平成２５年度経済産業省委託事業 平成２５年度化学物質安全対策 （in vitro 簡易有害性試験法の利用促進調査） 調査報告書 平成２６年３月 住友化学株式会社 目 次 まえがき ・・・3 I. 事業の成果 ・・・4 1. 要約 ・・・4 2. 本文 ・・・5 （1）催奇形性スクリーニング in vitro 試験法の調査 ・・・5 （2）皮膚感作性スクリーニング in vitro 試験法の調査 ・・・29 （3）発がん性スクリーニング in vitro 試験法の調査 ・・・42 II. 研究発表・講演、文献、特許の状況（共同研究、再委託研究も含む。 ） ・・・50 1. 研究発表・講演 ・・・50 2. 文献 ・・・51 参考資料 ・・・53 2 まえがき 化学物質の適正管理のためには、様々な有害性評価を実施し、その毒性を把握すること が重要である。数万物質におよぶ化学物質の有害性を評価していくためには、精度の高い、 迅速かつ効率的なスクリーニングが欠かせず、特に、細胞や細菌等を活用した in vitro 試験 法は、動物試験の前段階のスクリーニングとして、短期間・低コストに実施できる手法と して期待されている。 現在、化学物質管理規制の中で、有害性物質検出のためのスクリーニング法として、in vitro 試験法が一部用いられている。しかしながら、化学物質の有害性は様々であり、それ ぞれの物質の有害性を評価するための、毒性メカニズム発現に基づく in vitro 試験法の開発 は未だ研究途上にあるといってよい。 このような状況の中で、我が国では最先端のバイオ技術を活用し、催奇形性、免疫毒性、 発がん性等をスクリーニングする新たな in vitro 試験法の開発が進められている。こうして 開発された新たな試験手法は、試験データの蓄積、専門家によるコンセンサス、試験従事 者の経験が十分でない等、開発には費用と試験期間を要すことから、民間ですぐに利用で きる段階まで進んでいない。特に、民間で普及・促進するためには開発された新しい試験 法の客観的評価と信頼性の確保が欠かせず、一定程度の試験データの蓄積、及びその試験 法の頑健性や予測性、再現性など、有用性に関する専門家のコンセンサスを得る必要があ る。 そこで、化学物質の迅速かつ効率的なスクリーニングに資する試験法として、新たに開 発された in vitro 試験法の民間利用を促進するため、催奇形性、免疫毒性、発がん性のそれ ぞれの in vitro 試験法についてバリデーション等を実施し、多くの化学物質を用いたデータ の蓄積を図るとともに、専門家による評価により新規試験法の有用性確認を進める。 3 Ⅰ．事業の成果 1. 要約 経済産業省では、化学物質の適正管理に向けた取組の一環として、有害性評価を迅速か つ効率的に実施できる試験法の開発や標準化に向けた取組を推進している。これまで我が 国で開発された有害性試験法である、催奇形性スクリーニング in vitro 試験法（マウス ES 細胞を用いた Hand1-Luc EST 法）を始め、皮膚感作性スクリーニング in vitro 試験法 （THP-G8 細胞による 2 色発光 IL-8 Luc アッセイ）や発がん性スクリーニング in vitro 試 験法（Bhas 42 細胞を用いた形質転換試験法）等は、低コスト・短期間にスクリーニング試 験を行うことが出来る方法として期待されている。平成 25 年度委託事業においては、住友 化学株式会社が受託し、これら試験法のバリデーション等テストガイドライン化に向けた 調査、取組等を行った。 催奇形性スクリーニング in vitro 試験法の調査については、現状の動物試験法及び in vitro 試験法について現状整理を行った。また、Hand1-Luc EST 法の国際バリデーション 試験を開始し、データの蓄積、催奇形性試験法、又は代替試験法等の国内外の専門家の評 価等を踏まえたプロトコール（案）を作成した。また、民間での利用促進に向けて、事業 者において今後必要となる取組等を整理し、とりまとめた。 皮膚感作性スクリーニング in vitro 試験法の調査については、現状の動物試験法及び in vitro 試験法について現状整理を行った。また、2 色発光 IL-8 Luc アッセイの国際バリデー ション試験を継続し、データの蓄積、催奇形性試験法、又は代替試験法等の国内外の専門 家の評価等を踏まえたプロトコール（案）を作成した。また、民間での利用促進に向けて、 事業者において今後必要となる取組等を整理し、とりまとめた。 発がん性スクリーニング in vitro 試験法の調査については、現状の動物試験法及び in vitro 試験法について現状整理を行った。一方 Bhas 42 細胞を用いた形質転換試験法（Bhas 42 CTA）について平成 25 年 9 月 OECD にテストガイドライン案を提出し、関係者から収 集された意見を平成 26 年 1 月開催の専門家会議（Expert meeting）にて回答し、理解を得 た。この他、この専門家会議では SHE cell を用いた形質転換試験法と共に、技術的内容等 について討議された。 4 2. 本文 （1）催奇形性スクリーニング in vitro 試験法の調査 ① 催奇形性試験法の現状整理 我々の身の回りには、工業製品、化粧品、農薬、医薬品などの多数の化学物質が存在す る。現在、これらのヒトへの安全性を高精度に評価するために、多くの実験動物を用いた 毒性試験が行われている。安全性評価の中で生殖発生毒性試験が対象としている項目は、 配偶子（精子、卵子）の形成および生殖機能（受胎、妊娠維持、分娩、哺育）ならびに次 世代の発生および成長といった世代を越えた幅広いものである。1961 年に発生したアザラ シ肢症などを主徴としたサリドマイド禍をきっかけに、世界各国で化合物の登録申請の際 に生殖発生毒性試験の重要性が認識されるようになった。特に、次世代（胎児）の形態的 な異常を検出する催奇形性試験は生殖発生毒性試験の中で最も重要視されている。毒性試 験の中でも胎児への影響を評価する催奇形性試験は特に時間と多くの動物や経費が必要な 試験の一つである。 近年、動物福祉の観点から、動物細胞や微生物などを用いた in vitro 試験法は、短期間に 少量の化合物で評価できる簡便な試験法であり、化学物質の安全性評価の効率化に大きく 貢献するため、世界的に研究が進められている。胎児は子宮の中で胎盤を介して母体と密 接に関わっており、 個体発生は母体の代謝や生理学的変動に影響されるため、 単純な in vitro 系で評価するには限界がある。しかしながら評価項目としての重要性から、in vitro 試験系 を開発初期の段階でスクリーニングレベルで利用することができれば、開発の効率化を図 る上で非常に有効であるため、古くから様々な手法が考案されてきた。以下ように複数の in vitro 試験方が開発されている（表 1-1） 。 表 1-1 催奇形性 in vitro 試験法 Cells lines Study MOT Assay Material Ascitic mouse ovarian tumour cells Index Inhibition of cell attachment to lectin-coated sufases Reference Braun et al., 1982 HEPM Assay Human embryonic palatal Cell proliferation mesenchyme cellsmesenchyme cells Pratt et al., 1985 Gap junction Chinese hamster ovary cells Inhibition of gap junction Trosko et al., 1982 Embryonic Stem cell test Mouse enbryonic stem cells Mouse fibroblast cells Cardiomyocyte differentiation Cell proliferation Spielmann et al., 1997 Primary Cultures Micromass culture Rat embryo (limb bud) Cartilage differentiation Cell proliferation Flint et al., 1984 Organs Palate culture Mouse embryo (palate) Fusion of secondary palate Shiota et al., 1990 Limb bud culture Mouse embryo (limb bud) Cartilage differentiation Morphology Freedman et al., 1982 Whole embryo culture Rat embryo Morpholgenetic differentiation Schmid et al., 1985 Whole embryo culture 一方で、個体発生は様々な分化の過程を経て成立するため、一般的な培養細胞等を用いた 5 方法で発生毒性を評価することは難しく、現在も良い in vitro 試験は存在しないため、世界 的に開発が切望されていた。In vitro 発生毒性試験においては、ガイドライン化を目指し 1996～2000 年に欧州代替法センター（ECVAM）が中心となりマウス ES 細胞を用いた試 験法（EST: Embryonic Stem cell Test）、ラット胎児の肢芽を用いる小塊培養法(Micromass test)及びラットの初期胚を用いた全胚培養法(rat whole-embryo culture test )の検証試験 が実施された（ATLA 30, 151-176, 2002, Elke Genschow et al.） 。3 種の試験は動物実験と 70%以上の一致率が認められ、小塊培養法および全胚培養法は動物を使用するが、EST は 培養細胞を用いる点で汎用性の高い方法として注目された。しかし、EST は 2001 年に ECVAM の諮問機関から代替試験法として提案されたが、OECD のガイドライン化には至 らなかった。その理由として、①様々な毒性メカニズムをもつ多種類の被験物質の検証、 ②判定法の改良、③神経や骨など心筋以外の細胞への分化誘導系の導入、④代謝の評価の 導入等の必要性があげられた。さらに、EST の評価方法が心筋細胞の拍動の有無を顕微鏡 下で観察するため、煩雑かつ熟練した技術・ノウハウを必要とし汎用性に欠けることや、 多数の検体を同時に扱うことが難しいという欠点も指摘された。 このような状況下、世界的に EST の改良化の研究が実施された。改善面ごとにまとめ以 下に示した。 ・心筋分化の定量指標の改善 心筋特異的タンパクの FACS による分化阻害効果の定量化 （Toxicol Sci. 2009 108(2): p389-400 Buesen R. et al ） 心筋の活動電位を指標とした分化阻害効果の定量化 （J Toxicol Sci. 2010 : p899-909 Koseki N. et al） ・神経および骨細胞分化誘導の導入 Toxicol Appl Pharmacol. 2012 262(3): p330-40 Methods Mol Biol. 2012 889: p147-79. Theunissen PT. et al Kuske B. et al しかしながら、何れも少数の被験物質数の結果を例示するのみで催奇形性を予測するまで に至っておらず、ガイドライン化を目的としたバリデーション試験は実施されていない。 このような背景下、簡便で汎用性の高い発生毒性試験法の開発を目指し、2006-2010 年 NEDO 委託事業「高機能簡易型有害性評価手法の開発／培養細胞を用いた有害性評価手法 の開発／催奇形性予測試験法の開発」において原理的、手法的にも異なる新しい NEDO-EST 法を開発した。 NEDO-EST 法は、発生毒性の指標となるマーカー遺伝子の探索により同定した Hand1 や Cmya1 遺伝子（Suzuki et al., J.Toxicol.Sci.,2011）の発現量を、ルシフェラーゼの発光 量で簡便に測定可能な組換え ES 細胞（Hand1-ES および Cmya1-ES 細胞）を用いて発生 6 毒性を予測する方法である。化合物の心筋細胞への分化に対する影響、ES 細胞の増殖に対 する影響の検討、繊維芽細胞の増殖に対する影響の検討の 3 試験を実施して ID50 値、IC50 値を算出し、Prediction model（PM）式に代入して発生毒性を予測する。心筋分化を用い る Hand1-および Cmya1-EST 法は従来の EST 法と同等以上の予測性を有し、短期間(約１ 週間)に多数の化合物を評価可能な試験であった（Suzuki et al., Toxicol. Sci.,2011)。レポー タージーンアッセイにより化合物の影響を簡便に測定できることから、従来型の EST 法や、 他研究者らによって提案されている改良 EST よりも多数の被験物質を一度に評価すること ができる有用な試験法である。一方、Hand1-EST を含めた従来の EST は、代謝や胎盤移 行性の影響を考慮できない等の点から、発生毒性の動物実験代替法としてのテストガイド ライン化には改良の必要性が指摘されていた。そこで、我々はテストガイドライン化を視 野に入れた精度向上を目的に Hand1-EST 法に改良を加え、新たな予測試験法である Hand1-Luc EST を開発中である。従来、ES-D3 細胞株を親細胞として安定形質転換細胞 株 Hand1-ES 細胞を樹立したが（Suzuki et al., Toxicol. Sci., 2011） 、将来、第三者機関に 配布しやすいように権利上に問題のないマウス ES 細胞（KOB1-ES 株）を親細胞として Hand1-ES (KOB1) 細胞を作製した。Hand1-ES(KOB1)細胞は心筋分化後、ルシフェラー ゼ活性を測定した結果、Hand1-ES 細胞と同等の性質を有することが確認されたため、本 細胞をバリデーション試験に利用することとした。 ② データの蓄積 1) 国際バリデーション試験の実施概要 Hand1-Luc EST の国際バリデーション開始に先立ち、バリデーションの組織委員会 （Validation management team: VMT）を組織した。国内外の催奇形性試験法または代替 法試験の専門家からなる VMT メンバー、およびコンサルティングメンバーは以下のとおり である（表 1-2 および 1-3）。 表 1-2 Hand1-Luc EST VMT メンバー Name Role and expertise Affiliation Trial Coordinator Noriho Tanaka VMT Chair, HRI/FDSC and OTIP Lead Laboratory Koichi Saito Hirohisa Nagahori Noriyuki Suzuki Le Coz Florian Developer of this assay and expertise embryonic toxicty Sumitomo Chemical Co. Ltd., Kazuhiko Matsumoto QC and toxicology expertise Consultant, Gakushuin univ. 7 Makiko Kuwagata Chemical selection and reprotox expertise HRI/FDSC Hajime Kojima Management of quality control, Chemical selection and chemical supply JaCVAM, NIHS (JaCVAM representative) Takashi Omori Data analysis and biostatistics dossier Doshisha Univ. Liaison members ICCVAM liaison - Lori Rinckel - David Allen - Warren Casey -Multi-study validation expertise -Multi-study validation expertise -Director (NICEATM) NICEATM / ICCVAM, USA ECVAM liaison Michael Schaeffer Multi-study validation expertise EURL ECVAM, Italy KoCVAM liaison Eui-Bae Jeung Test system expertise KoCVAM, Korea ZEBET liaison Andrea Seiler Multi-study validation expertise, BfR / ZEBET, Germany Reproductive toxicity expertise - HRI/FDSC：Hatano Research Institute/Food and Drug Safety Center - JaCVAM：Japanese Center for the Validation of Alternative Methods - NICEATM / ICCVAM：The National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) and the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) - EURL ECVAM：The European Union Reference Laboratory for alternatives to animal testing - KoCVAM：Korean Center for the Validation of Alternative Methods - BfR / ZEBET：Bundesinstitut für Risikobewertung /The Centre for Documentation and Evaluation of Alternatives to Animal Experiments - AIST：The National Institute of Advanced Industrial Science and Technology 表 1-3 コンサルティングメンバー 氏名 Hajime Kojima Makiko Kuwagata Kazuhiko Matsumoto 役割 所属 Management of Chemicals selection, repository, coded and distribution 8 JaCVAM HRI/FDSC Consultant Yoshihiro Ohmiya Document checking for *bioluminescenece measurement by luminometer and for **the other data in data sheets AIST Takashi Omori Mayumi Kobayashi Aoi Maruya Azusa Mori Manabu Nishio Data cleaning and data analysis Doshisha Univ., Japan The strategic decisions will be taken by the VMT only. 2013 年 2 月に初めて VMT 会議を開催し、試験プロトコールの確認、study plan の策定、 評価被験物質数について議論を重ねた。会議開催日と主な議題を以下に示した。 内容 実施年月 2012 年 12 月～ 2013 年 2 月 バリデーション委員会の組織、試験評価施設の決定、study plan の策定、被験物質の選定、試験プロトコール準備 2013 年 2 月 21 日～2 月 22 日 1st VMT 会議 (大阪): 試験スケジュールの確認、study plan の策定、被験物質の選 定、試験プロトコールの改善点、試験成立条件の必要性 2013 年 4 月 30 日～5 月 1 日 2013 年 6 月 18 日～6 月 19 日 2nd VMT 会議 (京都): 試験スケジュールの確認、試験プロトコールの改善点、試験 成立条件 3rd VMT 会議 (京都): 試験プロトコールの改善点、暫定試験成立条件、試験スケジ ュールの確認、Phase 0 試験開始の合意 2013 年 6 月 19 日 実施施設への技術移転（大阪） 2013 年 7 月～8 月 Phase 0 試験: 3 施設、陽性対照および 3 物質による transferability の確認 2013 年 9 月 22 日～9 月 23 日 4th VMT 会議(京都): Phase 0 試験の検証および phase 1 試験の計画 2013 年 11 月 6 日 電話会議: プロトコールの最適化に関する報告 2013 年 11 月～ 2014 年 1 月 Phase 1 試験: 3 施設、陽性対照およびコード化 3 物質による施設内再現性 の確認 リードラボによる陽性陰性の判定式の構築 2014 年 2 月 19 日～2 月 21 日 5th VMT 会議(京都): Phase 1 試験の検証および phase 2 試験の計画 9 2) Hand1-Luc EST の特徴およびプロトコールの修正点 国内外専門家を含めた会議をする過程で試験プロトコールを改善した。主な改善点を以下 にまとめた。 1. 96well plate デザインの変更 96 well プレートの周縁部は中心部に比べ培地が蒸発し、細胞の状態や反応に影響を及ぼす ことが知られている（edge 効果と呼ぶ） 。本誌試験では 37℃、CO2 インキュベーターで 5 から 6 日間培養することから、培地量が周縁部のウェルと中心のウェルで異なることが懸 念される。従来法では、周縁部も利用した試験を計画していたが、edge 効果の懸念を排除 するため、周辺部を試験には使用しないように修正した。さらに培地の蒸発を防止するた め、周縁部には培地と等量の PBS(-)緩衝液を入れることとした。さらに、溶媒対照（Vehicle control: VC）と被験物質以外に、試験細胞の状態を確認する目的で、培地のみで培養する 培地コントロール（Medium control: MC）を設定することとした。この修正に伴い、2 化 合物／1 プレート、試験濃度 7 点の計画を修正し、1 化合物／1 プレート、試験濃度 7 点で 実施することとした（図 1-1） 。 【変更前】 Chemical 2 Chemical 1 A B C D E F G H 1 VC Conc.1 Conc.2 Conc.3 Conc.4 Conc.5 Conc.6 Conc.7 2 → → → → → → → → 3 → → → → → → → → 4 → → → → → → → → 5 → → → → → → → → 6 → → → → → → → → 7 VC Conc.1 Conc.2 Conc.3 Conc.4 Conc.5 Conc.6 Conc.7 8 → → → → → → → → 9 → → → → → → → → 10 → → → → → → → → 11 → → → → → → → → 12 → → → → → → → → 1 2 3 4 5 6 7 8 9 10 11 12 VC: Vehicle control Conc.: Concentration 【変更後】 A B MC VC Conc.1 Conc.2 Conc.3 Conc.4 Conc.5 Conc.6 Conc.7 BG MC: Mediume control C ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ VC: Vehicle control D ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ Conc.: Concentration E ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ BG.: Background F ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ G ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ H Cell (+) Cell (-) PBS (-) 図 1-1 96 well plate デザインの変更概要 2. 1 プレート法による IC50 と ID50 の同時測定 バリデーション開始当初、Hand1-ES 細胞により胚葉体（EB）を形成させて培養する分化 10 阻害試験（differentiation assay）と、EST 法に準じ ES 細胞の平面培養による細胞毒性試 験（cytotoxicity assay）を計画した（図 1-2） 。 Hand1-ES (KOB1) cells Test compounds Day 0 Cytotoxicity assay Differentiation assay 96well plate white U96 well plate Day 6 Cell viability assay (luminescence) Luc-activity (luminescence) ES-ID50 (Differentiation toxicity) ES-IC50 (ES/cytotoxicity) 図 1-2 Hand1-Luc EST の概要 VMT メンバーより推奨された 20 化合物をリードラボにて 3 回試験し IC50、ID50 値を集計 した結果、ID50 よりも IC50 が小さいケースが散見された。この結果を受け、VMT メンバー からは、異なる培養条件で ID50 と IC50 を比較するよりも、分化阻害試験と同じ条件にて培 養した細胞に対して IC50 を取得するように提言を受けた。この提案に従い、2 つの実験条 件を検討した。2 枚のプレートに播種したサンプルをルシフェラーゼアッセイと生細胞の ATP 量を CellTiter Glo（プロメガ）により定量する方法（2 プレート法）と、1 枚のプレ ートに播種したサンプルに、CellTiter Fluor（プロメガ）を用いて、生細胞に取り込まれた 蛍光物質の量を測定し、その後、同一のサンプルを用いてルシフェラーゼ活性を測定する 方法（1 プレート法）を比較した（図 1-3） 。 11 Day 0 Day 6 Hand1-ES cells Hand1-ES cells Test compounds Test compounds Two plates One plate white U96 well plate white U96 well plate CellTiter Glo IC50 CellTiter Fluor IC50 Steady-Glo (luciferase) ID50 Steady-Glo (luciferase) ID50 図 1-3 検討した 2 試験法の概要 その結果、表 1-4 に示すとおり、1)改良したいずれの化合物でも ID50 は IC50 値よりも同等 または小さく、2)1 および 2 プレート法の IC50 はいずれの測定方法でも同程度の結果にな ることが確認された。 表 1-4 1 プレート法および 2 プレート法の結果 Previous method IC50 (ug/mL) ID50 (ug/mL) Steady GLO In vivo Category Test chemicals CellTiter GLO positive positive positive positive positive negative negative 5-Fluorouracil Hydroxyurea Ara C VPA Boric acid Acrylamide Isoniazid 0.06 5.69 0.03 151.56 135.39 43.02 99.86 = 0.06 > 4.31 < 0.06 > 134.69 > 52.59 < 71.60 < 384.22 Modified method 1 plate 2 plates IC50 ID50 (ug/mL) (ug/mL) IC50 (ug/mL) ID50 (ug/mL) CellTiterFluor CellTiter + Fluor SteadyGLO CellTiter GLO Steady GLO 0.04 6.39 0.06 213.93 65.49 >200 446.25 0.06 10.65 0.10 160.04 99.39 >200 520.20 > 0.03 > 4.04 = 0.06 > 66.42 > 53.56 > 130.56 > 346.06 > > = > > > > 0.03 4.35 0.06 84.59 61.38 199.08 320.55 プレート間での誤差を解消し、 上述のように 1 つのプレートで IC50 と ID50 が求められれば、 実験操作も簡略化することができる。そのため、バリデーション試験では 1 つのプレート に播種し分化誘導した Hand1-ES 細胞を用いて、CellTiter Fluor による細胞毒性を測定し た後、Steady-Glo luminescent assay kit（プロメガ）によりルシフェラーゼ活性を測定す ることとした。 12 3. 測定時間の変更 Hand1 遺伝子は分化誘導に伴い約 6 日目に上昇し、7 日目以降は減弱する。同様に Hand1-ES 細胞を分化誘導した場合ルシフェラーゼ活性は約 6 日目に上昇し、以降は減弱 するため、分化 6 日目に測定する計画であった。バリデーション試験において施設内、施 設間差を最小限にするためには、より厳密に最適な測定時間を決定する必要がある。詳細 にルシフェラーゼ活性の時間推移を検討した結果、分化 120 時間後が最も活性が高く、そ れ以降では減少期にあることが分かった（図 1-4） 。 2000000 1800000 1600000 1400000 1200000 1000000 800000 600000 400000 200000 0 Mean value of the 3 experiments± SD CellTiter Fluor 3500 Steady-Glo Day 5 Day 4 Day 6 3000 2500 2000 1500 1000 500 0 24 96 120 Time after seeding 144 192 (hrs) 0 図 1-4 ルシフェラーゼ活性および細胞数の経時的変動 そこで、測定時間を分化 120 時間後のプロトコールに決定し、代表化合物 20 点について Hand1-Luc EST 試験を実施した。Methoxyacetic acid、Lithium chloride 、Hydroxyurea 6-Aminonicotinamide の結果を以下に示した（図 1-5） 。 Day 6 protocol Differentiation tox Relative activity 150 300 100 200 100 0 0 50 0 15.6 31.3 62.5 125 250 500 1000 0 15.62531.2562.5 125 250 500 1000 300 100 200 50 100 0 IC50/ID50 = 1.28(486.50/379.24) 150 400 Relative activity Relative activity IC50/ID50 = 0.83(396.31/476.07) 0 0 15.6 31.3 62.5 125 250 500 1000 0 15.62531.2562.5 125 250 500 1000 Relative activity IC50/ID50 = 0.88(414.13/471.33) 500 400 100 300 200 100 0 IC50/ID50 = 1.50(490.44/327.72) 150 Relative activity Cytotoxicity Relative activity Methoxyacetic acid 120 hrs protocol 400 0 15.6 31.3 62.5 125 250 500 1000 50 0 0 15.62531.2562.5 125 250 500 1000 IC50/ID50 = 0.81(390.46/480.16) IC50/ID50 = 1.65(599.33/362.44) 13 Differentiation tox 200 Relative activity Cytotoxicity 120 hrs protocol Day 6 protocol 900 800 700 600 500 400 300 200 100 0 Relative activity LiCl 0 150 100 50 0 15.62531.25 62.5 125 250 500 1000 0 IC50/ID50 = 1.02(1000/984.80) 150 Relative activity Relative activity 200 150 100 100 50 0 15.62531.25 62.5 125 250 500 1000 IC50/ID50 = 1.86(686.97/ 369.98) 0 50 0 15.62531.25 62.5 125 250 500 1000 0 15.62531.25 62.5 125 250 500 1000 IC50/ID50 = 1.84(635.29/ 345.69) IC50/ID50 = 1.11(1000/903.93) 150 Relative activity Relative activity 500 400 100 300 200 100 0 0 50 0 15.62531.25 62.5 125 250 500 1000 0 Cytotoxicity Differentiation tox 350 300 250 200 150 100 50 0 15.62531.25 62.5 125 250 500 1000 IC50/ID50 = 1.96(681.93/ 347.68) 120 hrs protocol Day 6 protocol 150 Relative activity Hydroxyurea Relative activity IC50/ID50 = 1.04(1000/961.81) 100 50 0 0 0.31250.625 1.25 2.5 5 10 0 20 Relative activity Relative activity 200 50 50 0 0 0.31250.625 1.25 2.5 5 10 0 20 150 100 50 50 0.31250.625 1.25 2.5 5 10 0 20 IC50/ID50 = 1.25(4.98/3.99) 5 10 20 120 hrs protocol Day 6 protocol 150 Relative activity Relative activity 0.31250.625 1.25 2.5 IC50/ID50 = 1.65(4.63/2.80) 100 50 0 0 0.078125 0.15625 0.3125 0.6251.25 2.5 5 0 0.078125 0.15625 0.31250.625 1.25 2.5 5 IC50/ID50 = 1.84(1.56/0.85 ) IC50/ID50 = 1.00(1.10/1.10) 150 Relative activity 150 Relative activity 20 0 0 100 50 0 0.078125 0.15625 0.3125 0.6251.25 2.5 100 50 0 5 0 0.078125 0.15625 0.31250.625 1.25 2.5 IC50/ID50 = 1.27(1.15/0.90) 5 IC50/ID50 = 1.53(1.28/0.84) 150 Relative activity Relative activity 150 100 50 0 10 100 0 0 5 150 Relative activity Relative activity 200 Differentiation tox 0.31250.625 1.25 2.5 IC50/ID50 = 2.16(7.40/3.43) IC50/ID50 = 1.32(6.28/4.77 ) Cytotoxicity 20 100 100 250 200 150 100 50 0 10 150 150 6-Aminonicotinamide 300 5 200 250 0 0.31250.625 1.25 2.5 IC50/ID50 = 2.78(5.99/2.15) IC50/ID50 = 1.17(5.56/4.75 ) 0 0.078125 0.15625 0.3125 0.6251.25 2.5 100 50 0 5 IC50/ID50 = 1.31(1.15/0.88) 0 0.078125 0.15625 0.31250.625 1.25 2.5 5 IC50/ID50 = 1.28(1.12/0.87) 14 図 1-5 代表化合物の試験結果（従来法と 120 時間プロトコールの比較） 分化 6 日後測定のプロトコールでは、ルシフェラーゼ活性が濃度非依存的な上昇を示して いたが、分化 120 時間後の測定プロトコールに変更した場合、いずれの化合物でも先述の 現象が解消された。20 化合物の IC50、ID50 をまとめ、3 試験の平均値、標準偏差、CV（標 準偏差／平均値）をまとめた（表 1-5） 。IC50 および ID50 の CV 値が低いことから細胞毒性 試験および分化阻害試験の施設内再現性が高く、発生毒性陽性の化合物は、ID50 が IC50 よ りも小さくなる傾向が認められた。 表 1-5 従来法と 120 時間プロトコールの比較 Day 6 IC50(µg/mL) in vivo Posi Posi Posi Posi Posi Posi Posi Posi Posi Posi Posi Posi Posi Posi Posi Posi Nega Nega Nega Nega Nega Chemicals 5-FU 5-FU Hydroxyurea 6-AN Cytosine arabinoside Methotrexate RA 5-Bromo-2'-deoxyuridine VPA Dexamethasone Boric acid Methoxyacetic acid Lithium chloride Dimethadione Phenytoin Caffeine Isoniazid Ascorbic Acid Diphenhydramine HCl Dimethyl phthalate Acrylamide > > > > 1st 0.03 0.02 5.56 1.10 0.05 0.03 0.005 0.43 106.54 27.54 65.77 396.31 1000.00 1000.00 250.00 203.96 397.60 463.93 27.14 391.67 117.97 120hours (Day 5) in vivo Posi Posi Posi Posi Posi Posi Posi Posi Posi Posi Posi Posi Posi Posi Posi Posi Nega Nega Nega Nega Nega > > > > > 2nd 0.03 0.03 6.28 1.15 0.05 0.04 0.005 0.33 147.41 24.83 71.12 414.13 1000.00 1000.00 250.00 250.00 278.59 706.68 19.05 406.18 112.59 Chemicals 1st 2nd 5-FU 0.03 0.04 5-FU 0.02 0.04 Hydroxyurea 5.99 7.40 6-AN 1.56 1.28 Cytosine arabinoside 0.06 Methotrexate 0.04 0.04 RA > 0.005 > 0.005 5-Bromo-2'-deoxyuridine 0.40 0.29 VPA 217.49 160.99 Dexamethasone 23.74 26.90 Boric acid 83.03 85.09 Methoxyacetic acid 486.50 490.44 Lithium chloride 686.97 635.29 Dimethadione > 1000.00 > 1000.00 Diphenylhydantoin(=phenyto> 500.00 > 500.00 Caffeine > 250.00 245.90 Isoniazid 316.56 190.93 Ascorbic Acid 51.499 41.47 Diphenhydramine HCl 35.39 20.51 Dimethyl phthalate > 500.00 353.00 Acrylamide 75.72 57.43 > > > > > 3rd 0.03 0.04 4.98 1.15 0.05 0.05 0.005 0.35 97.19 26.08 58.34 390.46 1000.00 1000.00 250.00 195.15 343.67 732.19 16.95 500.00 119.73 mean 0.03 0.03 5.61 1.13 0.05 0.04 0.005 0.37 117.05 26.15 65.08 400.30 1000.00 1000.00 250.00 216.37 339.95 634.27 21.05 432.62 116.76 > > > > ID50(µg/mL) SD 0.002 0.01 0.65 0.03 0.002 0.01 0.06 26.71 1.35 6.42 12.33 29.46 59.59 148.07 5.38 58.80 3.72 CV 8 27 12 3 3 21 15 23 5 10 3 14 18 23 26 14 3 1st 0.04 0.03 4.75 1.10 0.06 0.02 0.000001 0.44 78.46 27.94 51.56 476.07 984.80 > 1000.00 10.33 136.69 387.06 596.66 11.89 24.47 86.99 2nd 0.04 0.04 4.77 0.90 0.08 0.04 0.00010 0.40 136.52 28.89 88.77 471.33 903.93 > 1000.00 14.39 86.61 377.81 938.44 20.87 59.86 83.25 1st 0.03 0.02 2.15 0.85 0.02 0.000031 0.15 132.80 17.97 69.03 379.24 369.98 410.68 14.61 170.43 363.88 46.16 3.82 64.16 88.09 2nd 0.04 0.03 3.43 0.84 0.03 0.02 0.00008 0.22 78.90 30.10 61.38 327.72 345.69 767.54 16.27 156.83 214.54 42.85 6.78 69.93 71.90 IC50(µg/mL) > > > > 3rd 0.03 0.04 4.63 1.12 0.05 0.04 0.005 0.39 173.99 27.36 83.50 599.33 681.93 1000.00 500.00 193.06 241.32 33.90 46.13 500.00 66.05 > > > mean 0.03 0.03 6.01 1.32 0.06 0.04 0.005 0.36 184.15 26.00 83.87 525.42 668.06 1000.00 500.00 229.65 249.60 42.29 34.01 451.00 66.40 3rd 0.04 0.04 3.99 0.88 0.06 0.03 0.000085 0.39 71.22 37.36 49.51 480.16 961.81 > 1000.00 10.05 109.92 467.86 877.82 10.37 38.34 88.18 mean 0.04 0.04 4.50 0.96 0.07 0.03 0.000063 0.41 95.40 31.40 63.28 475.85 950.18 > 1000.00 11.59 111.07 410.91 804.31 14.38 40.89 86.14 IC50/ID50 SD 0.002 0.01 0.45 0.12 0.01 0.01 0.000054 0.03 35.80 5.18 22.10 4.42 41.67 2.43 25.06 49.53 182.36 5.67 17.83 2.57 CV 5 16 10 13 12 31 86 6 38 17 35 1 4 21 23 12 23 39 44 3 1st 0.72 0.71 1.17 1.00 0.81 1.51 3356.79 0.98 1.36 0.99 1.28 0.83 1.02 1.00 24.19 1.49 1.03 0.78 2.28 16.00 1.36 ID50(µg/mL) SD 0.006 0.012 1.388 0.224 0.011 0.002 0.064 29.590 1.968 1.079 64.038 28.496 31.756 63.223 8.827 12.865 84.869 9.151 CV 16 36 23 17 19 5 18 16 8 1 12 4 14 25 21 38 19 14 3rd 0.03 0.03 2.80 0.87 0.03 0.02 0.000037 0.19 88.13 26.75 67.88 362.44 347.68 402.33 12.94 125.85 320.14 42.37 7.58 104.67 75.31 2nd 0.91 0.84 1.32 1.27 0.63 1.10 48.92 0.81 1.08 0.86 0.80 0.88 1.11 1.00 17.37 2.89 0.74 0.75 0.91 6.79 1.35 3rd 0.79 0.96 1.25 1.31 0.73 1.36 58.66 0.89 1.36 0.70 1.18 0.81 1.04 1.00 24.88 1.78 0.73 0.83 1.63 13.04 1.36 mean 0.80 0.85 1.25 1.18 0.72 1.28 79.39 0.89 1.23 0.83 1.03 0.84 1.05 21.57 1.95 0.83 0.79 1.46 10.58 1.36 IC50 / ID50 mean 0.03 0.03 2.79 0.85 0.03 0.02 0.00 0.18 99.94 24.94 66.10 356.47 354.45 526.85 14.61 151.04 299.52 43.79 6.06 79.59 78.43 SD 0.005 0.008 0.641 0.020 0.004 0.002 0.000 0.036 28.825 6.261 4.123 26.277 13.489 208.487 1.666 22.846 76.775 2.064 1.982 21.916 8.539 CV 17 30 23 2 12 9 51 19 29 25 6 7 4 40 11 15 26 5 33 28 11 1st 1.02 1.08 2.78 1.84 1.83 161.93 2.76 1.64 1.32 1.20 1.28 1.86 2.43 34.22 1.47 0.87 1.12 9.27 7.79 0.86 2nd 1.08 1.28 2.16 1.53 1.93 1.98 65.80 1.33 2.04 0.89 1.39 1.50 1.84 1.30 30.72 1.57 0.89 0.97 3.03 5.05 0.80 3rd 1.22 1.20 1.65 1.28 1.76 1.77 133.67 2.08 1.97 1.02 1.23 1.65 1.96 2.49 38.63 1.53 0.75 0.80 6.09 4.78 0.88 mean 1.10 1.21 2.15 1.55 1.85 1.86 103.98 1.96 1.84 1.04 1.27 1.47 1.88 1.90 34.23 1.52 0.83 0.97 5.61 5.67 0.85 IC50 / ID50 < 1.0 3) フェーズ 0 試験 リードラボ（住友化学株式会社）による技術移管説明会を実施した後、試験プロトコール の技術易移転性（transferability）の確認と、試験成立条件の基礎データを取得しプロトコ ールを最適化する目的で、陽性対照物質 5-Fruorouracil （5-FU）および非コード化 3 物質 （Ascorbic acid：AA、Boric acid：BA、 Hydroxyurea：HU）の 1 判定試験を 3 回実施し た。 1. 被験物質 15 非コード化の 3 物質は、化合物管理チームより配布され、各施設で適切に保管した。プロ トコールに従い、一部を秤量して希釈溶媒 PBS (-) を選択し、最大溶解濃度を決定した。 2. 試験方法 共通プロトコール（Hand1 Luc EST バリデーションプロトコール ver. 04E）に準じて独立 した 4 回（dose finding study を 1 回、definitive study を 3 回）の試験を実施した。1 試 験に陽性対照物質および非コード物質を実施した。 化合物添加直後および約 6 日目後に、CellTiter Fluor Cell Viability Assay (Promega, Cat. #G6082)を添加し、30 分インキュベートした後、各施設の保有する機器にて蛍光測定した。 その後、Steady-Glo Luciferase Assay System (Promega, Cat. #X1006) を添加し、30 分 インキュベートし、1 sec/well の条件下で化学発光測定した。測定終了後、測定値を計算用 エクセルデータシートに代入し、蛍光と化学発光値を算出し、自動計算により IC50 および ID50 の算出を行った。何れの試験においても陽性対照物質 5-FU を評価し、試験成立基準を 満たすことを確認した。試験ごとに、予め指定されたチェック項目（試薬管理の記録、細 胞調製手順、被験物質の調製手順、析出有無の確認、測定手順）を Hand1-Luc EST 記録 用紙（QC チェックシート（Hand1-Luc Ph0 study）ver1.1.xls）に記載した。 3. Hand1-Luc EST における試験成立の判定基準 (1) Performance standard of assay それぞれ一回の実験で，陽性対象物質 5-FU が下記の条件を満たすことを絶対条件 とする。 IC50 : 0.008 – 0.200 μg/mL ID50 : 0.006 – 0.150 μg/mL (2) Quality control-1 トリパンブリ―染色による細胞生存率が 90%以上 (3) Quality control-2 陽性対象物質 5-FU およびコード物質曝露時の各 96 ウェルプレート内で下記の条 件を全て満たす。 A. 化合物添加直後 (Day0) と約 6 日目（Day6）の蛍光測定の結果，medium control (MC) in Day6 / MC in Day0 比の平均値の 95%信頼区間の下限が 10 より 大きい B. 上記 A の蛍光測定後の化学発光測定の結果，MC in Day6/ MC in Day0 比の平 均値の 95%信頼区間の下限が 100 より大きい 16 C. Day6 の蛍光測定および化学発光測定の結果，vehicle control (VC) / MC 比の平 均値が 0.7 より大きい D. Day6 の蛍光測定および化学発光測定の結果，vehicle control (VC) / BG 比の 95%信頼区間の下限が 0.2 より大きい 全試験結果は、確証として出力データとともにデータ管理チーム（同志社大学大森研究室） に提出され、適切に解析された。 4.結果 陽性対照 5-FU、非コード 3 物質の各施設（A～C）の結果を以下に示した。5-FU はいずれ の施設においても、リードラボの IC50 値 0.03±0.01 μg/mL、および ID50 値 0.04 ± 0.004 μg/mL に近接した値が得られた（表 1-6）。 表 1-6 各施設の 5-Fu の結果 Cytotoxicity IC50 Lab. n Mean SD Min Median Max Missing A 3 0.0357 0.0071 0.0285 0.0361 0.0426 - B 4 0.0334 0.0037 0.0289 0.0336 0.0374 - C 5 0.0347 0.0081 0.0256 0.0337 0.0466 - Total 12 0.0345 0.0061 0.0256 0.0344 0.0466 - Differentiation assay ID50 Lab. n Mean SD Min Median Max Missing A 3 0.0325 0.0128 0.0203 0.0314 0.0458 - B 4 0.038 0.0028 0.0350 0.0375 0.0418 - C 5 0.0352 0.0028 0.0309 0.0361 0.0382 - Total 12 0.0355 0.0063 0.0203 0.0363 0.0458 - 単位：μg/mL つぎに Ascorbic acid はリードラボの IC50 値 634.3±148.1 μg/mL、ID50 値 804.3±182.4 μg/mL と近接する 1 施設と、それよりも低い 2 施設に分かれた（表 1-7）。 表 1-7 各施設の AA の結果 17 Cytotoxicity IC50 Lab. n Mean SD Min Median Max Missing A 3 212.8 155.6 101.5 146.3 390.6 0 B 3 552.3 153.9 440.1 489.1 727.7 0 C 3 289.5 76.5 215.9 284.0 368.6 0 Total 9 351.5 192.9 101.5 368.6 727.7 0 Differentiation assay ID50 Lab. n Mean SD Min Median Max Missing A 3 178.8 154.7 4.4 232.4 299.5 0 B 1 798.6 . 798.6 798.6 798.6 2 C 3 251.3 96.6 154.2 252.3 347.4 0 Total 7 298.4 247.1 4.4 252.3 798.6 2 単位：μg/mL Boric acid はいずれの施設においても、リードラボの IC50 値 65.1±6.4 μg/mL、および ID50 。 値 63.3±22.1μg/mL に近接した値が得られた（表 1-8） 表 1-8 各施設の Boric acid の結果 Cytotoxicity IC50 Lab. n Mean SD Min Median Max Missing A 3 88.2 3.3 84.4 89.7 90.6 0 B 3 58.8 4.1 55.3 57.8 63.3 0 C 3 83.7 10.4 74.7 81.4 95.0 0 Total 9 76.9 14.9 55.3 81.4 95.0 0 Differentiation assay ID50 Lab. n Mean SD Min Median Max Missing A 3 109.4 6.8 101.7 111.9 114.5 0 B 3 87.6 6.5 81.1 87.5 94.1 0 C 3 92.1 6.6 86.6 90.3 99.4 0 Total 9 96.3 11.5 81.1 94.1 114.5 0 単位：μg/mL 同様に Hydroxyurea はいずれの施設においても、 リードラボの IC50 値 5.61±0.65 μg/mL、 。 および ID50 値 4.50±0.45μg/mL に近接した値が得られた（表 1-9） 表 1-9 各施設の Hydroxyurea の結果 18 Cytotoxicity IC50 Lab. n Mean SD Min Median Max Missing A 3 5.23 1.43 3.95 4.96 6.77 0 B 3 4.44 0.33 4.25 4.25 4.83 0 C 3 4.30 0.27 3.99 4.39 4.50 0 Total 9 4.66 0.86 3.95 4.39 6.77 0 Differentiation assay ID50 Lab. n Mean SD Min Median Max Missing A 3 5.54 0.45 5.02 5.77 5.82 0 B 3 3.47 0.86 2.55 3.61 4.25 0 C 3 4.91 0.43 4.63 4.70 5.41 0 Total 9 4.64 1.06 2.55 4.70 5.82 0 単位：μg/mL Ascorbic acid のみ施設間で異なったが、陽性対照および他 2 物質では施設間再現性が高く 近接した IC50、ID50 値が測定されたため、本フェーズの目的である transferability は確認 され、フェーズ 1 試験を進めることにバリデーション実行委員全員の合意が得られた。ま た、本フェーズの結果を検証し、暫定的に試験成立条件を最適化した。 4) フェーズ 1 試験 フェーズ 0 試験の結果より、リードラボからの技術移管は成功したため、細かなプロトコ ールおよび判定基準を修正した。その修正を反映し、施設内再現性とプロトコールの頑健 性を確認する目的でコード化 9 物質（3 種類を 3 回繰り返して実施する）の 1 判定試験を 3 セットに分けて実施した。 1. 被験物質 コード化した 9 物質は、バリデーション化合物管理チームより配布され、各施設で適切に 保管した。プロトコールに従い、一部を秤量して希釈溶媒（PBS (-) または DMSO）を選 択し、最大溶解濃度を決定した（表 1-10）。 表 1-10 フェーズ 1 試験のコード化物質の内訳 19 2. 試験方法 共通プロトコール（Hand1-Luc EST バリデーションプロトコール ver. 05-1E）に準じて独 立した 3 回の試験を実施した （1 試験：1 回の dose finding study と 1 回の definitive study） 。 1 試験に陽性対照物質および 3 コード物質を実施した。化合物添加 120 時間後に、CellTiter Fluor Cell Viability Assay (Promega, Cat. #G6082)を添加し、30 分インキュベートした後、 各施設保有の装置にて蛍光測定した。その後、Steady-Glo Luciferase Assay System (Promega, Cat. #X1006) を添加し、30 分インキュベートし、1 sec/well の条件下で化学発 光 測 定 し た 。 測 定 終 了 後 、 測 定 値 を 計 算 用 エ ク セ ル デ ー タ シ ー ト （ VerE05-1 (Hand1-Luc_EST)20131118.xlsx）に代入し、蛍光と化学発光値を算出し、自動計算により IC50 および ID50 の算出を行った。何れの試験においても陽性対照物質 5-FU を評価し、試 験成立基準を満たすことを確認した。試験ごとに、予め指定されたチェック項目（試薬管理 の記録、細胞調製手順、被験物質の調製手順、析出有無の確認、測定手順）を Hand1-Luc EST 記録用紙（20131118 Ver.2.xlsx）に記載した。 3. 判定基準 (1) Performance standard of assay それぞれ一回の実験で，陽性対象物質 5-FU が下記の条件を満たすことを絶対条件 とする。 IC50 : 0.003 – 0.065 μg/mL ID50 : 0.003 – 0.067 μg/mL (2) Quality control 20 陽性対象物質 5-FU およびコード物質曝露時の各 96 ウェルプレート内で下記の条 件を全て満たす。 A. 蛍光測定の結果，medium control (MC) / background (BG) 比の 95%信頼区間 の下限が 2 より大きい B. 化学発光測定の結果，MC / BG 比の 95%信頼区間の下限が 10 より大きい C. 蛍光測定の結果，vehicle control (VC) / BG 比の 95%信頼区間の下限が 0.2 よ り大きい D. 化学発光測定の結果，vehicle control (VC) / BG 比の 95%信頼区間の下限が 0.2 より大きい E. VC の精度 (CV% = 標準偏差/平均値 × 100) が 100%以内 全試験結果は、確証として出力データとともにデータ管理チーム（同志社大学大森研究室） に提出され、適切に解析された。 4. 結果 陽性対照 5-FU の結果 いずれの試験でも試験成立条件を満たし、近接した IC50、ID50 値が得られた。 コード 01 化合物の結果 施設 A、B および C の各 3 回の IC50、ID50 値を図 1-6 に示した。何れの施設も IC50 や ID50 は同程度の結果が得られた。また、それぞれのグラフをまとめた（図 1-7） 。 図 1-6 各施設によるコード 01 化合物の結果 21 図 1-7 各施設によるコード 01 化合物の細胞毒性および分化阻害試験の結果 コード 02 化合物の結果 施設 A、B および C の各 3 回の IC50、ID50 値を図 1-8 に示した。施設 A および B では IC50 や ID50 は同程度の結果が得られたが、施設 A では低い ID50 が認められた。また、それぞれ のグラフをまとめた（図 1-9） 22 図 1-8 各施設によるコード 02 化合物の結果 23 図 1-9 各施設によるコード 02 化合物の細胞毒性および分化阻害試験の結果 コード 03 化合物の結果 全 9 試験の ID50 は近接した値が得られたが、IC50 値では、7 試験は最大溶解濃度以上の IC50 となったが、2 つの試験は極めて低い IC50 が認められた（図 1-10 の case1 および case2） 。 図 1-11 に示すように、全 9 試験のグラフパターンはほぼ同等であるが、case1 および case2 の場合だけ中濃度域で 50%阻害率を若干下回ったため、計算上の IC50 値が低く算出された ものと考えられた。また、最高濃度にてルシフェラーゼ活性が再上昇するケースが認めら れた（図 1-11）。DMSO に溶解したコード 03 化合物は培地に添加した際に、最高濃度域で 析出するため、試験濃度に達していないことが考えられた。 24 >250 µg/ml(top dose) >500 µg/ml(top dose) Case 1 Case2 図 1-10 各施設によるコード 03 化合物の結果 Case 1 Case2 25 precipitation 図 1-11 各施設によるコード 03 化合物の細胞毒性および分化阻害試験の結果 以上のデータをもとに、陽性陰性判定予測式（非公開）に IC50 および ID50 を代入し、P（陽 性）または N（陰性）を予測した。施設 B のコード 03 化合物以外は施設内で一致しており、 高い施設内再現性（89 %；8/9 試験）となった（表 1-11） 。 表 1-11 フェーズ 1 試験の予測結果まとめ Code 01 02 03 Lab. A B C A B C A B C 1 P P P P N N P P P Set No. 2 P P P P N N P P P 3 P P P P N N P N P final judgment P P P P N N P P P in vivo class P N P P；陽性 N；陰性 ③ 専門家による評価 フェーズ 1 の結果を元に 5th VMT 会議を以下の要領で実施した。日時、参加者等は以下の 26 とおりである。 ------------------------------------------------------------------------------------------------------------------------5th VMT 会議 2014 年 2 月 19 日（同志社大学 今出川キャンパス神学館 1 階会議室） リードラボラトリーによる検討結果の報告ならびに予測式についての討論 2014 年 2 月 20 日（同志社大学 室町キャンパス寒梅館 6 階会議室） フェーズ 1 試験の結果および次フェーズに向けたプロトコールの修正 フェーズ 2 試験の化合物選定（VMT メンバーのみ参加） 電話会議（フェーズ 1 試験の結果報告およびフェーズ 2 試験の計画） 2014 年 2 月 21 日（同志社大学 室町キャンパス寒梅館 6 階会議室） 電話会議（フェーズ 1 試験の結果報告およびフェーズ 2 試験の計画） 参加者： （敬称略） 田中憲穂 (食薬センター、Chair)、 小島 肇(JaCVAM、 Co-chair、Management of QC)、 斎藤幸一、長堀博久、鈴木紀之、Florian Le Coz (住友化学)、大森 崇、 小林真弓、森 梓、西尾 学、丸谷あおい (同志社大学)、近江谷克裕(産業技術総 合研究所)、松本一彦(学習院大学)、桑形真希子 (食薬センター)、Liu Shujie、池 田直弘(花王株式会社) 、柳 和則、泉川 健(住化分析センター)、生悦住茉友、渡 辺美香、山崎晶次郎 (食薬センター) Andrea Seiler (BfR/ZEBET), Eui-Bae Jeung (KoCVAM), Michael Schaeffer (EURL ECVAM), Warren Casey (ICCVAM) ------------------------------------------------------------------------------------------------------------------------内容 バリデーション実行委員である海外メンバーにフェーズ 1 試験の結果を報告した際、IC50 と ID50 の計算方法が議論となった。