E-09
2.00E-09
6.00E-10
6.00E-10
6.00E-10
2.00E-10
2.00E-10
2.00E-10
2.00E-11
2.00E-11
2.00E-11
6.00E-05
6.00E-05
6.00E-05
2.00E-05
2.00E-05
2.00E-05
6.00E-06
6.00E-06
6.00E-06
2.00E-06
2.00E-06
2.00E-06

40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40

160
160
160
-

40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40

80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80

160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160

1.0E-06
1.0E-06
1.0E-06
1.0E-06
1.0E-06
1.0E-06
1.0E-07
1.0E-07
1.0E-07
1.0E-08
1.0E-08
1.0E-083.0E-09
3.0E-09
3.0E-09
1.0E-09
1.0E-09
1.0E-09
3.0E-103.0E-103.0E-101.0E-101.0E-101.0E-101.0E-11
1.0E-11
1.0E-11
3.0E-05
3.0E-05
3.0E-05
1.0E-05
1.0E-05
1.0E-05
3.0E-06
3.0E-06
3.0E-06
1.0E-06
1.0E-06
1.0E-06© OECD, (2015)

72

34

OECD/OCDE

493

Concentration CodeCompetitor Initial
Concentration (M)hrER stock (uL)

NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
H
H
H
H
H
H
BC
BC
BC
BC
BC
..BC

WP5
WP5
WP5
WP6
WP6
WP6
WP7
WP7
WP7
WP8
WP8
WP8
OTES1
OTES1
OTES1
OTES2
OTES2
OTES2
OTES3
OTES3
OTES3
OTES4
OTES4
OTES4
OTES5
OTES5
OTES5
OTES6
OTES6
OTES6
OTES7
OTES7
OTES7
OTES8
OTES8
OTES8
H1
H2
H3
H4
H5
H6
BC1
BC2
BC3
BC4
BC5
BC6

6.00E-07
6.00E-07
6.00E-07
2.00E-07
2.00E-07
2.00E-07
6.00E-08
6.00E-08
6.00E-08
6.00E-09
6.00E-09
6.00E-09
2.00E-03
2.00E-03
2.00E-03
2.00E-04
2.00E-04
2.00E-04
2.00E-05
2.00E-05
2.00E-05
2.00E-06
2.00E-06
2.00E-06
2.00E-07
2.00E-07
2.00E-07
2.00E-08
2.00E-08
2.00E-08
2.00E-09
2.00E-09
2.00E-09
2.00E-10
2.00E-10
2.00E-10
-

40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40

80
80
80
80
80
80

Competitor Final
Concentration (M)Well Codenorethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
hot
hot
hot
hot
hot
hot
buffer control
buffer control
buffer control
buffer control
buffer control
buffer control

Final Volume (ul)Well type

1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3

Volume from dilution
plate( uL)

Replicate

E1
E2
E3
E4
E5
E6
D7
E8
E9
E10
E11
E12
F1
F2
F3
F4
F5
F6
F7
F8
F9
F10
F11
F12
G1
G2
G3
G4
G5
G6
G7
G8
G9
G10
G11
G12
H1
H2
H3
H4
H5
H6
H7
H8
H9
H10
H11
H12

Tracer (Hot E2) Volume(uL)

Position

S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S

Buffer Volume (uL)

PlateCompetitive Binding Assay Well Layout

40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40

80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
-

160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
40
40
40
40
40
40
160
160
160
160
160
160

3.0E-073.0E-073.0E-07
1.0E-07
1.0E-07
1.0E-073.0E-083.0E-083.0E-083.0E-09
3.0E-09
3.0E-09
1.0E-03
1.0E-03
1.0E-03
1.0E-04
1.0E-04
1.0E-04
1.0E-05
1.0E-05
1.0E-05
1.0E-06
1.0E-06
1.0E-06
1.0E-07
1.0E-07
1.0E-07
1.0E-08
1.0E-08
1.0E-08
1.0E-09
1.0E-09
1.0E-09
1.0E-101.0E-101.0E-10-Note that the "hot" wells are empty during incubation. The 40 µl are added only for scintillation counting.

© OECD, (2015)

35

73

493
493

OECD/OCDE
OECD/OCDE

74

VolumeVolumefrom
fromdilution
dilution
plate(
plate(uL)
uL)

Final
FinalVolume
Volume(ul)(ul)

Competitor
CompetitorFinal
Final
Concentration
Concentration(M)
(M)

TB
TB
TB
TB
TB
TB
TB
TB
TB
NSB
NSB
NSB
NSB
NSB
NSB
NSB
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC1
TC2
TC2
TC2
TC2
TC2
TC2
TC2
TC2
TC2
TC2
TC2
TC2
TC2
TC2
TC2
TC2
TC2
TC2
TC2
TC2
TC2
TC2
TC2
TC2
TC2
TC2

TracerTracer(Hot(HotE2)
E2)Volume
Volume
(uL)
(uL)

Well
WellCode
Code

total binding
binding
total
total binding
binding
total
total binding
binding
total
total binding
total binding
total binding
cold E2 (high)cold E2 (high)cold E2 (high)cold E2 (high)cold E2 (high)cold
cold E2
E2 (high)(high)
Test
Test Chemical
Chemical 1
1
Test Chemical
Chemical 1
1
Test
Test
Test Chemical
Chemical 1
1
Test
Test Chemical
Chemical 1
1
Test Chemical
Chemical 1
1
Test
Test
Test Chemical
Chemical 1
1
Test Chemical 1
Test Chemical 1
Test Chemical 1
Test Chemical 1
Test Chemical 1
Test Chemical 1
Test Chemical 1
Test Chemical 1
Test Chemical 1
Test Chemical 1
Test Chemical 1
Test Chemical 1
Test Chemical 1
Test Chemical
Chemical 1
1
Test
Test
Test Chemical
Chemical 1
1
Test
Test Chemical
Chemical 1
1
Test
Test Chemical
Chemical 1
1
Test
Test Chemical
Chemical 1
1
Test Chemical
Chemical 1
1
Test
Test Chemical
Chemical 1
1
Test
Test
Test Chemical
Chemical 1
1
Test Chemical 1
Test Chemical 1
Test Chemical 1
1
Test Chemical 2
Test Chemical 2
Test Chemical 2
Test Chemical 2
Test Chemical 2
Test
Test Chemical
Chemical 22
Test
Test Chemical
Chemical 22
Test
Test Chemical
Chemical 22
Test
Test Chemical
Chemical 22
Test
Test Chemical
Chemical 22
Test
Test Chemical
Chemical 22
Test
Test Chemical
Chemical 22
Test
Test Chemical
Chemical 2
2
Test Chemical 2
Test Chemical 2
Test Chemical 2
Test Chemical 2
Test Chemical 2

BufferBufferVolume
Volume(uL)
(uL)

Well
Welltype
type

11
22
33
1
2
3
1
2
3
1
2
33
11
22
33
11
22
33
1
1
2
2
3
3
1
1
2
2
3
3
1
21
23
31
12
23
31
12
23
31
21
32
31
21
32
13
21
32
13
21
32
13
21
32
13
2
1
3
2
3

hrER
hrERstockstock(uL)(uL)

Replicate
Replicate

A1
A1
A2
A2
A3
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
A12
B1
B1
B2
B2
B3
B3
B4
B4
B5
B5
B6
B6
B7
B7
B8
B8
B9
B9
B10
B10
B11
B11
B12
B12
C1
C1
C2
C2
C3
C3
C4
C4
C5
C5
C6
C6
C7
C7
C8
C8
C9
C9
C10
C10
C11
C11
C12
C12
D1
D2
D1
D3
D2
D4
D3
D5
D4
D6
D5
D7
D6
D8
D7
D9
D8
D10
D9
D11
D10
D12
D11
E1
D12
E2
E1
E3
E2
E3

Competitor
CompetitorInitial
InitialConcentration
Concentration(M)(M)

Position
Position

P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1

Concentration
ConcentrationCode
Code

Plate
Plate

Competitive
Competitive Binding
Binding Assay
Assay Well
Well Layout
Layout

T
T
T
B
T
B
B
B
T
T
2
1
B
T
B
3
B
T
1
4
B
T
5
B
S
6
0
S

---2.00E-06
2.00E-06
2.00E-06
2.00E-06
2.00E-06
2.00E-06
2.00E-06
2.00E-03
2.00E-03
2.00E-03
2.00E-03
2.00E-03
2.00E-03
2.00E-04
2.00E-04
2.00E-04
2.00E-04
2.00E-04
2.00E-04
2.00E-05
2.00E-05
2.00E-05
2.00E-05
2.00E-05
2.00E-05
2.00E-06
2.00E-06
2.00E-06
2.00E-06
2.00E-06
2.00E-06
2.00E-07
2.00E-07
2.00E-07
2.00E-07
2.00E-07
2.00E-07
2.00E-08
2.00E-08
2.00E-08
2.00E-08
2.00E-08
2.00E-08
2.00E-09
2.00E-09
2.00E-09
2.00E-09
2.00E-09
2.00E-09
2.00E-10
2.00E-10
2.00E-10
2.00E-10
2.00E-03
2.00E-03
2.00E-03
2.00E-04
2.00E-03
2.00E-04
2.00E-04
2.00E-04
2.00E-05
2.00E-04
2.00E-05
2.00E-05
2.00E-05
2.00E-05
2.00E-06
2.00E-05
2.00E-06
2.00E-06
2.00E-06
2.00E-06
2.00E-07
2.00E-06
2.00E-07
2.00E-07
2.00E-07
2.00E-07
2.00E-07