現状は 50%を跨ぐ 2 濃度間の値のみを用いて計算して いるため、コード 03 物質の例（case1 および case2）のような現象が生じた。そのため、 直線モデルにより IC50 や ID50 を求めるのではなくカーブフィッティングによる濃度依存的 なグラフ化、および算出方法への改善を検討することとした。また、化合物が析出する場 合は析出した濃度を試験対照外とすることや、50%を 2 度跨ぐような濃度非依存的なグラ フパターンが得られた場合には、再試験を行うことを推奨された。 フェーズ 1 試験では施設内再現性が確認されたため、次のフェーズでは施設間再現性の 確認を行う。ただし、1 回の試験結果で 1 つの判定をすべきか、複数回の試験結果の多数決 により 1 つの判定を行うべきとの意見もあった。そのため、用量設定試験（dose finding study）と本試験（definitive study）の結果を組み合わせて判定する方法を次のフェーズで 確認することとした。フェーズ 2 を 2a および 2b に分割し、2a ではコード化した被験化合 物 4 点を 3 施設で評価し、修正したプロトコールの頑強性の確認と施設間再現性の確認を 27 目的とする。さらに 2b では化合物数を増やして施設間再現性のさらなる確認を行う（表 1-12） 。 表 1-12 フェーズ 2 での予測判定（案） Dose finding Definitive Definitive Final study study 1 study 2 judgment Judgment case 1 P P - P Judgment case 2 P N P P Judgment case 3 P N N N Judgment case 4 N N - N Judgment case 5 N P P P Judgment case 6 N P N N P；陽性 N；陰性 ④ プロトコールの整備 フェーズ 2 に向けて修正した現時点の最終プロトコール（案）を Appendix (1)に添付した。 ⑤ 民間利用促進に向けた今後の取組 これまでに論文発表や、日本毒性学会、日本実験動物代替法学会、日本先天異常学会での 発表を通じて成果や進捗を公表した。そのため、学会やメールにて国内外の多数の製薬企 業や試験受託施設から、細胞の配布を要望されている。細胞を用いた in vitro 試験法の課 題のひとつは、試験細胞の品質を維持し続けることであり、特に ES 細胞は継続培養によ り分化能が低下する可能性が指摘されている。そのため、分配先は品質を検査確認できる 体制を有する細胞配布機関を予定しており、すでに興味を示した機関と交渉中である。 次年度以降もフェーズ 2 の試験を継続した後、平成 27 年度に SPSF（standard protocol submission form）の提出、およびバリデーションレポートを作製し、民間利用可能な有用 性の高い試験プロトコールを作成する予定である。 28 （2）皮膚感作性スクリーニング in vitro 試験法の調査 ① 皮膚感作性試験法の現状整理 １） 皮膚感作性試験法の現状 現在、感作性試験は主に動物を用いた試験法が一般的に行われており、国内外の行政に よ る 規 制 も 動 物 実 験 法 に 依 存 し て い る 。 動 物 を 用 い た 検 査 法 の 一 つ は 、 1969 年 に Magnusson and Kligman(1)によりに提案されたモルモットを用いた Maximization test と 呼ばれる試験法で、Day0 に化学物質、アジュバントを皮下注射し、その 1 週間後に同部位 に化学物質を貼付し感作、さらにその２週間後に別の部位に化学物質を貼付し、皮膚の反 応を見るという実験系である。ガイドライン上は「被験物質処置群には少なくとも 10 匹、 対照群には少なくとも 5 匹を用いる。」と記載されている。その後、用いる実験動物数、試 験期間の短縮を目的に、1992 年に Kimber and Basketter(2)により Local Lymph Node Assay が開発された。この方法では、まず 3 日間連続でマウスの耳介に化学物質を塗布、6 日目に放射線同位元素であるトリチウム-チミジンを全身投与し、５時間後に所属リンパ節 を摘出、そのトリチウム-チミジン取り込み量を測定する。いずれも感作性試験法としては 優れた方法であり、これまで医薬品、化粧品開発などの分野で多くの実績を残している。 上記の動物試験法の価格調査結果を示した。 動物試験法（1 検体の場合） Maximization test モルモット 約 20 匹 160～200 万円 Local Lymph Node Assay マウス 約 25 匹 80～100 万円 2.5 か月～ 1.5～2 か月 しかし近年の EU における化粧品開発における動物実験廃止の決定により、動物実験を用 いない感作性試験法の開発が強く求められるようになった。 その流れをうけて、様々な感作性試験代替法が開発された(図 2-1)。 29 図 2-1 in vitro 感作性試験の現状 その中で、昨年 Direct peptide reactivity assay (DPRA)と Kerainotsens が OECD test guideline に承認された。DPRA は、2004 年に Gerberick ら(3)が報告した、lysine, cysteine, histidine 含有ペプチドとの反応性の有無により化学物質の感作性を予測するという in chemico の方法である。これまでの感作性に関する多くの研究により、親電子性を有するこ とが感作性物質のもっとも共通した特性であることを明らかにしており(図 2-2)、感作性試 験の adverse outcome pathways (AOP)の観点からも妥当な検査法と思われる。 30 図 2-2 免疫反応誘導メカニズムの概要 一方 Keratinosens(4)においては、感作性物質のチオール反応性あるいは ROS 産生能を指 標に感作性予測をおこなう。具体的には、チオール反応性や ROS により活性化する Nrf2 という転写因子が結合する DNA 配列をプロモーター領域に含むルシフェラーゼ遺伝子を 表皮細胞由来培養細胞株 HaCaT に導入した安定細胞株を用い、ルシフェラーゼ活性誘導能 を指標に感作性を判定する。 この方法は、 必ずしも AOP における意味づけは明確ではなく、 また DPRA が対象にしている化学物質の特性を別な視点で評価しているにすぎないように も思われるが、精度、感度、特異度などその優れた特性から OECD の test guideline に採 択された。 両試験法以外に、現在 OECD においては、h-CLAT が test guide line の候補として検討さ れている(5, 6)。h-CLAT は感作性物質の樹状細胞活性化作用に注目した試験法で、ヒト単 球由来細胞株 THP-1 が感作性物質に反応して CD54 や CD86 という T 細胞刺激に関連する 副刺激分子の発現を増強することを指標に感作性を予測する。これも他の試験方法同様、 単独では偽陰性の化学物質が少なからず存在する。 以上現時点で、すでに 2 つの方法が OECD test guideline として承認され、もう一つが承 認に向けて検討中である。しかし、いずれの方法も単独ではほぼ 80%程度の精度、感度、 特異度であり、依然より優れた感作性試験法の開発が望まれている。また、現状では単独 の試験法では偽陰性をゼロにすることは不可能で、複数の感作性試験法の組み合わせが必 要と考えられている。特に、最近社会的に大きな話題と成ったロドデノール含有化粧品使 31 用者に白斑患者が多発した事例は、化粧品業界はもちろん行政、一般市民にいたるまで確 実な感作性試験法の必要性をあらためて再認識させる機会となった。 以上、感作性試験をとりまく国内外の情勢のなかで、現在開発中の IL-8 Luc assay は すでに提案されている方法とは後述するように、 全く異なった AOP に基づく試験法であり、 また評価に必要な時間が他の検査法と比較し短く、また化学物質と細胞添加以外の操作は ほぼ自動化されているという特徴を有している。 ２） IL-8 Luc assay の特徴 IL-8 Luc assay は、感作性物質が単球細胞に作用した際の IL-8 mRNA 発現増強を指標にし た試験法である。Takahashi ら(7)の報告に基づけば、精度 86％、感度 83％、特異度 90％ の試験法で、上述した 3 つの試験法と比較して遜色がない。しかし、これまで皮膚感作 AOP における IL-8 の位置づけは明確ではなかった。その最大の理由は、マウスの IL-8 ホモログ が特定されておらず遺伝子欠損マウスを用いた解析が行えないことに由来する。ところが 最近、感作性物質が樹状細胞に作用した際に IL-8 と共調して CCL2 (MCP-1)の発現が増強 することが報告された(8)。また、そのメカニズムとして IL-8 と CCL2 が共通して GGAATTTCC という NF-kB 結合配列を有し、EGR-1、p65 を含む同一の転写因子により 活性化されることも明らかにされた(9)。したがって、 IL-8 の転写活性を指標とする IL-8 Luc assay は見方を変えれば、CCL2 の転写活性を定量しているとも考えられる。CCL2 はマウ スにおいても同一の遺伝子が存在するが、これまでに CCL2 遺伝子欠損マウスを用いた接 触皮膚炎の解析は行われていない。しかし、CCL2 が樹状細胞を局所に引き寄せる作用があ ること(10)、また CCL2 により刺激された樹状細胞が NF-kB の活性化を介して種々の副刺 激分子や所属リンパ節への遊走に必要な CCR7 などの発現を増強することが報告されてい る(11)。さらに、CCL2 の発現を抑制する siRNA の投与により接触皮膚炎が感作相、惹起 相いずれにおいても抑制されることも報告されている(12)。これらの所見は、CCL2 が接触 皮膚炎の誘発に重要なケモカインであることを示すものであり、またそれらは同時に IL-8 の接触皮膚炎における関与も示唆する。加えて、2012 年に Kish らにより接触性皮膚炎の 成立に T 細胞浸潤に先行する好中球の皮膚への遊走が不可欠であることが報告された(13)。 以上の報告を総合すると、IL-8 Luc assay は接触皮膚炎の AOP のメカニズムに則った検査 法と思われる ② データの蓄積 １）バリデーション（Phase IIb）(図 2-3)の結果をふまえて Phase IIc への対応 Phase IIb においては、改訂プロトコール（IL-8 Luc assay protocol Ver. 015E）に従い、 施設内再現性評価を目的に 5 検体の 3 回測定を実施した。 32 図 2-3 Phase IIb の結果 しかし残念ながら 1 施設（Lab A）における発光測定において、被験物質濃度ゼロのポイン トで発光強度マイナスが測定されという結果が被検物質全体の 1/3 を占めていたことが quality check において明らかとなった。測定器の不具合か試験試薬のいずれかの問題であ ることが示唆されたが、いずれにしてもこれらの数値に関しては試験不成立と判断し、再 測定を行う事とした。しかし、再検査結果を総合しても図に示すように施設内再現性は 0.60 〜080、施設間再現性 0.86 となり必ずしも満足のいく値は得られなかった。また 5th VMT meeting の委員、特に海外メンバーからは、この試験法自体は high-throughput で代替試 験法としては素晴らしい方法であるので、今回の問題を確実に解決し Phase III へ進むよう にとコメントを頂いた。 そこで、施設内再現性のさらに高めること目的に試験方法、プロトコールを以下の様に改 変した。 １．これまでのプロトコールにおいては、FlnSLO-LA と Inhibition index (I.I)の 2 つのパ ラメーターを判定に用いていたが、その設定により施設内再現性が低下している可能性が 懸念されたため、新たなプロトコールでは I.I による判定をクライテリアから削除した。 ２．これまで FlnSLO-LA の判定に際しては、IL-8 Luc assay 開発当初から 1.4 という基準 を設けて陽性、陰性の判断を下していたが、Phase IIb 以降 THP-G8 細胞と化学物質の反 応時間を 18 時間に変更したところ FInSLO-LA の感作性物質処理後の値が大きくなり統計 的に陽性、陰性を判定することが可能となった。そこで PhaseIIc においては、これまでの FlnSLO-LA>=1.4 のクライテリア以外に FInSLO-LA を統計的に解析し 95％信頼区間が１ を超えた際に陽性と判断するクライテリアを設けて検討することにした。また、その際に FlnSLO-LA>=1.4 と統計的判定の両者を満たすか否かのクライテリアも検討項目に加えた。 33 以上のクライテリアに基づく判定方法を図 2-4 に示した。 図 2-4 フェーズⅡc 試験の判定方法 2）Phase IIc 改訂プロトコールにのっとり、コード化された 5 検体の 3 回測定（感作性物質/非感作性物 質判定に必要な 3 回測定を 1 試験とする）を 3 施設にて実施した。被検物質に関しては、 5th VMT meeting での被験物質選定会議にて決定した。 目的：施設内再現性を評価するため 実施期間：平成 24 年 11 月～平成 25 年 1 月 結果の評価に関しては、今回は phase IIb および Phase IIc が一部クライテリアの見直しを 除いてはほぼ同様のプロトコールで実験をおこなっているため、その両者を総合しておこ なった。結果は、図 2-5 および Appendix (2)a, (2)b, (2)c に示すようにクライテリア 1、2、 3 いずれにおいてもほぼ 80％の施設内再現性、クライテリア 1、2 においてはやはり 80％ を超える施設間再現性を示した。 34 図 2-5 Phase IIb and IIc のまとめ 3）6th VMT meeting における評価 ------------------------------------------------------------------------------------------------------------------------2014 年 2 月 26 日 場所：同志社大学今出川キャンパス寧静館 5 階大会議室 出席者： David Allen (NICEATM: Senior Scientist), Emanuela Corsini (University of Milan: Immuunotoxicology and Immunopharmacology Toxlab, Associate professor), Ai-Young Lee (Dongguk University Ilsan Hospital: Department of Dermatology, Professor) Noriho Tanaka (HRI, FDSC/OTIP: VMT, chair), Hajime Kojima (JaCVAM: Management of quality control), Setsuya Aiba, Yutaka Kimura (Tohoku University: Lead laboratory), Takashi Omori (Doshisha University: Biostatistician, data analysis), Aoi Maruya, Mayumi Kobayashi, Azusa Mori (Doshisha University: Data cleaning and analysis), Yoshihiro Ohmiya (AIST: QC monitoring of chemiluminescence measurement), Kohji Yamakage, Mika Watanabe (HRI, FDSC: Test facility), Koichi Saito, Noriyuki Suzuki, (Sumitomo Chemical Co., Ltd.: Test facility), Yoshihiro Nakajima (AIST: Test facility), Shojiro Yamazaki (HRI, FDSC/OTIP: Management office) ------------------------------------------------------------------------------------------------------------------------上述したように phase IIb and IIc の結果からは、クライテリア 1 ないし 2 を用いた IL-8 Luc assay は施設内、施設間ともにほぼ 80％の再現性を有しており試験法としての再現性の基 準をみたしていた。そこで今後は、Phase III としてさらに評価化学物質を増やし、評価法 の精度、感度、特異度などを検討することが提案された。 35 4）Lead laboratory による 80 被検物質の IL-8 Luc assay による評価結果 Lead laboratory では、昨年度に引き続き IL-8 Luc assay による化学物質評価を継続してお こない、年度内に 80 を超える化学物質の IL-8 Luc assay protocol Ver. 017E に基づく検討 を完了した。その結果を図 2-6 に示す。評価した化学物質の内訳は、extremely strong 7、 strong 16、moderate 20、weak 13、non-sensitizer 24 で、それらを評価した結果はクラ イテリア 1 では精度 0.788、感度 0.857、特異度 0.625、クライテリア 2 では精度 0.833、 感度 0.941、特異度 0.871、クライテリア 3 では精度 0.778、感度 0.857、特異度 0.609 で あった。 36 37 図 2-6 IL-8 Luc assay による 80 被検物質の感作性評価と他の試験法との比較 5）他評価法との比較 次に Lead laboratory で行った 80 被検物質の評価に基づく IL-8 Luc assay の特性を Ashikaga et al (14)、Nukada et al(15)、Emter et al (16)の論文を参考に h-CLAT、DPRA、 KeratinoSens の特性と比較した。クライテリア 2 に基づく IL-8 Luc assay に関しては、精 度はほぼ h-CLAT、KeratinoSens と同等で DPRA を上まわっていた。また感度に関しては、 IL-8 Luc assay は他のいずれの検査法よりも優れていたが、 特異度に関しては劣っていた。 また DPRA による評価結果とさらに詳細な比較検討をおこなったところ、IL-8 Luc assay は DPRA 評価における感作性物質のシステイン反応性と弱い相関性を認めたが、グルタチ オン、リジン、ヒスチジンとの反応性との相関は認められなかった。この結果は、IL-8 Luc assay と DPRA が異なった感作性物質の特性を評価していることを意味し両者を併用して 化学物質感作性を評価する有用性を示唆している（図 2-7） 。 図 2-7 DPRA と IL-8 Luc assay の相関性 38 ③ プロトコールの整備 IL-8 Luc assay protocol Ver. 016E の作成 今回の一連の validation study においてプロトコールの改変を重ね、現在 IL-8 Luc assay protocol Ver. 017E(Appendix (2)d)を作成し Phase IIc において使用した。 ④ 民間利用促進に向けた今後の取組 すでに IL-8 Luc assay に用いる THP-G8 細胞を鳥取大学医学部付属病院分子制御内科、渡 辺仁成講師らが用い、PM2.5 および黄砂の生物学的毒性評価への応用を検討している。 参考文献 1. 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Toxicology and applied pharmacology 245:281-290. 41 3) 発がん性スクリーニング in vitro 試験法の調査 ① 発がん性試験法の現状整理 発がん性は、正常な細胞をがん化（悪性腫瘍）させる性質で、発がん性物質とは、発が ん性を示す化学物質のことである。発がん性の試験法としては普通、マウスやラットなど のげっ歯類または必要に応じてその他の哺乳類）に検体を連続投与して各臓器のがんの有 無を検索する試験（長期発がん性試験）が行われている。これには 1 年以上の長期間を要 するため、より短期間で結果の出る方法として「中期発がん性試験」も用いられている。 これは 3～4 ヶ月程度の連続投与後、過形成などの前がん病変を検索する方法である。 現在、発がん性試験は主に動物を用いた試験法が一般的に行われており、国内外の行政 による規制も変異原性のデータを考慮しつつ、最終的には動物実験のデータに依存してい る。しかし、その試験期間が 1 年以上と長期に亘り、また、その費用も１検体 1～2 億円程 度と高額であるため、発がん性予測のためのスクリーニングを目的に in vitro 試験法の開発 がなされるようになった。 発がん性物質スクリーニングのための in vitro 試験法としては、これまで主として DNA に傷害をもたらして突然変異や染色体異常をおこす物質（遺伝毒性物質）を標的として変 異原性試験が実施されてきた。しかしながら、発がん性物質の中には非変異発がん物質（約 20％）も含まれていることから、これらの非遺伝毒性発がん性物質も検出する必要がある。 その方法として、細胞のがん化を指標とした形質転換試験法があり、Bhas 42 形質転換試験 法では、遺伝毒性発がん物質と非遺伝毒性発がん物質の両方を検出する事が可能である。 現在、OECD に提案されている形質転換試験法として、SHE 細胞、BALB/c 3T3 細胞 および、Bhas 42 細胞を用いたそれぞれ 3 種の試験法がある。これら in vitro 試験法の費 用は、動物を用いた試験法に比べて 1/100 以下と低い。 この形質転換試験には、下記に示すように大きく分けて初代細胞と株細胞を用いる 2 つの 方法がある。 1)SHE 細胞（シリアンハムスターの胎児細胞）を用いる方法 初代細胞を用いる方法で、検体を処理し、1 週間の培養で細胞の形態観察により、異常形 態を示す形質転換したコロニーを識別しカウントする。 2)BALB/c 3T3 細胞や C3H10T1/2 細胞、Bhas42 細胞を用いる方法 株細胞を用いる方法で、細胞を播種し、イニシエーターの検出では細胞の増殖期に検体 を処理し、プロモーターの検出では細胞増殖の休止期に処理し、約 6 週間（BALB/c 3T3） 又は 3 週間（Bhas42)培養後に出現するフォーカスの数をカウントする。 SHE 細胞を用いる方法については、1993 年にフランスとアメリカにより OECD に提案 された。その後、加盟国によるレビュー過程で、特に我が国は、細胞株を用いる試験系を 加えるべきと主張し、2006 年、厚労省より BALB/c 3T3 細胞を用いる方法について SPSF 42 （ Standard Project Submission Form）を提出した。 その後、US/EPA で専門家会議が招集され、SHE 細胞も BALB/c 3T3 細胞もバリデーシ ョンスタディの必要性が指摘され、ECVAM 主催でバリデーションが行われた。得られた結 果について、2010 年 10 月に ECVAM の ESAC（ECVAM Scientific Advisory Committee） でピアレビューがなされ評価をうけた。その結果、両方法ともバリデーションとしては、 検体数が不十分であることや、Balb/c 3T3 細胞に関してはプロトコールを検討することも 指摘された。 日本としては、同じ株細胞を用いる BALB/c 3T3 細胞とこの細胞に ras 遺伝子を導入し た Bhas 42 細胞に関して、どちらを OECD ガイドラインとして推進するかについて、専門 家と厚生労働省および経済産業省の担当者を交えて協議した結果、より簡便で感度に優れ る Bhas 42 細胞を用いる形質転換試験法を強力に推進することとし、2010 年、NEDO 委 託 事 業 で バ リ デ ー シ ョン さ れ た Bhas 42 細胞 を 用 い る 簡 便 で 精 度の 高 い 試 験 系 を OECD/TG として、経産省より申請するに至った。 なお、形質転換試験法の 3 法について、長所短所を以下の表 3-1 にまとめた。 表 3-1 形質転換試験法の 3 法の短所と長所 3種の形質転換試験法の短所長所 試験系 提案窓口 細胞種 SHE 初代細胞 仏、米 培養期間 １ ws 観察の難易 コスト やや難 形態での評価 低 易 フォーカスカウント 中 易 フォーカスカウント 低 ＊短期間でできる Balb/3T3 厚労省 株細胞 6-5 ws ＊培養期間が長い Bhas42 経産省 株細胞 3 ws ＊短期間で、感度・精度が高い 43 ② 標準化に向けた取組 Bhas 42 細胞形質転換試験法の背景・経緯 (独)新エネルギー･産業技術総合開発機構（NEDO）プロジェクト「高機能簡易型有害性 評価手法の開発」 の研究開発テーマの一つとして、 Bhas 42 細胞形質転換試験（Bhas 42 CTA） 法の有用性評価の研究開発が行なわれた。当該プロジェクトでは、Bhas 42 CTA 法に対し、 既存の発がん及び非発がん物質を有する 98 物質を適用した結果、遺伝毒性試験と同等以上 の性能を有することが判明し、特に遺伝毒性試験では不検出あるいは検出が不確かな発が ん物質を相当数検出できることが明らかになった。また、従来の 6-ウェルマイクロプレー トを使用した Bhas 42 CTA 法を効率的に実施するため、試験の自動化に適した 96-ウェル マイクロプレートを用いる方法の開発についても行なわれ、96-ウェルマイクロプレート法 （96 ウェル法）が 6-ウェルマイクロプレート法（6 ウェル法）とほぼ同等の結果を与える ことが確認された（1） 。 NEDO プロジェクトで Bhas 42 CTA 法の有用性が確認され、従来試験法に比べ秀でた試 験法であることが確認されたことから、Bhas 42 CTA 法を経済協力開発機構（OECD）の 毒性試験ガイドライン（以下「テストガイドライン」という。）化を目指し、平成 22 年（2010 年）1 月に、Bhas 42 CTA 法の SPSF（Standard Project Submission Form）を経済産業 省から OECD 事務局へ提出した。第 22 回テストガイドラインプログラム・ナショナルコ ーディネーターズ会合（WNT：the Working Group of National Cordinaters of the Test Guidlinese Programme、同年 3 月開催）において、Bhas 42 CTA 法のテストガイドライ ン化に向けた検討を開始することが了承された。テストガイドライン化に向けて必要とな るバリデーションスタディを実施し各種レポートを作成するため、国内外の形質転換試験 法 等 の 専 門 家 か ら 構 成 さ れ る Bhas 42 CTA の バ リ デ ー シ ョ ン マ ネ ー ジ メ ン ト （VMT:Validation Management Team）を立ち上げ、作業を開始した。 経済産業省では、平成 23 年度から Bhas 42 CTA 法のテストガイドライン化を推進する ため、テストガイドライン化に向けた調査研究を実施してきた。テストガイドライン化に 向けては、試験法のバリデーションレポートやバックグラウンドレビュードキュメント等 の作成を行い、評価機関により公式な評価を受ける必要がある。Bhas 42 CTA 法の評価機 関については、日本動物実験代替法評価センター（JaCVAM：Japanese Center for the Validation of Alternative Methodes）の協力・調整により、欧州代替法評価センター（EURL ECVAM：European Center for the Validation of Alternative Methods）が受け入れ、ピア レビューを行うことが決定された。 平成 24 年度では、EURL ECVAM が実施するピアレビューに向けて、ピアレビュー用の バリデーションレポート等を作成するとともに、ピアレビューの対応を実施した（表 3-2） 。 44 表 3-2 Bhas 42 CTA の背景・経緯①：平成 18～24 年度 6 ウェル法のバリデーション研究の経緯 2007 年 8 月 ・6 ウェル法のバリデーション研究を開始 2009 年 10 月 ・6 ウェル法のバリデーション研究参加機関からのデータ提出を完了 2010 年 10 月 ・6 ウェル法のバリデーション研究結果データの解析･まとめを終了 ・第 3 回 VMT 会議において 6 ウェル法バリデーション結果について VMT メンバーの同意を得た 96 ウェル法のバリデーション研究の経緯 2008 年 10 月 ・96 ウェル法のバリデーション研究を開始、段階的にプレバリデーシ ョン、Phase I、Phase II を進めた 2010 年 7 月 ・96 ウェル法のバリデーション研究協力機関のデータ提出を完了 2010 年 10 月 ・96 ウェル法のバリデーション研究結果データの解析･まとめを終了 ・第 3 回 VMT 会議において 96 ウェル法バリデーション結果について VMT メンバーの同意を得た ・Bhas 42 CTA を 6 ウェル法と 96 ウェル法の２つの手法を備えた発が ん性予測試験法として OECD テストガイドライン化する方針が決ま った 2011 年 7 月 ・Bhas 42 CTA のテストガイドライン化に向けた調査研究を開始 2011 年 9 月 ・EURL ECVAM の Dr. Raffaella Corvi を通して、ECVAM に Bhas 42 CTA のピアレビューを打診 2011 年 10 月 ・ECVAM が Bhas 42 CTA のピアレビューを正式に受入 2011 年 12 月 ・6 ウェル法バリデーション研究及び 96 ウェル法バリデーション研究 のデータを整理･解析 ・Bhas 42 CTA バリデーションレポート草案(Ver. 1)を作成 ・Bhas 42 CTA バリデーションレポート草案(Ver. 1)を VMT メンバー へ回覧 2012 年 1 月 ・Bhas 42 CTA バリデーションレポート草案(Ver. 1)を修正した上でこ れに追記を加えた草案(Ver. 2)を VMT メンバーへ回覧 2012 年 4 月 ・Bhas 42 CTA バリデーションレポートの草案(Ver. 2)に対して、VMT からの意見を検討、整理 ・VMT の意見に従って草案(Ver. 2)を修正して、草案(Ver. 3)を作成 2012 年 5 月 ・草案(Ver. 3)を VMT メンバー全員へ e-メールにて送付 2012 年 6 月 ・Dr. Raffaella Corvi（EURL ECVAM）並びに小島肇氏（JaCVAM、 国立衛研）と食品薬品安全センターとが会合を持ち、草案(Ver. 3)の内 45 容を審議し、概ね合意に達した ・Dr. Corvi からのコメントに対応した上で、森田氏による Bhas アッ セイバリデーションの被験物質選択報告書を Annex 14: Chemical properties and classes of 12 coded test chemicals and positive controls for the 6-well method validation study (Table 58)及び Annex 15: Chemical properties and classes of test chemicals for the pre-validation phase, phase I and phase II of 96-well method validation study (Table 59)として添付し、草案(Ver. 4)を作成 2012 年 7 月 ・草案(Ver. 4)を VMT メンバー全員に e-メールで送付 ・草案(Ver.5)を、小島氏を経て Dr. Patric Amcoff（EURL ECVAM）へ e-メールにて送付 ・各草案(Ver.3～5)の作成には、元米国動物実験代替法検証コーディネ ー ト 委 員 会 （ Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM)） 委員長である Dr. Leonard Schechtman にコンサルテーションを得た。 2012 年 7 月 ・JaCVAM の小島氏を通じて Dr. Patric Amcoff（EURL ECVAM）へ VMT 同意 Bhas 42 CTA バリデーションレポートを送付。ESAC ワ ーキンググループの各メンバーへ回付 2012 年 9 月 ・ESAC ワーキンググループ各メンバーは、バリデーションレポート の検討を開始、電話会議を開催 2012 年 10 月 ・ECVAM において会合を持ち、ピアレビューを実施 その他、電話会議も 2 回開催 46 Bhas 42 形質転換試験法のテストガイドライン案の作成及び OECD への提出 平成 25 年度は、Bhas 42 CTA 法のテストガイドライン(案)の作成を行い、平成 25 年 9 月 OECD にテストガイドライン案を提出した。OECD 事務局では加盟各国のコメントを収 集するため、このガイドライン案を各国のナショナルコーディネーターヘ配布し、各国の 専門家の意見を収集した。得られた加盟各国からのコメントは、平成 25 年 12 月半ばに提 案国のリードラボ（一般財団法人食品薬品安全センター 秦野研究所）へ回覧され、コメン トに対する回答とコメントに従った改定ガイドライン案の作成を求められた。その回答書 は平成 26 年 1 月 9 日に、リードラボ秦野研究所より OECD へ提出され、その改定ガイド ライン案とコメントの回答の一部について平成 26 年 1 月 14～16 日にパリで開催された専 門家会議（Expert meeting）で議論した。参考までにテストガイドライン(案)最新版とコメ ントへの回答書を Appendix (3)1~2 に示す。 平成 26 年 1 月 14 日～16 日に OECD 本部で開催された形質転換試験の専門家会議の目 的、概要は以下のとおりである。 参加者： 日本からの参加者は、秦野研の 3 人（佐々木、山影、田中）と小島（国立衛研）と林（安 評センター）の 5 人。海外の参加者は、フランス（3 名）、イギリス、イタリア、オランダ、 EU、ICAPO、OECD（各 2 名）アメリカ、オーストリア、カナダ、ドイツ、BIAC（1 名） の参加者が SHE CTA 法の会議に参加し、Bhas 42 CTA 法のセッションでは、ドイツとオ ランダを除き、同じ参加者が参加した。すでにこれまで数回開催された形質転換試験の専 門家会議に参加した顔ぶれのメンバーが大部分であった。会議の議事進行は、担当の Nathalie Delrue（OECD、Environment Directorate）によって進められた。（ICAPO は 動物愛護団体の代表、BIAC は工業会の代表） 目的： － Syrian Hamster Embryo（SHE）のセッションは、昨年（2013 年 4 月）開催された WNT 会議のフォローアップとして開催され、（i）SHE アッセイに関して懸案となって いる点に関しての対応について討議すること、（ii）テストガイドライン案を先に進める ためにはどのような計画で進めるかについての具体的な作業プランをつめること、であ った。 － Bhas42 CTA のセッションでの主な目的は、2013 年 12 月に回収された最初のコメン ト収集で WNT よりあがった技術的なコメントについて討議すること、であった。 概要： 47 会議の前半（1 日半）は、すでに昨年の WNT 会議（2013 年 4 月）にあげられ、保留に なった SHE CTA 法の問題点についての討議があり、2 月中にあと 1 回、専門家による電 話会議を開催し、今年（2014 年 4 月）の WNT 会議にあげる基本方針を最終確認して提 案することとした。うまくいけばガイドライン化できる可能性があるが、この WNT 会議 は全会一致なので、昨年主に反対した国が、今回の専門家会議でどのように発言するか が注目された。OECD の担当者としては、今年の WNT 会議で SHE CTA 法を通し、来 年の WNT 会議で Bhas CTA 法を通したい意向のようである。 会議の後半（1 日半）での Bhas CTA 法の討議では、まず、ECVAM の Dr. Raffaella Corvi が Bhas CTA 法に関する ECVAM のピアレビューの結果を説明し、結論として、本法に 関して、OECD ガイドライン化を図るべきである、との結論に至る経過をプレゼンした。 食薬センターによるプレゼン（Appendix (3)-3）においても、発がん物質の簡便なスクリ ーニング法として、より優れた方法であることを印象付けることができた。会議外の参 加者との懇談の中でも、メンバーの皆さんの Bhas CTA 法に関する期待感と皆さんが好 意的に受け取ってくれていることを感じた。下記に、討議の中で特に考慮すべき点とさ れた項目を記載した。 Bhas 42 CTA 法に関して、専門家会議における特に考慮すべき点として挙げられる内容 は以下のようにまとめられる。 提示された主な項目： Bhas CTA の討議の中で今後の検討課題となった点を挙げた。これに関しては、費用も 絡むと考えられが、 できるだけ完璧な回答を準備し、 2015 年 4 月の WNT 会議において、 スムーズにガイドライン化が可能となるように準備を進める必要がある。 １．Bhas 細胞の薬物代謝能の有無に関しての課題 我々は、体内で代謝して発がん性を示すような物質のいくつかが Bhas CTA 法で陽 性になっていることから、Bhas 42 細胞でその物質の代謝に関与した酵素が発現してい ると類推している。しかしながら、それを証明するため、Bhas 細胞が具体的にどのよ うな薬物代謝酵素が発現しているかの生化学的データがなく、明確でないことから、 分子生化学的な方法で関与する代謝酵素の発現を確認することが必要である。 対応案：この課題に明確に答えるには、分子生物学的な手法でデータを取らないと説得 力がないことから、薬物代謝の専門家とも相談し、場合によっては専門業者に委託し てデータを取得しておくのが良いと考えている。 48 ２．Bhas CTA 法での統計処理に関しては、SHE CTA 法の場合と異なる方法を用いてお り、先行している SHE CTA 法と統一化できないか、の要請があった。 対応案： Bhas CTA 法の場合、連続する 2 濃度で有意に陽性になった場合を、陽性と判 定している。これに関しては、バリデーションの際の VMT 会議の統計専門家（Dr. Sebastian Hoffman）の指示に従ったわけであるが、SHE CTA 法の場合、有意差検定 と trend test を用いている。Bhas CTA 法で実施したバリデーション・データや 98 物 質のデータについて、SHE CTA 法の方法と同じ方法を適用して、発がん性予測性の再 評価を行う必要があり、これも統計専門家と相談して進めなければならない。 ３．遺伝毒性や発がん性の定義を明確にして、評価した化学物質の分類（遺伝毒性発が ん物質、非遺伝毒性発がん物質）を再評価する必要がある。 対応案：バリデーション試験や 98 物質のリードラボでの in house データについては、 遺伝毒性物質と非遺伝毒性物質の定義を明確化（SHE で作成した分類結果を基準にす る）し、その定義に従って再分類する必要がある。また、in vivo の発がん性のデータ についても同様に、定義を明確にしたうえで、再分類する必要があるが、その際にプロ モーター作用についても文献調査し、再分類結果に反映させる必要がある。 ４．その他、会議では各国からよせられたプロトコール案のコメントに関して、質疑応 答を行い、修正可能な部分は修正を行った（Appendix (3)1~2 参照） 。 参考文献 1) Arai S, Sakai A, Hayashi K, Sasaki K, Muramatsu D, Endou N, Umdea M, Tanaka N. A high-throughput cell transformation assay applicable to automation for detecting potential chemical carcinogens using Bhas 42 cells. AATEX (2013) 18:1-19. 49 Ⅱ. 外部発表・講演、文献、特許の状況 1. 研究発表・講演 1) 台湾動物実験学会第 13 回総会、台北（中華民国） (2013 年 12 月 12－13 日) 特別講演：経産省/NEDO プロジェクトにおける発癌物質検出に関する代替試験法開発 Special Lecture: ESTABLISHMENT OF ALTERNATIVE ASSAY FOR CHEMICAL CARCINOGENS PERFORMED IN THE NEDO/METI PROJECT 2) 第 40 回 日本毒性学会学術年会（千葉／幕張メッセ国際会議場） 2013 年 6 月 17 日～19 日 演題 ：マウス ES 細胞を利用した発生毒性予測試験法 Hand1-Luc Embryonic Stem Cell Test(Hand1-Luc EST) 発表者 ：鈴木 紀之、永堀 博久、斎藤 幸一 所属 ：住友化学（株）生物環境科学研究所 3) 第 53 回 日本先天異常学会学術集会（大阪／千里ライフサイエンスセンター） 2013 年 7 月 22 日 演題 ：マウス ES 細胞を利用した発生毒性予測試験法 Hand1-Luc Embryonic Stem Cell Test(Hand1-Luc EST) 発表者 ：鈴木 紀之 所属 ：住友化学（株）生物環境科学研究所 4）日本動物実験代替法学会 第 26 回大会（京都／京都テルサ） 2013 年 12 月 19 日～21 日 演題 ：IL-8 Luc assay の施設間差試験およびデータセットの作製 発表者 ：木村 裕 1、藤村 千鶴 1、渡辺 美香 2、齋藤 るみ子 2,3、鈴木 紀之 4、岩城 知 子 5、山影 康次 2、斎藤 幸一 4、中島 芳浩 5、近江谷 克裕 6、酒井 綾子 2、丸谷 あおい 7、大森 崇 7、山崎 晶次郎 8、小島 肇 9、田中 憲穂 8、相場 節也 1 所属 ：1 東北大学大学院医学系研究科皮膚科学講座、2(一財)食薬センター秦野研究所、 3 東北大学 東北メディカルバンク・メガバンク機構、4 住友化学(株) 生物環境科 学研究所、5(独)産総研・健康工学研究部門、6(独)産総研・バイオメディカル研究 部門、7 同志社大学、8（公財）鳥取県産業振興機構、9 国立医薬品食品衛生研究所 50 5）日本動物実験代替法学会 第 26 回大会（京都／京都テルサ） 2013 年 12 月 19 日～21 日 演題 ：IL-8 Luc assay におけるばらつきを考慮した判定基準の提案 発表者 ：丸谷 あおい 1、相場 節也 2、木村 裕 2、渡辺 美香 3、鈴木 紀之 4、岩城 知子 5、山影 康次 3、斎藤 幸一 4、中島 芳浩 5、近江谷 克裕 6、山崎 晶次 郎 3、小島 肇 7、田中 憲穂 3、小林 眞弓 1、森 梓 1、大森 崇 1 所属 ：1 同志社大学 文化情報学部、2 東北大学大学院医学系研究科皮膚科学講座、3(財) 食品薬品安全センター 秦野研究所、4 住友化学(株) 生物環境科学研究所、5(独) 産業技術総合研究所・健康工学研究部門、6(独)産業技術総合研究所・バイオメデ ィカル研究部門、7 国立医薬品食品衛生研究所 6）日本動物実験代替法学会 第 26 回大会（京都／京都テルサ） 2013 年 12 月 19 日～21 日 演題 ：代替法のデータに基づいた構成法の異なる比の 95％信頼区間の比較 発表者 ：森 梓、丸谷あおい、小林眞弓、大森 崇 所属 ：同志社大学 文化情報学部 7) 日本動物実験代替法学会 第 26 回大会（京都／京都テルサ） 2013 年 12 月 19 日～21 日 演題 ： Hand1-Luc EST 試験法における IC50 と ID50 の関係の検討 発表者 ：小林 眞弓 1、鈴木紀之 2、 Le Coz Florian2、永堀博久 2，斎藤幸一 2、森 梓 1、 丸谷あおい 1、大森 崇 1 所属 ：1 同志社大学 文化情報学部、 2 住友化学(株) 生物環境科学研究所 2. 文献（発表論文） 1) Arai S, Sakai A, Hayashi K, Sasaki K, Muramatsu D, Endou N, Umdea M, Tanaka N. A high-throughput cell transformation assay applicable to automation for detecting potential chemical carcinogens using Bhas 42 cells. AATEX (2013) 18:1-19. 2) Sasaki K, Sakai A, Tanaka N. High-throughput quantification of morphologically transformed foci in Bhas 42 cells (v-Ha-ras transfected Balb/c 3T3) using spectrophotometry. In: Steinberg P. (ed) High-Throughput Screening Methods in Toxicity Testing. (2013) Wiley, New York, NY, pp 317-339. 51 3) Sasaki K, Sakai A, Yamazaki S, Umeda M, Tanaka N. Transformation assay in Bhas 42 cells: a model of initiated cells to study mechanisms of carcinogenesis and predict carcinogenicity of chemicals. (submitted) 4) Sasaki K, Huk A, El Yamani N, Tanaka N, Dusinska M. Bhas 42 cell transformation assay for genotoxic and non- genotoxic carcinogens. (submitted) 52 参考資料 - Appendix (1) - Appendix (2) a - Appendix (2) b - Appendix (2) c - Appendix (2) d - Appendix (3) 1 - Appendix (3) 2 - Appendix (3) 3 53 Appendix (1) Sumitomo Chemicals Co. Ltd. Ver.06E 1 2 3 4 5 6 7 8 Hand1-Luc Embryonic Stem Cell Test (Hand1-Luc EST) 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 Draft protocol ver. 06 E 10th March, 2014 Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd. Noriyuki Suzuki, Ph.D. Florian Le Coz, M.Sc. Hirohisa Nagahori, Ph.D. Koichi Saito, Ph.D. 1 Sumitomo Chemicals Co. Ltd. Ver.06E 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 TABLE OF CONTENTS INTRODUCTION ............................................................................................ 3 MATERIALS AND METHODS ....................................................................... 3 EXPERIMENT 1 .................................................................................... 3 1-1 Calculation of molecular weight of test chemicals .................... 3 1-2 Measurement of actual value of pKa or in silico method .......... 3 EXPERIMENT 2........................................................................................ 4 2-1 Measurement of actual value of water solubility or in silico method .......................................................................................................... 4 2-2 Measurement of ID50 and IC50 values in the Hand1-Luc EST ..... 4 A. Materials ........................................................................................ 4 Cells (Hand1-ES cells) ....................................................................... 4 Reagents and preparation methods ................................................... 4 Equipments ........................................................................................ 6 Expendable supplies .......................................................................... 6 B. Experimental Methods .................................................................. 7 Thawing of Hand1-ES cells ................................................................ 7 Maintenance of cells .......................................................................... 7 Preparation of test chemicals ........................................................... 8 Serial dilution of test chemicals and arrangement of plate format ...... 9 Dose finding study ........................................................................... 10 Definitive study ................................................................................ 13 Data analysis ................................................................................... 13 Tentative acceptability criteria (for phase 1 study) ........................... 14 EXPERIMENT 3 .................................................................................... 15 3-1 Measurement of metabolic stability of test chemicals ............. 16 JUDGMENT (Prediction of test chemicals) .............................................. 16 UPDATE RECORD ...................................................................................... 17 2 Sumitomo Chemicals Co. Ltd. Ver.06E 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 INTRODUCTION Hand1-Luc Embryonic Stem Cell Test (Hand1-Luc EST) is a novel short term test for predicting embryonic toxic chemicals using transgenic engineering mouse embryonic stem (ES) cells with consideration of pharmacokinetic property of test chemicals. This protocol describes how to prepare, determine the pharmacokinetic property and analyze cytotoxicity and differentiation toxicity of chemicals． MATERIALS AND METHODS EXPERIMENT1 1-1 Calculation of molecular weight of test chemicals Molecular weight can be calculated by commercial software or available tools on the internet such as ChemDraw or Stoichiometry Add-In for Excel (http://chemistry-in-excel.jimdo.com/). 1-2 Measurement of actual value of pKa or in silico methods Actual value can be measured by titration, spectrophotometric and conductometric methods. Detailed protocol is shown in OECD Test Guideline (112, http://www.oecd.org/env/ehs/testing/oecdguidelinesforthetestingofchemicals.h tm) The value can be calculated by commercial software or available tools on the internet such as ADMET Predictor, ACD/labs or Mervin (http://www.chemaxon.com/marvin/sketch/index.jsp). 3 Sumitomo Chemicals Co. Ltd. Ver.06E 1 2 3 4 5 6 7 8 9 10 11 12 13 14 EXPERIMENT 2 2-1 Measurement of actual value of water solubility or in silico methods Actual value can be measured via the OECD guideline 105 “Water solubility”. www.oecd.org/dataoecd/17/13/1948185.pdf The value can be calculated by commercial software or available tools on the internet such as ChemDraw, ADMET predictor or EPI suite. http://www.epa.gov/opptintr/exposure/pubs/episuitedl.htm 2-2 Measurement of ID50 and IC50 values in the Hand1-Luc EST Schema of the Hand1-Luc EST procedure is described in Figure 1. Dose finding study Definitive study Pre-culture of the cells Passage of the cells Vehicle selection Chemical preparation (diluted with ratio 1:10) 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Chemical preparation carry out with a smaller dilution factor covering the relevant of dose response) Cell preparation Cell preparation Addition of the chemicals to the plate Addition of the chemicals to the plate Measurement of 2-endpoints Measurement of 2-endpoints Data analysis Data analysis Figure.1 Outline of the Hand1-Luc EST A. Materials Cells (Hand1-ES) Transgenic mouse embryonic stem (ES) cell, named as Hand1-ES, containing firefly luciferase genes under the control of Hand1 (heart and neural crest derivatives expressed transcript 1) promoters were established by Sumitomo Chemical Co., Ltd. as a previous method with minor modification (Suzuki et al., Toxicol. Sci., 124, 460–471, 2011). An embryonic stem cell line (named as KOB1-ES) from C57/B6 mouse established by Sumitomo Chemical Co. Ltd. was used for development of the Hand1-ES cells. Reagents and preparation methods For maintenance of Hand1-ES cells StemMedium (Serum Free Media for Mouse ES Cell) (DS Pharma 4 Sumitomo Chemicals Co. Ltd. Ver.06E 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Biomedical, Cat. DSRK100) ▲Note Dissolved medium is stored at -20 °C (in an appropriate volume in tubes). Gelatin from porcine, type A (SIGMA, Cat. G2500) ESGRO mLIF (107 units/mL) (Millipore, Cat. ESG1107) Distilled water (SIGMA, Cat. W3500-500mL) 0.25 % Trypsin /1mM EDTA solution (Nacalai tesque, Cat.35554-64) 50 mg/mL Geneticin Disulfate (G418) solution (Nacalai tesque, Cat. 16513-84) Trypan blue solution (SIGMA, Cat.T8154-20ML or equivalents, regardless of makers, are acceptable ) For chemical exposure, positive control, solvents 5-Fluorouracil (5-FU) (SIGMA, Cat. F6627-1G, purity 99 %) Dimethyl sulfoxide (DMSO)（MP Biomedicals, Inc. (WAKO Pure Chemicals), Cat. 594-03771） PBS(-) (Invitrogen, Cat No. 10010-023 (500mL)) For differentiation or cytotoxicity assay DMEM (Invitrogen, Cat. 11965-092) FBS (Hyclone, Cat 533-90935, Lot. KTC30729) ▲Note The quality of the serum is very important for mESC growth and differentiation. Several lots of serum have to be tested before performing the assay. GlutaMaxI solution (100×conc.) (Invitrogen, Cat. 35050-061) 100 mM Non-Essential Amino Acid solution (NEAA) (Invitrogen, Cat. 11140-050) Penicillin-Streptomycin, liquid (PS solution) (Nacalai tesque, Cat. 26252-94) 2-Mercaptoethanol (2-ME) (Nacalai tesque, Cat. 21438-82) Steady-Glo® Luciferase Assay System（Promega, Cat No. E2510, E2520 and E2550） ▲Note Dissolved reagents stored at -80 °C (in an appropriate volume in tubes). CellTiter-FluorTM Cell Viability Assay（Promega, Cat No. G6080, G6081 and G6082） Maintenance medium (Culture medium for maintenance of Hand1-ES cells) Content Volume StemMedium 10 mL 100mM 2-ME solution 10 L ESGRO (107unit/mL) 1 L 50 mg/mL G418 solution 20 L Note - Supplemented medium is stored for no longer than 1 week at 4 °C. - Before use, supplemented medium should be prewarmed to 37 °C in a water bath or incubator. Assay medium 5 Sumitomo Chemicals Co. Ltd. Ver.06E 1 2 3 4 Content Volume DMEM 82 mL FBS (Heat inactivated) 15 mL GlutaMaxI solution (100× conc.) 1 mL 100mM NEAA solution 1 mL PS solution 1 mL 100mM 2-ME solution 100 L Note - Supplemented medium is stored for no longer than 2 weeks at 4 °C. - Before use, supplemented medium should be prewarmed to 37 °C in a water bath or incubator. 0.1% Gelatin solution Content Gelatin Distilled water Volume 0.5 g 500 mL Note - Autoclaved solution is stored for no longer than 2 months at 4 °C. - 60mm dishes should be coated with gelatin solution (37°C, over 30min). 2-Mercaptoethanol (2-ME) solution Content 2-ME (14.3 M solution) Distilled water Volume 70 L 10 mL Note Stock solutions are stored at -80 °C or prepared at time of use. 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Fetal Bovine Serum, Heat-Inactivated Content FBS Note Incubate at 56 °C for 30 min and store at -20 °C (appropriate volume in tubes). Dissolved FBS is kept at 4 °C and use within 1 month. Equipments Measuring device of micro-plate type luminometer Measuring device of micro-plate type fluorometer Plate shaker Water bath CO2 incubator (5%, 37°C) Single and 8 channel micropipette Appropriate cell counter (hemocytometer) Expendable supplies 60 mm Cell culture dish (BD Falcon, Cat.353004) 6 Sumitomo Chemicals Co. Ltd. Ver.06E 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 Storage Plate 96 well Round Bottom (Corning, Cat.3359) 96 well Assay Blocks 2mL/well (Corning, Cat.3960) PrimeSurface® 96well white plate (Sumitomo Bake Lite, Co., Ltd. MS-9096W） Plate seal (for PrimeSurface® 96well white plate) (Watson, Cat. 547-KTS-HC) Plate seal “TopSeal-A” (PerkinElmer No. 6050195) Cell strainer (40m, BD Falcon, Cat.352340) Reservoir Pipette B. Experimental Methods Thawing of Hand1-ES cells 1. 2. 3. 4. 5. 6. Coat the 60 mm dishes with the 0.1 % gelatin solution. Add 5 ml of the gelatin solution in the dishes and incubate at 37 °C during 30 min. Thaw frozen cells in 37 °C water bath, and add to the 9 mL of maintenance medium. Centrifuge the tube at 900 to 1,400 rpm for 5 min at room temperature, discard supernatant and resuspend in 5 mL of maintenance medium. Count cell number using aliquot of solution. Remove the gelatin solution from the dishes (step 1) and seed the cells at a concentration of approx. 0.5 to 1.0 ×106 cells/ gelatin-coated 60 mm dish in 5mL of maintenance medium. Incubate the cells at 37°C in a humidified atmosphere with 5% CO2. Maintenance of cells Grown cells should be passaged 2 or 3 days after thawing. 1. Prepare the 60 mm dishes coated with the 0.1 % gelatin solution before maintenance of cells. 2. Remove the supernatant from culture dish, wash with 5 mL of PBS(-). 3. Add 2 mL of 0.25% trypsin/1 mM EDTA and remove immediately. Then, cells are incubated at 37 °C for 1 to 2 min. 4. Add to 2 mL of maintenance medium to the dish, suspend the cells using micropipette (1000 L). 5. Count cell number 6. Remove the gelatin solution from the dishes(step 1) 7. For assay, seed the cells at a concentration of 2.0×106cells/ gelatin-coated 60 mm dish in 5mL of maintenance medium. Incubate the cells at 37°C with 5% CO2 for 1 day. 8. For passage, seed the cells at a concentration of 0.2, 0.5 and 1.0×106cells/ gelatin-coated 60 mm dish in 5mL of maintenance medium. Incubate the cells at 37°C with 5% CO2 for 2 or 3 days. Cells should be used until the cells reach 80–90% confluence. ▲Note; - Cells should be passed until the cells reach 80–90% confluence. - Cells should be used within 2 passages. 7 Sumitomo Chemicals Co. Ltd. Ver.06E 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 Preparation of test chemicals, choice of solvent and precipitation evaluation Test chemicals are dissolved in an appropriate solvent such as PBS(-) and DMSO. The recommended maximum final concentrations are 1 % (v/v) for PBS(-) or 0.1 % (v/v) for DMSO. The chemicals must be weighed and dissolved in solvent before each experiment, including 5-FU for the positive-reference control. The highest test concentration of any chemicals is 1000 g/mL. Dilution series of 7 concentrations of 5-FU (0.0003, 0.0006, 0.0013, 0.0025, 0.005, 0.01 and 0.02 mg/mL; with a common ratio of 1:2) as positive-reference control is dissolved from a 0.02 mg/mL prepared stock solution in PBS(-). A flowchart for selection of appropriate vehicle, preparation of chemicals and precipitation evaluation is shown in Figure 2. Dissolve the test chemical first in PBS(-) to obtain a concentration of 100 mg/mL. For example, weigh 10 mg of the test chemical in an appropriate tube and add PBS(-) up to 100 L. If the chemical is not soluble at 100 mg/mL, dilute with a common ratio of 1:2 to obtain a concentration of 50 mg/mL. If the chemical is not soluble at 50 mg/mL, dilute with a common ratio of 1:2 to obtain a concentration of 25 mg/mL. Other final volumes are also possible but the minimum required one should be 100 L depending on the needs of test chemical. If the chemical is not soluble at 25 mg/mL in PBS(-), the chemical should be dissolved in DMSO at 1000 mg/mL. For example, weigh 100 mg of the test chemical in an appropriate tube and add DMSO up to 100L. At this step, a precipitation test should be undertaken as followed: add 2μl of the DMSO dissolved chemical in the well of 96 well Assay Block containing 998μl of assay medium using an 8 channel micropipette. Mix thoroughly the medium with the chemical with the micropipette. If the precipitate is not observed at 1000μg/ml, check water solubility over 1000 μg/ml. For example, the solubility of the chemical for the following concentrations (12.5, 6.25, 3.125 and 1.56 mg/mL) should be evaluated and recorded since the value for the one the chemical is soluble will be used for the prediction model. If the chemical is not soluble at 1000 mg/mL, the highest soluble concentration should be determined by diluting the solution from 500 mg/ml at a common ratio of two (250 mg/ml -> 125 mg/ml if needed) with DMSO. If the chemical is not soluble at 250 mg/mL in DMSO, prepare solution of test chemical using PBS(-) or DMSO with 2-fold dilution factor (ex; 125 mg/mL in DMSO or 12.5 mg/mL in PBS(-) if needed). Other final volumes are also possible but the minimum required one should be 100 L depending on the needs of test chemical. Sonication and vortexing may be used if needed，and attempt to dissolve the chemical for at least 5 minutes. For each highest concentration where the chemical is soluble, the precipitation test should be provided. For example, if the chemical is soluble in DMSO at 500 mg/mL, then from this concentration and with a with 2-fold dilution factor, the possibility of precipitation in the assay medium should be verified. For this purpose, add 2 μL of the DMSO dissolved chemical in the 8 Sumitomo Chemicals Co. Ltd. Ver.06E 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 well of 96 well Assay Block containing 998μl of assay medium using an 8 channel micropipette. Mix thoroughly the medium with the chemical with the micropipette. If a precipitate appears on the tips of the 8 channel micropipette or remains in the medium after mixing, then the corresponding concentrations should not be taken into account for the experiment. For example, if the last precipitate is observed at 250μg/mL then the highest concentration used for the experiment will be 125μg/mL. Figure 2 Vehicle selection and preparation of test chemicals Serial dilution of test chemicals and arrangement of plate format Generally, the test chemicals should be prepared as follows; - Maximum concentration (final concentration): 1000 g/mL (depend on the solubility of test chemicals) - Serially dilute with the same solvent at a common ratio of 1:10, basically in the dose finding assay. Carry out the definitive study with smaller dilution factor (ex; 1:1.5, 1:2, 1:3 or 1:5) covering the relevant range of dose response. 1. Prepare dissolved 5-FU as positive-reference control and test chemicals according to the procedure (described in section “Preparation of test chemicals”) using 96 well microplates (Corning, Cat. 3359) (cf. Figure 3, row 1A - 1H, 2A - 2H) before assay. 9 Sumitomo Chemicals Co. Ltd. Ver.06E Positive Test control chemical (5-FU) 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 3 A PBS(-) B 0 . 0 0 0 3 m g /CmoLn c . 1 C 0 . 0 0 0 6 m g /CmoLn c . 2 D 0 . 0 0 1 3 m g /CmoLn c . 3 E 0 . 0 0 2 5 m g /CmoLn c . 4 F 0.005 mg/m CLo n c . 5 G 0.01 mg/mL Conc.6 H 0.02 mg/mL Conc.7 4 5 6 7 8 9 10 11 12 VC ratio 1:10 ratio 1:10 ratio 1:10 ratio 1:10 ratio 1:10 ratio 1:10 VC: Vehicle control (DMSO or PBS(-)) Figure 3 2. 3. Serial dilution and arrangement of test chemicals (1) Add 20 L of diluted 5-FU to the well of 96 well Assay Block containing 980 L of assay medium using an 8 channel micropipette (cf. Figure 4, row 1A – 1H). Add 20L of chemicals to the well of 96 well Assay Block containing 980 L of assay medium using an 8 channel micropipette if chemicals are dissolved in PBS(-) (cf. Figure 4, row 2A – 2H). Add 2 L of chemicals to the well of 96 well Assay Block containing 998 L of assay medium using an 8 channel micropipette if chemicals are dissolved in DMSO. Store solution at room temperature (20 30°C ) until next steps. 1 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 2 A B C D E F G H Figure 4 2 3 4 5 6 7 8 9 10 11 12 ← 9 8 L 0 o r 9 9L8 o f a s s a y m e d i u m Serial dilution and arrangement of test chemicals (2) Dose finding study Preparation procedure of cells for the assays. 1. 2. 3. Dispense 100 L of PBS(-) into the peripheral wells and 100 L of assay medium into row 11B – 11G (cf. Figure 5). Prewarm assay medium and PBS(-) at 37 °C. 0.25% Trypsin /1mM EDTA solution is kept at room temperature (20 - 30°C) by bathing in water or ambient air. Remove the supernatant from culture dish, wash with 5 mL of PBS(-). 10 Sumitomo Chemicals Co. Ltd. Ver.06E 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 4. Add 1 - 2 mL of 0.25% trypsin/1 mM EDTA and remove immediately. Then, cells are incubated at 37 °C for 1 - 2 min. 5. Add to 2 mL of assay medium to the dish, and suspend the cells using a micropipette (1000L). 6. Use cell strainer with 50mL centrifuge tubes to obtain more uniform single-cell suspensions. 7. Check viability by staining an aliquot of the cell suspension with Trypan blue. ▲Note A viability of 90 % or higher is acceptable. 8. Count cell number. 9. Dilute the cells in assay medium at cell density of 15,000 cells/mL (750 cells /50 l/well). 10. Transfer the cells to a reservoir, and dispense 50 L of cell suspension to PrimeSurface® 96well white plate (Sumitomo Bake Lite, Co. Ltd., MS-9096W） (cf. Figure 5; row 2B – 10G in the plates for 5-FU or test chemicals). 11. Incubate the cells at 37°C with 5% CO2. 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H : Cells 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 : 100uL of PBS(-) : 100uL of assay medium Figure 5 Preparation of the cells for the assay Treatment of the test chemicals for the assays. 1. After more than 2 hrs of incubation, mix prepared solution of 5-FU and test chemicals by pipetting thoroughly. 2. Add 50 Lof assay medium to a plate (cf. Figure 6; row 2B –2G). 3. Dispense 50 L of solution of chemicals to 96 well-plate containing the cells using an 8 channel micropipette (cf. Figure 6; row 3B –10G). 4. Shake the plate with a plate shaker for a few seconds. 5. Incubate the cells at 37°C with 5% CO2. 11 Sumitomo Chemicals Co. Ltd. Ver.06E 1 A B C D E F G H 2 3 4 MC VC conc.1 conc.2 conc.3 conc.4 conc.5 conc.6 conc.7 MC VC conc.1 conc.2 conc.3 conc.4 conc.5 conc.6 conc.7 MC VC conc.1 conc.2 conc.3 conc.4 conc.5 conc.6 conc.7 MC VC conc.1 conc.2 conc.3 conc.4 conc.5 conc.6 conc.7 MC VC conc.1 conc.2 conc.3 conc.4 conc.5 conc.6 conc.7 MC VC conc.1 conc.2 conc.3 conc.4 conc.5 conc.6 conc.7 : Cells 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 5 6 7 8 9 10 11 12 : 100uL of PBS(-) : 100uL of assay medium Figure 6 Arrangement of chemicals and vehicles for assay Measurement of fluorescence and luminescence ▲Note Switch on measuring equipments 30 min before starting the measurement. 120 hours after seeding the Hand1-ES cells, measure the two following endpoints as described below: For cytotoxicity assay 1. Completely thaw the CellTiter-Fluor™ Cell Viability Assay components in a 37°C water bath. Vortex the GF-AFC substrate (▲Note avoid exposure to light) to ensure homogeneity, then briefly centrifuge for complete substrate volume recovery. 2. Transfer the GF-AFC Substrate into the Assay Buffer container to form a complete reagent (cf; Assay buffer 1mL : GF-AFC substrate 10 L) . Mix by vortexing the contents until the substrate is thoroughly dissolved. 3. Add 10 L of CellTiter-Fluor™ complete reagent to each well (cf. Figure 6; row 2B - 11G). 4. Mix briefly by orbital shaking, then incubate for at least 30 minutes at 37°C. Seal the surface of the plates. ▲Note Don’t incubate longer than 1hour, and be sure to shield plates from ambient light. 5. Measure resulting fluorescence using a fluorometer (380–400nmEx /505nmEm). ▲Note You may need to adjust instrument gains. For differentiation assay 1. Thaw the Steady-Glo® Luciferase Assay System and equilibrate to 25 °C prior to use (▲Note avoid exposure to light). 2. Dispense 100 L of Steady-Glo® Luciferase Assay System to each well of 96 well plates by an 8 channel micropipette (cf: Figure 6; row 2B – 11G). 3. Shake the plates gently for over 30 min at room temperature (20 – 30 °C) with a plate shaker. Seal the surface of the plates. 12 Sumitomo Chemicals Co. Ltd. Ver.06E 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 4. ▲Note Don’t incubate longer than 1hour, and be sure to shield plates from ambient light. Place the plate in luminometer. Luminescence is measured with the appropriate time (cf. 1sec for validation study) according to the manufacturer’s instructions. Definitive study Calculate endpoints according to Steps “Data analysis”. For the definitive study, choose seven dilutions. Carry out the definitive study with a smaller dilution factor covering the relevant range of dose response according to dose finding study. Data analysis Use an Excel (Microsoft) spreadsheet for data recording (Result format ver. XXE). Calculation method and the acceptance criteria for these assays are as follows; Determination of ID50 and IC50 values Relative viabilities (surviving rate) or relative activities for each concentration of test chemicals against vehicle control can be calculated using Excel (Microsoft) spreadsheet. Calculation formulas are described as follows; For differentiation assay a : Mean values of vehicle control b : Mean values of test chemicals c : Mean values of background d : NET values of vehicle control : a-c e : NET values of test chemicals : b-c A : Relative activity of vehicle control : 100 (%) B : Relative activity of test chemicals : e/d X 100 (%) For cytotocxicity assay a : Mean values of vehicle control b : Mean values of test chemicals c : Mean values of background d : NET values of vehicle control : a-c e : NET values of test chemicals : b-c A : Relative viability of cells for vehicle control : 100 (%) B : Relative viability of cells for test chemicals : e/d X 100 (%) Inhibition of differentiation is expressed as the concentration of the test chemical that reduced the luminescence by 50% (ES-ID50, calculated from the concentration-response curve). Cytotoxicity is expressed as the concentration of the chemical reducing the viability of cells to 50% of the control level (ES-IC50 determined from concentration-response curves). Statistical analysis of the dose-response curves from differentiation toxicity and cytotoxicity assays is carried out using an Excel (Microsoft) spreadsheet 13 Sumitomo Chemicals Co. Ltd. Ver.06E 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 for data recording (Result format ver.XXE). The full concentration-response curve is required for the calculation of the ID50 and IC50 values, but this may not always be achievable or practical due to limitations of the test concentration range (for example due to cytotoxicity or solubility problems). In the dose finding study, if IC50 and ID50 values cannot be obtained at the highest concentration of each assay, definitive study is not required. Tentative acceptance criteria (for phase 1 study) Quality control i) Quality control of cell condition Check viability of the cells by staining an aliquot of the cell suspension with Trypan blue. A viability of 90% or greater is acceptable. ii) Quality control of differentiation and cytotoxicity assays To verify the cell growth and cell differentiation, the comparison of the medium control (MC) and background (BG) is used. - The Lower limit of the 95% confidence intervals of the ratio of MC / BG should be above 1 for the cytotoxicity assay - The Lower limit of the 95% confidence intervals of the ratio of MC / BG should be above 10 for differentiation assay iii) Performance standard of the assay The quality of the assay must be controlled using 5-FU as a positive-reference chemical. -The range of ID50 for 5-FU should be within 0.003 and 0.067 g/mL (according to phase 0 study). -The range of IC50 for 5-FU should be within 0.003 and 0.065 g/mL (according to phase 0 study). iv) Quality control for effect of vehicle To verify the effect of the vehicle, the comparison of the medium control (MC) and vehicle control (VC) is used. - The Lower limit of the 95% confidence intervals of the ratio of VC / MC should be above 0.2 for the cytotoxicity and differentiation assays. - In addition to the above criterion CV (SD/mean X 100%) of VC should be below 100%. V) Dose dependency In case of a cytotoxicity and/or differentiation toxicity curve that crosses the 50% line more than 2 times, the test should be redone since no conclusion can be drawn. 14 Sumitomo Chemicals Co. Ltd. Ver.06E 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 EXPERIMENT 3 3-1 Measurement of metabolic stability of test chemicals Liver S9 fractions Rat liver S9 fractions are commercially available (Male, pool of 400 livers, XenoTech). The brief procedure for preparation is as follows. A portion of liver from male SD rats is homogenized in 4 volumes of Tris/HCl buffer (pH7.4) with a homogenizer. Liver homogenate is centrifuged at 9,000 × g for about 20 min at 4 °C to separate S9 fractions. The protein level in the S9 fraction can be measured by commercially available protein assay kit. Metabolic stability Substrate solution is prepared by mixing 100 µM substrate in acetonitrile with 100 fold volume of 6 mM β-NADPH (Oriental Yeast) in 125 mM potassium phosphate buffer (pH7.4). The reaction is initiated by mixing 50 µL of substrate solution with 50 µL of diluted liver S9 fractions (0.5 mg protein/mL) in 125 mM potassium phosphate buffer (pH7.4). After incubation for 35 min at 37 ºC, the reaction was stopped by addition of 400 µL methanol. After centrifugation at 3,000 g for 10 min, the supernatant is analyzed by the triple quadrupole LC/MS and the peak area of the target ion is calculated. The control sample is prepared without liver S9 fractions. The metabolic stability is expressed as the ratio of peak area with liver S9 to that of control. 15 Sumitomo Chemicals Co. Ltd. Ver.06E 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 JUDGMENT In the Hand1-Luc EST, the embryotoxic potency of test chemicals was predicted using a biostatistically based prediction model shown in Figure 7. non-disclosure Figure 7 Prediction method for Hand1-Luc EST 16 Sumitomo Chemicals Co. Ltd. Ver.06E 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 UPDATE RECORD Ver.01E, 2013, Feb, 20th distributions Ver.02E, 2013, April, 30th distributions Major modification - Plate format for the assays - Positive reference chemicals - Preparation of test chemicals - Maximum final concentrations of vehicle (PBS(-) and DMSO) - Quality control and acceptability criteria Ver.03E, 2013, July, 18th distributions - Plate format for the assays - Detection method for cytotoxicity Ver.04E, 2013, July 23th distributions - Minor modification - Tentative acceptance criteria Ver.05-1 E, 2013, November 6th distributions Major modification - Measurement at 120 hr - Tentative acceptance criteria or phase 1 study. Ver.06-1 E, 2014, March 10th distributions Major modification: - Measurement of the water solubility, suppression of the measurement of the logP - Inclusion of a precipitation evaluation step - Modification of the prediction model -Tentative acceptance criteria modified for phase 2. 17 Appendix (2) a SAS Output file:///Users/aibasetsuya/Dropbox/感作性試験関連/P... IILL--88 LLuucc A Assssaayy V Vaalliiddaattiioonn SSttuuddyy P Phhaassee 22bb aanndd cc LLaabb..:: A A ((M Meetthhoodd::KKII)) RRuunn 11 1/4 C Chheem miiccaall O Orriigg.. C Cooddee 01-1 003 01-2 010 01-3 22 33 44 JJuuddggee((KKII)) P P Sens. P P Sens. 007 N P Gray 02-1 005 P P P 02-2 011 P P N Sens. 02-3 008 P P P Sens. 03-1 014 P P P 03-2 006 P P P 03-3 001 P P P 04-1 009 N N N Non-Sens. 04-2 013 N N N Non-Sens. 04-3 015 N P N Gray 05-1 012 N N N Non-Sens. 05-2 004 N N N Non-Sens. 05-3 002 N N P 08-1 021 P P 08-2 029 P N P P Sens. 08-3 032 N P N N Non-Sens. 12-1 022 P P Sens. 12-2 030 P P Sens. 12-3 033 P P Sens. 19-1 023 N N N 19-2 026 P N N N Non-Sens. 19-3 034 P N N N Non-Sens. 21-1 024 N P N N Non-Sens. 21-2 027 N N N Non-Sens. 21-3 035 N N N Non-Sens. 22-1 025 N N N Non-Sens. 22-2 028 P N N 22-3 031 N N N P P Sens. Sens. Sens. P N Sens. Non-Sens. Sens. Non-Sens. P Sens. Non-Sens. 14/03/03 17:19 SAS Output file:///Users/aibasetsuya/Dropbox/感作性試験関連/P... IILL--88 LLuucc A Assssaayy V Vaalliiddaattiioonn SSttuuddyy P Phhaassee 22bb aanndd cc LLaabb..:: B B ((M Meetthhoodd::KKII)) RRuunn 11 2/4 22 33 44 C Chheem miiccaall O Orriigg.. C Cooddee JJuuddggee((KKII)) 01-1 010 P P Sens. 01-2 001 P P Sens. 01-3 009 P P Sens. 02-1 005 P P P 02-2 013 P P P 02-3 003 P P Sens. 03-1 015 P P Sens. 03-2 007 P P Sens. 03-3 012 P P P 04-1 002 P N N 04-2 006 N N N Non-Sens. 04-3 014 N N N Non-Sens. 05-1 011 N N N Non-Sens. 05-2 004 P P 05-3 008 N P P 08-1 024 N P N P Sens. 08-2 027 N N P P Sens. 08-3 035 P P 12-1 025 P P 12-2 028 P P Sens. 12-3 031 P P Sens. 19-1 021 N N N 19-2 029 N N N 19-3 032 P P N 21-1 022 P P Sens. 21-2 030 P P Sens. 21-3 033 N N P 22-1 023 N N N 22-2 026 P N N N Non-Sens. 22-3 034 N N P N Non-Sens. P Sens. Sens. Sens. N Non-Sens. Sens. Sens. Sens. P Sens. N Non-Sens. Non-Sens. N P Sens. Sens. Non-Sens. 14/03/03 17:19 SAS Output file:///Users/aibasetsuya/Dropbox/感作性試験関連/P... IILL--88 LLuucc A Assssaayy V Vaalliiddaattiioonn SSttuuddyy P Phhaassee 22bb aanndd cc LLaabb..:: C C ((M Meetthhoodd::KKII)) RRuunn 11 3/4 22 33 44 C Chheem miiccaall O Orriigg.. C Cooddee JJuuddggee((KKII)) 01-1 011 P P Sens. 01-2 004 P P Sens. 01-3 006 P P Sens. 02-1 010 P P P 02-2 002 P P P Sens. 02-3 015 P P P Sens. 03-1 008 P P P 03-2 003 P P P Sens. 03-3 014 P P P Sens. 04-1 007 N N N Non-Sens. 04-2 013 N N P N Non-Sens. 04-3 005 N N P N Non-Sens. 05-1 009 N P N 05-2 012 N P N N Non-Sens. 05-3 001 N P N P Sens. 08-1 022 P P N N Sens. 08-2 030 N P N N Non-Sens. 08-3 033 P N P Sens. 12-1 023 P P P Sens. 12-2 026 P P Sens. 12-3 034 P P Sens. 19-1 024 P N N 19-2 027 N N N 19-3 035 N N N N Non-Sens. 21-1 025 N N P N Non-Sens. 21-2 028 N N P P Sens. 21-3 031 N P N P Sens. 22-1 021 N N N 22-2 029 N N N 22-3 032 N N N P P Sens. Sens. Gray N Non-Sens. Non-Sens. Non-Sens. N Non-Sens. Non-Sens. 14/03/03 17:19 SAS Output file:///Users/aibasetsuya/Dropbox/感作性試験関連/P... IILL--88 LLuucc A Assssaayy V Vaalliiddaattiioonn SSttuuddyy P Phhaassee 22bb aanndd cc ((M Meetthhoodd::KKII)) LLaabb.. 4/4 AA B B C C C Chheem miiccaall JJuuddggee((AAVVEE)) JJuuddggee((AAVVEE)) JJuuddggee((AAVVEE)) 01 SSG SSS SSS 02 SSS SSS SSS 03 SSS SSS SSS 04 NNG NNN NNN 05 NNN NSS GNS 08 SSN SSS SNS 12 SSS SSS SSS 19 NNN NNS NNN 21 NNN SSS NSS 22 NSN NNN NNN 14/03/03 17:19 Appendix (2) b SAS Output file:///Users/aibasetsuya/Dropbox/感作性試験関連/P... IILL--88 LLuucc A Assssaayy V Vaalliiddaattiioonn SSttuuddyy P Phhaassee 22bb aanndd cc LLaabb..:: A A ((M Meetthhoodd::O OM M)) RRuunn 11 1/4 C Chheem miiccaall O Orriigg.. C Cooddee 01-1 003 01-2 010 01-3 22 33 44 JJuuddggee((O OM M)) N P Gray P P Sens. 007 N P Gray 02-1 005 P P P 02-2 011 P P N Sens. 02-3 008 P P P Sens. 03-1 014 P P P 03-2 006 P P P 03-3 001 P P P 04-1 009 P N N Gray 04-2 013 N N N Non-Sens. 04-3 015 N N N Non-Sens. 05-1 012 N P P Sens. 05-2 004 N N N Non-Sens. 05-3 002 N N P 08-1 021 P P 08-2 029 P N N P Sens. 08-3 032 N P P P Sens. 12-1 022 P P Sens. 12-2 030 P P Sens. 12-3 033 P P Sens. 19-1 023 N N N 19-2 026 P P N P Sens. 19-3 034 P N N N Non-Sens. 21-1 024 N P P N Sens. 21-2 027 N P P Sens. 21-3 035 N N N Non-Sens. 22-1 025 N N N Non-Sens. 22-2 028 P N N 22-3 031 N N N P P Sens. Sens. Sens. P N Sens. Non-Sens. Sens. Non-Sens. P Sens. Non-Sens. 14/03/03 17:20 SAS Output file:///Users/aibasetsuya/Dropbox/感作性試験関連/P... IILL--88 LLuucc A Assssaayy V Vaalliiddaattiioonn SSttuuddyy P Phhaassee 22bb aanndd cc LLaabb..:: B B ((M Meetthhoodd::O OM M)) RRuunn 11 2/4 22 33 44 C Chheem miiccaall O Orriigg.. C Cooddee JJuuddggee((O OM M)) 01-1 010 P N Gray 01-2 001 P P Sens. 01-3 009 P P Sens. 02-1 005 P P P 02-2 013 P P P 02-3 003 P P Sens. 03-1 015 P P Sens. 03-2 007 P P Sens. 03-3 012 P P P 04-1 002 N N N 04-2 006 N N N Non-Sens. 04-3 014 N N N Non-Sens. 05-1 011 N N N Non-Sens. 05-2 004 N N 05-3 008 N N P 08-1 024 N P P P Sens. 08-2 027 N P N P Sens. 08-3 035 P P 12-1 025 N N 12-2 028 P P Sens. 12-3 031 P P Sens. 19-1 021 N N P 19-2 029 N N N 19-3 032 N P N 21-1 022 P P Sens. 21-2 030 P P Sens. 21-3 033 N N P 22-1 023 N N N 22-2 026 N N P N Non-Sens. 22-3 034 N N P N Non-Sens. P Sens. Sens. Sens. N Non-Sens. Gray Gray Sens. P Gray P Sens. Non-Sens. N P Non-Sens. Sens. Non-Sens. 14/03/03 17:20 SAS Output file:///Users/aibasetsuya/Dropbox/感作性試験関連/P... IILL--88 LLuucc A Assssaayy V Vaalliiddaattiioonn SSttuuddyy P Phhaassee 22bb aanndd cc LLaabb..:: C C ((M Meetthhoodd::O OM M)) RRuunn 11 3/4 22 33 44 C Chheem miiccaall O Orriigg.. C Cooddee JJuuddggee((O OM M)) 01-1 011 P P Sens. 01-2 004 P P Sens. 01-3 006 P P Sens. 02-1 010 P P P 02-2 002 P P P Sens. 02-3 015 P P P Sens. 03-1 008 P P P 03-2 003 P P P Sens. 03-3 014 P P P Sens. 04-1 007 N N N Non-Sens. 04-2 013 N N N P Non-Sens. 04-3 005 N N P N Non-Sens. 05-1 009 N P N 05-2 012 N N N N Non-Sens. 05-3 001 N P N N Non-Sens. 08-1 022 N P N P Sens. 08-2 030 N P P P Sens. 08-3 033 P P P Sens. 12-1 023 N P P Sens. 12-2 026 P P Sens. 12-3 034 P P Sens. 19-1 024 N N N 19-2 027 N N N 19-3 035 N N P N Non-Sens. 21-1 025 N N P P Sens. 21-2 028 P P P P Sens. 21-3 031 N P N P Sens. 22-1 021 N N N 22-2 029 P N N 22-3 032 N N N P P Sens. Sens. Gray N Non-Sens. Non-Sens. Non-Sens. N Non-Sens. Non-Sens. 14/03/03 17:20 SAS Output file:///Users/aibasetsuya/Dropbox/感作性試験関連/P... IILL--88 LLuucc A Assssaayy V Vaalliiddaattiioonn SSttuuddyy P Phhaassee 22bb aanndd cc ((M Meetthhoodd::O OM M)) LLaabb.. 4/4 AA B B C C C Chheem miiccaall JJuuddggee((AAVVEE)) JJuuddggee((AAVVEE)) JJuuddggee((AAVVEE)) 01 GSG GSS SSS 02 SSS SSS SSS 03 SSS SSS SSS 04 GNN NNN NNN 05 SNN NGG GNN 08 SSS SSS SSS 12 SSS GSS SSS 19 NSN SNN NNN 21 SSN SSS SSS 22 NSN NNN NNN 14/03/03 17:20 Appendix (2) c SAS Output file:///Users/aibasetsuya/Dropbox/感作性試験関連/P... IILL--88 LLuucc A Assssaayy V Vaalliiddaattiioonn SSttuuddyy P Phhaassee 22bb aanndd cc LLaabb..:: A A ((M Meetthhoodd::M MA A)) RRuunn 11 1/4 C Chheem miiccaall O Orriigg.. C Cooddee 01-1 003 01-2 010 01-3 22 33 44 JJuuddggee((M MAA)) N P Gray P P Sens. 007 N P Gray 02-1 005 P P P 02-2 011 P P N Sens. 02-3 008 P P P Sens. 03-1 014 P P P 03-2 006 P P P 03-3 001 P P P 04-1 009 N N N Non-Sens. 04-2 013 N N N Non-Sens. 04-3 015 N N N Non-Sens. 05-1 012 N N N Non-Sens. 05-2 004 N N N Non-Sens. 05-3 002 N N N 08-1 021 P P 08-2 029 P N N P Sens. 08-3 032 N P P P Sens. 12-1 022 P P Sens. 12-2 030 P P Sens. 12-3 033 P P Sens. 19-1 023 N N N 19-2 026 P N N N Non-Sens. 19-3 034 P N N N Non-Sens. 21-1 024 N P N N Non-Sens. 21-2 027 N N N Non-Sens. 21-3 035 N N N Non-Sens. 22-1 025 N N N Non-Sens. 22-2 028 P N N 22-3 031 N N N P P Sens. Sens. Sens. P N Sens. Non-Sens. Sens. Non-Sens. P Sens. Non-Sens. 14/03/03 17:20 SAS Output file:///Users/aibasetsuya/Dropbox/感作性試験関連/P... IILL--88 LLuucc A Assssaayy V Vaalliiddaattiioonn SSttuuddyy P Phhaassee 22bb aanndd cc LLaabb..:: B B ((M Meetthhoodd::M MA A)) RRuunn 11 2/4 22 33 44 C Chheem miiccaall O Orriigg.. C Cooddee JJuuddggee((M MAA)) 01-1 010 P N Gray 01-2 001 P P Sens. 01-3 009 P P Sens. 02-1 005 P P P 02-2 013 P P P 02-3 003 P P Sens. 03-1 015 P P Sens. 03-2 007 P P Sens. 03-3 012 P P P 04-1 002 N N N 04-2 006 N N N Non-Sens. 04-3 014 N N N Non-Sens. 05-1 011 N N N Non-Sens. 05-2 004 N N 05-3 008 N N P 08-1 024 N P N P Sens. 08-2 027 N N N P Non-Sens. 08-3 035 P P 12-1 025 N N 12-2 028 P P Sens. 12-3 031 P P Sens. 19-1 021 N N N 19-2 029 N N N 19-3 032 N P N 21-1 022 P P Sens. 21-2 030 P P Sens. 21-3 033 N N P 22-1 023 N N N 22-2 026 N N N N Non-Sens. 22-3 034 N N P N Non-Sens. P Sens. Sens. Sens. N Non-Sens. Gray Gray Sens. P Gray N Non-Sens. Non-Sens. N P Non-Sens. Sens. Non-Sens. 14/03/03 17:20 SAS Output file:///Users/aibasetsuya/Dropbox/感作性試験関連/P... IILL--88 LLuucc A Assssaayy V Vaalliiddaattiioonn SSttuuddyy P Phhaassee 22bb aanndd cc LLaabb..:: C C ((M Meetthhoodd::M MA A)) RRuunn 11 3/4 22 33 44 C Chheem miiccaall O Orriigg.. C Cooddee JJuuddggee((M MAA)) 01-1 011 P P Sens. 01-2 004 P P Sens. 01-3 006 P P Sens. 02-1 010 P P P 02-2 002 P P P Sens. 02-3 015 P P P Sens. 03-1 008 P P P 03-2 003 P P P Sens. 03-3 014 P P P Sens. 04-1 007 N N N Non-Sens. 04-2 013 N N N N Non-Sens. 04-3 005 N N P N Non-Sens. 05-1 009 N P N 05-2 012 N N N N Non-Sens. 05-3 001 N P N N Non-Sens. 08-1 022 N P N N Non-Sens. 08-2 030 N P N N Non-Sens. 08-3 033 P N P Sens. 12-1 023 N P P Sens. 12-2 026 P P Sens. 12-3 034 P P Sens. 19-1 024 N N N 19-2 027 N N N 19-3 035 N N N N Non-Sens. 21-1 025 N N P N Non-Sens. 21-2 028 N N P P Sens. 21-3 031 N P N P Sens. 22-1 021 N N N 22-2 029 N N N 22-3 032 N N N P P Sens. Sens. Gray N Non-Sens. Non-Sens. Non-Sens. N Non-Sens. Non-Sens. 14/03/03 17:20 SAS Output file:///Users/aibasetsuya/Dropbox/感作性試験関連/P... IILL--88 LLuucc A Assssaayy V Vaalliiddaattiioonn SSttuuddyy P Phhaassee 22bb aanndd cc ((M Meetthhoodd::M MA A)) LLaabb.. 