40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40

----00
00
00
00
00
00
0
0
0
0
0
0
0
0
0
0
0
0
0
00
00
00
00
00
00
00
00
0
0
0
0
0
0
0
0
00
00
00
00
00
00
00
00
0
0
0
0
0

40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40

80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80

160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160

---1.0E-06
1.0E-06
1.0E-06
1.0E-06
1.0E-06
1.0E-06
1.0E-06
1.0E-03
1.0E-03
1.0E-03
1.0E-03
1.0E-03
1.0E-03
1.0E-04
1.0E-04
1.0E-04
1.0E-04
1.0E-04
1.0E-04
1.0E-05
1.0E-05
1.0E-05
1.0E-05
1.0E-05
1.0E-05
1.0E-06
1.0E-06
1.0E-06
1.0E-06
1.0E-06
1.0E-06
1.0E-07
1.0E-07
1.0E-07
1.0E-07
1.0E-07
1.0E-07
1.0E-08
1.0E-08
1.0E-08
1.0E-08
1.0E-08
1.0E-08
1.0E-09
1.0E-09
1.0E-09
1.0E-09
1.0E-09
1.0E-09
1.0E-101.0E-101.0E-101.0E-101.0E-03
1.0E-03
1.0E-03
1.0E-04
1.0E-03
1.0E-04
1.0E-04
1.0E-04
1.0E-05
1.0E-04
1.0E-05
1.0E-05
1.0E-05
1.0E-05
1.0E-06
1.0E-05
1.0E-06
1.0E-06
1.0E-06
1.0E-06
1.0E-07
1.0E-06
1.0E-07
1.0E-07
1.0E-07
1.0E-07
1.0E-07

0
S
0
S
0
S
00
S
S
1001
11
11
22
22
22
3
3
3
3
3
3
4
4
4
4
4
4
5
55
55
56
66
66
67
77
77
78
8
8
81
1
1
21
2
22
32
33
33
43
44
44
54
5
5
5
5
5

© OECD, (2015)36© OECD, (2015)36

OECD/OCDE

493

Position

Replicate

Well Code

Concentration CodeCompetitor Initial
Concentration (M)hrER stock (uL)

Buffer Volume (uL)

Tracer (Hot E2) Volume(uL)

Volume from dilution
plate( uL)

Final Volume (ul)Competitor Final
Concentration (M)

P1
P1
P1
P1
P1
P1
P1
P1
P1

E4 1
E5 2
E6 3
E7 1
E8 2
E9 3
E10
E11
E12

Test Chemical 2
Test Chemical 2
Test Chemical 2
Test Chemical 2
Test Chemical 2
Test Chemical 2
1
Test Chemical 2
2
Test Chemical 2
3
Test Chemical 2

TC2
TC2
TC2
TC2
TC2
TC2
TC2
TC2
TC2

6
6
6
7
7
7
8
8
8

2.00E-08
2.00E-08
2.00E-08
2.00E-09
2.00E-09
2.00E-09
2.00E-10
2.00E-10
2.00E-10

40
40
40
40
40
40
40
40
40

0
0
0
0
0
0
0
0
0

40
40
40
40
40
40
40
40
40

80
80
80
80
80
80
80
80
80

160
160
160
160
160
160
160
160
160

1.0E-08
1.0E-08
1.0E-08
1.0E-09
1.0E-09
1.0E-09
1.0E-101.0E-101.0E-10

P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1

F1 1
F2 2
F3 3
F4 1
F5 2
F6 3
F7 1
F8 2
F9 3
F10
F11
F12
G1
G2
G3
G4
G5
G6
G7
G8
G9
G10
G11
G12
H1
H2
H3
H4
H5
H6
H7
H8
H9
H10
H11
H12

Test Chemical 3
Test Chemical 3
Test Chemical 3
Test Chemical 3
Test Chemical 3
Test Chemical 3
Test Chemical 3
Test Chemical 3
Test Chemical 3
1
Test Chemical 3
2
Test Chemical 3
3
Test Chemical 3
1
Test Chemical 3
2
Test Chemical 3
3
Test Chemical 3
1
Test Chemical 3
2
Test Chemical 3
3
Test Chemical 3
1
Test Chemical 3
2
Test Chemical 3
3
Test Chemical 3
1
Test Chemical 3
2
Test Chemical 3
3
Test Chemical 3
1norethynodrel
2
norethynodrel
3
norethynodrel
1
norethynodrel
2
norethynodrel
3
norethynodrel
1
cold E2 S
IC50
2cold E2 S
IC50
3cold E2 S
IC50
1cold E2 S
1.00E-7.
2
cold E2 S
1.00E-73
cold E2 S
1.00E-7

TC3
TC3
TC3
TC3
TC3
TC3
TC3
TC3
TC3
TC3
TC3
TC3
TC3
TC3
TC3
TC3
TC3
TC3
TC3
TC3
TC3
TC3
TC3
TC3
NE
NE
NE
NE
NE
NE

1
2.00E-03
1
2.00E-03
1
2.00E-03
2
2.00E-04
2
2.00E-04
2
2.00E-04
3
2.00E-05
3
2.00E-05
3
2.00E-05
4
2.00E-06
4
2.00E-06
4
2.00E-06
5
2.00E-07
5
2.00E-07
5
2.00E-07
6
2.00E-08
6
2.00E-08
6
2.00E-08
7
2.00E-09
7
2.00E-09
7
2.00E-09
8
2.00E-10
8
2.00E-10
8
2.00E-10
IC50
IC50
IC50
1.00E-4.5
1.00E-4.5
1.00E-4.5
40
0
40
0
40
0
40
0
40
0
40
0

40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
80
80
80
80
80
80

40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
160
160
160
160
160
160

80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80

160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160
160

1.0E-03
1.0E-03
1.0E-03
1.0E-04
1.0E-04
1.0E-04
1.0E-05
1.0E-05
1.0E-05
1.0E-06
1.0E-06
1.0E-06
1.0E-07
1.0E-07
1.0E-07
1.0E-08
1.0E-08
1.0E-08
1.0E-09
1.0E-09
1.0E-09
1.0E-101.0E-101.0E-10© OECD, (2015)

Well type

Plate

Competitive Binding Assay Well Layout

37

75

493

OECD/OCDEANNEX 3

The Chemical Evaluation and Research Institute (CERI) In Vitro Estrogen Receptor Binding
Assay Using a Human Recombinant ERα Ligand Binding Domain Protein
INITIAL CONSIDERATIONS AND LIMITATIONS (See also GENERAL INTRODUCTION, page1)
1.
This in vitro Estrogen Receptor (ERα) saturation and competitive binding test method uses aligand binding domain (LBD) of the human ERα (hrERα). This protein construct was produced by the
Chemicals Evaluation Research Institute (CERI), Japan, and exists as a glutathione-S-transferase (GST)
fusion protein, and is expressed in E. coli. The CERI protocol underwent an international multi-laboratory
validation study (2) which has demonstrated its relevance and reliability for the intended purpose of the test
method.
2.
This test method is a screening procedure for identifying substances that can bind to the hrERα. It
is used to determine the ability of a test chemical to compete with 17β-estradiol for binding to hrERα-LBD.
Quantitative assay results may include the IC50 (a measure of the concentration of test chemical needed todisplace half of the [3H]-17β-estradiol from the hrERα) and the relative binding affinities of test chemicals forthe hrERα compared to 17β-estradiol. For chemical screening purposes, acceptable qualitative assay results
may include classifications of test chemicals as either hrERα binders, non-binders, or equivocal based uponcriteria described for the binding curves.
3.
The test method uses a radioactive ligand that requires a radioactive materials license for thelaboratory. All procedures with radioisotopes and hazardous chemicals should follow the regulations andprocedures as described by national legislation.
4.
The “GENERAL INTRODUCTION” and “hrER BINDING TEST METHODCOMPONENTS” (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.

PRINCIPLES OF THE TEST METHOD (See also GENERAL INTRODUCTION, page 1)
5. The hrERα binding assay measures the ability of a radiolabeled ligand ([ 3H]17β-estradiol) to bind with
the ER in the presence of increasing concentrations of a test chemical (i.e. competitor). Test chemicals thatpossess a high affinity for the ER compete with the radiolabeled ligand at a lower concentration ascompared with those chemicals with lower affinity for the receptor.
6. This test method consists of two major components: a saturation binding experiment to characterise
receptor-ligand interaction parameters, followed by a competitive binding experiment that characterises thecompetition between a test chemical and a radiolabeled ligand for binding to the ER.
7. The purpose of the saturation binding experiment is to characterise a particular batch of receptors forbinding affinity and number in preparation for the competitive binding experiment. The saturation binding
experiment measures, under equilibrium conditions, the affinity of a fixed concentration of the estrogen© OECD, (2015)76

38

OECD/OCDE

493

receptor for its natural ligand (represented by the dissociation constant, Kd), and the concentration of active
receptor sites (Bmax).
8. The competitive binding experiment measures the affinity of a substance to compete with [3H]17βestradiol for binding to the ER. The affinity is quantified by the concentration of test chemical that, atequilibrium, inhibits 50% of the specific binding of the [ 3H]17β-estradiol (termed the “inhibitory
concentration 50%” or IC50). This can also be evaluated using the relative binding affinity (RBA, relative
to the IC50 of estradiol measured separately in the same run). The competitive binding experimentmeasures the binding of [3H]17β-estradiol at a fixed concentration in the presence of a wide range (eightorders of magnitude) of test chemical concentrations. The data are then fit, where possible, to a form of the
Hill equation (Hill, 1910) that describes the displacement of the radioligand by a one-site competitive
binder. The extent of displacement of the radiolabeled estradiol at equilibrium is used to characterise thetest chemical as a binder, non-binder, or generating an equivocal response.
PROCEDURE
Demonstration of Acceptable hrERα Protein Performance9.
Prior to routinely conducting the saturation and competitive binding assays, each new batch of
hrERα should be shown to be performing correctly in the laboratory in which it will be used. A two-stepprocess should be used to demonstrate performance. These steps are the following:Conduct a saturation [3H]-17β-estradiol binding assay to demonstrate hrERα specificity andsaturation. Nonlinear regression analysis of these data (e.g. BioSoft; McPherson, 1985; Motulsky,
1995) and the subsequent Scatchard plot should document hrERα binding affinity of the [ 3H]-17βestradiol (Kd) and the number of receptors (Bmax) for a particular batch of hrERα.



Conduct a competitive binding assay using the control substances (reference estrogen (17βestradiol), a weak binder (e.g. norethynodrel or norethindrone), and a non-binder(octyltriethoxysilane, OTES). Each laboratory should establish an historical database to document
the consistency of IC50 and the relevant values for the reference estrogen and weak binder amongexperiments and different batches of hrERα. In addition, the parameters of the competitive binding
curves for the control substances should be within the limits of the 95% confidence interval (seeTable 1) that were developed using data from laboratories that participated in the validation study
for this test method (2).

© OECD, (2015)

39

77

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OECD/OCDE

Table 1. Performance criteria developed for the reference estrogen and weak binder, CERI hrER
Binding Assay.

Substance

17β-estradiol

Norethynodrel

NorethindroneC

ParameterMeanaStandard
Deviation(n)95% Confidence Intervalsb
Lower Limit Upper LimitTop

104.7413.12 (70)

101.6

107.9

Bottom0.85

2.41 (70)

0.28

1.43

HillSlope-1.22

0.20 (70)

-1.27

-1.17

LogIC50-8.93

0.23 (70)

-8.98

-8.87

Top

101.31

10.55 (68)98.76

103.90Bottom2.39

5.01 (68)

1.18

3.60

HillSlope-1.04

0.21 (68)

-1.09

-0.99

LogIC50-6.19

0.40 (68)

-6.29

-6.10

Top

92.27

7.79 (23)

88.90

95.63

Bottom

16.52

10.59 (23)

11.94

21.10

Hill Slope-1.18

0.32 (23)

-1.31

-1.04

LogIC50-6.01

0.54 (23)

-6.25

-5.78

a

Mean ± Standard Deviation (SD) with (sample size (n) were calculated using curve fit estimates (4parameter Hill equation) for control runs conducted in four laboratories during the validation study (seeAnnex N of reference 2).
b
The 95% confidence are provided as a guide for acceptability criteria.
cTesting of norethindrone was optional for Subtask 4 during validation study (see Reference 2, see Subtask
4). Thus, the mean ± SD (n) were calculated using curve fit estimates (4-parameter Hill equation) for
control runs conducted in two laboratories.
The range for the IC50 will be dependent upon the Kd of the receptor preparation and concentration ofradiolabeled ligand used within each laboratory. Appropriate adjustment for the range of the IC50 based
upon the conditions used to conduct the test method will be acceptable.

Demonstration of laboratory proficiency
10.
See paragraphs 17 and 18 and Table 2 in “ hrER BINDING TEST METHODCOMPONENTS” of this Test Guideline. Each assay (saturation and competitive binding) should consist of
three independent runs (i.e. with fresh dilutions of receptor, chemicals, and reagents) on different days, andeach run should contain three replicates.
Determination of Receptor (hrERα) Concentration
11.
The concentration of active receptor varies slightly by batch and storage conditions. For thisreason, the concentration of active receptor as received from the supplier should be determined. This will
yield the appropriate concentration of active receptor at the time of the run.
40
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12.
Under conditions corresponding to competitive binding (i.e. 0.5 nM [3H]-estradiol), nominal
concentrations of 0.1, 0.2, 0.4 and 0.6 nM receptor should be incubated in the absence (total binding) andpresence (non-specific binding) of 1 µM unlabeled estradiol. Specific binding, calculated as the differenceof total and non-specific binding, is plotted against the nominal receptor concentration. The concentrationof receptor that gives specific binding values corresponding to 40% of added radiolabel is related to thecorresponding receptor concentration, and this receptor concentration should be used for saturation andcompetitive binding experiments. Frequently, a final hrER concentration of 0.2 nM will comply with thiscondition.
13.
If the 40% criterion repeatedly cannot be met, the experimental set up should be checked forpotential errors. Failure to achieve the 40% criterion may indicate that there is very little active receptor in
the recombinant batch, and the use of another receptor batch should then be considered.
Saturation assay
14. Eight increasing concentrations of [3H]17β-estradiol should be evaluated in triplicate, under thefollowing three conditions (see Table 2):
a. In the absence of unlabelled 17β-estradiol and presence of ER. This is the determinationof total binding by measure of the radioactivity in the wells that have only [3H]17βestradiol.
b. In the presence of a 2000- fold excess concentration of unlabelled 17β-estradiol overlabelled 17β-estradiol and presence of ER. The intent of this condition is to saturate theactive binding sites with unlabelled 17β-estradiol, and by measuring the radioactivity inthe wells, determine the non-specific binding. Any remaining hot estradiol that can bind to
the receptor is considered to be binding at a non-specific site as the cold estradiol should beat such a high concentration that it is bound to all of the available specific sites on thereceptor.
c. In the absence of unlabelled 17β-estradiol and absence of ER (determination of total
radioactivity)
Preparation of [3H]-17β-estradiol, unlabelled 17β-estradiol solutions and hrERα
A 40 nM solution of [3H]-17β-estradiol should be prepared from a 1 µM stock solution of [3H]-17βestradiol in DMSO, by adding DMSO (to prepare 200 nM) and assay buffer at room temperature (toprepare 40 nM). Using this 40 nM solution, the series of [3H]-17β-estradiol dilutions prepared, ranging from
0.313 nM to 40 nM with assay buffer at room temperature (as represented in lane 12 of Table 2).
15.
The final assay concentrations, ranging from 0.0313 to 4.0 nM, will be obtained by adding 10 µLof these solutions to the respective assay wells of a 96-well microtiter plate (see Tables 2 and 3).
Preparation of assay buffer, calculation of the original [3H]-17β-estradiol stock solution based on its
specific activity, preparation of dilutions and determination of the concentrations are described in depth in
the CERI protocol (2).
16.
Dilutions of unlabeled 17β-estradiol solutions should be prepared from a 1 nM 17β-estradiol
stock solution by adding assay buffer to achieve eight increasing concentrations initially ranging from0.625 µM to 80 µM. The final assay concentrations, ranging from 0.0625 to 8 µM, will be obtained byadding 10 µL of these solutions to the respective assay wells of a 96-well microtiter plate dedicated to themeasurement of non-specific binding (see Tables 2 and 3). Preparation of unlabelled 17β-estradiol dilutions
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is described in depth in the CERI protocol (2).
17.
The concentration of receptor that gives 40±10% specific binding should be used (see paragraphs
12-13). The hrERα solution should be prepared with ice-cold assay buffer immediately prior to use, i.e.
after all wells for total binding, non-specific binding and hot ligand alone have been prepared.
18.
The 96-well microtiter plates are prepared as illustrated in Table 2, with 3 replicates per [3H]-17βestradiol concentration. Volume assignment of [3H]-17β-estradiol, unlabeled 17β-estradiol, buffer andreceptor are provided in Table 3.
Table 2: Saturation Binding Assay Microtiter Plate Layout

1*

2*

3*

For measurement of
TB

4*

5*

6*

For measurement of
NSB

7*

8*

9*

For determinationof hot ligand alone

0.0313 nM [ 3H] E2 0.0313 nM [3H] E2+ ER
0.0313 nM
A+ 0.0625 μM E2+ ER
0.0625 nM [3H] E2
0.0625 nM [3H] E2+ ER
0.0625 nM+ 0.125 μM E2
B+ ER
30.125 nM [3H] E2
0.125 nM [3H] E2+
ER
0.125 nM
+ 0.25 μM E2
C+ ER
0.250 nM [3H] E2
0.250 nM [3H] E2+ ER
0.250 nM
+ 0.5 μM E2
D+ ER
0.50 nM [ H] E2
0.50 nM [3H] E2+
ER0.50 nM+ 1 μM E2
E+ ER
1.00 nM [3H] E2
1.00 nM [3H] E2+ ER
1.00 nM+ 2 μM E2F+ ER
2.00 nM [3H] E2
2.00 nM [3H] E2+ ER
2.00 nM+ 4 μM E2
G+ ER
4.00 nM [3H] E2
4.00 nM [3H] E2+ ER
4.00 nM+ 8 μM E2
H+ ER
TB: total binding,
NSB: non-specific binding[3H] E2: [3H]17β-estradiol
E2: unlabelled 17β-estradiol*The indicated concentrations here are the final concentrations in each well.
**The dilutions of unlabeled E2 and [ H]E2 can be prepared in a different plate.
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10