4/4 AA B B C C C Chheem miiccaall JJuuddggee((AAVVEE)) JJuuddggee((AAVVEE)) JJuuddggee((AAVVEE)) 01 GSG GSS SSS 02 SSS SSS SSS 03 SSS SSS SSS 04 NNN NNN NNN 05 NNN NGG GNN 08 SSS SNS NNS 12 SSS GSS SSS 19 NNN NNN NNN 21 NNN SSS NSS 22 NSN NNN NNN 14/03/03 17:20 Appendix (2) d IL-8 Luc assay protocol ver. 017E November 11th, 2013 This protocol describes how to maintain cells, how to prepare test chemicals, and how to measure the luciferase activity of a THP-1-derived IL-8 reporter cell line, THP-G8, for immunotoxity assay. Department of Dermatology, Tohoku University Graduate School of Medicine Yutaka Kimura, M.D., Ph.D. Setsuya Aiba, M.D., Ph.D. 1 Contents 1. Materials ...................................................................................................................5 1-1 Cells ....................................................................................................................5 1-2 Reagents and equipment .....................................................................................5 1-3 Culture medium ..............................................................................................7 1-3-1 A medium: for maintenance of THP-G8 cells .............................................7 1-3-2 B medium: for luciferase assay ...................................................................7 1-3-3 C medium: for thawing THP-G8 cells .........................................................7 1-4 Cobalt chloride (CoCl2): CoCl2 is used as a positive control 1-5 Thawing of THP-G8 cells 1-6 Maintenance of THP-G8 cells ...........................8 ................................................................................8 ..........................................................................8 2. Preparation of cells for assay ................................................................................10 3. Preparation of chemicals and cell treatment with chemicals ............................11 3-1 Dissolution by vehicle ...................................................................................... 11 3-2 When the chemical is prepared as 25, 50 or 100 mg/ml in distilled water ......14 3-2-1 Arrangement of chemicals and vehicle (1st experiment) .............................14 3-2-2 Serial dilution (1st experiment) ....................................................................14 3-2-3 2 step dilution (1st experiment) ..................................................................15 3-2-4 Final constituents in each well of the plate (1st experiment) .......................17 3-2-5 Arrangement of chemicals and solvent (2nd , 3rd and 4th experiment) .........18 3-2-6 Serial dilution (2nd , 3rd and 4th experiment) ................................................18 3-2-7 2 step dilution (2nd , 3rd and 4th experiment) ..............................................19 3-2-8 3-3 Final constituents in each well of the plate (2nd , 3rd and 4th experiment) ...21 When the chemical is prepared as a 500 mg/ml DMSO solution ....................22 3-3-1 Arrangement of chemicals and vehicle (1st experiment) .............................22 3-3-2 Serial dilution (1st experiment) ....................................................................22 2 3-3-3 Dilution of DMSO solution with the B medium (1st experiment) .............23 3-3-4 2 step dilution (1st experiment) ..................................................................24 3-3-5 Final constituents in each well of the plate (1st experiment) .......................26 3-3-6 Arrangement of chemicals and vehicle (2nd , 3rd and 4th experiment) .........27 3-3-7 Serial dilution (2nd , 3rd and 4th experiment) ................................................27 3-3-8 Dilution of DMSO solution with the B medium (2nd , 3rd and 4th experiment) ......................................................................28 3-3-9 2 step dilution (2nd , 3rd and 4th experiment) ................................................29 3-3-10 Final constituents in each well of the plate (2nd , 3rd and 4th experiment) .31 4. Preparation of Cobalt chloride (CoCl2) and treatment of THP-G8 cells with CoCl2 ............................................................................................................ 32 4-1 Preparation of cells ...........................................................................................32 4-2 Preparation of Cobalt chloride (CoCl2) and treatment of THP-G8 cells with CoCl2 (2 step dilution) .....................................................................................33 4-3 Final constituents in each well of the plate ......................................................36 4-4 Measurement ....................................................................................................36 5. Validation of reagents and equipment ................................................................38 5-1 Measurement of transmittance of optical filter for multicolor measurement .............................................................................38 5-1-1 Reagents ....................................................................................................38 5-1-2 Calibration .................................................................................................38 5-1-2-1 Preparation of luminescence reaction solution ..................................38 5-1-2-2 5-2 Bioluminescence measurement .........................................................39 Quality control of equipment ...........................................................................40 5-2-1 Light source ..............................................................................................40 5-2-2 Data collection (an example using TRIANT® by ATTO) ........................40 6. Measurement of luciferase activity .....................................................................42 7. Criteria ..................................................................................................................43 3 7-1 Definition of the parameters used in IL-8 Luc assay .......................................43 7-2 The criteria to identify sensitizers in IL-8 Luc assay .......................................43 8. Update record .......................................................................................................46 4 1. Materials 1-1 Cells The human macrophage-like cell line, THP-1, was obtained from the American Type Culture Collection (Manassas, VA, USA). A THP-1-derived IL-8 reporter cell line, THP-G8, that harbors SLO and SLR luciferase genes under the control of IL-8 and GAPDH promoters, respectively, was established by Dept. of Dermatology, Tohoku University School of Medicine. (Takahashi T. et al. An in vitro test to screen skin sensitizers using a stable THP-1-derived IL-8 reporter cell line, THP-G8. Toxicol Sci, 124(2), 359-369, 2011) (International patent publication No.；WO2012/002507A1) 1-2 Reagents and equipment For maintenance of THP-G8 cells ・RPMI-1640 (GIBCO Cat#11875-093, 500 ml) ・FBS (Biological Industries Cat#04-001-1E Lot:715004) ・Antibiotic-Antimycotic (GIBCO Cat#15240-062) ・G418 (Nacalai tesque Cat#16513-84) ・Puromycin (InvivoGen Cat#ant-pr-1) For chemical exposure, positive control, solvents ・Cobalt chloride (Hexahydrate) (Sigma Cat#255599) ・DMSO (Sigma Cat#D5879) ・Distilled water (GIBCO Cat#10977-015) For measurement of luciferase activity ・Tripluc® Luciferase assay reagent (TOYOBO Cat#MRA-301) 5 < Expendable supplies> • T-75 Flask Tissue Culture Treated (e.g. BD Falcon Cat#35-3136) • 96 well μ clear black plate (flat-bottom, for measurement of the luciferase activity, e.g. Greiner bio-one Cat#655090) • 96 well clear plate (round-bottom, for preparation chemicals and CoCl2) • 96 well Assay Block, 2ml (e.g. costar Cat#3960) • Reservoir • Pipette <Equipment for measurement of luciferase activity> • Measuring device: a microplate-type luminometer with a multi-color detection system which can equip an optical filter e.g. Phelios AB-2350 (ATTO), ARVO (PerkinElmer), Tristar LB941 (Berthold) • Optical filter: 600 nm long pass filter, 600～700 nm band pass filter • Measuring time: set 1～5 sec/well measuring time <Others> • Pipetman • 8 channel or 12 channel pipetman (optimized for 20~100 μl and 0.5~10 μl) • Plate shaker (for 96 well plate) • CO2 incubator (37°C, 5% ) • Water bath • Cell counter: hemocytometer, trypanblue 6 1-3 Culture medium 1-3-1 A medium: for maintenance of THP-G8 cells (500 ml, stored at 2-8°C) Final Required Reagent Company Concentration concentration amount in medium RPMI-1640 GIBCO #11875-093 FBS Biological Industries Cat#04-001-1E - - 445 ml - 10 % 50 ml Lot:715004 Antibiotic-Antimycotic GIBCO #15240-062 100× 1× 5 ml Puromycin InvivoGen # ant-pr-1 10 mg/ml 0.15 μg/ml 7.5 μl G418 Nacalai tesque #16513-84 50 mg/ml 300 μg/ml 3 ml 1-3-2 B medium: for luciferase assay (30 ml, stored at 2-8°C) Final Required Reagent Company Concentration concentration amount in medium RPMI-1640 GIBCO #11875-093 FBS Biological Industries Cat#04-001-1E - - 27 ml - 10 % 3 ml Lot:715004 1-3-3 C medium: for thawing THP-G8 cells (30 ml, stored at 2-8°C) Final Required Reagent Company Concentration concentration amount in medium RPMI-1640 GIBCO #11875-093 FBS Biological Industries Cat#04-001-1E - - 26.7 ml - 10 % 3 ml 100× 1× 0.3 ml Lot:715004 Antibiotic-Antimycotic GIBCO #15240-062 7 1-4 Cobalt chloride (CoCl2) CoCl2 is used as a positive control <Stock solution preparation> • Dissolve 25 mg CoCl2 in 1 ml of distilled water (25mg/ml). <Storage> • Stored at -20°C <How to use> • Make one twentieth, one tenth and one fifth dilutions of the stock solution with distilled water (1.25, 2.5, 5 mg/ml), and then dilute 25 times with B medium and add 50 μl of this solution to well (final concentration; 25, 50, 100 μg/ml). Reagent CoCl2 Concentration of Concentration before Final stock solution adding to cell concentration 25 mg/ml 1.25, 2.5, 5 mg/ml Company Sigma Cat#255599 Distilled GIBCO water Cat#10977-015 25, 50, 100 μg/ml 1-5 Thawing of THP-G8 cells • Prewarm 9 ml of C medium in 15 ml polypropylene conical tube in 37°C water bath (for centrifuge) and 15 ml of C medium in T-75 Flask at 37°C, 5% CO2 incubator (for culture). • Thaw frozen cells (2x106 cells / 0.5 ml of Freezing Medium) in 37°C water bath, and add to the 15 ml polypropylene conical tube with 9 ml of prewarmed C medium. Centrifuge the tube at 1,400 rpm at room temperature for 5 min, discard supernatant, and resuspend in 15 ml of prewarmed C medium in T-75 Flask. Cells are incubated in 37°C, 5% CO2. 1-6 Maintenance of THP-G8 cells • Prewarm A medium in T-75 Flask at 37°C, 5% CO2 incubator. Culture medium should be changed to A medium 3 or 4 days after thawing. At that time, count cell number, centrifuge the tube at 1,400 rpm for 5 min, discard supernatant, and resuspend in prewarmed A medium in T-75 Flask. Cells are passaged at 2～ 8 5x105/ml depending on condition of cells and incubated in 37°C, 5% CO2. • Interval of subculture should be 3～4 days. Cells can be used between one and six weeks after thawing. 9 2. Preparation of cells for assay The cell passage should be done 2-4 days before the assay. Use cells between 1 and 6 weeks after thawing. Prewarm B medium in 37°C water bath. Count cell number, and collect cells needed (2.5 x 106 cells for one chemical are required, but for safety, 3.75 x 106 cells for one chemical should be prepared), centrifuge the tube at 1,400 rpm, 5 min. Resuspend in prewarmed B medium at cell density of 1×106/ml. Transfer cell suspension to a reservoir, and add 50 μl of cell suspension to 96 well μclear black plate (flat bottom) using an 8 channel or 12 channel pipetman. (cf. Figure.1, row C-F) Figure 1. flatbottom black A B C D E F 1 2 3 4 5 6 7 8 9 10 11 12 Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul G H 10 3. Preparation of chemicals and cell treatment with chemicals Prepare chemicals and add them to wells after cell preparation. 3-1 Dissolution by vehicle (cf. Figure 2) Dissolve the chemical first in distilled water. Namely, weigh 0.025 g of the test chemical in a volumetric flask and add distilled water up to 1 ml. If the chemical is soluble at 25 mg/mL, weigh 0.050 g of the test chemical in a volumetric flask and add distilled water up to 1 ml. If the chemical is not soluble at 50 mg/mL, 25 mg/mL is the highest soluble concentration. If the chemical is soluble at 50 mg/mL, weigh 0.100 g of the test chemical in a volumetric flask and add distilled water up to 1 ml. If the chemical is not soluble at 100 mg/mL, 50 mg/mL is the highest soluble concentration. If the chemical is soluble at 100 mg/mL, 100 mg/mL is the highest soluble concentration. If the chemical is not soluble in water, the chemical should be dissolved in DMSO at 500 mg/ml. Namely, weigh 0.5 g of the test chemical in volumetric flask and add DMSO up to 1 ml. If the chemical is not soluble at 500 mg/mL, the highest soluble concentration should be determined by diluting the solution from 500 mg/ml at a common ratio of two (250 mg/ml 125 mg/ml continued if needed) with DMSO. Sonication and vortex may be used if needed，and attempt to dissolve the chemical for at least 5 minutes. The chemical should be used within 4 hours after being dissolved in distilled water or DMSO. 11 Figure 2. In the first experiment (1st experiment), when the chemical is prepared in distilled water, conduct 11 serial dilutions at a common ratio of 2 from the highest concentration using distilled water. When the chemical is prepared as a DMSO solution, conduct 11 serial dilutions at a common ratio of 2 from the highest concentration using DMSO. In the second, third or fourth experiment (2nd , 3rd or 4th experiment), determine the minimum concentration at which I.I.-SLR-LA (mentioned later in 7-1) became lower than 0.05 in the 1st experiment, use the concentration one step (2-times) higher than this determined concentration as the highest concentration of the chemical to examine, and conduct 11 serial dilutions at a common ratio of 1.5 from the highest concentration. If I.I.-SLR-LA did not become lower than 0.05 or became lower than 0.05 at the highest 12 concentration in the 1st experiment, conduct 11 serial dilutions at a common ratio of 1.5 from the highest concentration in the 1st experiment. For example, in Figure 3 below, the minimum concentration at which I.I.-SLR-LA became lower than 0.05 is 7.81g/ml. The highest concentration of the chemical to examine is the concentration one step (2-times) higher than 7.81g/ml, which is 15.63 g/ml. In Figure 4 below, I.I.-SLR-LA did not become lower than 0.05. In such a case, the highest concentration of the chemical to examine is the highest concentration in the 1st experiment, namely 500 g/ml. 0.05 g/ml Final concentration in 2nd , 3rd and 4th experiment Figure 3. 0.05 g/ml Final concentration in 2nd , 3rd and 4th experiment Figure 4 13 3-2 When the chemical is prepared as 25, 50 or 100 mg/ml in distilled water If the chemical is prepared at 50 or 100 mg/ml in distilled water, use the prepared concentration instead of the 25 mg/ml distilled water solution. 3-2-1 Arrangement of chemicals and vehicle (1st experiment) Add 100 μl of the 25 mg/ml distilled water solution of the chemical to well #A12, and 50 μl of the distilled water to wells #A1-#A11of the 96 well clear plate (round bottom). 3-2-2 Serial dilution (1st experiment) Conduct 11 serial dilutions at a common ratio of 2 as indicated in Figure 5 from well #A11 to well #A2. (Transfer 50 μl to the next (left) well) round bottom clear A 1 Distilled water 50ul 2 Distilled water 50ul Distilled water 50ul B C D E F G H 4 Distilled water 50ul 5 Distilled water 50ul 6 Distilled water 50ul 7 Distilled water 50ul 8 Distilled water 50ul 9 Distilled water 50ul 10 Distilled water 50ul 11 Distilled water 50ul 12 Chemical 25mg/ml in distilled water 100ul Serial dilution at a common ratio of 2 : transfer 50 ul （pipetman、yellow tip） round bottom clear A 3 1 Distilled water 50ul 2 3 4 5 6 7 8 9 10 11 12 Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical 0.025mg/ml 0.05mg/ml in 0.10mg/ml in 0.20mg/ml in 0.39mg/ml in 0.78mg/ml in 1.56mg/ml in 3.13mg/ml in 6.25mg/ml in 12.5mg/ml in 25mg/ml in in distilled distilled distilled distilled distilled distilled distilled distilled distilled distilled distilled water water water water water water water water water water water 100ul 50ul 50ul 50ul 50ul 50ul 50ul 50ul 50ul 50ul 50ul B C D E F G H Figure 5. 14 3-2-3 2 step dilution (1st experiment) Add 20 μl of the diluted chemical to 480 μl of the B medium prepared in the assay block. And add 50 μl to THP-G8 in a 96 well plate using an 8 channel or 12 channel pipetman after pipetting 20 times. Shake the plate with a plateshaker, and incubate in a CO2 incubator for 16 hours (37°C, 5%). round bottom clear A 1 2 3 4 5 6 8 9 10 11 12 Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical 0.025mg/ml 0.05mg/ml in 0.10mg/ml in 0.20mg/ml in 0.39mg/ml in 0.78mg/ml in 1.56mg/ml in 3.13mg/ml in 6.25mg/ml in 12.5mg/ml in 25mg/ml in in distilled distilled distilled distilled distilled distilled distilled distilled distilled distilled distilled water water water water water water water water water water water 100ul 50ul 50ul 50ul 50ul 50ul 50ul 50ul 50ul 50ul 50ul Distilled water 50ul B C D E F G H 20ul Assay Block A 7 1 B medium 480ul 2 B medium 480ul 3 B medium 480ul 4 B medium 480ul 5 B medium 480ul 6 B medium 480ul B C D E F G H Figure 6. 15 7 B medium 480ul 8 B medium 480ul 9 B medium 480ul 10 B medium 480ul 11 B medium 480ul 12 B medium 480ul Assay Block 1 B medium 500ul A 2 Chemical 1.0ug/ml in B medium 500ul 3 Chemical 2.0ug/ml in B medium 500ul 4 Chemical 3.9ug/ml in B medium 500ul 5 Chemical 7.8ug/ml in B medium 500ul 6 7 8 9 10 11 12 Chemical Chemical Chemical Chemical Chemical Chemical Chemical 15.6ug/ml in 31.3ug/ml in 62.5ug/ml in 125ug/ml in 250ug/ml in 500ug/ml in 1000ug/ml in B medium B medium B medium B medium B medium B medium B medium 500ul 500ul 500ul 500ul 500ul 500ul 500ul B C D E F G H 50ul flatbottom black 1 2 3 4 5 6 7 8 9 10 11 12 Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul A B C D E F G H Figure 7 16 3-2-4. Final constituents in each well of the plate. (1st experiment) flatbottom black 1 2 3 4 5 6 7 8 9 10 11 12 Cell 5x10^4 Chemical 0ug/ml 100ul B-medium Cell 5x10^4 Chemical 0ug/ml 100ul B-medium Cell 5x10^4 Chemical 0ug/ml 100ul B-medium Cell 5x10^4 Chemical 0ug/ml 100ul B-medium Cell 5x10^4 Chemical 0.5ug/ml 100ul B-medium Cell 5x10^4 Chemical 0.5ug/ml 100ul B-medium Cell 5x10^4 Chemical 0.5ug/ml 100ul B-medium Cell 5x10^4 Chemical 0.5ug/ml 100ul B-medium Cell 5x10^4 Chemical 1.0ug/ml 100ul B-medium Cell 5x10^4 Chemical 1.0ug/ml 100ul B-medium Cell 5x10^4 Chemical 1.0ug/ml 100ul B-medium Cell 5x10^4 Chemical 1.0ug/ml 100ul B-medium Cell 5x10^4 Chemical 2.0ug/ml 100ul B-medium Cell 5x10^4 Chemical 2.0ug/ml 100ul B-medium Cell 5x10^4 Chemical 2.0ug/ml 100ul B-medium Cell 5x10^4 Chemical 2.0ug/ml 100ul B-medium Cell 5x10^4 Chemical 3.9ug/ml 100ul B-medium Cell 5x10^4 Chemical 3.9ug/ml 100ul B-medium Cell 5x10^4 Chemical 3.9ug/ml 100ul B-medium Cell 5x10^4 Chemical 3.9ug/ml 100ul B-medium Cell 5x10^4 Chemical 7.8ug/ml 100ul B-medium Cell 5x10^4 Chemical 7.8ug/ml 100ul B-medium Cell 5x10^4 Chemical 7.8ug/ml 100ul B-medium Cell 5x10^4 Chemical 7.8ug/ml 100ul B-medium Cell 5x10^4 Chemical 15.6ug/ml 100ul B-medium Cell 5x10^4 Chemical 15.6ug/ml 100ul B-medium Cell 5x10^4 Chemical 15.6ug/ml 100ul B-medium Cell 5x10^4 Chemical 15.6ug/ml 100ul B-medium Cell 5x10^4 Chemical 31.3ug/ml 100ul B-medium Cell 5x10^4 Chemical 31.3ug/ml 100ul B-medium Cell 5x10^4 Chemical 31.3ug/ml 100ul B-medium Cell 5x10^4 Chemical 31.3ug/ml 100ul B-medium Cell 5x10^4 Chemical 62.5ug/ml 100ul B-medium Cell 5x10^4 Chemical 62.5ug/ml 100ul B-medium Cell 5x10^4 Chemical 62.5ug/ml 100ul B-medium Cell 5x10^4 Chemical 62.5ug/ml 100ul B-medium Cell 5x10^4 Chemical 125ug/ml 100ul B-medium Cell 5x10^4 Chemical 125ug/ml 100ul B-medium Cell 5x10^4 Chemical 125ug/ml 100ul B-medium Cell 5x10^4 Chemical 125ug/ml 100ul B-medium Cell 5x10^4 Chemical 250ug/ml 100ul B-medium Cell 5x10^4 Chemical 250ug/ml 100ul B-medium Cell 5x10^4 Chemical 250ug/ml 100ul B-medium Cell 5x10^4 Chemical 250ug/ml 100ul B-medium Cell 5x10^4 Chemical 500ug/ml 100ul B-medium Cell 5x10^4 Chemical 500ug/ml 100ul B-medium Cell 5x10^4 Chemical 500ug/ml 100ul B-medium Cell 5x10^4 Chemical 500ug/ml 100ul B-medium A B C D E F G H Figure 8 17 3-2-5 Arrangement of chemicals and vehicle (2nd , 3rd and 4th experiment) Add 150 μl of the distilled water solution of the chemical prepared at the highest concentration defined by the 1st experiment (A mg/ml) to well #A12, and 50 μl of distilled water to wells #A1-#A11of 96 well clear plate (round bottom). 3-2-6 Serial dilution (2nd , 3rd and 4th experiment) Conduct 11 serial dilutions at a common ratio of 1.5 as indicated in Figure 9 from well #A11 to well #A2.(Transfer 100 μl to the next (left) well) round bottom clear A 1 Distilled water 50ul Distilled water 50ul B C D E F G H 3 Distilled water 50ul 4 Distilled water 50ul 5 Distilled water 50ul 6 Distilled water 50ul 7 Distilled water 50ul 8 Distilled water 50ul 9 Distilled water 50ul 10 11 Distilled water 50ul Distilled water 50ul 12 Chemical Amg/ml in distilled water 150ul Serial dilution at a dilution common: transfer ratio of 1.5 : transfer 100 ul （pipetman、yellow tip） 2-fold 50 ul （pipetman、yellow tip） round bottom clear A 2 1 Distilled water 50ul 2 3 4 5 6 7 8 9 10 Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical A/1.510 mg/ml in distilled water 150ul A/1.59 mg/ml in distilled water 50ul A/1.58 mg/ml in distilled water 50ul A/1.57 mg/ml in distilled water 50ul A/1.56 mg/ml in distilled water 50ul A/1.55 mg/ml in distilled water 50ul A/1.54 mg/ml in distilled water 50ul A/1.53 mg/ml in distilled water 50ul A/1.52 mg/ml in distilled water 50ul B C D E F G H Figure 9 18 11 12 Chemical Chemical A/1.5 mg/ml A mg/ml in in distilled distilled water water 50ul 50ul 3-2-7 2 step dilution (2nd , 3rd and 4th experiment) Add 20 μl of the diluted chemical to 480 μl of the B medium prepared in the assay block. And add 50 μl to THP-G8 in a 96 well plate using an 8 channel or 12 channel pipetman after pipetting 20 times. Shake the plate with a plateshaker, and incubate in a CO2 incubator for 16 hours (37°C, 5% ). round bottom clear A 1 Distilled water 50ul 2 3 4 5 6 8 9 10 Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical A/1.510 mg/ml in distilled water 150ul A/1.59 mg/ml in distilled water 50ul A/1.58 mg/ml in distilled water 50ul A/1.57 mg/ml in distilled water 50ul A/1.56 mg/ml in distilled water 50ul A/1.55 mg/ml in distilled water 50ul A/1.54 mg/ml in distilled water 50ul A/1.53 mg/ml in distilled water 50ul A/1.52 mg/ml in distilled water 50ul B C D E F G H 11 12 Chemical Chemical A/1.5 A mg/ml in mg/ml in distilled water distilled water 50ul 50ul 20ul Assay Block A 7 1 B medium 480ul 2 B medium 480ul 3 B medium 480ul 4 B medium 480ul 5 B medium 480ul 6 B medium 480ul B C D E F G H Figure 10 19 7 B medium 480ul 8 B medium 480ul 9 B medium 480ul 10 B medium 480ul 11 B medium 480ul 12 B medium 480ul Assay Block 1 2 Chemical 10 B medium 500ul A A/1.5 /25 mg/ml in B medium 500ul 3 Chemical 9 A/1.5 /25 mg/ml in B medium 500ul 4 Chemical 8 A/1.5 /25 mg/ml in B medium 500ul 5 Chemical 7 A/1.5 /25 mg/ml in B medium 500ul 6 Chemical 6 A/1.5 /25 mg/ml in B medium 500ul 7 Chemical 5 A/1.5 /25 mg/ml in B medium 500ul B C D E F G H 8 Chemical 4 A/1.5 /25 mg/ml in B medium 500ul 9 Chemical 3 10 Chemical 2 A/1.5 /25 mg/ml in B medium 500ul A/1.5 /25 mg/ml in B medium 500ul 11 12 Chemical Chemical A/1.5/25 A/25 mg/ml mg/ml in B in B medium medium 500ul 500ul 50ul flatbottom black 1 2 3 4 5 6 7 8 9 10 11 12 Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul A B C D E F G H Figure 11 20 3-2-8. Final constituents in each well of the plate. (2nd , 3rd and 4th experiment) flat-bottom black 1 2 3 4 5 6 7 8 9 10 11 12 A B C Cell 5x10^4 Chemical 0mg/ml 100ul B medium Cell Cell Cell Cell Cell Cell Cell Cell Cell 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical A/1.510/50mg/ml A/1.59/50mg/ml A/1.58/50mg/ml A/1.57/50mg/ml A/1.5 6/50mg/ml A/1.55/50mg/ml A/1.54/50mg/ml A/1.53/50mg/ml A/1.52/50mg/ml 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul B medium B medium B medium B medium B medium B medium B medium B medium B medium Cell 5x10^4 Chemical A/1.5/50mg/ml 100ul B medium Cell 5x10^4 Chemical A/50mg/ml 100ul B medium D Cell 5x10^4 Chemical 0mg/ml 100ul B medium Cell Cell Cell Cell Cell Cell Cell Cell Cell 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical A/1.510/50mg/ml A/1.59/50mg/ml A/1.58/50mg/ml A/1.57/50mg/ml A/1.5 6/50mg/ml A/1.55/50mg/ml A/1.54/50mg/ml A/1.53/50mg/ml A/1.52/50mg/ml 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul B medium B medium B medium B medium B medium B medium B medium B medium B medium Cell 5x10^4 Chemical A/1.5/50mg/ml 100ul B medium Cell 5x10^4 Chemical A/50mg/ml 100ul B medium E Cell 5x10^4 Chemical 0mg/ml 100ul B medium Cell Cell Cell Cell Cell Cell Cell Cell Cell 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical A/1.510/50mg/ml A/1.59/50mg/ml A/1.58/50mg/ml A/1.57/50mg/ml A/1.5 6/50mg/ml A/1.55/50mg/ml A/1.54/50mg/ml A/1.53/50mg/ml A/1.52/50mg/ml 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul B medium B medium B medium B medium B medium B medium B medium B medium B medium Cell 5x10^4 Chemical A/1.5/50mg/ml 100ul B medium Cell 5x10^4 Chemical A/50mg/ml 100ul B medium F Cell 5x10^4 Chemical 0mg/ml 100ul B medium Cell Cell Cell Cell Cell Cell Cell Cell Cell 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical A/1.510/50mg/ml A/1.59/50mg/ml A/1.58/50mg/ml A/1.57/50mg/ml A/1.5 6/50mg/ml A/1.55/50mg/ml A/1.54/50mg/ml A/1.53/50mg/ml A/1.52/50mg/ml 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul B medium B medium B medium B medium B medium B medium B medium B medium B medium Cell 5x10^4 Chemical A/1.5/50mg/ml 100ul B medium Cell 5x10^4 Chemical A/50mg/ml 100ul B medium G H Figure 12 21 3-3 When the chemical is prepared as a 500 mg/ml DMSO solution. If the chemical is prepared at a lower concentration, use the prepared concentration instead of 500 mg/ml DMSO solution. 3-3-1 Arrangement of chemicals and vehicle (1st experiment) Add 100 μl of the 500 mg/ml DMSO solution of the chemical to well #A12, 50 μl of DMSO to wells #A1-#A11, and 90 μl of the B medium to wells #B1-#B12 of the 96 well clear plate (round bottom) 3-3-2 Serial dilution (1st experiment) Conduct 11 serial dilutions at a common ratio of 2 as indicated in Figure 13 from well #A11 to well #A2.(Transfer 50 μl to the next (left) well) round bottom clear 1 2 3 4 5 6 7 8 9 10 11 12 A DMSO 100% 50ul DMSO 100% 50ul DMSO 100% 50ul DMSO 100% 50ul DMSO 100% 50ul DMSO 100% 50ul DMSO 100% 50ul DMSO 100% 50ul DMSO 100% 50ul DMSO 100% 50ul DMSO 100% 50ul Chemical 500mg/ml in DMSO 100ul B B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul C D E F G H Serial dilution at a common ratio of 2 : transfer 50 ul （pipetman、yellow tip） round bottom clear 1 A DMSO 100% 50ul B B medium 90ul 2 3 4 5 6 7 8 9 Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical 0.5mg/ml in 1.0mg/ml in 2.0mg/ml in 3.9mg/ml in 7.8mg/ml in 15.6mg/ml 31.3mg/ml 62.5mg/ml DMSO DMSO DMSO DMSO DMSO in DMSO in DMSO in DMSO 100ul 50ul 50ul 50ul 50ul 50ul 50ul 50ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul C D E F G H Figure 13. 