11**

12**

unlabeled
E2 dilutions
for platecolumn 4-6[3H]E2
dilutionsfor platecolumn 1-9

0.625 μM

0.313 nM

1.25 μM

0.625 nM

2.5 μM

1.25 nM

5 μM

2.5 nM

10 μM

5 nM

20 μM

10 nM

40 μM

20 nM

80 μM

40 nM

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Table 3. Reagent Volumes for Saturation Microtiter Plate
Lane Number

1

Preparation Steps
Volume ofcomponents forreaction wells
above and orderto add

2

3

4

5

6

7*

8*

9*

TB Wells

NSB Wells

Hot Ligand Alone

Buffer

60 µL

50 µL

90 µL

unlabeled E2 fromlane 11 in Table2

-

10 μL

-

[3H]E2 fromlane12 in Table2

10 µL

10 µL

10 µL

hrERα

30 µL

30 µL

-

100 µL100 µL100 µL
Quantification of
the radioactivity justafter thepreparation. No
incubation

Total reaction volume

Incubation

FOLLOWING 2 HOUR INCUBATIONREACTION

Treatment with 0.4% DCC

Yes

Yes

No

Volume of 0.4% DCC

100 µL100 µL

-

Yes

Yes

No

100 µL**

50 µL

Filtration

MEASURING THE DPMS
Quantification volume added toscintillation cocktail

100 µL*** If an LSC for microplates is used for measuring dpms, the preparation of hot ligand alone in the same assay plateof TB and NSB wells is not appropriate. The hot ligand alone should be prepared in a different plate.
** If centrifugation is used to separate DCC, the 50 μL of supernatant should be measured by LSC in order to avoid
contamination of DCC.

19.
Assay microtiter plates for the determination of total binding and non-specific binding should be
incubated at room temperature (22°C to 28°C) for two hours.
Measurement of [3H]-17β-Estradiol bound to hrERα20.
Following the two hour incubation period, [3H]-17β-Estradiol bound to hrERα should be
separated from free [3H]-17β-Estradiol by adding 100µL an ice cold 0.4% DCC suspension to the wells.
The plates should then be placed on ice for 10 minutes and the reaction mixture and DCC suspension
should be filtered, by transfer to a mictotiter plate filter, to remove DCC. A 100 µL of the filtrate should
then be added to scintillation fluid in LSC vials for determination of disintegration per minute (dpms) per
vial by liquid scintillation counting.
21.
Alternatively, if a microplate filter is not available, removal of DCC can be obtained bycentrifugation. A 50 µL of supernatant containing the hrERα-bound [3H]-17β-estradiol should then be takenwith extreme care, to avoid any contamination of the wells by touching DCC, and should be used for
scintillation counting.
22.
The hot ligand alone condition is used for determining the disintegration per minute (dpm) of[3H]-17β-estradiol added to the assay wells. The radioactivity should be quantified just after preparation.
These wells should not be incubated and should not be treated with DCC suspension but their content
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should be delivered directly into the scintillation fluid. These measures demonstrate how much [ 3H]-17βestradiol in dpms was added to each set of wells for the total binding and non-specific binding.
Competitive binding assay
23.
The competitive binding assay measures the binding of a single concentration of [3H]-17βestradiol in the presence of increasing concentrations of a test chemical. Three concurrent replicates should
be used at each concentration within one run. In addition, three non-concurrent runs should be performed
for each chemical tested. The assay should be set up in one or more 96-well microtiter plates.
Controls
24.
When performing the assay, concurrent solvent and controls (i.e. reference estrogen, weakbinder, and non-binder) should be included in each experiment. Full concentration curves for thereference estrogen and controls (i.e. weak binder and non-binder) should be used in one plate during eachrun. All other plates should contain (i) a high- (maximum displacement i.e. approximately full
displacement of radiolabeled ligand) and medium- (approximately, the IC50) concentration of E2 andweak binder in triplicate; (ii) solvent control and non-specific binding, each in triplicate. Procedures forthe preparation of assay buffer, [3H]-17β-estradiol, hrERα and test chemical solutions are described in
depth in the CERI protocol (2).
- Solvent control:25.
The solvent control indicates that the solvent does not interact with the test system and alsomeasures total binding (TB). DMSO is the preferred solvent. Alternatively, if the highest concentration of
the test chemical is not soluble in DMSO, ethanol may be used. The concentration of DMSO in the final
assay wells should be 2.05% and could be increased up to 2.5% in case of lack of solubility of the test
chemical. Concentrations of DMSO above 2.5% should not be used because of interference of higher
solvent concentrations with the assay. For test chemicals that are not soluble in DMSO, but are soluble inethanol, a maximum of 2% ethanol may be used in the assay without interference.
- Buffer control:
26.
The buffer control (BC) should contain neither solvent nor test chemical, but all of the othercomponents of the assay. The results of the buffer control are compared to the solvent control to verify thatthe solvent used does not affect the assay system.
- Strong binder (reference estrogen)27.
17β-estradiol (CAS 50-28-2) is the endogenous ligand and binds with high affinity to the ER,
alpha subtype. A standard curve using unlabeled 17β-estradiol should be prepared for each hrERαcompetitive binding assay, to allow for an assessment of variability when conducting the assay over timewithin the same laboratory. Eight solutions of unlabeled 17β-estradiol should be prepared in DMSO andassay buffer, with final concentrations in the assay wells to be used for the standard curve spaced asfollows: 10-6, 10-7, 10-8, 10-8.5, 10-9, 10-9.5, 10-10, 10-11 M. The highest concentration of unlabeled 17βestradiol (1 µM) should serve as the non-specific binding indicator. This concentration is distinguished by
the label “NSB” in Table 4 even though it is also part of the standard curve.
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28.
A weak binder (norethynodrel (CAS68-23-5), or alternate, norethindrone (CAS 68-22-4)) should
be included to demonstrate the sensitivity of each experiment and to allow an assessment of variability
when conducting the assay over time. Eight solutions of the weak binder should be prepared in DMSO andassay buffer, with final concentrations in the assay wells as follows: 10-4.5, 10-5.5, 10-6, 10-6.5, 10-7, 10-7.5, 10-8
and 10-9 M.
- Non binder
29.
Octytriethoxysilane (OTES, CAS 2943-75-1) should be used as the negative control (non-binder).
It provides assurance that the assay as run, will detect test chemicals that do not bind to the hrERα. Eightsolutions of the non-binder should be prepared in DMSO and assay buffer, with final concentrations in theassay wells as follows: 10-3,10-4, 10-5, 10-6, 10-7, 10-8, 10-9, 10-10 M. Di-n-butyl phthalate (DBP, CAS 84-722) can be used as an alternative non-binder, but only tested up to 10-4M. The maximum solubility of DBP
in the assay has been demonstrated to be 10-4M.
hrERα concentration
30.
The amount of receptor that gives specific binding of 40±10% should be used (see paragraphs 1213 of Annex 3). The hrERα solution should be prepared by dilution of the functional hrERα into ice cold
assay buffer, immediately prior to use.
[3H]-17β-estradiol
31.

The final concentration of [3H]-17β-estradiol in the assay wells should be of 0.5 nM.

Test Chemicals
32.
In the first instance, it is necessary to conduct a solubility test to determine the limit of solubility
for each test chemical and to identify the appropriate concentration range to use when conducting the test
protocol. The limit of solubility of each test chemical is to be initially determined in the solvent and thenfurther confirmed under assay conditions. The final concentration tested in the assay should not exceed
1mM. Range finder testing includes a solvent control along with at least 8 log serial dilutions, starting at
maximum acceptable concentration (e.g. 1 mM or lower, based upon the limit of solubility), and thepresence of cloudiness or precipitate noted (see also paragraph 35 of Annex 3). Once the concentration
range for testing has been determine, a test chemical should be tested using 8 log concentrations spaced
appropriately as defined in the preceding range finding test. Concentrations tested in the second and third
experiments should be further adjusted as appropriate to better characterise the concentration response
curve, if necessary.
33.
Dilutions of the test chemical should be prepared in the appropriate solvent (see paragraph 25 ofAnnex 3). If the highest concentration of the test chemical is not soluble in either DMSO or ethanol, andadding more solvent would cause the solvent concentration in the final tube to be greater than theacceptable limit, the highest concentration may be reduced to the next lower concentration. In this case, anadditional concentration may be added at the low end of the concentration series. Other concentrations inthe series should remain unchanged.
34.
The test chemical solutions should be closely monitored when added to the assay well, as the test
chemical may precipitate upon addition to the assay well. The data for all wells that contain precipitateshould be excluded from curve-fitting, and the reason for exclusion of the data noted.
35.
If there is prior existing information from other sources that provide a log(IC50) of a test chemical,
it may be appropriate to geometrically space the dilutions more closely around the expected log(IC50) (i.e.
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0.5 log units). The final results should show enough sufficient spread of concentrations on either side of thelog(IC50), including the “top” and “bottom”, such that the binding curve can be adequately characterised.
Assay plate organisation
36.
Labeled microtiter plates should be prepared using sextuple incubations for the solvent control, thehighest concentration the reference estrogen (E2) which also serves as the non-specific binding (NSB)
indicator, the buffer control, the eight concentrations of the non-binding control (octyltriethoxysilane), theseven lower concentrations for the reference estrogen (E2), the eight concentrations of the weak binder(norethynodrel or norethindrone), and the eight concentrations of each test chemical (TC). An example
layout of the plate layout diagram for the full concentration curves for the reference estrogen and controls isgive below in Table 4. Additional microtiter plates are used for the test chemical and should contain platecontrols (i.e. (i) a high- (maximum displacement) and medium- (approximately, the IC50) concentration ofE2 and weak binder in triplicate; (ii) solvent control (as total binding) and non-specific binding, each insextuple (Table 5). An example of a competitive assay microtiter plate layout worksheet using three
unknown test chemicals is provided in Appendix 3 of Annex 3. The concentrations indicated in theworksheet as well as in Tables 4 and 5 refer to the final concentrations used in each assay well. The
maximum concentration for E2 should be 1×10-7 M and for the weak binder, the highest concentration used
for the weak binder on plate 1 should be used. The IC50 concentration has to be determined by thelaboratory based on their historical control database. The expectation is that this value would be similar tothat observed in the validation studies (see table 1).
Table 4: Competitive Binding Assay Microtiter Plate Layout1,2, Full Concentration Curves for
Reference Estrogen and Controls (Plate 1)
1

2

3

4

5

6

7

8

9

Buffer Control andPositive Control (E2)

Weak Positive(Norethynodrel)

Negative Control(OTES)

A

Blank*

1×10-9 M

1×10-10 M

BE2 (1×10-11 M)

1×10-8 M

1×10-9 MC

E2 (1×10-10 M)

1×10-7.5 M

1×10-8 MD

E2 (1×10-9.5 M)

1×10-7 M

1×10-7 ME

E2 (1×10-9 M)

1×10-6.5 M

1×10-6 MF

E2 (1×10

M)

1×10 M

1×10 MG

E2 (1×10-8 M)

1×10-5.5 M

1×10-4 M

-8.5

-6

10

11

12

TB and NSB
TB (solvent control)(2.05% DMSO)NSB (10-6 M E2)

Buffer control-5

H

Blank (for hot)**

E2 (1×10-7 M)
1×10-4.5 M
1×10-3 M
Sample set up for the standards microtiter plate to be run with each experiment.
2
Note that this microtiter plate is made using the dilutions made in the dilution plate described for the standards inthe previous sections.
In this example, the weak binder is norethinodrel (NE)*real blank, well not used**blank, not used during the incubation, but used to confirm the total radioactivity added.

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Table 5: Competitive Binding Assay Microtiter Plate Layout, Additional Plates for Test Chemicals(TC) and Plate Controls.
1
2
3
4
5
6
7
8
9
10
11
12
Test Chemical-1(TC-1)

Test Chemical-2

Test Chemical-3

Controls(TC-2)(TC-3)

A

TC-1 (1×10-10 M)

TC-2 (1×10-10 M)

TC-3 (1×10-10 M)E2 (1×10-7M)

B

TC-1 (1×10-9 M)

TC-2 (1×10-9 M)

TC-3 (1×10-9 M)

E2 (IC50)

C

TC-1 (1×10-8 M)

TC-2 (1×10-8 M)

TC-3 (1×10-8 M)

NE (1×10-4.5M)

D

TC-1 (1×10-7 M)

TC-2 (1×10-7 M)

TC-3 (1×10-7 M)

NE (IC50)

E

TC-1 (1×10-6 M)

TC-2 (1×10-6 M)

TC-3 (1×10-6 M)

F

TC-1 (1×10-5 M)

TC-2 (1×10-5 M)

TC-3 (1×10-5 M)

G

TC-1 (1×10-4 M)

TC-2 (1×10-4 M)

TC-3 (1×10-4 M)

H

TC-1 (1×10-3 M)

TC-2 (1×10-3 M)

TC-3 (1×10-3 M)

NSB (10-6 M E2)

TB (Solvent control)

In this example, the weak binder is norethinodrel (NE)

Completion of competitive binding assay
37.
Excepting wells for total binding and blanks (for hot), as shown in Table 6, 50 μL of the assay
buffer should be placed in each well, and should be mixed with 10 µL of the solvent control, reference
estrogen (E2), weak binder, non-binder, and test chemicals, respectively, 10 µL of a 5 nM [3H]-17βestradiol solution. Then, 30µL of ice cold receptor solution was added to each plate and mixed gently. The
hrERα solution should be the last reagent to be added. Assay microtiter plates should be incubated at room
temperature (22° to 28°C) for 2 hours.
Table 6: Volume of Assay Components for hrER Competitive Binding Assay, Microtiter Plates
Lane Number Preparation Steps
Volume ofcomponents forreaction wells
above and orderto addRoom Temperature assay Buffer
Unlabeled E2, weak binder, non-binder,
solvent and test chemicals*
3[ H]-17β-estradiol to yield final
concentration of 0.5 nM (i.e. 5 nM)
rERα concentration as determined (seeparagraphs 12-13)

Other than TB
wells

TB wells

Blank (for hot)

50 µL

60 µL

90 µL

10 µL

-

-

10 µL

10 µL

10 µL

30 µL

30 µL

-

Total volume in each assay well
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The quantification of [3H]-17β-Estradiol bound to hrERα, following separation of [3H]-17βEstradiol bound to hrERα from free [3H]-17β-Estradiol by adding 100 μL of ice-cold DCC suspension toeach well, should then be performed as described in paragraphs 21-23 of Annex 3 for the saturation binding
assay.
39.
Wells G10-12 and H10-12 (identified as blank (for hot) in Table 4) represent the dpms of the [3H]labeled-estradiol in 10 μL. The 10 μL aliquot should be delivered directly into the scintillation fluid.
Acceptability criteria
Saturation binding assay
40.
The specific binding curve should reach a plateau as increasing concentrations of [ 3H]-17βestradiol were used, indicating saturation of hrERα with ligand.
41.
The specific binding at 0.5 nM of [3H]-17β-estradiol should be inside the acceptable range 30% to
50% of the average measured total radioactivity added across runs. Occasional slight excursions outside ofthis range are acceptable, but if runs are consistently outside this range or a particular run is significantly
outside this range, the protein concentration should be adjusted and the saturation assay repeated.
42.

The data should produce a linear Scatchard plot.

43.
The non-specific binding should not be excessive. The value for non-specific binding should
typically be <35% of the total binding. However, the ratio might occasionally exceed this limit whenmeasuring very low dpm for the lowest concentration of radiolabeled 17β-estradiol tested.
Competitive binding assay
44.
Increasing concentrations of unlabeled 17β-estradiol should displace [3H]-17β-estradiol from thereceptor in a manner consistent with a one-site competitive binding.
45.
The IC50 value for the reference estrogen (i.e. 17β-estradiol) should be approximately equal to themolar concentration of [3H]-17β-estradiol plus the Kd determined from the saturation binding assay.
46.
The total specific binding should be consistently within the acceptable range of 40 ± 10 % when
the average measured concentration of total radioactivity added to each well was 0.5 nM across runs.
Occasional slight excursions outside of this range are acceptable, but if runs are consistently outside thisrange or a particular run is significantly outside this range, the protein concentration should be adjusted.
47.
The solvent should not alter the sensitivity or reproducibility of the assay. The results of thesolvent control (TB wells) are compared to the buffer control to verify that the solvent used does not affectthe assay system. The results of the TB and Buffer control should be comparable if there is no effect of thesolvent on the assay.
48.
The non-binder should not displace more than 25% of the [3H]-17β-estradiol from the hrERα
when tested up to 10-3 M (OTES) or 10-4 M (DBP).
49.

Performance criteria were developed for the reference estrogen and two weak binders (e.g.