22 B medium 90ul B medium 90ul B medium 90ul 10 11 12 Chemical 125mg/ml in DMSO 50ul Chemical 250mg/ml in DMSO 50ul Chemical 500mg/ml in DMSO 50ul B medium 90ul B medium 90ul B medium 90ul Dilution of DMSO solution with the B medium (1st experiment) 3-3-3 Dilute 10 μl of the DMSO solution of the chemical in wells #A1-#A12 with 90 μl of the B medium using an 8-12 channel pipetman. (cf. Figure 14) round bottom clear 1 A DMSO 100% 50ul B B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul round bottom clear 1 2 3 4 5 A DMSO 100% 40ul 2 3 4 5 6 7 8 9 Chemical 15.6mg/ml in DMSO 50ul Chemical 31.3mg/ml in DMSO 50ul Chemical 62.5mg/ml in DMSO 50ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul 6 7 8 9 10 11 12 Chemical 15.6mg/ml in DMSO 40ul Chemical 31.3mg/ml in DMSO 40ul Chemical 62.5mg/ml in DMSO 40ul Chemical Chemical Chemical Chemical Chemical 0.5mg/ml in 1.0mg/ml in 2.0mg/ml in 3.9mg/ml in 7.8mg/ml in DMSO DMSO DMSO DMSO DMSO 100ul 50ul 50ul 50ul 50ul 10 11 12 Chemical Chemical Chemical 125mg/ml in 250mg/ml in 500mg/ml in DMSO DMSO DMSO 50ul 50ul 50ul C D E F G H B Chemical Chemical Chemical Chemical Chemical 0.5mg/ml in 1.0mg/ml in 2.0mg/ml in 3.9mg/ml in 7.8mg/ml in DMSO DMSO DMSO DMSO DMSO 90ul 40ul 40ul 40ul 40ul Chemical Chemical Chemical 125mg/ml in 250mg/ml in 500mg/ml in DMSO DMSO DMSO 40ul 40ul 40ul Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical 0mg/ml 0.05mg/ml 0.10mg/ml 0.20mg/ml 0.39mg/ml 0.78mg/ml 1.56mg/ml 3.13mg/ml 6.25mg/ml 12.5mg/ml 25mg/ml 50mg/ml DMSO 10% DMSO 10% DMSO 10% DMSO 10% DMSO 10% DMSO 10% DMSO 10% DMSO 10% DMSO 10% DMSO 10% DMSO 10% DMSO 10% in B medium in B medium in B medium in B medium in B medium in B medium in B medium in B medium in B medium in B medium in B medium in B medium 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul C D E F G H Figure 14. 23 10ul 3-3-4 2 step dilution (1st experiment) Add 10 μl of the diluted chemical to 490 μl of the B medium prepared in the assay block. And add 50 μl to THP-G8 in a 96 well plate using an 8 channel or 12 channel pipetman after pipetting 20 times. Shake the plate with a plateshaker and incubate in a CO2 incubator for 16 hours (37°C, 5% ). round bottom clear 1 A DMSO 100% 40ul B 2 3 4 5 6 Chemical Chemical Chemical Chemical Chemical 0.5mg/ml in 1.0mg/ml in 2.0mg/ml in 3.9mg/ml in 7.8mg/ml in DMSO DMSO DMSO DMSO DMSO 90ul 40ul 40ul 40ul 40ul 8 9 10 Chemical 15.6mg/ml in DMSO 40ul Chemical 31.3mg/ml in DMSO 40ul Chemical 62.5mg/ml in DMSO 40ul 11 12 Chemical Chemical Chemical 125mg/ml in 250mg/ml in 500mg/ml in DMSO DMSO DMSO 40ul 40ul 40ul Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical 0mg/ml 0.05mg/ml 0.10mg/ml 0.20mg/ml 0.39mg/ml 0.78mg/ml 1.56mg/ml 3.13mg/ml 6.25mg/ml 12.5mg/ml 25mg/ml 50mg/ml DMSO 10% DMSO 10% DMSO 10% DMSO 10% DMSO 10% DMSO 10% DMSO 10% DMSO 10% DMSO 10% DMSO 10% DMSO 10% DMSO 10% in B medium in B medium in B medium in B medium in B medium in B medium in B medium in B medium in B medium in B medium in B medium in B medium 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul C D E F G H 10ul Assay Block A 7 1 B medium 490ul 2 B medium 490ul 3 B medium 490ul 4 B medium 490ul 5 B medium 490ul 6 B medium 490ul B C D E F G H Figure 15. 24 7 B medium 490ul 8 B medium 490ul 9 B medium 490ul 10 B medium 490ul 11 B medium 490ul 12 B medium 490ul Assay Block 1 DMSO 0.2% in B medium 500ul A 2 Chemical 1.0ug/ml DMSO 0.2% in B medium 500ul 3 Chemical 2.0ug/ml DMSO 0.2% in B medium 500ul 4 Chemical 3.9ug/ml DMSO 0.2% in B medium 500ul 5 Chemical 7.8ug/ml DMSO 0.2% in B medium 500ul 6 Chemical 15.6ug/ml DMSO 0.2% in B medium 500ul 7 8 Chemical Chemical 31.3ug/ml 62.5ug/ml DMSO 0.2% DMSO 0.2% in in B medium B medium 500ul 500ul B C D E F G H 9 10 11 12 Chemical 125ug/ml DMSO 0.2% in B medium 500ul Chemical 250ug/ml DMSO 0.2% in B medium 500ul Chemical 500ug/ml DMSO 0.2% in B medium 500ul Chemical 1000ug/ml DMSO 0.2% in B medium 500ul 50ul flat-bottom black 1 2 3 4 5 6 7 8 9 10 11 12 Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium Cell 5x10^4 NAC (-) 50ul medium A B C D E F G H Figure 16. 25 3-3-5. Final constituents in each well of the plate. (1st experiment) flat-bottom black 1 2 3 4 5 6 7 8 9 10 11 12 C Cell 5x10^4 Chemical 0ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 0.5ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 1.0ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 2.0ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 3.9ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 7.8ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 15.6ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 31.3ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 62.5ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 125ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 250ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 500ug/ml DMSO 0.1% 100ul medium D Cell 5x10^4 Chemical 0ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 0.5ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 1.0ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 2.0ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 3.9ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 7.8ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 15.6ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 31.3ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 62.5ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 125ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 250ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 500ug/ml DMSO 0.1% 100ul medium E Cell 5x10^4 Chemical 0ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 0.5ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 1.0ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 2.0ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 3.9ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 7.8ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 15.6ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 31.3ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 62.5ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 125ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 250ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 500ug/ml DMSO 0.1% 100ul medium F Cell 5x10^4 Chemical 0ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 0.5ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 1.0ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 2.0ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 3.9ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 7.8ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 15.6ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 31.3ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 62.5ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 125ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 250ug/ml DMSO 0.1% 100ul medium Cell 5x10^4 Chemical 500ug/ml DMSO 0.1% 100ul medium A B G H Figure 17. 26 3-3-6 Arrangement of chemicals and vehicle (2nd , 3rd and 4th experiment) Add 150 μl of the DMSO solution of the chemical prepared at the highest concentration defined by the 1st experiment (A mg/ml) to well #A12, 50 μl of DMSO to wells #A1-#A11, and 90 μl of the B medium to wells #B1-#B12 of a 96 well clear plate (round bottom). 3-3-7 Serial dilution (2nd , 3rd and 4th experiment) Conduct 11 serial dilutions at a common ratio of 1.5 as indicated in Figure 18 from well #A11 to well #A2. (Transfer 100 μl to the next (left) well) round bottom clear 1 2 3 4 5 6 7 8 9 10 11 12 A DMSO 100% 50ul DMSO 100% 50ul DMSO 100% 50ul DMSO 100% 50ul DMSO 100% 50ul DMSO 100% 50ul DMSO 100% 50ul DMSO 100% 50ul DMSO 100% 50ul DMSO 100% 50ul DMSO 100% 50ul Chemical A mg/ml in DMSO 150ul B B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul C D E F G H Serial dilution at a common ratio of 1.5 : transfer 100 ul （pipetman、yellow tip） round bottom clear 1 A DMSO 100% 50ul B B medium 90ul 2 3 4 5 6 7 8 9 10 Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical A/1.510 mg/ml in DMSOr 150ul A/1.59 mg/ml in DMSO 50ul A/1.58 mg/ml in DMSO 50ul A/1.57 mg/ml in DMSO 50ul A/1.56 mg/ml in DMSO 50ul A/1.55 mg/ml in DMSO 50ul A/1.54 mg/ml in DMSO 50ul A/1.53 mg/ml in DMSO 50ul A/1.52 mg/ml in DMSO 50ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul C D E F G H Figure 18. 27 11 12 Chemical Chemical A/1.5 mg/ml A mg/ml in in DMSO DMSO 50ul 50ul B medium 90ul B medium 90ul 3-3-8 Dilution of DMSO solution with the B medium (2nd , 3rd and 4th experiment) Dilute 10 μl of the DMSO solution of the chemical in wells #A1-#A12 with 90 μl of the B medium using an 8-12 channel pipetman. (cf. Figure 19) round bottom clear 1 2 3 4 5 6 7 8 9 10 Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical 10 9 8 7 6 5 4 3 11 2 12 Chemical Chemical A/1.5 mg/ml A mg/ml in in DMSO DMSO 50ul 50ul A DMSO 100% 50ul A/1.5 mg/ml in DMSOr 150ul A/1.5 mg/ml in DMSO 50ul A/1.5 mg/ml in DMSO 50ul A/1.5 mg/ml in DMSO 50ul A/1.5 mg/ml in DMSO 50ul A/1.5 mg/ml in DMSO 50ul A/1.5 mg/ml in DMSO 50ul A/1.5 mg/ml in DMSO 50ul A/1.5 mg/ml in DMSO 50ul B B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul B medium 90ul round bottom clear 1 2 3 4 5 6 7 8 9 10 11 12 Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical A DMSO 100% 40ul A/1.510 mg/ml in DMSOr 140ul A/1.59 mg/ml in DMSO 40ul A/1.58 mg/ml in DMSO 40ul A/1.57 mg/ml in DMSO 40ul A/1.56 mg/ml in DMSO 40ul A/1.55 mg/ml in DMSO 40ul A/1.54 mg/ml in DMSO 40ul A/1.53 mg/ml in DMSO 40ul A/1.52 mg/ml in DMSO 40ul B Chemical 0mg/ml DMSO 10% in B medium 100ul Chemical A/1.510/10 mg/ml DMSO 10% in B medium 100ul Chemical A/1.59/10 mg/ml DMSO 10% in B medium 100ul Chemical A/1.58/10 mg/ml DMSO 10% in B medium 100ul Chemical A/1.57/10 mg/ml DMSO 10% in B medium 100ul Chemical A/1.56/10 mg/ml DMSO 10% in B medium 100ul Chemical A/1.55/10 mg/ml DMSO 10% in B medium 100ul Chemical A/1.54/10 mg/ml DMSO 10% in B medium 100ul Chemical A/1.53/10 mg/ml DMSO 10% in B medium 100ul Chemical A/1.52/10 mg/ml DMSO 10% in B medium 100ul C D E F G H C D E F G H Figure 19. 28 Chemical Chemical A/1.5 mg/ml A mg/ml in in DMSO DMSO 40ul 40ul Chemical A/1.5/10 mg/ml DMSO 10% in B medium 100ul Chemical A/10 mg/ml DMSO 10% in B medium 100ul 3-3-9 2 step dilution (2nd , 3rd and 4th experiment) Add 10 μl of the diluted chemical to 490 μl of the B medium prepared in the assay block. And add 50 μl to THP-G8 in a 96 well plate using an 8 channel or 12 channel pipetman after pipetting 20 times. Shake the plate with a plateshaker and incubate in a CO2 incubator for 16 hours (37°C, 5% ). round bottom clear 1 A DMSO 100% 40ul B Chemical 0mg/ml DMSO 10% in B medium 100ul 2 3 Chemical 4 Chemical 10 5 Chemical 9 6 Chemical 8 7 Chemical 7 Chemical 6 9 Chemical 5 10 Chemical 4 11 Chemical 3 2 A/1.5 mg/ml in DMSOr 140ul A/1.5 mg/ml in DMSO 40ul A/1.5 mg/ml in DMSO 40ul A/1.5 mg/ml in DMSO 40ul A/1.5 mg/ml in DMSO 40ul A/1.5 mg/ml in DMSO 40ul A/1.5 mg/ml in DMSO 40ul A/1.5 mg/ml in DMSO 40ul A/1.5 mg/ml in DMSO 40ul Chemical A/1.510/10 mg/ml DMSO 10% in B medium 100ul Chemical A/1.59/10 mg/ml DMSO 10% in B medium 100ul Chemical A/1.58/10 mg/ml DMSO 10% in B medium 100ul Chemical A/1.57/10 mg/ml DMSO 10% in B medium 100ul Chemical A/1.56/10 mg/ml DMSO 10% in B medium 100ul Chemical A/1.55/10 mg/ml DMSO 10% in B medium 100ul Chemical A/1.54/10 mg/ml DMSO 10% in B medium 100ul Chemical A/1.53/10 mg/ml DMSO 10% in B medium 100ul Chemical A/1.52/10 mg/ml DMSO 10% in B medium 100ul C D E F G H 12 Chemical Chemical A/1.5 mg/ml A mg/ml in in DMSO DMSO 40ul 40ul Chemical A/1.5/10 mg/ml DMSO 10% in B medium 100ul Chemical A/10 mg/ml DMSO 10% in B medium 100ul 10ul Assay Block A 8 1 B medium 490ul 2 B medium 490ul 3 B medium 490ul 4 B medium 490ul 5 B medium 490ul 6 B medium 490ul B C D E F G H Figure 20. 29 7 B medium 490ul 8 B medium 490ul 9 B medium 490ul 10 B medium 490ul 11 B medium 490ul 12 B medium 490ul Assay Block 1 2 4 5 6 7 8 9 10 Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical 10 A/1.5 /500 mg/ml DMSO 0.2% in B medium 500ul 9 A/1.5 /500 mg/ml DMSO 0.2% in B medium 500ul 8 A/1.5 /500 mg/ml DMSO 0.2% in B medium 500ul 7 A/1.5 /500 mg/ml DMSO 0.2% in B medium 500ul 6 A/1.5 /500 mg/ml DMSO 0.2% in B medium 500ul 5 A/1.5 /500 mg/ml DMSO 0.2% in B medium 500ul 4 A/1.5 /500 mg/ml DMSO 0.2% in B medium 500ul 3 DMSO 0.2% A/1.5 /500 mg/ml in B DMSO 0.2% medium in B 500ul medium 500ul A 3 Chemical B C D E F G H 11 12 A/1.5 /500 mg/ml DMSO 0.2% in B medium 500ul Chemical A/1.5/500 mg/ml DMSO 0.2% in B medium 500ul Chemical A/500 mg/ml DMSO 0.2% in B medium 500ul 2 50ul flatbottom black 1 2 3 4 5 6 7 8 9 10 11 12 Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul Cell 5x10^4 B medium 50ul A B C D E F G H Figure 21. 30 3-3-10. Final constituents in each well of the plate. (2nd , 3rd and 4th experiment) flatbottom black 1 2 3 4 5 6 7 8 9 10 11 12 A B C Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical 0 A/1000 A/1.5 10/1000 A/1.5 9/1000 A/1.5 8/1000 A/1.57/1000 A/1.5 6/1000 A/1.5 5/1000 A/1.5 4/1000 A/1.53/1000 A/1.5 2/1000 A/1.5/1000 mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul medium medium medium medium medium medium medium medium medium medium medium medium D Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical 10 9 8 7 6 5 4 3 2 0 A/1000 A/1.5 /1000 A/1.5 /1000 A/1.5 /1000 A/1.5 /1000 A/1.5 /1000 A/1.5 /1000 A/1.5 /1000 A/1.5 /1000 A/1.5 /1000 A/1.5/1000 mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul medium medium medium medium medium medium medium medium medium medium medium medium E Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical 0 A/1000 A/1.5 10/1000 A/1.5 9/1000 A/1.5 8/1000 A/1.57/1000 A/1.5 6/1000 A/1.5 5/1000 A/1.5 4/1000 A/1.53/1000 A/1.5 2/1000 A/1.5/1000 mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul medium medium medium medium medium medium medium medium medium medium medium medium F Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell Cell 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 5x10^4 Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical 0 A/1000 A/1.5 10/1000 A/1.5 9/1000 A/1.5 8/1000 A/1.57/1000 A/1.5 6/1000 A/1.5 5/1000 A/1.5 4/1000 A/1.53/1000 A/1.5 2/1000 A/1.5/1000 mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul 100ul medium medium medium medium medium medium medium medium medium medium medium medium G H Figure 22. 31 4. Preparation of Cobalt chloride (CoCl2) and treatment of THP-G8 cells with CoCl2. 4-1 Preparation of cells Add 50 μl of the cell suspension to wells #C1-#C4, #D1-#D4, #E1-#E4, #F1-#F4 of a 96 well μ clear black plate (flat bottom) (cf. Figure 23). flatbottom black 1 2 3 4 Cell 5x10^4 50ul medium Cell 5x10^4 50ul medium Cell 5x10^4 50ul medium Cell 5x10^4 50ul medium Cell 5x10^4 50ul medium Cell 5x10^4 50ul medium Cell 5x10^4 50ul medium Cell 5x10^4 50ul medium Cell 5x10^4 50ul medium Cell 5x10^4 50ul medium Cell 5x10^4 50ul medium Cell 5x10^4 50ul medium Cell 5x10^4 50ul medium Cell 5x10^4 50ul medium Cell 5x10^4 50ul medium Cell 5x10^4 50ul medium 5 6 A B C D E F G H Figure 23. 32 7 8 9 10 11 12 4-2 Preparation of Cobalt chloride (CoCl2) and treatment of THP-G8 cells. Prepare CoCl2 and add them to wells after cell preparation. Dilute 25 mg/ml of the CoCl2 solution with distilled water as described below in a 96 well clear plate (round bottom): #A1: distilled water 200 l; #A2: distilled water 190 l + CoCl2 10 l; #A3: distilled water 180 l + CoCl2 20 l; #A4: distilled water 160 l + CoCl2 40 l round bottom clear A 1 2 3 4 Distilled water 200ul Distilled water 190ul + 25mg/ml CoCl2 10ul Distilled water 180ul + 25mg/ml CoCl2 20ul Distilled water 160ul + 25mg/ml CoCl2 40ul 5 6 B C D E F G H Figure 24. 33 7 8 9 10 11 12 Add 20 μl of the diluted chemical to 480 μl of the B medium prepared in the assay block, vigorously pipet with an 8 channel or 12 channel pipetman at least 20 times, and then, immediately add 50 μl of the solution to THP-G8 in a 96 well plate using an 8 channel or 12 channel pipetman. Shake the plate with a plateshaker, and incubate in a CO2 incubator for 16 hours (37°C, 5%). round bottom clear A 1 2 3 4 Distilled water 200ul Distilled water 190ul + 25mg/ml CoCl2 10ul Distilled water 180ul + 25mg/ml CoCl2 20ul Distilled water 160ul + 25mg/ml CoCl2 40ul B C D E F G H 6 7 8 9 10 11 12 20ul Assay Block A 5 1 B medium 480ul 2 B medium 480ul 3 B medium 480ul 4 5 B medium 480ul B C D E F G H Figure 25. 34 6 7 8 9 10 11 12 Assay Block A 1 B medium 500ul 2 3 4 5 6 7 8 9 10 11 12 CoCl2 CoCl2 CoCl2 50ug/ml in 100ug/ml in 200ug/ml in B medium B medium B medium 500ul 500ul 500ul B C D E F G H 50ul flatbottom black 1 2 3 4 C Cell 5x10^4 50ul medium Cell 5x10^4 50ul medium Cell 5x10^4 50ul medium Cell 5x10^4 50ul medium D Cell 5x10^4 50ul medium Cell 5x10^4 50ul medium Cell 5x10^4 50ul medium Cell 5x10^4 50ul medium E Cell 5x10^4 50ul medium Cell 5x10^4 50ul medium Cell 5x10^4 50ul medium Cell 5x10^4 50ul medium F Cell 5x10^4 50ul medium Cell 5x10^4 50ul medium Cell 5x10^4 50ul medium Cell 5x10^4 50ul medium 5 A B G H Figure 26. 35 6 7 8 9 10 11 12 4-3 Final constituents in each well of the plate flatbottom black 1 2 3 4 5 6 7 8 9 10 11 12 A B C Cell Cell Cell Cell 5x10^4 5x10^4 5x10^4 5x10^4 Chemical CoCl2 CoCl2 CoCl2 0ug/ml 25ug/ml 50ug/ml 100ug/ml 100ul medium 100ul medium 100ul medium 100ul medium D Cell Cell Cell Cell 5x10^4 5x10^4 5x10^4 5x10^4 Chemical CoCl2 CoCl2 CoCl2 0ug/ml 25ug/ml 50ug/ml 100ug/ml 100ul medium 100ul medium 100ul medium 100ul medium E Cell Cell Cell Cell 5x10^4 5x10^4 5x10^4 5x10^4 Chemical CoCl2 CoCl2 CoCl2 0ug/ml 25ug/ml 50ug/ml 100ug/ml 100ul medium 100ul medium 100ul medium 100ul medium F Cell Cell Cell Cell 5x10^4 5x10^4 5x10^4 5x10^4 Chemical CoCl2 CoCl2 CoCl2 0ug/ml 25ug/ml 50ug/ml 100ug/ml 100ul medium 100ul medium 100ul medium 100ul medium G H Figure 27. 4-4 Measurement Thaw Tripluc® Luciferase assay reagent (Tripluc) and keep it at room temperature by bathing in water or ambient air. Start luminometer 30 min before starting the measurement for stabilization of the photomultiplier. Transfer 100 μl of pre-warmed Tripluc to each well containing the reference samples of the plate using a pipetman. Shake the plate for 10 min at room temperature (about 25°C) with a plate shaker. Remove bubbles on the solutions in the wells if they appear. Place the plate into the luminometer of under-test to measure the luciferase activity. Bioluminescence is measured for 3 sec each in the absence (F0) and presence (F2) of 36 the optical filter. Copy the result of F0 and F2 measurement (values are expressed as counts) and paste it to the appropriate area in the “Data Input” sheet of the data sheet. Calculated factors and graphs will appear on the “Cobalt chloride” sheet of the data sheet. The acceptance criterion is FInSLO-LA 1.4. Continue the experiment if these criteria are accepted. 37 5. Validation of reagents and equipment 5-1 Measurement of transmittance of optical filter for multicolor measurement For color discriminations in the multi-color reporter assay, detectors (luminometer and plate reader) are usually equipped with optical filters, such as sharp-cut (long-pass) filters and band-pass filters. The transmittance factors of these filters for each bio-luminescence signal color have to be calibrated prior to all experiments by following the protocols below. 5-1-1 Reagents ・Single reference samples: Lyophilized luciferase enzyme reagent of SLO Lyophilized luciferase enzyme reagent of SLR ・Mixed reference samples: Lyophilized mixed luciferase enzyme reagent of SLO and SLR ・Reference sample storing buffer ・Assay reagent: Tripluc® Luciferase assay reagent（TOYOBO Cat#MRA-301） ・B medium: for luciferase assay (30 ml, stored at 2 – 8°C) Conc Reagent Final conc. Required Company . RPMI-1640 GIBCO #11875-093 FBS Biological Industries Cat#04-001-1E in medium amount - - 27 ml - 10 % 3 ml Lot:715004 5-1-2 Calibration 5-1-2-1 Preparation of luminescence reaction solution Thaw Tripluc® Luciferase assay reagent (Tripluc) and keep it at room temperature by bathing in water or ambient air. Start the luminometer 30 min before starting the measurement for stabilization of the photomultiplier. Add 200 μl of the reference sample storing buffer to each tube of the lyophilized reference samples to dissolve the enzymes, followed by separating them into 1.5 ml 38 disposable tubes at 10 μl each and storing in a freezer at -80°C. The stored frozen solution of the reference samples can be used for one half year. Add 1 mL of B medium to each tube of the frozen reference sample (10 μl in a tube) and label them as SLO1/1, SLR1/1, and SLO/SLR1/1. Keep the reference samples on ice to prevent deactivation. Prepare dilution series of the single reference samples of SLO and SLR as follows. Dilute 0.3 ml of each 1/1 solution with 0.9 ml of B medium to make SLO1/4 and SLR1/4. In the same manner, prepare 1/16 and 1/64 solution of each. Keep diluted reference samples on ice. Prepare dilution series of the mixed reference sample of SLO and SLR as follows. Dilute 0.1 ml of the 1/1 solution with 0.9 ml of B medium (SLO/SLR1/10). Keep the diluted reference samples on ice. 5-1-2-2 Bioluminescence measurement Transfer 100 μl of the diluted reference samples to a black 96 well plate (flat bottom) as shown below. 1 2 3 4 5 6 7 8 9 10 11 12 A SLO SLO SLO SLO SLO SLO SLO SLO SLO SLO SLO SLO B 1/1 1/1 1/1 1/4 1/4 1/4 1/16 1/16 1/16 1/64 1/64 1/64 SLR SLR SLR SLR SLR SLR SLR SLR SLR SLR SLR SLR 1/1 1/1 1/4 1/4 1/4 1/16 1/16 1/16 1/64 1/64 1/64 C D 1/1 E SLO/ SLO/ SLO/ SLO/ SLO/ F SLR SLR 1/1 1/1 SLO/ SLR SLR SLR SLR 1/1 1/10 1/10 1/10 G H Figure 28. 39 Transfer 100 μl of pre-warmed Tripluc to each well containing the reference samples of the plate using a pipetman. Shake the plate for 10 min at room temperature (about 25°C) with a plate shaker. Remove bubbles on the solutions in wells if they appear. Place the plate into the luminometer of the under-test to measure the luciferase activity. Bioluminescence is measured for 3 sec each in the absence (F0) and presence (F2) of the optical filter. Copy the results of the F0 and F2 measurement (values are expressed as counts) and paste it to the appropriate area in the “Data Input” sheet of the data sheet for data analyses. Calculated transmittance factors will appear on the “transmittance” sheet of the excel file. Here, check the line a dynamic range of the luminometer used. Calculated transmittance factors will be used for all the measurements executed using the same luminometer. Check the color discrimination result of the mixed reference sample by comparing it with the reference value attached in the lyophilized sample. Record all the results for quality control. 5-2 Quality control of equipment In order to confirm the detector stability as the quality control, the reference luciferase sample, optical property, the protocol described here should be performed at the beginning of the experiments every day. 5-2-1 Light source LED Plate: Reference LED light source plates equipped with stabilized red, green, and blue LEDs are commercially available. For example, TRIANT® (wSL-0001) by ATTO (Tokyo, Japan) L12367 by Hamamatsu Photonics (Shizuoka, Japan) 5-2-2 Data collection (an example using TRIANT® by ATTO) 1) Start luminometer 30 min before starting the measurement for stabilization of the photomultiplier. 2) Start LED plate and select “PMT” mode. 3) Select three-color (BRG) mode and adjust light intensity to 1/10 (1E-1). 4) Place the LED plate into the luminometer. Light intensity is measured for 3 sec each 40 in the absence (F0) and presence (F2) of the optical filter. 5) Blue, green, and red LEDs are located at the position of F6, E6, and D6, respectively. Copy the collected data of each position to the appropriate area on Sheet “LED” in the excel file of “IL-8 Luc Assay Datasheet”. 6) Check the photo-detector performance by comparing with old data of the LED plate. For quality control purpose, every collected data should be accumulated. 41 6. Measurement of luciferase activity Thaw Tripluc® Luciferase assay reagent (Tripluc) and keep it at room temperature by bathing in water or ambient air. Start the luminometer 30 min before starting the measurement for stabilization of the photomultiplier. Transfer 100 μl of pre-warmed Tripluc from the reservoir to each well containing the reference samples of the plate using an 8 channel or 12 channel pipetman. Shake the plate for 10 min at room temperature (about 25°C) with a plate shaker. Remove bubbles on the solutions in the wells if they appear. Place the plate in the luminometer of the under-test to measure the luciferase activity. Bioluminescence is measured for 3 sec each in the absence (F0) and presence (F2) of the optical filter. Copy the result of F0 and F2 measurement (values are expressed as counts) and paste it to the appropriate area in the “Data Input” sheet of the data sheet. And input the transmittance factors calculated at 5-1-2-2 (#I33, #I34 of the “Transmittance” sheet) to #G4, #G5 of the “Data Input” sheet. Calculated factors and graphs will appear on the “Result Format” sheet and the “Graph” sheet of the data sheet, respectively. 42 7. Criteria 7-1 Definition of the parameters used in IL-8 Luc assay 7-2 The criteria to identify sensitizers in IL-8 Luc assay There are three temporary criteria to identify sensitizers. We are going adopt one of these criteria after phase IIc varidation. 1. The criteria which use FInSLO-LA and I.I.-SLR-LA In each experiment: Criteria: chemicals that demonstrate FInSLO-LA 1.4 and I.I.-SLR-LA 0.05 are judged as positive. 2. The criteria which use the lower limit of the 95% confidence interval of FInSLO-LA In each experiment: Criteria: chemicals that demonstrate the lower limit of the 95% confidence interval of FInSLO-LA 1.0 are judged as positive. 3. The criteria which use FInSLO-LA and the lower limit of the 95% confidence 43 interval of FInSLO-LA In each experiment: Criteria: chemicals that demonstrate FInSLO-LA 1.4 and the lower limit of the 95% confidence interval of FInSLO-LA 1.0 are judged as positive. Criteria 2 Criteria 3 Negative Negative Positive Negative Negative Negative Positive Positive 3 2.5 FInSLO-LA 2 1.5 1 0.5 0 vehicle conc.1 conc.2 3 2.5 FInSLO-LA 2 1.5 1 0.5 0 vehicle conc.1 conc.2 3 2.5 FInSLO-LA 2 1.5 1 0.5 0 vehicle conc.1 conc.2 5 4.5 4 FInSLO-LA 3.5 3 2.5 2 1.5 1 0.5 0 vehicle conc.1 conc.2 1st experiment : THP-G8 cells are stimulated with chemicals serially diluted at a common ratio of 2 from the highest soluble concentration. 2nd, 3rd and 4th experiments : Determine the minimum concentration at which 44 I.I.-SLR-LA became lower than 0.05 in the 1st experiment. Use the concentration one step (2-times) higher than this determined concentration as the highest concentration of the chemical to examine, and conduct 11 serial dilutions at a common ratio of 1.5 from the highest concentration. If I.I.-SLR-LA did not become lower than 0.05, or I.I.-SLR-LA became lower than 0.05 at the highest final concentration in the 1st experiment, conduct 11 serial dilutions at a common ratio of 1.5 from the highest concentration in the 1st experiment. Chemicals that make 2 positive results in the 1st, 2nd, 3rd or 4th experiments are considered sensitizers and chemicals that make 3 negative results in the 1st, 2nd, 3rd or 4th experiments are considered non-sensitizers. You can skip the follow-on experiment once the judgement by all three criteria is made. Criteria to determine sensitizer or non-sensitizer. 1st Positive 2nd 3rd 4th Judge Positive - - Sensitizer Positive - Sensitizer Positive Sensitizer Negative Non-sensitizer - Sensitizer Positive Sensitizer Negative Non-sensitizer Positive Sensitizer Negative Non-sensitizer - Non-sensitizer Negative Negative Positive Positive Negative Negative Negative Positive Negative 45 8. Update record Ver.017E, 2013, Nov 11, distribution Modify the criteria Ver.016E, 2013, Oct, 29 distribution Delete the criteria I.I.0.80 Ver.015E, 2012, Nov, 12 distribution Change preparation of chemicals Ver.014E, 2012, Oct, 26 distribution Change preparation of water-soluble chemicals Change preparation of CoCl2 Delete the description concerned to LPS Change incubation time (5 hours to 16 hours) Change the Quality control of equipment Change the criteria Ver.013E, 2012, Aug, 03 distribution Ver.012E, 2012, Jul, 12 distribution Ver.011E, 2012, Jun, 05 distribution Ver.008E, 2011, Dec, 19 distribution IL-8 Luc assay detailed protocol, 2011, Nov. 11 distribution 46 Appendix (3) 1 HRI draft ver 8, January 9/ 2014 Appendix (3) 1 DRAFT TEST GUIDELINE In Vitro Carcinogenicity: Bhas 42 Cell Transformation Assay INTRODUCTION 1. In vitro cell transformation refers to the induction of phenotypic alterations in cultured cells which are characterized as the change from non-transformed to transformed phenotype, the latter being considered typical aberrations associated with cells exhibiting neoplastic potential in vivo (1, 2). Transformed cells with the characteristics of malignant cells have the ability to induce tumors in susceptible animals (3, 4, 5); this supports the use of phenotypic alterations in vitro as criteria for predicting carcinogenic potential in vivo. 2. Various types of cell transformation assays (CTAs) have been developed for detection of carcinogenic stimuli. The Syrian hamster embryo (SHE) CTA is a primary cell system, and BALB/c 3T3, C3H10T1/2 and Bhas 42 cell CTAs are systems using established cell lines. OECD reviewed the performances of CTAs based on retrospective data (OECD Detailed Review Paper 31, DRP 31) (6).An international validation study of Bhas 42 CTA was performed by the New Energy and Industrial Technology Development Organization (NEDO) in conjunction with the Japanese Center for the Validation of Alternative Methods (JaCVAM). This validation study ensured the use of a standardized Bhas 42 CTA protocol, confirmed its transferability within and between laboratories, and established its intra- and inter-laboratory reproducibility. 