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norethynodrel, norethindrone) using data from the validation study for the CERI hrER Binding Assay(Annex N of reference 2). 95% confidence intervals are provided for the mean ± SD (n) of all control runs
across four laboratories that participated in the validation study. 95% conference intervals were calculated
for the curve fit parameters (i.e. top, bottom, Hillslope and Log IC50) for the reference estrogen and weakbinders, and the Log10RBA of the weak binders relative to the reference estrogen. Table 1 provides
expected ranges for the curve fit parameters that can be used as performance criteria. In practice, the rangeof the IC50 may vary slightly based upon the experimentally derived Kd of the receptor preparation andligand concentration used for the test method.
50.
No performance criteria were developed for curve fit parameters for the test chemicals because of
the wide array of existing potential test chemicals and variation in potential affinities and outcomes (e.g.
Full curve, partial curve, no curve fit). However, professional judgment should be applied when reviewing
results from each run for a test chemical. A sufficient range of concentrations of the test chemical should be
used to clearly define the top (e.g. 90 - 100% of binding) of the competitive curve. Variability among
replicates at each concentration of test chemical as well as among the 3 non-concurrent runs should bereasonable and scientifically defensible. Controls from each run for a test chemical should approach themeasures of performance reported for this CERI test method and be consistent historical control data fromeach respective laboratory.
ANALYSIS OF DATA
Saturation binding assay
51.
Both total and non-specific binding are measured. From these values, specific binding ofincreasing concentrations of [3H]-17β-estradiol under equilibrium conditions is calculated by subtracting
non-specific from total. A graph of specific binding versus [3H]-17β-estradiol concentration should reach aplateau for maximum specific binding indicative of saturation of the hrERα with the [3H]-17β-estradiol. In
addition, analysis of the data should document the binding of the [3H]-17β- estradiol to a single, high-affinity
binding site. Non-specific, total, and specific binding should be displayed on a saturation binding curve.
Further analysis of these data should use a non-linear regression analysis (e.g. BioSoft; McPherson, 1985;
Motulsky, 1995) with a final display of the data as a Scatchard plot.
52.
The data analysis should determine Bmax and Kd from the total binding data alone, using theassumption that non-specific binding is linear, unless justification is given for using a different method. In
addition, robust regression should be used when determining the best fit unless justification is given. The
method chosen for robust regression should be stated. Correction for ligand depletion (e.g. using the methodof Swillens 1995) should always be used when determining Bmax and Kd from saturation binding data.
Competitive binding assay
53.
The competitive binding curve is plotted as specific [3H]-17β- estradiol binding versus theconcentration (log10 units) of the competitor. The concentration of the test chemical that inhibits 50% of
the maximum specific [3H]-17β-estradiol binding is the IC50 value.
54.
Estimates of log(IC50) values for the positive controls (e.g. reference estrogen and weak binder)
should be determined using an appropriate nonlinear curve fitting software to fit a four parameter Hill
49
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equation (e.g. BioSoft; McPherson, 1985; Motulsky, 1995). The top, bottom, slope, and log(IC50) should
generally be left unconstrained when fitting these curves. Robust regression should be used when
determining the best fit unless justification is given. Correction for ligand depletion should not be used.
Following the initial analysis, each binding curve should be reviewed to ensure appropriate fit to the model.
The relative binding affinity (RBA) for the weak binder should be calculated as a percent of the log (IC 50)
for the weak binder relative to the log (IC50) for 17β-estradiol. Results from the positive controls and thenon-binder control should be evaluated using the measures of the test method performance in paragraphs
44-49 of this Annex 3.
55.
Data for all test chemicals should be analyzed using a step-wise approach to ensure that data areappropriately analyzed and that each competitive binding curve is properly classified. It is recommended
that each run for a test chemical initially undergo a standardized data analysis that is identical to that used
for the reference estrogen and weak binder controls (see paragraph 54 of this Annex 3). Once completed, atechnical review of the curve fit parameters as well as a visual review of how well the data fit thegenerated competitive binding curve for each run should be conducted. During this technical review, theobservations of a concentration dependent decrease in the percent [3H]-17β-estradiol specifically bound,low variability among the technical replicates at each test chemical concentration, and consistency in fit
parameters among the three runs are a good indication that the assay and data analyses were conducted
appropriately.
Data interpretation
56.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be a binder forthe hrERα if a binding curve can be fit and the lowest point on the response curve within the range of thedata is less than 50% (Figure 1).
57.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be a nonbinder for the hrERα if: A binding curve can be fit and the lowest point on the fitted response curve within the range of thedata is above 75%, or A binding curve cannot be fit and the lowest unsmoothed average percent binding among theconcentration groups in the data is above 75%.
58.
Test chemicals are considered equivocal if none of the above conditions are met (e.g. the lowest
point on the fitted response curve is between 76 – 51%).

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493

Table 7. Criteria for assigning classification based upon competitive binding curve for a test
chemical.
Classification

Criteria

BinderaA binding curve can be fit.

Non-binderb

The lowest point on the response curve within the range of thedata is less than 50%.
If a binding curve can be fit,the lowest point on the fitted response curve within the range ofthe data is above 75%.
If a binding curve cannot be fit,the lowest unsmoothed average percent binding among theconcentration groups in the data is above 75%.
Any testable run that is neither a binder nor a non-binder

Equivocalc(e.g. The lowest point on the fitted response curve is between 76 – 51%).

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Figure 1. Examples of test chemical classification using competitive binding curve.

% Radioligand Bound

125
Non-Binder

100

75

Equivocal

50
Binder25

Binder

0
-12

-11

-10

-9

-8

-7

-6

-5

Concentration (log Molar)

-4

-3

59.
Multiple runs conducted within a laboratory for a test chemical are combined by assigning
numeric values to each run and averaging across the runs as shown in Table 8. Results for the combined
runs within each laboratory are compared with the expected classification for each test chemical.

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493

Table 8. Method for classification of test chemical using multiple runs within a laboratory
To assign value to each run:Classification

Numeric Value

Binder2

Equivocal

1

Non-binder0

To classify average of numeric value across runs:
Classification

Numeric Value

BinderAverage ≥ 1.5

Equivocal

0.5 ≤ Average < 1.5

Non-binder

Average < 0.5

TEST REPORT
60. See paragraph 24 of “hrER BINDING TEST METHOD COMPONENTS” of this Test Guideline.

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OECD/OCDEAppendix 1: List of Terms[3H]E2: 17β-Estradiol radiolabeled with tritium
DCC: Dextran-coated charcoal
E2: Unlabeled 17β-estradiol (inert)
Assay buffer: 10 mM Tris-HCl, pH 7.4, containing 1 mM EDTA, 1mM EGTA, 1 mM NaVO3, 10 %Glycerol, 0.2 mM Leupeptin, 1 mM Dithiothreitol and 10 mg/mL Bovine Serum Albumin
hrERα: Human recombinant estrogen receptor alpha (ligand binding domain)
Replicate: One of multiple wells that contain the same contents at the same concentrations and are assayedconcurrently within a single run. In this protocol, each concentration of test chemical is tested in triplicate;that is, there are three replicates that are assayed simultaneously at each concentration of test chemical.
Run: A complete set of concurrently-run microtiter plate assay wells that provides all the informationnecessary to characterize binding of a test chemical to the hrERα (viz., total [3H]-17β-estradiol added to theassay well, maximum binding of [3H]-17β-estradiol to the hrERα, nonspecific binding, and total binding at
various concentrations of test chemical). A run could consist of as few as one assay well (i.e. replicate) per
concentration, but since this protocol requires assaying in triplicate, one run consists of three assay wells perconcentration. In addition, this protocol requires three independent (i.e. non-concurrent) runs per chemical.

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Appendix 2

Plate

Position

Replicate

Well type

Well Code

Concentration CodeCompetitor Initial
Concentration (M)hrER stock (uL)

Buffer Volume (uL)

Tracer (Hot E2) Volume(uL)

Volume from dilution
plate( uL)

Final Volume (ul)Competitor Final
Concentration (M)Competitive Binding Assay Well Layout

S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S

A1
A2
A3
B1
B2
B3
C1
C2
C3
D1
D2
D3
E1
E2
E3
F1
F2
F3
G1
G2
G3
H1
H2
H3
A4
A5
A6
B4
B5
B6
C4
C5
C6
D4
D5
D6
E4
E5
E6
F4
F5
F6
G4
G5
G6

1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3

Blank
Blank
Blank
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
cold E2
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel
norethynodrel

BK
BK
BK
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE

BK1
BK2
BK3
S1
S1
S1
S2
S2
S2
S3
S3
S3
S4
S4
S4
S5
S5
S5
S6
S6
S6
S7
S7
S7
WP1
WP1
WP1
WP2
WP2
WP2
WP3
WP3
WP3
WP4
WP4
WP4
WP5
WP5
WP5
WP6
WP6
WP6
WP7
WP7
WP7

—
—
—
1.00E-10
1.00E-10
1.00E-10
1.00E-09
1.00E-09
1.00E-09
3.16E-09
3.16E-09
3.16E-09
1.00E-08
1.00E-08
1.00E-08
3.16E-08
3.16E-08
3.16E-08
1.00E-07
1.00E-07
1.00E-07
1.00E-06
1.00E-06
1.00E-06
1.00E-08
1.00E-08
1.00E-08
1.00E-07
1.00E-07
1.00E-07
3.16E-07
3.16E-07
3.16E-07
1.00E-06
1.00E-06
1.00E-06
3.16E-06
3.16E-06
3.16E-06
1.00E-05
1.00E-05
1.00E-05
3.16E-05
3.16E-05
3.16E-05

—
—
—
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30

—
—
—
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50

—
—
—
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10

—
—
—
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10

—
—
—
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100

—
—
—
1.0E-11
1.0E-11
1.0E-11
1.0E-101.0E-101.0E-10
3.2E-10
3.2E-10
3.2E-101.0E-09
1.0E-09
1.0E-09
3.2E-09
3.2E-09
3.2E-09
1.0E-08
1.0E-08
1.0E-08
1.0E-07
1.0E-07
1.0E-07
1.0E-09
1.0E-09
1.0E-09
1.0E-08
1.0E-08
1.0E-08
3.2E-08
3.2E-08
3.2E-08
1.0E-07
1.0E-07
1.0E-07
3.2E-07
3.2E-07
3.2E-07
1.0E-06
1.0E-06
1.0E-06
3.2E-06
3.2E-06
3.2E-06© OECD, (2015)

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OECD/OCDE

1
2
3
4
5
6
1

56

Tracer (Hot E2)
Volume(Volumefromu
Dilution
L plate( uL)
)

50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
60
60
60
60
60
60
50
50
50
50
50
50
100
100
100
100
100
100
-

10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10

10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
-

100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100

3.2E-05
3.2E-05
3.2E-05
1.0E-101.0E-101.0E-101.0E-09
1.0E-09
1.0E-09
1.0E-08
1.0E-08
1.0E-08
1.0E-07
1.0E-07
1.0E-07
1.0E-06
1.0E-06
1.0E-06
1.0E-05
1.0E-05
1.0E-05
1.0E-04
1.0E-04
1.0E-04
1.0E-03
1.0E-03
1.0E-03
1.0E-06
1.0E-06
1.0E-06
1.0E-06
1.0E-06
1.0E-06-

Competitor Final
Concentration (M)

30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
90

Final Volume (ul)

3.16E-04
3.16E-04
3.16E-04
1.00E-09
1.00E-09
1.00E-09
1.00E-08
1.00E-08
1.00E-08
1.00E-07
1.00E-07
1.00E-07
1.00E-06
1.00E-06
1.00E-06
1.00E-05
1.00E-05
1.00E-05
1.00E-04
1.00E-04
1.00E-04
1.00E-03
1.00E-03
1.00E-03
1.00E-02
1.00E-02
1.00E-02
1.00E-05
1.00E-05
1.00E-05
1.00E-05
1.00E-05
1.00E-05-

Buffer Volume
(uL)

WP8
WP8
WP8
OTES1
OTES1
OTES1
OTES2
OTES2
OTES2
OTES3
OTES3
OTES3
OTES4
OTES4
OTES4
OTES5
OTES5
OTES5
OTES6
OTES6
OTES6
OTES7
OTES7
OTES7
OTES8DBP7
OTES88
OTES8
TB1
TB2
TB3
TB4
TB5
TB6
S1
S2
S3
S4
S5
S6
BC1
BC2
BC3
BC4
BC5
BC6
H1

hrER stock (uL)

NE
NE
NE
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
TB
TB
TB
TB
TB
TB
NSB
NSB
NSB
NSB
NSB
NSB
BC
BC
BC
BC
BC
BC
Hot

Competitor Initial
Concentration (M)norethynodrel
norethynodrel
norethynodrel
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
OTES
total binding
total binding
total binding
total binding
total binding
total binding
cold E2 (high)cold E2 (high)cold E2 (high)cold E2 (high)cold E2 (high)cold E2 (high)Buffer control
Buffer control
Buffer control
Buffer control
Buffer control
Buffer control
Blank (for hot)© OECD, (2015)94

Well type

Replicate

1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
4
5
6
1
2
3
4
5
6

Concentration
Code

H4
H5
H6
A7
A8
A9
B7
B8
B9
C7
C8
C9
D7
D8
D9
E7
E8
E9
F7
F8
F9
G7
G8
G9
H7
H8
H9
A10
A11
A12
B10
B11
B12
C10
C11
C12
D10
D11
D12
E10
E11
E12
F10
F11
F12
G10*

Well Code

S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S

Position

Plate

Competitive Binding Assay Well Layout

OECD/OCDE
S
S
S
S
S

G11*
G12*
H10*
H11*
H12

2
3
4
5
6

Blank (for hot)Blank (for hot)Blank (for hot)Blank (for hot)Blank (for hot)

Hot
Hot
Hot
Hot
Hot

H2
H3
H4
H5
H6

-

493

90
90
90
90
90

-

10
10
10
10
10

-

100
100
100
100
100

-

*: Note that the "hot" wells are empty during incubation. The 10 µl are added only for scintillation counting.

© OECD, (2015)

30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30

50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50

6
6
7
7
7
8
8
8
1
1
1
2
2
2
3
3
3
4
4
4
5
5
5

10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10

Competitor Final
Concentration (M)1.00E-09
1.00E-09
1.00E-09
1.00E-08
1.00E-08
1.00E-08
1.00E-07
1.00E-07
1.00E-07
1.00E-06
1.00E-06
1.00E-06
1.00E-05
1.00E-05
1.00E-05
1.00E-04
1.00E-04
1.00E-04
1.00E-03
1.00E-03
1.00E-03
1.00E-02
1.00E-02
1.00E-02
1.00E-09
1.00E-09
1.00E-09
1.00E-08
1.00E-08
1.00E-08
1.00E-07
1.00E-07
1.00E-07
1.00E-06
1.00E-06
1.00E-06
1.00E-05
1.00E-05
1.00E-05

Final Volume (ul)

2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3

1
1
1
2
2
2
3
3
3
4
4
4
5
5
5
6

Volume from dilution
plate( uL)

U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U2
U2
U2
U2
U2
U2
U2
U2
U2
U2
U2
U2
U2
U2
U2

Tracer (Hot E2) Volume(uL)

Well CodeUnknown 1
Unknown 1
Unknown 1
Unknown 1
Unknown 1
Unknown 1
Unknown 1
Unknown 1
Unknown 1
Unknown 1
Unknown 1
Unknown 1
Unknown 1
Unknown 1
Unknown 1
Unknown 1
Unknown 1
Unknown 1
Unknown 1
Unknown 1
Unknown 1
Unknown 1
Unknown 1
Unknown 1
Unknown 2
Unknown 2
Unknown 2
Unknown 2
Unknown 2
Unknown 2
Unknown 2
Unknown 2
Unknown 2
Unknown 2
Unknown 2
Unknown 2
Unknown 2
Unknown 2
Unknown 2

Buffer Volume (uL)

Well type

1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1

hrER stock (uL)Replicate

A1
A2
A3
B1
B2
B3
C1
C2
C3
D1
D2
D3
E1
E2
E3
F1
F2
F3
G1
G2
G3
H1
H2
H3
A4
A5
A6
B4
B5
B6
C4
C5
C6
D4
D5
D6
E4
E5
E6

Competitor Initial
Concentration (M)Position

P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1

Concentration Code

Plate

Competitive Binding Assay Well Layout

10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10

100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100

1.0E-101.0E-101.0E-101.0E-09
1.0E-09
1.0E-09
1.0E-08
1.0E-08
1.0E-08
1.0E-07
1.0E-07
1.0E-07
1.0E-06
1.0E-06
1.0E-06
1.0E-05
1.0E-05
1.0E-05
1.0E-04
1.0E-04
1.0E-04
1.0E-03
1.0E-03
1.0E-03
1.0E-101.0E-101.0E-101.0E-09
1.0E-09
1.0E-09
1.0E-08
1.0E-08
1.0E-08
1.0E-07
1.0E-07
1.0E-07
1.0E-06
1.0E-06
1.0E-06

57

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P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1
P1

F4
F5
F6
G4
G5
G6
H4
H5
H6
A7
A8
A9
B7
B8
B9
C7
C8
C9
D7
D8
D9
E7
E8
E9
F7
F8

1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2

P1
P1
P1
P1
P1
P1
P1

F9
G7
G8
G9
H7
H8
H9

3
1
2
3
1
2
3

P1

A10

1

P1

A11

P1

A12

P1
P1

U2
U2
U2
U2
U2
U2
U2
U2
U2
U3
U3
U3
U3
U3
U3
U3
U3
U3
U3
U3
U3
U3
U3
U3
U3
U3

6
6
6
7
7
7
8
8
8
1
1
1
2
2
2
3
3
3
4
4
4
5
5
5
6
6

1.00E-04
1.00E-04
1.00E-04
1.00E-03
1.00E-03
1.00E-03
1.00E-02
1.00E-02
1.00E-02
1.00E-09
1.00E-09
1.00E-09
1.00E-08
1.00E-08
1.00E-08
1.00E-07
1.00E-07
1.00E-07
1.00E-06
1.00E-06
1.00E-06
1.00E-05
1.00E-05
1.00E-05
1.00E-04
1.00E-04

30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30

50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50

10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10

10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10

100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100

1.0E-05
1.0E-05
1.0E-05
1.0E-04
1.0E-04
1.0E-04
1.0E-03
1.0E-03
1.0E-03
1.0E-101.0E-101.0E-101.0E-09
1.0E-09
1.0E-09
1.0E-08
1.0E-08
1.0E-08
1.0E-07
1.0E-07
1.0E-07
1.0E-06
1.0E-06
1.0E-06
1.0E-05
1.0E-05

U3
U3
U3
U3
U3
U3
U3

6
7
7
7
8
8
8

1.00E-04
1.00E-03
1.00E-03
1.00E-03
1.00E-02
1.00E-02
1.00E-02

30
30
30
30
30
30
30

50
50
50
50
50
50
50

10
10
10
10
10
10
10

10
10
10
10
10
10
10

100
100
100
100
100
100
100

1.0E-05
1.0E-04
1.0E-04
1.0E-04
1.0E-03
1.0E-03
1.0E-03

Control E2 (max)S

E2max1

1.00E-06

30

50

10

10

100

1.00E-07

2

Control E2 (max)S

E2max2

1.00E-06

30

50

10

10

100

1.00E-07

3

Control E2 (max)S

E2max3

1.00E-06

30

50

10

10

100

1.00E-07

B10

1

Control E2 (IC50)

S

E2IC501

E2IC50x10

30

50

10

10

100

E2IC50

B11

2

Control E2 (IC50)

S

E2IC502

E2IC50x10

30

50

10

10

100

E2IC50P1

B12

3

Control E2 (IC50)