3. Since DNA damage and mutation are known to be initiating events for carcinogenesis, several short-term in vitro and in vivo genotoxicity tests are commonly used to predict chemical carcinogenicity. Not all carcinogens, however, are known to be genotoxicants; animal carcinogenesis studies have clearly demonstrated that there exists a promotion process distinct from an initiation process (two-stage carcinogenesis) for agents that are not direct acting carcinogens (8). In addition, it has long been knownthat some CTAs can reproduce the two-stage carcinogenesis progression and therefore detect and distinguish between the promoting activity and the initiating activity of carcinogens (9). 4. The Bhas 42 cell line was established by the transfection of the v-Ha-ras oncogene into the BALB/c 3T3 A31-1-1 cell line and because of its resulting desirable phenotypic properties and responsiveness to chemical carcinogens was selected from among other such transfected cell lines for the CTA (10, 11). Similar to the parental BALB/c 3T3 cell line, untransformed Bhas 42 cells grow to confluence forming a contact-inhibited monolayer and such cells lack tumorigenicity upon transplantation in vivo. After exposure to carcinogenic stimuli, such cells become morphologically altered and form independent aberrant colonies, referred to as transformed foci, capable of invading the surrounding non-transformed contact-inhibited monolayer. This focus formation is the endpoint of the Bhas 42 CTA. 5. The current protocol for the Bhas 42 CTA consists of two assay components, the initiation assay and promotion assay for examining tumor-initiating activity and tumor-promoting activity of chemicals, respectively (12, 13). It is acknowledged that mutation induced by chemical insult is fixed after several cell replication cycles (14, 15). Thus, in the initiation assay the cells are treated at the beginning of growth phase to allow for fixation of the induced DNA 1 HRI draft ver 8, January 9/ 2014 Appendix (3) 1 damage, and in the promotion assay the cells are repeatedly treated at stationary phase to provide a growth advantage for anomalous cells. 6. Several comprehensive studies were performed to assess the relevance and predictive reliability of the Bhas 42 CTA. These included (a) an extensive analysis of 98 chemicals (13), (b) a multi-laboratory collaborative study (16), (c) a prevalidation study (17), and (d) two international validation studies (18, 19). For the latter, the Validation Advisory Committee and the Validation Management Team were comprised of international experts from European Centre for the Validation of Alternative Methods (ECVAM), the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), NTP Interagency Center for the Evaluation of Alternative Toxicological Method (NICEATM) and JaCVAM. The results of all of these studies confirmed the applicability, transferability, reproducibility and reliability of the Bhas 42 CTA protocol and the assay was found to be sufficiently sensitive to predict both initiating activity and promoting activity of carcinogens. 7. Test results derived from the Bhas 42 CTA are expected to be used as part of a testing strategy (rather than a stand-alone assay) and/or in a weight-of-evidence approach to predicting carcinogenic potential. When employed in combination with other information such as genotoxicity data, structure-activity analysis and pharmaco/toxicokinetic information, CTAs in general and the Bhas 42 CTA specifically can contribute to the assessment of carcinogenic potential (20) and may reduce the use of in vivo testing. CTAs may be particularly useful for evaluating chemicals for which in vivo testing is not allowed (e.g. regulation on cosmetics in the European Union [Regulation (EC) 1223/2009 of the European Parliament and of the Council of 30 November 2009 on cosmetic products]), is limited, or is only required for chemicals identified as genotoxic (21). 8. This Test Guideline (TG) provides an in vitro procedure using the Bhas 42 CTA, which can be used for hazard identification of chemical carcinogens having initiating and/or promoting activity. The test method described is based upon the protocol reported for this assay in Sakai et al. (18). 9. In this Test Guideline, the two formats using both 6-well plates and 96-well plates are described. After initial development of the Bhas 42 CTA 6-well format, the assay was adapted to a 96-well format which was designed for high-throughput analyses. Although the number of cells plated and expression of transformation frequency differ between the 6-well and 96-well formats, the overall results obtained are similar and the formats can be used interchangeably (19, 22). INITIAL CONSIDERATIONS AND LIMITATIONS 10. The Bhas 42 cell line was established by transfection of the v-Ha-ras oncogene into the BALB/c 3T3 A31-1-1 cloned cell line, for which stable integration of the v-Ha-ras gene was demonstrated (10, 11). Like the parental BALB/c 3T3 A31-1-1 cells, the Bhas 42 cell line still maintains its non-transformed morphological properties, including that of density dependent inhibition of cell growth (contact-inhibition). It is considered to be an initiated cell line; that is, the cells are considered to have advanced beyond a “normal” condition toward a more atypical pathological state, having progressed to a certain extent along the multi-step carcinogenesis process (23). This attribute makes Bhas 42 cells highly sensitive to carcinogenic stimuli and accounts for the short latency period for expression of the transformed focus phenotype. 2 HRI draft ver 8, January 9/ 2014 Appendix (3) 1 11. Due to their initiated state and their sensitivity to carcinogenic stimuli, Bhas 42 cells may spontaneously transform under inappropriate culture conditions. Therefore, it is important to maintain strict quality control of cells, assay components, and test conditions, including the use of low passage target cells, maintainenance of a sub-confluent cell population density (≤ 70% confluence) among cell stocks to be used for treatment, and use of suitable pre-screened batches of foetal bovine serum (FBS). It should be noted that spontaneous transformation is a common intrinsic occurrence and is expressed at different relative frequencies among the available target cell systems used in CTAs. Irrespective of the CTA system, those spontaneous transformation rates are moderated by adhering to the strict quality control measures described above. In this way, the spontaneous and chemically induced transformation frequencies are readily distinguishable. 12. The chromosome number is ranging from 58 to 72 and modal chromosome number is 60 (42%), it shows hyper triploidy. Bhas 42 cells have similar marker chromosomes as BALB/c 3T3 cells. Bhas 42 cells respond to polycyclic aromatic hydrocarbons, 2-acetylaminofluorene, and cyclophosphamide. Since these chemicals require metabolic activation (13), Bhas 42 cells contain some level of the cytochrome P450 family of enzymes including at least CYP1A1 for polycyclic aromatic hydrocarbons (24), CYP1A2 for 2-acetylaminofluorene (25) and CYP2B6 for cyclophosphamide (26). 13. Initiating activity and promoting activity of carcinogens can be distinguished in the in vivo carcinogenicity studies using the two-stage carcinogenesis model but this distinction is not generally pursued. In its evaluation of the relative performance of CTAs, OECD reported on CTA responsiveness to 260 carcinogens in its DRP 31. Only 9 in vivo tumor promoters (3.5%) were included in the review, and all of them showed positive results in all or either of the SHE, BALB/c 3T3 and C3H10T1/2 CTAs. As to the performance of the in vitro promotion assay using the Bhas 42 CTA, 14 in vivo tumor promoters were investigated, 13 (92.9%) of which were positive in the Bhas 42 cell promotion assay (13, 19, 27). These results indicate that the Bhas 42 cell promotion assay can be a valuable in vitro system for identifying potential in vivo tumor promoters.. 14. Morphologically, various types of transformed foci are observed (refer to Paragraph 47 and Annex 2). For this reason, adequate training of laboratory personnel engaged in the identification and scoring of transformed foci is essential. A photo catalog of various examples of untransformed and transformed foci has been found to be a valuable tool with which to assist in the recognition of such transformed foci and in distinguishing them from non-transformed foci (see Annex 2). PRINCIPLE OF THE TEST METHOD 15. Bhas 42 cells proliferate exponentially and when they reach confluence, they form a contact-inhibited monolayer. Appropriate numbers of Bhas 42 cells are plated into each well of 6-well plates or 96-well plates. In the initiation assay, the cells are treated with a given test chemical at a low cell density for three days (from Day 1 to Day 4), allowed to replicate and then fixed and stained on Day 21 after plating. In the promotion assay, the treatment with the test chemical is commenced at sub-confluence and continued for 10 days (from Day 4 to Day 14). The cells are then fixed and stained on Day 21 after plating. Plates are coded and scored; the resulting foci are evaluated for their morphological phenotype. The evaluation of the morphological phenotype is by stereomicroscope. 16. Transformation frequency is quantified using stereomicroscopy as follows: (a) for the 6-well format transformed foci in each well are scored; (b) for the 96-well format the number of wells with transformed foci are counted. The latter counting method eliminates the imprecision that could result from attempting to score multiple foci forming in the 3 HRI draft ver 8, January 9/ 2014 Appendix (3) 1 smaller wells of the 96-well plates. It is important, however, to be cognizant not only of those foci that form on the bottom of the wells, but also those that may adhere to the side-wall of such wells (19, 22). 17. Cytotoxicity is evaluated colorimetrically by estimating the amount of dye (crystal violet) extracted from the treated cells (28). For this purpose, the relative optical density (OD) is obtained by calculating the ratio of the OD determined for the treated cells to the OD of solvent control cells. The transformation index is statistically determined from the relative increase in the number of morphologically transformed foci observed in the treated group compared to the number of such foci appearing in the solvent controls. PROCEDURE Culture media, reagents and solutions 18. The culture media, reagents and solutions are described in Annex 1. Culture conditions and preparation of cell suspension 19. Minimum essential medium supplementing 10 %FBS (M10F) is used for population expansion of cells so as to generate master cell stocks and working cell stocks, all of which are stored frozen in a liquid nitrogen tank. Cell cultures used for cytotoxicity and transformation assays are derived from those frozen cell stocks. Dulbecco’s modified Eagle’s medium/F12 supplementing 5 %FBS (DF5F) is used for the cell growth assays and transformation assays as well as routine maintenance and subculturing of cells. 20. Bhas 42 cells are incubated at 37ºC in a humidified atmosphere of 5% CO2 and air. It is important that all cell stocks and working cultures be maintained at a sub-confluent density at all times prior to use in transformation assays, such that they do not exceed 70% confluence and thereby retain their property of density dependent inhibition of cell growth. This ensures that loss of cell-to-cell contact inhibition is the result of treatment with chemical carcinogens and not a function of failure to maintain the necessary pre-assay cell culture conditions. The necessity of this becomes clear when it is realized that those cells that are no longer contact-inhibited and exhibit unrestricted growth are those that are transformed and preferentially form aberrant foci atop the confluent cell monolayer. Preparation and cryopreservation of Bhas 42 cell stocks 21. Bhas 42 cells should be obtained from a reliable source, specifically, JCRB Cell Bank, National Institute of Biomedical Innovation (NIBIO, Osaka, Japan) [http://cellbank.nibio.go.jp/english/] and shown to be free of adventitious contaminating agents (e.g. mycoplasma). 22. If the Bhas 42 cells are cultured with sufficient care using acceptable pre-screened lots of FBS and proper attention paid to maintenance of sub-confluent cell density, the cells can be passaged 2-3 times without losing the properties that make them suitable for use as a CTA target cell system. The most practical solution to ensure the uninterrupted availability of such suitable cell populations is to have available a large stock of frozen early passage cells. For this purpose, initial master cell stocks are generated and cryopreserved in a liquid nitrogen tank in aliquots that will eventually serve to generate working cell stocks. Cells are cultured with M10F in a 100- or 150-mm dish or in a 75- or 4 HRI draft ver 8, January 9/ 2014 Appendix (3) 1 150-cm2 flask to a cell density not to exceed 70% confluence. They are then suspended at a cell density of 5 x 105 cells/mL in cold fresh M10F containing a suitable cryoprotective agent (e.g. 5% dimethyl sulfoxide) to make a master cell stock from which 0.5 mL aliquots are cryopreserved and stored in liquid nitrogen. Cells from one master stock are cultured for 1-2 passages in M10F before cryopreservation. From this cell population, approximately 100 aliquots are prepared and cryopreserved so as to provide sufficient working cell stocks. The quality of those cells is then confirmed for their ability to fulfill the acceptance criteria described in Paragraph 23, which are the same as those criteria for accepting a given lot of FBS. 23. In order to identify suitable lots of FBS for the transformation assay, several batches of FBS are checked using the cells from one master stock. The acceptance criteria for a given lot of FBS include (a) adequate plating efficiency of Bhas 42 cells (≥50%), (b) low background of spontaneous transformation, and (c) ability to facilitate Bhas 42 cell transformation by positive controls (1 µg/mL MCA and 0.05 µg/mL TPA (refer to Paragraph 27). FBS batches which fulfill the criteria in Paragraph 52 for the 6-well format and Paragraph 53 for the 96-well format are those that are selected for use in subsequent transformation assays. 24. Freshly prepared 2-3 passage cells derived from the cryopreserved cell stocks are used for each transformation assay. 25. The cells at higher passages (within 10 passages) can be used for dose setting. Controls 26. The solvent for a test chemical is used as the negative control. 27. For positive controls a known tumor-initiator, 3-methylcholanthrene (MCA, final concentration of 1 µg/mL), is used in the initiation assay, and a known tumor-promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA, final concentration of 50 ng/mL), is used in the promotion assay. MCA and TPA are dissolved in dimethyl sulfoxide (DMSO), which serves as the solvent for these two control agents. When the solvent for the test chemical is not DMSO, DMSO is still necessary as the negative control for MCA or TPA. The stock solutions of MCA and TPA in DMSO can be stored in frozen aliquots at -20oC for at least two years. Preparation of test chemical solutions 28. Test chemicals are dissolved or suspended in an appropriate solvent and diluted if appropriate, prior to the treatment of the cells. Distilled water, DMSO, acetone, and ethanol can be used to dissolve test chemicals, and the final solvent concentrations in the medium should not exceed 5%, 0.5%, 0.5% and 0.1%, respectively. Although the concentration of DMSO can be as high as 0.5%, 0.1% is recommended when possible. If solvents other than the above well-established ones are employed, their use should be supported by data indicating their compatibility with the test chemical and the test system, as well as their lack of transforming activity. In such cases, untreated controls devoid of the solvent of choice should also be included. 29. The maximum concentrations to be tested in cell transformation assay depend on test chemical solubility and cytotoxicity. For test chemicals of defined composition, the highest dose level should be 0.01 M, 2 mg/mL or 2 μL/mL, 5 HRI draft ver 8, January 9/ 2014 Appendix (3) 1 whichever is the lowest. For test chemical of undefined composition, e.g. complex mixtures (plant extracts, tars, environmental extracts, etc.), the top concentration should be at least 5 mg/mL. Poorly soluble chemicals should be tested up to the first concentration producing a visible opacity (precipitation) in the final test medium observable by the unaided eye. At least four concentrations must be tested and these are determined according to the results of the cell growth assay. Paragraphs 34, 39 – 41 provide further details on the top concentration that should be tested. Experimental design 30. The design of the two formats used, i.e. the 6-well format and a 96-well format, are fundamentally similar in that their experimental procedures are basically the same except for the number of cells plated and the expression of transformation frequency. In the following sections, details are presented for the 6-well format and those modifications associated with the 96-well format are indicated. 31. Both formats consist of an initiation assay component and a promotion assay component. These assays can detect initiating activity and promoting activity of carcinogens, respectively. In the initiation assay, the cells are treated with chemicals in the beginning of growth phase and in the promotion assay the treatment is started at sub-confluence of cell growth. Both assays consist from two steps. In the first step, concentrations are selected in a preliminary cell growth assay using a large dose range. In the second step of the transformation assay, a cell growth assay is concurrently performed to verify that the selected doses meet acceptation criteria for the assays (Fig. 1). Bhas 42 cell transformation assay (Initiation assay and promotion assay, respectively) First step Cell growth assay to set doses Second step Transformation assay plus Concurrent cell growth assay Figure 1: General Scheme of the Bhas 42 CTA Initiation assay ▪ Cell growth assay to set doses 32. The cells at ≤ 70% confluence in DF5F are suspended with trypsin and 4,000 cells are seeded into a well with 2 mL of DF5F (Day 0). Wells containing medium alone are also prepared for the blank control in the colorimetric analysis (the blank control can be shared among different assays performed simultaneously). At 20-24 hours (Day 1) after cell seeding, the culture medium is replaced with fresh medium containing various concentrations of a test chemical, or concentrated test chemical solutions are added to each well without medium replacement. The medium is changed with fresh medium on Day 4. On Day 7 the cultures are fixed with methanol or 10% formalin for 6 HRI draft ver 8, January 9/ 2014 Appendix (3) 1 approximately 10 min, washed and dried. The cells are stained with crystal violet (CV) solution for approximately 15 min, rinsed well with water and dried (Fig. 2). Three wells are prepared in each group. 0 1 4 7 Fix：Methanol or formaldehyde Stain：Crystal violet : DMEM/F12 + 5% FBS (DF5F) : Chemical treatment : Cell seeding : Chemical addition : Medium change Figure 2: Time line of cell growth assay in the initiation assay 33. The CV is extracted from the stained cells with 2 mL of dye extraction solution, and the OD is measured at a wavelength between 540-570 nm. The relative cell growth of cultures treated with a chemical is calculated as follows: Relative cell growth (%) = [(Treatment – Blank)/(Control – Blank)] x 100 “Treatment”, “Control” and “Blank” refer to the absorbance of the CV extracts of each treatment group, the solvent control group and the medium only group, respectively. 34. Five to nine concentrations are set up based on the results of the cell growth assay. These concentrations cover a range from little or no toxicity to the highest acceptable level of toxicity (less than 20% survival compared to the negative control). Ideally, those concentrations that are included are: (a) at least one concentration below the no observed effect level (NOEL, around 80-120% of cell growth), (b) two concentrations between the NOEL and the 50% inhibitory concentration (IC50), and (c) two concentrations between the IC50 and the IC90. The ratio between neighboring concentrations should be less than square root of 10 (Fig. 3). Some test chemicals exhibit a steep concentration–response curve. With these test chemicals, test concentrations should be spaced at much closer intervals. In addition, it may become necessary to set up one or two more additional test concentrations below and above the expected dose range in order to allow for possible unanticipated cytotoxic fluctuations among experiments. ----------------------------NOEL--------------------------------IC50-------------------------IC90 At least one dose Two doses Two doses Figure 3: Dose setting for the initiation assay component of the transformation assay 35. In the 96-well format, the cell growth assay is carried out in the same manner as the 6-well format except the following conditions. - Into each well, 200 cells are seeded with 0.05 mL of DF5F (Day 0). 7 HRI draft ver 8, January 9/ 2014 Appendix (3) 1 - The cultures in 0.05 mL of medium are treated by the addition of another 0.05 mL of medium containing a test chemical or solvent alone at two times the final desired concentrations, so that the final volume of the medium becomes 0.1 mL (Day 1). - The volumes of CV solution and dye extraction solution are 0.1 mL/well. - For each group, eight wells are prepared. ▪ Transformation assay 36. The frozen working stock cells are rapidly thawed, suspended in M10F and cultured in 100-mm culture plates at a volume of 10 mL medium. When the cells reach approximately 70% confluence, they are trypsinized, plated in DF5F at an appropriate density (70,000 to 100,000 cells) per 100-mm culture plates (Day -3). When these cells reach approximately 70% confluence, they are again trypsinized and suspended in DF5F at 2,000 cells/mL. The cell suspension is seeded into each well at a volume of 2 mL (4,000 cells/well) for the transformation assay and the concurrent cell growth assay (Day 0). Twenty to 24 hours (Day 1) after seeding, the cells are treated for three days in the same way as the cell growth assay (Day 1-4). The medium is changed with fresh DF5F on Day 4, 7, 10 (or 11) and 14. On Day 7, the cultures for concurrent cell growth assay are fixed with methanol or 10% formalin for approximately 10 min, washed and dried. The cells are stained with crystal violet (CV) solution for approximately 15 min, rinsed well with water and dried (Fig. 2). Three wells are prepared in each group. On Day 21, the cells for transformation assay are fixed with methanol and stained with 5% Giemsa solution for approximately 15 min (Fig. 4). The positive control (1 µg/mL MCA) and the negative (solvent) control(s) are included in the transformation assay for each test chemical. When the solvent used for the test chemical is not DMSO, DMSO is still necessary as the negative control for MCA and thus, two solvent controls (one for the test article and one for the positive control) are required. In addition, an untreated control devoid of the solvent used for the test article should also be included in cases when the solvent used is one other than those commonly employed (see Paragraph 28). Nine wells are prepared for each group (one plate of six wells for the transformation assay and three wells for the concurrent cell growth assay). Mother culture Day -7 or -6 -3 Transformation assay 0 1 4 7 Cell growth assay 10 or 11 14 21 Fix：Methanol or formaldehyde Stain：Crystal violet Transformation assay Fix：Methanol Stain：Giemsa : MEM10 + 10% FBS (M10F) : Cell seeding : DMEM/F12 + 5% FBS (DF5F) : Chemical addition : Chemical treatment : Medium change Figure 4: Time line for the initiation assay component of the transformation assay 37. In the 96-well format, the transformation assay is carried out in the same manner as in the transformation assay for the 6-well format except that 200 cells are seeded/well in 0.05mL of medium on Day 0, to which is added 0.05 mL medium containing twice the desired final concentration of test chemical on Day 1. One 96-well plate (96 wells) for each group is prepared for the transformation assay and eight wells are prepared for the concurrent cell growth assay, respectively. 8 HRI draft ver 8, January 9/ 2014 Appendix (3) 1 Promotion assay ▪ Cell growth assay to set doses 38. The experimental procedure is basically the same as the initiation assay, except for the number of cells plated and timing of chemical treatment. Cells are plated at 14,000 cells/well in 2 mL of DF5F on Day 0, and chemical treatment is started on Day 4 by exchanging existing medium with fresh medium containing the test chemical solution (Fig. 5). Three wells are prepared for each group. 0 4 7 Fix：Methanol or formaldehyde Stain：Crystal violet : DMEM/F12 + 5% FBS (DF5F) : Chemical treatment : Cell seeding : Medium change containing a test chemical Figure 5: Time line of cell growth assay in the promotion assay 39. There are two different types of chemicals that exhibit promoting activity. One group includes those chemicals that enhance cell growth more than 20% of the control. With these test chemicals, concentrations are selected to cover the range from little or no growth enhancement effect to concentrations that enhance cell growth. In practice, one concentration below the NOEL, three concentrations in the range of growth enhancement and one concentration in the range of weak growth inhibition are assessed (Fig. 6). --------------------NOEL----- Growth enhancement------ Growth inhibition One dose Three doses One dose Figure 6: Dose setting for the promotion assay component of the transformation assay for chemicals that exhibit marked growth enhancement 40. The second chemical group that exhibits promoting activity is that which inhibits cell growth. For these test chemicals, concentrations are selected to cover the range from the NOEL to a level below the IC50. Ideally, at least two concentrations below the NOEL, two concentrations between the NOEL and the IC50 and one concentration between IC50 and IC90 are evaluated (Fig. 7). -----------------------NOEL----------------------------------------IC50---------------- IC90 At least two doses Two doses One dose Figure 7: Dose setting for the promotion assay component of the transformation assay for chemicals that inhibit growth 9 HRI draft ver 8, January 9/ 2014 Appendix (3) 1 41. It is noteworthy that there are chemicals that induce more pronounced growth inhibition in the transformation assay than would otherwise be observed in the concurrent cell growth assay. This phenomenon can be attributed to the difference in the duration of the treatment periods for each, i.e. 10 days for the transformation assay versus 3 days for the cell growth assay (29). 42. When test chemicals exhibit a steep concentration–response curve, considerations similar to those described in the initiation assay (refer to Paragraph 34) may need to be taken into account, e.g. inclusion of additional test concentrations and test concentration intervals, thereby ensuring an acceptable assay outcome. 43. In the 96-well format, the cell growth assay is carried out in the same manner as the 6-well format except for the plating of 400 cells in 0.1 mL of DF5F (Day 0). Eight wells are prepared for each group. ▪ Transformation assay 44. 8): The transformation assay is carried out in the same manner as the initiation assay except for the following (Fig. - The cells are suspended in DF5F at 7,000 cells/mL of which a volume of 2 mL is plated into each well (14,000 cells/well) on Day 0. Nine wells are prepared for each group (one plate of six wells for the transformation assay and three wells for the concurrent cell growth assay). - The cells are exposed to the test chemical for 10 days, from Day 4 to Day 14. - The chemical treatment is carried out on Day 4, 7 and 10 (or 11) by exchanging existing medium with fresh medium containing the chemical solution or solvent alone. - On Day 14, the medium is changed with the fresh medium containing neither solvent nor test chemical. - TPA (50 ng/mL) is used for the positive control. Mother culture Day -7 or -6 -3 Transformation assay 0 4 7 Cell growth assay 10 or 11 14 21 Fix：Methanol or formaldehyde Stain：Crystal violet Transformation assay Fix：Methanol Stain：Giemsa : MEM10 + 10% FBS (M10F) : Cell seeding : DMEM/F12 + 5% FBS (DF5F) : Medium change containing a test chemical : Chemical treatment : Medium change Figure 8: Time line for the promotion assay component of the transformation assay 10 HRI draft ver 8, January 9/ 2014 Appendix (3) 1 45. In the 96-well format, the transformation assay is carried out in the same manner as that for the 6-well format except for the plating of 400 cells in 0.1 mL of DF5F (Day 0). One 96-well plate (96 wells) for each group is prepared for the transformation assay and eight wells for each group are employed for the concurrent cell growth assay. Evaluation of the results ▪ Determination of transformation frequency 46. Transformed foci are scored using a stereomicroscope. If a given concentration results in a failure of the target cells to reach confluence because of cytotoxicity, that concentration is considered not acceptable for transformation assessment and is excluded from focus-counting. For such situations, “toxicity” is recorded in the data sheet. 47. Transformed foci are characterized by the following morphological properties: (a) more than 100 cells, (b) spindle-shaped cells differing in appearance from the contact-inhibited monolayer cells, (c) deep basophilic staining, (d) random orientation of cells, especially visible at the edge of foci (criss-cross misalignment of individual cells), (e) dense multi-layering of cells (piling up), and (f) invasive growth into the surrounding confluent monolayer of contact-inhibited cells. It should be noted that all transformed foci need not necessarily exhibit all of these morphological characteristics to be regarded as transformed, but that observation of clear-cut morphological aberrations such as these is generally sufficient to classify transformed foci as such (see Annex 2). For quantification, the number of transformed foci in each well are recorded for each group. 48. In the 96-well format, transformed foci are judged using the same criteria as in the 6-well format. For assay scoring, the number of wells having transformed foci relative to the number of wells observed is recorded for every group. Thus, a well having one focus is counted as one and a well having two or more foci is likewise counted as one. ▪ Statistical analysis 49. Statistical unit in the 6-well format is a number of transformed foci per well. Test chemical-induced transformation frequency is statistically analyzed by multiple comparison using the one-sided Dunnett test (p<0.05, upper-sided). For the positive controls, the statistical significance is evaluated by the one-sided t-test or Aspin-Welch test (p<0.05, upper-sided) depending on the results of F-test for homoscedasticity (homogeneity of variance). 50. Statistical unit in the 96-well format is a number of wells with one or more transformed foci. Test chemical-induced transformation frequency is statistically analyzed using the chi-square test with Bonferroni adjustment (p-value<0.05, upper-sided). For the positive controls, the statistical significance is evaluated by the one-sided chi-square test (p<0.05, upper-sided). ▪ Assay acceptance criteria 51. The following criteria (Paragraphs 52 and 53) must be fulfilled for a given assay to be considered valid. When considered invalid, the initiation or promotion assay is repeated independently, as needed, to satisfy the assay acceptance criteria. 11 HRI draft ver 8, January 9/ 2014 Appendix (3) 1 52. In the 6-well format, the following criteria must be fulfilled for a given assay to be considered valid: - When contamination or technical problems are observed in wells, a minimum of two undamaged wells per group is necessary in the concurrent cell growth assay and a minimum of five undamaged wells per group is necessary in the transformation assay. In cases where such technical difficulties are encountered, “contamination”, “accident”, “technical error”, etc. are recorded in the data sheet. - In the negative control, the number of (spontaneous) transformed foci must be 10 or less per well in the initiation assay and 12 or less per well in the promotion assay. - In the positive control, there must be a statistically significant increase in the number of transformed foci per well compared to the corresponding negative control. - A transformation assay is considered acceptable if four test chemical concentrations persist and the following conditions are satisfied. If it can not be satisfied the following conditions due to characteristics of the test chemical such as a steep concentration–response curve and precipitation etc, the rationale should be justified and documented. - In the initiation assay, the results of the concurrent cell growth assay include at least one concentration near the NOEL and three concentrations in the range between the NOEL and the IC90. -In the promotion assay showing growth enhancement, the results of the concurrent cell growth assay include at least one concentration near the NOEL and two concentrations in the range of growth enhancement. - In the promotion assay showing growth inhibition, the results of the concurrent cell growth assay include at least two concentrations below the NOEL and two concentrations between the NOEL and the IC50. 53. In the 96-well format, the following criteria must be fulfilled for a given assay to be considered valid: - When contamination or technical problems are observed, a minimum of four undamaged wells per group is necessary in the concurrent cell growth assay and a minimum of 90 undamaged wells per group is necessary in the transformation assay. In cases where such technical difficulties are encountered, “contamination”, “accident”, “technical error”, etc. are recorded in the data sheet. - In the initiation assay, the number of wells in the negative control plates having (spontaneous) transformed foci must be 15 wells/plate or less; if damaged wells are present, the number of undamaged wells with transformed foci must be ≤15.625%). In the promotion assay, the number of wells in the negative control plates having (spontaneous) transformed foci must be 20 wells/plate or less; if damaged wells are present, the number of undamaged wells with transformed foci must be ≤20.833%. - In the positive control, there must be a statistically significant increase in the proportion of wells having transformed foci. 12 HRI draft ver 8, January 9/ 2014 Appendix (3) 1 - A transformation assay is considered acceptable if four test chemical concentrations persist and the following conditions are satisfied. If it can not be satisfied the following conditions due to characteristics of the test chemical tested such as a steep concentration–response curve and precipitation etc, the rationale should be justified. - In the initiation assay, the results of the concurrent cell growth assay include at least one concentration near the NOEL and three concentrations in the range between the NOEL and the IC90. -In the promotion assay showing growth enhancement, the results of the concurrent cell growth assay include at least one concentration near the NOEL and two concentrations in the range of growth enhancement. - In the promotion assay showing growth inhibition, the results of the concurrent cell growth assay include at least two concentrations below the NOEL and two concentrations between the NOEL and the IC50. 54. In the initiation and promotion assays, when cytotoxicity from chemical treatment results in an inhibition of confluence at the end of transformation assay such that at least four test chemical concentrations are not available to be evaluated, the following criteria can be invoked in deciding whether or not to repeat such an experiment: - If a minimum of two sequential doses induce statistically significant increases in transformation frequency, then a repeat experiment is not necessary and the result is judged as positive. - Other outcomes, e.g. one test chemical concentration induces a statistically significant increase in transformation frequency and two concentrations do not, would necessitate a repeat experiment at lower concentrations (i.e. those that would not inhibit confluence). - Other experimental results in which an insufficient number of test chemical concentrations remains available for scoring should be evaluated on a case-by-case basis to determine the design for a repeat study. ▪ Data interpretation criteria 55. The assay results in the 6-well format and 96-well format are judged as follows: - The results in the initiation and promotion assays are judged positive if there are two or more sequential doses that induce statistically significant increases in the number of transformed foci per well relative to the corresponding solvent control. - The results in the initiation and promotion assays are judged negative if there is no dose showing a statistically significant increase in the number of transformed foci per well. - If the statistically significant increase occurs at only one or non-sequential doses, the assay result is regarded as equivocal, in which case the initiation or promotion assay should be repeated. Modification of experimental conditions in which a broader or narrower range of test chemical concentrations, as appropriate, should be considered in such follow-up experiments to eliminate such equivocality. 13 HRI draft ver 8, January 9/ 2014 Appendix (3) 1 - When results in the initiation assay are determined to be positive based upon the above criteria, the test chemical is considered to have carcinogenic initiating activity in this test system. - When results in the promotion assay are determined to be positive based upon the above criteria, the test chemical is considered to have carcinogenic promoting activity in this test system. - A positive result in either the initiation assay or the promotion assay is taken to mean that the test chemical possesses potential carcinogenic activity in this test system, irrespective of which endpoint is positive. - A negative results in both assays are taken to mean that the test chemical dose not possess potential carcinogenic activity in this test system. - Final judgment is based on a comprehensive evaluation that also takes into account of the biological relevance. Laboratory Proficiency 56. In order to assure the proficiency of a given laboratory, the laboratory should perform tests using four positive chemicals, each acting via different mechanisms of action in both the initiation assay and the promotion assay, and two negative chemicals. Those chemicals recommended are listed in Table 2. During the course of these tests, the laboratory should establish: - A historical negative (untreated, solvent) control range and distribution. - A historical positive control range and distribution. Re-evaluation of laboratory proficiency is recommended if major changes to experimental conditions are introduced in the assay (e.g. use of automated instead of manual scoring techniques). Before using this Test Guideline, it is recommended that personnel be trained in a laboratory experienced in this assay. Table 2: Chemicals for Assessing Laboratory Proficiency (see reference 13) --------------------------------------------------------------------------------------------------------------------------------------------------Category Chemical CASRN --------------------------------------------------------------------------------------------------------------------------------------------------1. Positive chemicals for initiation assay (Genotoxic carcinogens) N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) 70-25-7 3-Methylcholanthrene (MCA) 56-49-5 2. Positive chemicals for promotion assay (Nongenotoxic carcinogens) 12-O-tetradecanoylphorbol-13-acetate (TPA) 16561-29-8 Methapyrilene HCl 135-23-9 3. Negative chemicals for both assays (Noncaricinogens) Caffeine (CFN) 58-08-2 Mannitol 69-65-8 --------------------------------------------------------------------------------------------------------------------------------------------------- 14 HRI draft ver 8, January 9/ 2014 Appendix (3) 1 REPORTING Test report 57. The test report should include the following information: Test chemical identification and CAS number. (if known) physical nature and purity physical properties relevant for conducting the assay stability of the test chemical (if known) Solvent (if appropriate) identification justification for choice of solvent concentrations tested and preparation of the dosing solutions signs of precipitation (if appropriate) Cells and media source of cells number of cell subcultures (passage number) maintenance of cell cultures absence of cell culture contamination, especially mycoplasma identification of media and serum (provider and batch number) used for cell culture cryopreservation, maintenance, and assays Test conditions rationale for selection of test chemical concentrations, including cytotoxicity data and solubility limitations composition of media serum concentration, origin, quality, selection criteria concentrations of test chemicals volume of solvent and test chemical added duration of treatment incubation temperature incubation atmosphere: percent CO2 and air number of cells plated for cell growth assays, concurrent cytotoxicity tests and transformation assays positive and negative controls: identification, CAS numbers, concentrations criteria for scoring morphologically altered foci Results results of the dose range finding test results of the concurrent cell growth assay solubility of test chemical, pH and signs of precipitation in medium number of total valid (quantifiable) wells, number of wells lost and the reason(s) for the loss 15 HRI draft ver 8, January 9/ 2014 Appendix (3) 1 - number of total foci (6-well format) transformation frequency: transformed foci/well (6-well format), the number of wells with transformed foci/ total number of wells (96-well format) dose-response relationship, where one exists statistical analyses: statistical test(s) employed, analytical results concurrent negative (solvent) control data, untreated control data where appropriate, and positive control data historical negative (solvent) and positive control data, with ranges, means and standard deviations Data should be presented in tabular form. The following values should be presented for each group (treated group, solvent and positive control groups): i. results of the concurrent cell growth assay ii number of total valid (quantifiable) wells iii number of total foci (6-well format) or the number of wells with transformed foci (96-well format) iv transformation frequency v statistical test(s) employed, analytical results Discussion of results Conclusion REFERENCES 1. Barrett, J.C. and Ts’o, P.O.P. (1978), Evidence for the progressive nature of neoplastic transformation in vitro, Proc. Natl. Acad. Sci. USA, 75, 3761-3765. 2. Kakunaga, T. and Yamasaki, H. (1985), Transformation Assay of Established Cell Lines: Mechanisms and Application, IARC Scientific Publications No. 67, International Agency for Research on Cancer, Lyon, 225p. 3. Berwald, Y. and Sachs, L. (1963), In vitro cell transformation with chemical carcinogens, Nature, 200, 1182-1184. 4. Newbold, R.F., Overell, R.W. and Connell, J.R. 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ECVAM (2012), Recommendation concerning the cell transformation assays (CTA) using Syrian Hamster Embryo cells (SHE) and the BALB/c 3T3 mouse fibroblast cell line for in vitro carcinogenicity testing, including the ESAC opinion (Annex 1) based on the ESAC peer review of an EURL ECVAM-coordinated validation study of three CTA protocols for in vitro carcinogenicity testing http://ihcp.jrc.ec.europa.eu/our_labs/eurl-ecvam/eurl-ecvam-recommendations/cta-recommendation 8. Berenblum, I. (1941), The mechanism of carcinogenesis: a study of the significance of cocarcinogenic action and related phenomenon, Cancer Res., 1, 807-814. 9. Mondal, S., Brankow, D.W. and Heidelberger, G. (1976), Two-stage chemical oncogenesis in cultures of C3H/10T1/2 cells, Cancer Res., 36, 2254-2260. 10. Sasaki, K., Mizusawa, H. and Ishidate, M. (1988), Isolation and characterization of ras-transfected BALB/3T3 clone showing morphological transformation by 12-O-Tetradecanoylphorbol-13-acetate, Jpn. J. Cancer Res., 79, 921-930. 11. Sasaki, K., Sakai, A., Yamazaki, S., Umeda, M. and Tanaka, N., Transformation assay in Bhas 42 cells: a model of initiated cells to study mechanisms of carcinogenesis and predict carcinogenicity of chemicals. (submitted) 12. Asada, S., Sasaki, K., Tanaka, N., Takeda, K., Hayashi, M. and Umeda, M. (2005), Detection of initiating as well as promoting activity of chemicals by a novel cell transformation assay using v-Ha-ras-transfected BALB/c 3T3 cells (Bhas 42 cells), Mutat. Res., 588, 7-21. 13. Sakai, A., Sasaki, K., Muramatsu, D., Arai, S., Endou, N., Kuroda, S., Hayashi, K., Lim, Y.M., Yamazaki, S., Umeda, M. and Tanaka, N. (2010), A Bhas 42 cell transformation assay on 98 chemicals: the characteristics and performance for the prediction of chemical carcinogenicity, Mutat. Res., 702, 100-122. 14. Chu, E. H. and Malling, H. V. (1968), Mammalian cell genetics. II. Chemical induction of specific locus mutations in Chinese hamster cells in vitro, Proc. Natl. Acad. Sci. US A., 61, 1306-1312. 15. Duncan, M.E. and Brookes, P. (1973), The induction of azaguanine-resistant mutants in cultured Chinese hamster cells by reactive derivatives of carcinogenic hydrocarbons. Mutat. Res. 21, 107-118. 16. Ohmori, K., Umeda, M., Tanaka, N., Takagi, H., Yoshimura, I., Sasaki, K., Asasda, S., Sakai, A., Araki, H., Asakura, M., Baba, H., Fushiwaki, Y., Hamada, S., Kitou, N., Nakamura, T., Nakamura, Y., Oishi, H., Sasaki, S., Shimada, S., Tsuchiya, T., Uno, Y., Washizuka, M., Yajima, S., Yamamoto, Y., Yamamura, E. and Yatsushiro, T., Non-Genotoxic Carcinogen Study Group in the Environmental Mutagen Society of Japan (2005), An inter-laboratory collaborative study by the Non-Genotoxic Carcinogen Study Group in Japan, on a cell transformation assay for tumour promoters using Bhas 42 cells, AT LA., 33, 619-639. 17 HRI draft ver 8, January 9/ 2014 Appendix (3) 1 17. Tanaka, N., Sasaki, K., Hayashi, K., Sakai, A., Asada, S., Muramatsu, D, Kuroda, S, Mizuhashi, F., Nagai, M., Suzuki, H., Imamura, T., Asakura, M., Satoh, H., Sakamoto, A., Nakao, R., Hirose, H., Ishii, N. and Umeda, M. (2009), An international collaborative study on a cell transformation assay using Bhas 42 cells, AATEX, 14, 831-848. 18. Sakai, A., Sasaki, K., Hayashi, K., Muramatsu, D., Arai, S., Endou, N., Kuroda, S., Poth, A., Bohnenberger, S., Kunkelmann, T., Asakura, M., Hirose, H., Ishii, N., Mizuhashi, F., Kasamoto, S., Nagai, M., Pant, K., Bruce, S.W., Sly, J.E., Yamazaki, S, Umeda, M, and Tanaka, N. (2011), An international validation study of a Bhas 42 cell transformation assay for the prediction of chemical carcinogenicity, Mutat. Res., 725, 57-77. 19. Hayashi, M., Kojima, H., Corvi, R., Stokes, W., Jacobs, A., Morita, T., Schechtman, L. and Suzuki, M. (2012), Bhas 42 cell transformation assay validation study report, (submitted to JaCVAM) 20. Creton, S., Aardema, M., Carmichael, P.L., Harvey, J.S., Martin, F.L., Newbold, R.F., O’Donovan, M.R., Pant, K., Poth, A., Sakai, A., Sasaki, K., Scott, A.D., Schechtman, L.M., Shen, R.R., Tanaka, N. and Yasaei, H. (2012), Cell transformation assays for prediction of carcinogenic potential: state of the science and future research needs, Mutagenesis, 27, 93–101. 21. Vanparys, P., Corvi, R., Aardema, M.J., Gribaldo, L., Hayashi, M., Hoffmann, S. and Schechtman, L. (2012), Application of in vitro cell transformation assays in regulatory toxicology for pharmaceuticals, chemicals, food products and cosmetics, Mutat. Res., 744, 111-116. 22. Arai, S., Sakai, A., Hayashi, K., Sasaki, K., Muramatsu, D., Endou, N., Umeda, M. and Tanaka, N. (2013), A high-throughput cell transformation assay applicable to automation for detecting potential chemical carcinogens using Bhas 42 cells, AATEX, 18 (in press) 23. Bozic, I., Antal, T., Ohtsuki, H., Carter, H., Kim, D., Chen, S., Karchin, R., Kinzler, K.W., Vogelstein, B. and Nowak, M.A. (2010), Accumulation of driver and passenger mutations during tumor progression, Proc. Natl. Acad. Sci. USA., 107, 18545-18550. 24. Shimada, T., Oda, Y., Gillam, E. M., Guengerich, F.P. and Inoue, K. (2001), Metabolic activation of polycyclic aromatic hydrocarbons and other procarcinogens by cytochromes P450 1A1 and P450 1B1 allelic variants and other human cytochromes P450 in Salmonella typhimurium NM2009, Drug. Metab. Dispos., 29, 1176-1182. 25. Schrenk, D., Gant, T.W., Michalke, A., Orzechowski, A., Silverman, J.A., Battula, N. and Thorgeirsson, S.S. (1994), Metabolic activation of 2-acetylaminofluorene is required for induction of multidrug resistance gene expression in rat liver cells, Carcinogenesis, 15, 2541-2546. 26. Roy, P., Yu, L.J., Crespi, C.L. and Waxman, D.J. (1999), Development of a substrate-activity based approach to identify the major human liver P-450 catalysts of cyclophosphamide and ifosfamide activation based on cDNA-expressed activities and liver microsomal P-450 profiles, Drug Metab Dispos., 27, 655-666. 18 HRI draft ver 8, January 9/ 2014 Appendix (3) 1 27. Sakai, A., Iwase, Y., Nakamura, Y., Sasaki, K., Tanaka, N. and Umeda, M. (2002), Use of a cell transformation assay with established cell lines, and a metabolic cooperation assay with V79 cells for the detection of tumour promoters: a review, AT LA., 30, 33-59. 28. Hayashi. K., Sasaki, K., Asada, S., Tsuchiya, T., Hayashi, M., Yoshimura, I., Tanaka, N. and Umeda, M. (2008), Technical modification of the Balb/c 3T3 cell transformation assay: the use of serum-reduced medium to optimise the practicability of the protocol, AT LA, 36, 653-665. 29. Arai, S., Tanaka, N., Sasaki, K. and Sakai, A. (2010), A Study on the Dose Setting of Test Chemicals for the Promotion Assay in Bhas 42 Cell Transformation Assay, AATEX, 15, 6-13. 19 HRI draft ver 8, January 9/ 2014 Appendix (3) 1 Annex 1: Culture media, reagents and solutions Media and supplements: - MEM: Minimum essential medium with 2.2 g/L NaHCO3 and 0.292 g/L L-glutamine. - DMEM/F12: Dulbecco’s modified Eagle’s medium/F12 with 1.2 g/L NaHCO3. - FBS: Fetal bovine serum, selected based upon a low frequency of spontaneous transformed focus formation and a high induced frequency of transformed focus formation in the positive control. - PS: Penicillin G sodium (10,000 units/mL) and streptomycin sulfate (10 mg/mL). - M10F: MEM + 10% FBS + 1% PS (500 mL MEM + 56 mL FBS + 5 mL PS): used for cell population expansion, cell storage, and the first culture after thawing. - DF5F: DMEM/F12 + 5% FBS + 1% PS (500 mL DMEM/F12 + 26.5 mL FBS + 5 mL PS): used for routine subculturing of cells, cell growth assays and transformation assays. Fixatives and staining solutions: - Formalin (37% formaldehyde): used for fixing cells. - Methanol: used for fixing cells. - 0.1% crystal violet (CV) solution: used for staining cells in cell growth assays. CV, 1 g, is dissolved in 50 mL of ethanol, and the total volume is adjusted to 1 L with distilled water/ultra-pure water. - Extraction solution: 0.02 mol/L HCl and 50% ethanol (480 mL distilled water/ultra-pure water + 500 mL ethanol + 20 mL 1 M HCl) used for extracting CV in cell growth assays.. - 5% Giemsa solution: used for staining cells in transformation assays. 20 HRI draft ver 8, January 9/ 2014 Appendix (3) 1 Annex 2: Photo catalog of foci in Bhas 42 CTA <Negative (Non-transformed) Foci> B- S- M- R- I- B+/- S+ M- R- I- The cells simply gather together. The morphology of the cells changes to spindle-shaped. However, all other characteristics are negative. B+/- S+ M+/- R- I+ B+ S+ M+ R+ I+ Piling up is scarcely observed and other positive characteristics are barely discernable. Morphological characteristics resembling transformation are observed but the focus is exceedingly small. <Positive (Transformed) Foci> B+ S++ M+/- R+ I+ B+ S++ M+ R+ I+ Piling up is limited. The cells comprising the focus are markedly spindle-shaped (tapered and elongated), displaying a swirling parallel arrangement. Some areas of piling up are observed within the focus. Foci consist of markedly spindle-shaped (tapered and elongated) cells generally aligned relative to each other. 21 HRI draft ver 8, January 9/ 2014 Appendix (3) 1 B+ S++ M+/- R++ I+ B+ S+ M+ R+ I+ Scattered areas of piling up and knotting (dense clustering) of cells is observed. Cells are exceedingly spindle-shaped (tapered and elongated) and randomly orientated. All properties that are characteristic of the transformed phenotype are moderately expressed. B+ S++ M++ R++ I+ B+ S+ M++ R+ I+ All aberrant phenotypic characteristics are clearly visible. With the exception of multilayered areas, which are too dense to discern individual cell morphology, the atypical properties of the transformed focus are readily observed at the periphery of the focus. The cells comprising the periphery of the focus are less densely packed and their spindle-shape and random orientation, although apparent, are less striking. The interlaced cells at the edge of the focus invade the surrounding monolayer. B+ S- M++ R- I+ B+ S++ M++ R++ I+ The multi-layering and density of cells are apparent throughout the focus. The cells comprising the periphery of the focus are not obviously spindle-shaped or randomly orientated but do invade. the contact-inhibited monolayer. Daughter (secondary) foci originating from a single parent focus and exhibiting typical and uniform transformed morphology. These are found in close proximity, are often connected by cellular appendages, and are scored as one transformed focus. Abbreviations: B, basophioic; S, spindle-shaped; M, multilayer; R, random orientated; I, invasive. 22 Appendix (3) 2 Table of WNT Comments /responses on the draft Test Guideline on a Bhas Cell Transformation Assay December 2013 - Comments were received from France, Germany, Japan, the US, ICAPO and BIAC. Comment France Japan Response General comments The procedure provides accurate instructions for a given laboratory which want to develop the assay. Some clarification could be made, see below. Transforming activities of aneuploidogens or estrogens have not yet tested. Applicability domain of this assay should be described in this test guideline. Some explanations were added in each comment. DES and -estradiol (Ohmori, 2004, Mutation Res 557, 191-202; Ohmori, 2005, ATLA, 33, 619-639, Sakai, 2010, Mutation Res, 702, 100-122) had been tested and showed the negative results in this system. As applicability domain of this assay, any types of chemicals including metal ions are applicable except volatile and gaseous chemicals. At the start, the cell growth assay is conducted for the dose setting of the test article. The concurrent cell growth assay is needed to evaluate Why should we need to carry out the concurrent cell growth assay when running the whether transformation assay is performed at adequate transformation assay? Isn't the concurrent assay omissible? concentrations of chemicals. Further, there are fluctuations among experiments. US (1) A very interesting and ingenious assay that can predict the tumor-initiating and tumor-promoting potentials of test chemicals with the ultimate purpose of judging their potential carcinogenic activity. The assay can help to predict genotoxic and nongenotoxic carcinogens. I agree with the Validation Management Team (VMT) that the assay has scientific backing, is reproducible within and between labs, is transferrable, and has satisfactory capacity to predict chemical carcinogenicity. However, I suggest that the VMT should look into the following, if they have not already considered these issues: a. The assay appears to predict nongenotoxic carcinogenic potential 1 a) We need to discuss on this issue since the definition based solely on tumor-promoting activity. There are many tumorpromoters that are not carcinogenic. Although sensitivity and specificity are satisfactory, it is not clear whether the chemicals selected for training and testing covered a reasonable representation of noncarcinogenic tumor-promoters. This is important because if noncarcinogenic tumor promoters are not distinguishable from carcinogenic tumor promoters, this assay may over-predict nongenotoxic carcinogens. of noncarcinogenic and carcinogenic tumour promoters is not clear. b. From the mechanistic point of view, the complete carcinogenic process involves initiation, promotion and progression. It is not clear whether this assay covers the progression aspect of the carcinogenic process. Nongenotoxic carcinogens have a variety of mechanisms. Have any attempts been made to determine whether Bhas 42 CTA can predict receptor-mediated (e.g., PPAR, Ah, RXR, CAR, etc.) nongenotoxic carcinogens? b) The final endpoint of Bhas 42 CTA is the induction of morphologically transformed foci. Induced transformed clones cause tumors in nude mice after implantation of 106 cells in each site. Therefore, we expect Bhas 42 CTA covers carcinogenic process including initiation and promotion. So far, the progression phenomena indicating progression of malignancy in vivo have not been clarified in in vitro study. Concerning receptor-mediated nongenotoxic carcinogens, we need more data. c. Each predictive assay is expected to have unique strengths and limitations. Has there been a systematic attempt to find the strengths and limitations of Bhas 42 CTA, particularly in areas where Bhas 42 is superior to similar CTAs, such as SHE cell CTA? Are there limitations for testing direct-acting genotoxic carcinogens? 