S

E2IC503

E2IC50x10

30

50

10

10

100

E2IC50P1

C10

1

Control NE (max)S

Nemax1

1.00E-3.5

30

50

10

10

100

1.00E-4.5

P1

C11

2

Control NE (max)S

Nemax2

1.00E-3.5

30

50

10

10

100

1.00E-4.5

P1

C12

3

Control NE (max)S

Nemax3

1.00E-3.5

30

50

10

10

100

1.00E-4.5

P1

D10

1

Control NE (IC50)S

NEIC501

NEIC50 x10 30

50

10

10

100

NEIC50P1

D11

2

Control NE (IC50)S

NEIC502

NEIC50 x10 30

50

10

10

100

NEIC50P1

D12

3

Control NE (IC50)S

NEIC503

NEIC50 x10 30

50

10

10

100

NEIC50P1

E10

1

cold E2 (high)

NSB

S1

1.00E-05

30

50

10

10

100

1.0E-06

P1

E11

2

cold E2 (high)

NSB

S2

1.00E-05

30

50

10

10

100

1.0E-06

P1

E12

3

cold E2 (high)

NSB

S3

1.00E-05

30

50

10

10

100

1.0E-06

P1

F10

4

cold E2 (high)

NSB

S4

1.00E-05

30

50

10

10

100

1.0E-06

Unknown 3
Unknown 3
Unknown 3
Unknown 3
Unknown 3
Unknown 3
Unknown 3© OECD, (2015)

96

Unknown 2
Unknown 2
Unknown 2
Unknown 2
Unknown 2
Unknown 2
Unknown 2
Unknown 2
Unknown 2
Unknown 3
Unknown 3
Unknown 3
Unknown 3
Unknown 3
Unknown 3
Unknown 3
Unknown 3
Unknown 3
Unknown 3
Unknown 3
Unknown 3
Unknown 3
Unknown 3
Unknown 3
Unknown 3
Unknown 3

58

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493

P1

F11

5

cold E2 (high)

NSB

S5

1.00E-05

30

50

10

10

100

1.0E-06

P1

F12

6

cold E2 (high)

NSB

S6

1.00E-05

30

50

10

10

100

1.0E-06

P1

G10

1

total binding

TB

TB1

-

30

60

10

-

100

-

P1

G11

2

total binding

TB

TB2

-

30

60

10

-

100

-

P1

G12

3

total binding

TB

TB3

-

30

60

10

-

100

-

P1

H10

4

total binding

TB

TB4

-

30

60

10

-

100

-

P1

H11

5

total binding

TB

TB5

-

30

60

10

-

100

-

P1

H12

6

total binding

TB

TB6

-

30

60

10

-

100

-

© OECD, (2015)

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OECD/OCDEANNEX 4
Considerations for the Analysis of Data from the hrER Competitive Binding Assay

1.
The hrERα competitive binding assay measures the binding of a single concentration of [ 3H]17β-estradiol in the presence of increasing concentrations of a test chemical. The competitive binding
curve is plotted as specific [3H]-17β- estradiol binding versus the concentration (log10 units) of thecompetitor. The concentration of the test chemical that inhibits 50% of the maximum specific [3H]-17βestradiol binding is the IC50.
Data Analysis for the Reference Estrogen and Weak Binder (1)2.
Data from the control runs are transformed (i.e. percent [3H]-17β-estradiol specific binding andthe log concentration of the control chemical) for further analysis. Estimates of log(IC50) values for the
positive controls (e.g. reference estrogen and weak binder) should be determined using an appropriate
nonlinear curve fitting software to fit a four parameter Hill equation i.e.(e.g. BioSoft; GraphPad Prism) (2).
The top, bottom, slope, and log(IC50) can typically be left unconstrained when fitting these curves. Robust
regression should be used when determining the best fit unless justification is given. The method chosen forrobust regression should be stated. Correction for ligand depletion was not needed for the FW or CERI
hrER test methods, but may be considered if needed. Following the initial analysis, each binding curveshould be reviewed to ensure an appropriate fit to the model. The relative binding affinity (RBA) for the
weak binder can be calculated as a percent of the log (IC50) for the weak binder relative to the log (IC50)
for 17β-estradiol. Results for the positive controls and the non-binder control should be evaluated using
measures of assay performance and acceptability criteria as described in the PBTG (paragraph 20), Annex
2 (FW Assay, paragraphs 41-51) and Annex 3 (CERI Assay, paragraphs 41-51). Examples of 3 runs forthe reference estrogen and weak binder are shown in Figure 1.
Figure 1. Examples of the competitive binding curves for the reference estrogen and the control weak binder.

% Radioligand Bound

100

75

17
 -estradiol

50

Norethynodrel

25

0
-12

-11

-10

-9

-8

-7

-6

Log Concentration (Molar)© OECD, (2015)
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Data Analysis for Test Chemicals
3.
Data for all test chemicals should be analyzed using a step-wise approach to ensure that data
are appropriately analyzed and that each competitive binding curve is properly classified. Each run
for a test chemical should initially undergo a standardized data analysis that is identical to that used
for the reference estrogen and weak binder controls. Once completed, a technical review of the curve
fit parameters as well as a visual review of how well the data fit the generated competitive binding
curve for each run should be conducted. During this technical review, the observations of aconcentration dependent decrease in the percent [3H]-17β-estradiol specifically bound, low variability
among the technical replicates at each chemical concentration, and consistency in fit parameters
among the three runs are a good indication that the assay and data analyzes were conducted
appropriately. Professional judgment should be applied when reviewing results from each run for atest chemical, and the data used to classify each test chemical as a binder or non-binder should bescientifically defensible.
4.
Occasionally, there may be examples of data that require additional attention in order toappropriately analyze and interpret the hrER binding data. Previous studies had shown cases where
the analysis and interpretation of competitive receptor binding data can be complicated by an upturnof the percent specific binding when testing chemicals at the highest concentrations (Figure 2). Thisis a well-known issue that has been encountered when using protocols for a number of competitive
receptor binding assays (3). In these cases, a concentration dependent response is observed at lower
concentrations, but as the concentration of the test chemical approaches the limit of solubility, thedisplacement of [3H]17β-estradiol no longer decreases. In these cases, data for the higher
concentrations indicate that the biological limit of the assay has been reached. For example, thisphenomena is many times associated with chemical insolubility and precipitation at highconcentrations, or may also be a reflection of exceeding the capacity of the dextran-coated charcoal totrap the unbound radiolabeled ligand during the separation procedure at the highest chemical
concentrations. Leaving such data points in when fitting competitive binding data to a sigmoid curve
can sometimes lead to a misclassification of the ER binding potential for a test chemical (Figure 2).
To avoid this, the protocol for the FW and CERI hrER binding assays includes an option to exclude
from the analyses data points where the mean of the replicates for the percent [ 3H]17β-estradiol
specific bound is 10% or more above that observed for the mean value at a lower concentration (i.e.
This is commonly referred to as the 10% rule). This rule can only be used once for a given curve, andthere must be data remaining for at least 6 concentrations such that the curve can be correctlyclassified.

© OECD, (2015)

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OECD/OCDE

Figure 2. Examples, Competitive Binding Curves with and without Use of the 10% Rule.
E x a m p le A , A m b ig u o u s C u rv e F it C o rre c te d100

A m b ig u o u s c u r v e fit% R a d io lig a n d B o u n d

80

60

D a ta P o in t

40

d e le t e d
10%
R u le
U sed

20

0

-1 1

-1 0

-9

-8

-7

-6

-5

-4

-3

L o g C o n c e n tra tio n (M o la r)

5.
The appropriate use of the 10% rule to correct these curves should be carefully considered andreserved for those cases where there is a strong indication of a hrER binder. During the conduct ofexperiments for the validation study of the FW hrER Binding Assay, it was observed that the 10% rulesometimes had an unintended and unforeseen consequence. Chemicals that did not interact with thereceptor (i.e. true non-binders) often showed variability around 100% radioligand binding that were greaterthan 10% across the range of concentrations tested. If the lowest value happened to be at a low
concentration, the data from all higher concentrations could potentially be deleted from the analysis byusing the 10% rule, even though those concentrations could be useful in establishing that the chemical is a
non-binder. Figure 3 show examples where the use of the 10% rule is not appropriate.

© OECD, (2015)
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Figure 3. Examples, Competitive Binding Data Where Use of the 10% Rule is Not Appropriate.

E x a m p le A , U s e o f 1 0 % R u le N o t A p p r o p r ia te
120% R a d io lig a n d B o u n d

100

80

60

40

20

0

-1 1

-1 0

-9

-8

-7

-6

-5

-4

-3

L o g C o n c e n tr a tio n (M o la r)

E x a m p le B , U s e o f 1 0 %R u le N o t A p p r o p r ia te

120% R a d io lig a n d B o u n d

100

80

60

40

20

0

-1 1

-1 0

-9

-8

-7

-6

-5

-4

-3

L o g C o n c e n tra tio n (M o la r)© OECD, (2015)

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References

1. OECD (2015), Integrated Summary Report: Validation of Two Binding Assays Using Human
Recombinant Estrogen Receptor Alpha (hrERα), Health and Safety Publications, Series on Testingand Assessment (No. 226), Organisation for Economic Cooperation and Development, Paris.
2. Motulsky H. and Christopoulos A. (2003), The law of mass action, In Fitting Models to Biological
Data Using Linear and Non-linear Regression. GraphPad Software Inc., San Diego, CA, pp 187191. Www.graphpad.com/manuals/Prism4/RegressionBook.pdf
3. Laws SC, Yavanhxay S, Cooper RL, Eldridge JC. (2006), Nature of the Binding Interaction for 50
Structurally Diverse Chemicals with Rat Estrogen Receptors. Toxicological Sci. 94(1):46-56.

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