2 c) Strengths: - The most important strength is that the induced transformed foci cause tumors in nude mice. - The results of Bhas 42 CTA show low incidence of false negative (27%) and false positive (16%). - Among 28 Ames negative carcinogens, 17 carcinogens were positive in Bhas 42 CTA (Sakai et al, 2010, Mutat. Res., 702, 100-122). - Bhas 42 CTA can differentially detect initiating and promoting activity of carcinogens. - Bhas 42 CTA is more sensitive to carcinogens than BALB/c3T3 CTA. - - Culture period (3 weeks) is shorter than conventional cell line CTAs (6-7 weeks) using BALB/c 3T3 and C3H10T1/2. Multilayered transformed foci which are formed on monolayered normal cells are counted objectively. Limitations: - Culture period is longer that SHE cell CTA (1week). - Metabolic activities of Bhas 42 cells are supposed to be lower than primary cells. - The response of Bhas 42 cells to receptormediated nongenotoxic carcinogens are unknown. There is no technically limitation for testing direct-acting genotoxic carcinogens. d. (2) Several genotoxic polycyclic aromatic hydrocarbons and aromatic amines owe their lack of carcinogenic activity to their lack of tumorpromoting activity. The consistent negative tumor-promoting activity for Dibenz[a,h]anthacene is surprising in view of its high carcinogenic potency. The inconsistent, equivocal assay result for the recently elevated human carcinogen o-toluidine is also puzzling. Carcinogenicity is a fundamental endpoint in the assessment of risks to humans from exposure to chemicals. The standard approach to this kind of testing is to conduct 2-year bioassays in rats and mice. The Bhas 42 cell linebased CTA could be an important component in the battery of tests for predicting chemical tumorogenicity. The Bhas 42 CTA uses clones of BALB/c 3T3 cells transfected with an oncogenic murine ras gene (v-Ha-ras), and is conducted over a time period of 21 days. The time needed to conduct 3 d) Further study is needed about inconsistent results between Bhas 42 CTA in vitro and carcinogenicity in vivo. the study is even shorter than the BALB/c 3T3 and SHE CTAs, and further reduces the large number of lab animals required in bioassays. The proposed guideline claims that the assay is capable of detecting the tumor-initiating and tumor-promoting activities of chemical carcinogens according to different test protocols, and thus will detect both genotoxic and nongenotoxic carcinogens. In light of this, the Bhas 42 CTA could provide additional information when compared to the classical Ames test. The Validation Management Team analyzed test results for known chemicals and concluded that the assay is reproducible and transferable within and between laboratories, and thus could be used for the prediction of chemical carcinogenicity. Nevertheless, the test reliability in concordance (78%), specificity (84%), positive predictivity (86%) and false positive results (16%), when compared with standard genotoxicity assays and test results with otoluidine, could raise some concerns. In summary, the Bhas 42 cell line based CTA could be a valuable addition to the toolbox of methods available for the characterization of carcinogenicity in a weight of evidence approach or within an integrated testing strategy.. However, some thoughts/suggestions that could potentially improve this method are presented for consideration: a. The assay procedures and methods are fundamentally the same between the 6-well and the 96-well format micro plates methods, except for the methodology of foci scoring and statistical methods used. It may be beneficial to use alternative terminology such as “6well format” and “96-well format” of assay to better understand the test. b. The 6-well format is one way to do the test, but the 96-well microplates format method has obvious advantages: all controls and test probes are on the same plates (minimize variation of incubation conditions); count of positive wells instead of colonies (could minimize human error and subjective judgment). The Bhas 42 CTA using 96-well micro-plates (96-well method) has been developed to be 4 a) Changed “method” to “format”. b) 96-well format is very promising for high throughput automated application. On the other hand, 6-well format is a conventional method which is easy for foci counting. We proposed both methods, since users can freely choose any format by own preference and object. utilized for high throughput automated applications using micro-plate readers. Utilizing H2O2 + WST-8 method could also improve the method. In order to simplify protocol and avoid misreading and confusions, we would like to recommend use the 96-well micro-plates format (96-well method) ONLY and establish a section in the protocol for high throughput automated applications. c. Method evaluation was performed on a limited number of known chemicals that did not include many classes of carcinogens (for example, metals such as cobalt, nickel, arsenic). Further testing with these and other chemical carcinogen classes are needed to better understand Bhas 42 CTA limitations. c) Metal ions are applicable in the Bhas 42 CTA. Some metals have been tested by Bhas 42 CTA system. The results of cobalt and nickel are negative and sodium arsenate, sodium arsenite and zinc were positive in the promotion assay. d. It was not obvious to us whether this test could discriminate between initiator and promoter oncogenic effect. This should be clarified by a systematic evaluation of the chemicals with known characteristics. Method limitations should be established. d) Almost of all direct-acting genotoxic carcinogens can be detected as initiators. Some indirect-acting genotoxic carcinogens also. However, receptor (absence in Bhas 42 cells) mediated promoters cannot be detected. Please see below minor suggestions for Annex 12: Recommended Protocol for the Bhas 42 Cell Transformation Assay (2012.7.27) NATIONAL POSITION: The Commenters raised some interesting issues on the domain of applicability (e.g. do they apply to metals) that should be addressed by the expert group. 5 BIAC ICAPO The TG is currently a good first draft, but will need several modifications. Based on Bhas 42 CTA is utilized; paper review, the validation report seems adequate to support an OECD TG - for detection of possible carcinogens presumed development. However, it is acknowledged that we do not have hands-on experience by structure-activity relationships. of the assay. It is also expected that the expert group will address the place of the TG - for screening of nongenotoxic carcinogens in a testing scheme. - for screening of genotoxic noncarcinogens - for mechanistic research The draft revised test guideline refers to “test substance”, while current OECD - for screening of anti-carcinogens terminology is “test chemical”. Similarly, in vitro TGs are using the word “solvent”, not “vehicle” or “solvent/vehicle”. These should be changed, and the document Changed the wording to OECD terminology. reviewed for any other discrepancy from currently adopted TG language and format. ICAPO is pleased to support the development of this test guideline. The Bhas 42 CTA Thank you for your valuable comments. We would like offers serious ethical and practical advantages over traditional animal carcinogenicity to discuss on your suggestions in the expert meeting. assays, and ICAPO hopes to see the guideline adopted quickly so that industry and regulators can gain more experience conducting the assay and interpreting the data it provides. According to the test guideline, the predictivity of the Bhas 42 CTA has been extensively verified using a large number of chemicals. The assay accurately detects carcinogens acting via several different mechanisms and the data it provides are relevant to the hazard assessment of chemicals. Therefore, we would argue against the use of language such as “…are expected to be used as part of a testing strategy (rather than a stand-alone assay) and/or in a weight-of-evidence approach…” (Paragraph 7). This phrasing tends to be used almost reflexively to refer to all in vitro assays, regardless of the potential use of the assay. Results of all assays (including in vivo) are used together with other evidence about a chemical’s toxic potential. This wording should be reconsidered for two reasons: 1. This language may discourage use of the test guideline, especially by those not familiar with the assay, because it implies the data will be of limited use unless other tests are also performed. 2. This language implies that the Bhas 42 CTA will always be accompanied by other assays or tests, when in fact in certain contexts it may be the only or decisive assay, as noted in paragraph 7. We suggest making the test guideline Introduction more clear and descriptive about the possible uses of the assay in a more constructive, neutral way, taking into account 6 US BIAC US BIAC US that in some cases the assay may be the decisive assay for a regulatory (or business development) decision. In this regard, although outside the scope of the TG itself, it would be helpful to develop an Adverse Outcome Pathway(s) for carcinogenicity that could provide context for the use of all assays that address this pathway, including CTAs. Finally, as context for the development of this assay, the Introduction should outline some of the documented limitations of the rodent cancer bioassay, including its cost, the large number of animals required to run it, the length of time required to run it, and its often limited applicability to humans. At the very least, the TG should highlight some of the advantages of the Bhas 42 CTA over the rodent cancer bioassay, including its much shorter time and smaller cost, and its use of a cell line rather than live animals or primary cells. Paragraph 1 Expert Comments: Line 13 – replace “can” with “could” We could not follow your question because there are no line 13, lines 33-36 in paragraph 1. Lines 33 – 36: The text within these lines state that steps of the 6-well method and the 6-well microplate method, as well as the 9-well method and the 96-well microplate In paragraph 30, composition of the following section method are the same, and “similar results are obtained with both methods.”. has been explaining. Therefore, we think it does not Throughout the document (Lines 49, 151, 160, 161, 162, 349, 447, 462, 494, 504, 508, need. 518, 523, and 594, only the “6-well method” and “96-well method” are used. We suggest adding the following to Line 3 6: “For simplicity, the following protocol refers to both methods as the “6-well method” and “96-well method.” Paragraph 2 It is questioned whether referring to the other than the Bhas 42 cell line and validation Rewrote. is appropriate in this TG. Suggest rewriting paragraph accordingly. Paragraph 2.1 Line 50 – replace “used” with “recommended” We could not find the place of line 50 in paragraph 2 Paragraph 3 Replace “it has become clear” with “it has long been known” since reference 9 is from Changed. year 1976 and almost 40 years old. Paragraph 3.1.1 to 3.2.3 Lines 185, 238, 269, and 331 – replace “Schematic protocol” with “Time line” Changed. Paragraph 4 7 Germany BIAC “After exposure to carcinogenic stimuli, such cells can become morphologically altered…” Paragraph 5 “The current protocol for the Bhas 42 CTA consists of two assay components…” (delete dash between “two” and “assay”) Paragraph 6 Please define ICCVAM (ECVAM and JaCVAM are defined in paragraph 2) It may be useful to provide some of the validation data in this paragraph. Paragraph 7 As discussed above, we suggest editing this paragraph: “Test results derived from the Bhas 42 CTA may, depending on the regulatory context, provide sufficient evidence to determine the carcinogenic potential of a substance, especially if considered are expected to be used as part of a testing strategy (rather than a stand-alone assay) and/or in a weight-of-evidence approach to predicting carcinogenic potential. When employed in combination with other information such as genotoxicity data, structure-activity analysis and pharmaco/toxicokinetic information, CTAs in general and the Bhas 42 CTA specifically can contribute to the assessment of carcinogenic potential (20). This test can and may reduce the use of in vivo testing. CTAs may be particularly useful for evaluating chemicals for which in vivo testing is not allowed (e.g. regulation on cosmetics in the European Union [Regulation (EC) 1223/2009 of the European Parliament and of the Council of 30 November 2009 on cosmetic products]), is limited, or is only required for chemicals identified as genotoxic (21); in other contexts a validated in vitro method must be considered preferentially to in vivo testing. Paragraph 8 Typographical error : Replace « The test method described is based upon the protocol for reported for this assay in Sakai et al. (13). » by « The test method described is based upon the protocol for reported for this assay in Sakai et al. (13). » Please strike out the reference Sakai et al. to be consistent in the document. Give abbreviation for test guideline (TG) since it is used here for the first time. ICAPO Would it be appropriate to add a last sentence after “the overall results obtained are The question may be for paragraph 9. We changed. similar” stating that the methods can be used interchangeably? “The test method described is based upon the protocol for reported for this assay in Corrected. ICAPO ICAPO BIAC ICAPO ICAPO France 8 Corrected. Corrected. Corrected. We think it does not need. We need to discuss on this point more with experts. Corrected. Changed Ref 13 to 18. Corrected. Sakai et al. (13).” Paragraph 9 Paragraph 10 France ICAPO France Japan BIAC ICAPO Paragraph 11 Precise the initials FBS (foetal bovine serum) “Therefore, it is important to maintain strict quality control of cells, assay components, and test conditions, including the use of low passage target cells…” Paragraph 12 Please, clarify if a metabolic supplementation is recommended in this assay. Otherwise, could you specify whether the activation ways of the three proposed positive controls cover the scope of a broad metabolic supplementation? ...”being tested,,” suppress one comma Corrected. Corrected. CYP1A1 is predominantly involved in activation of polycyclic aromatic hydrocarbons (Shimada T. et al., Drug. Metab. Dispos., 2001, 29, 1176-82.). CYP1A2 is predominantly involved in activation of 2acetylaminofluorene (Schrenk D. et al., Carcinogenesis, 1994, 15, 2541-6). CYP2B6 is involved in NATIONAL POSITION: The paragraph needs clarifications cyclophosphamide activation (Roy P. et al., Drug Metab. Dispos., 1999, 27, 655-66). Therefore, we more precisely described about CYP species supposed to be expressing in Bhas 42 cells. Karyotype of Bhas 42 cell line should be described. At least, modal number or ploidy The chromosome number is ranging from 55 to 65, it of this cell line need at this para. The all cytochrome P450 family enzymes confirmed shows hyper triploidy. Bhas 42 cells have similar at this cell line should be described. marker chromosomes as BALB/c 3T3 cells. Representative cytochrome P450 enzymes were described in Paragraph 12. There is an extra “,” after “tested” in the first sentence. Please delete. Deleted. The first part of this paragraph negatively overshadows the second part, which is Agree with your proposal. evidence that Bhas 42 cells maintain some cyp activity. We recommend deleting the Deleted the first sentence and revised the second first sentence. The second sentence could then read: “The fact that Bhas 42 cells sentence. respond to polycyclic aromatic hydrocarbons, 2-acetylamineofluorene, and cyclophosphamide. Since these substances , all of which require metablic activation (13), suggests that Bhas 42 cells contain some level of the cytochrome P450 family of enzymes including CYP1A1 and others.” 9 Consistent with recommendations from the VMG-non-animal and as described in Jacobs et al., 2013,1 we recommend that the metabolic capacity of this cell line be characterized. BIAC France BIAC Jacobs, M.N., Laws, SC., Willett, K., Schmieder, P., Odum, J., Bovee, T.F. 2013. In vitro metabolism and bioavailability tests for endocrine active substances: What is needed next for regulatory purposes? ALTEX. 30(3):331-51. Paragraph 13 Consider replacing “animal” with “in vivo” in the first sentence on the first row. Replaced and deleted. Delete the last part of the first sentence (“…in carcinogenicity studies in vivo”) as duplicative Delete the last sentence “Further studies of this kind…” since it is purely speculative. Paragraph 14 Replace “aberrant” by “transformed”. Replaced. Paragraph 15 Last sentence: Delete “blind” and add to the end of the sentence that the evaluation of Deleted. the morphological phenotype is by stereomicroscope. Paragraph 16 Paragraph 17 Paragraph 18 France Paragraph 19 Explain why is necessary to use two different media (question of specificity, cost..?). NATIONAL POSIITON: Agreed 1 DF5F is essential medium for high induction of cell transformation frequency. M10F medium is routinely used for cell culture of Bhas 42 cells because it shows low spontaneous transformation frequency (For BALB/3T3 CTA: Tsuchiya T. and Umeda M., Carcinogenesis, 1995, 16, 1887-1894. For Bhas 42 CTA: Ohmori K. et al., Mutat. Res., 2004, 557, 191- Jacobs, M.N., Laws, SC., Willett, K., Schmieder, P., Odum, J., Bovee, T.F. 2013. In vitro metabolism and bioavailability tests for endocrine active substances: What is needed next for regulatory purposes? ALTEX. 30(3):331-51. 10 202). Defined. Define “M10F” and “DF5F” Paragraph 20 BIAC BIAC Paragraph 21 It seems to be a restricting factor that the cells need to be obtained from a single source For uses we are going to provide Bhas 42 cells to in Japan. Is this limitation acceptable in an OECD TG for such a basic thing as the ATCC (American Type Culture Collection, USA), cells to be used in an in vitro assay? ECACC (European Collection of Cell Cultures, Europe) or other authorized cell banks without any restriction. Paragraph 22 It is said that the cells can be passed ‘several” times. Please add an upper limit for Added “2-3 passages” in text. passage that is still appropriate to use. Paragraph 23 France Paragraph 24 How is “low passage” defined? Please add a passage number. Paragraph 25 Explain why higher passages are tolerated in cell proliferation assays? BIAC How is “higher passages” defined? Please add a upper passage number. BIAC Added “2-3 passages” in text. Spontaneous transformed cells are increased by successive passages. However, short-term cytotoxicity assay is not affected by successive passages. It will be passaged within 10 passages. Added “within 10 passages” and deleted “The same cell source can be used for the cell growth assay, although”. Paragraph 26 BIAC Germany BIAC Paragraph 27 Define “MCA” and “TPA” when used the first time. Paragraph 28 The concentration 0.5% is mentioned twice. Please perform respective corrections by deletion or the addition of a further concentration. What is the guidance for non-soluble chemicals? Can suspensions be tested? What is the guidance for volatile and/or gaseous chemicals? Are the concentrations of the solvents correct? It seems odd that acetone can be used 11 Defined. We keep these concentrations, because it is easy to understand. We performed this assay using suspension of test chemical, but cannot apply volatile and/or gaseous chemicals without modification of this test protocol. France as a higher final concentration than ethanol, and that DMSO and acetone can be The concentrations of the solvent are correct, because tolerated at only 0.5% (not 1%). 1% DMSO and acetone inhibit cell growth slightly and high concentration of ethanol affect cell function (Pontes et al, 2008, Arch. Toxicol., 82, 197-198). Paragraph 29 “Five to nine concentrations...” precise that at least four concentrations must be tested “Five to nine concentrations should” was replaced with (according to paragraph 52). “At least four concentrations must”. NATIONAL POSIITON: The number of concentrations should be harmonized with paragraph 52 Paragraph 30 France BIAC France Paragraph 31 Contradiction between “a dose range-finding test is first conducted” and “concurrent Replaced with the sentence you recommended. cell growth assay”. Or precise: ex: In a first step, concentrations are selected in a preliminary cell growth assay using a large dose range. In the transformation assay, a cell growth assay is concurrently performed to verify that the selected doses meet acceptation criteria for the assays. NATIONAL POSIITON: The paragraph needs clarification. Figure 1 does not seem to add much in clarity, since both assays are more identical. Changed it to a single stream as your suggestion. Maybe simplify further by drawing a single stream under the assays instead of two separate? Or use Figure 1 from the Annex 12 Recommended Protocol for the Bhas 42 Cell Transformation Assay. The subtitle “Cell growth assay to set doses” needs to be more prominent. Now it is hardly visible. Paragraph 32 - See paragraph 36: Also describe the procedure from Day -7. - Explain why there are two ways to perform cell treatment (removing or not the medium). Does it depend on the test substance or something else? - Question: Can ethanol be used as a fixating agent instead of more hazardous chemicals such methanol or formalin? 12 Changed to Bold. -Paragraph 32 describes about the cell growth assay to set doses. The procedure from Day -7 (frozen cells) does not always need for cell growth assay to set doses. -Coincident results can be obtained in both procedures and you can choose any procedure depending on your experimental situations. Germany BIAC Japan BIAC France France BIAC France -Probably it is acceptable to use ethanol, but we do not have experience so far. Please provide a description in the text regarding the meaning of Day -3 in Figure 2: Deleted “day-3” for Figure 2 and Figure 5, because pre Schematic protocol of cell growth assay in the initiation assay. culture does not affect in the cell growth assay. Paragraph 33 Add “the” in front of “CV”. Added. Paragraph 34 NOEL is not clear to me. Data points often fluctuate. How can we be confident the NOEL means 80-120% cell growth in Bhas 42 CTA, point is NOEL? Is statistical aid needed? and statistical analysis does not need. Paragraph 35 The subtitle “Transformation assay” needs to be more prominent. Now it is hardly Changed to Bold. visible. Paragraph 36 - “(7,000 to 10,000 cells/mL)” : Precise the final volume of medium or the amount of Changed text. cells per well Added description about concurrent cell growth assay. - “concurrent cell growth assay “ : see note in paragraph 31 Paragraph 37 “One 96-well plate is prepared”: add “for each group” Added. The subtitle “Cell growth assay to set doses” needs to be more prominent. Now it is Changed to Bold. hardly visible Paragraph 38 Paragraph 39 “that markedly enhance”: precise, ex: that increase more than ..(ex: 25%, 50%...) cell growth Defined “markedly” as “more than 20% cell growth”, because NOEL is 80-120% cell growth. NATIONAL POSIITON: Please define “markedly enhance” France Paragraph 40 “one concentration above I50” : replace by “one concentration between IC50 and IC90” (as refer to paragraph 34) NATIONAL POSIITON: See consistency with paragraph 34 13 Replaced. Paragraph 41 Paragraph 42 BIAC France BIAC Paragraph 43 The subtitle “Transformation assay” needs to be more prominent. Now it is hardly visible Paragraph 44 Paragraph 45 “One 96-well plate is prepared”: add “for each group” “eight wells are employed”: add “eight wells for each group are employed” There are several subtitles as commented on previously that are hardly visible in the current format and need to be written more prominently. Paragraph 46 Changed to Bold. Added. Added. Changed to Bold. Paragraph 47 Paragraph 48 France Paragraph 49 In the 6-well method, what is the statistical unit? Is it the transformation frequency determined for each 6 wells per group (n = 6) in one experiment, or is it the transformation frequency in a total of 6 wells per group in n repeated experiment? Number of transformed foci per well is statistical unit. Clarified it in test. NATIONAL POSIITON: Agreed Paragraph 50 BIAC Paragraph 51 The statement “repeated independently, as needed, to satisfy the acceptance criteria” is Added in fourth item of paragraph 52 for the a bit puzzling in all its clarity. Are there any circumstances where one can stop description about the cases that it is difficult to satisfy repeating the assay(s) and draw the conclusion that no conclusion can be drawn? There the acceptance criteria. is a reference to paragraphs 52 and 53; paragraph 54 also seems to provide advice for when to repeat 9as does paragraph 55). 14 France Paragraph 52 -“must be 10 or less.....12 or less”: Do these numbers represent the mean of the values found in 6 wells? - “in the range of growth enhancement.” Add “and n concentration(s) inducing a weak inhibition (to be in agreement with paragraph 39) - “the NOEL and the IC50.” Add “and n concentration(s) above the IC50 (to be in agreement with paragraph 40). NATIONAL POSIITON: Agreed -It was determined based on the mean of the negative historical control data of an international collaborative study (Tanaka et al, 2009, AATEX, 14, 831-848) and biological relevance. -Acceptance criteria were determined by focussing at key concentrations at which the test chemicals induce growth enhancement, because such chemicals can exhibit promotion activity at key concentrations. Therefore, higher concentrations inducing inhibition of cell growth are not required for the test chemicals inducing growth enhancement. -For the test chemical inducing growth inhibition, concentrations above the IC50 are not required in promotion assay, because long treatment (10 days) can cause not to reach confluence at the end of transformation assay. Japan France Japan BIAC France Remove a phrase, “it is recommended…” Since it describes acceptance criteria, Deleted and added the description about the cases that it “recommendation” is inappropriate. is difficult to satisfy the acceptance criteria. Paragraph 53 See the two last comments for paragraph 52 See answer in paragraph 52. Remove a phrase, “it is recommended…” Since it describes acceptance criteria, See answer in paragraph 52. “recommendation” is inappropriate. Remove the closing parenthesis after ≤15.625% Deleted. Paragraph 54 Paragraph 55 The guideline states that, in case of positive results, it can be concluded that “..the test chemical possesses potential carcinogenic activity.”. Should it not be also indicated how to conclude in case of negative results (e.g: inconclusive, further data needed…)? 15 We described in the text how to conclude the results. Japan BIAC France BIAC ICAPO BIAC NATIONAL POSIITON: Agreed “ A positive result in either the initiation assay or the promotion assay is taken to mean that the test chemical possesses potential carcinogenic activity, irrespective of which endpoint is positive.” should be deleted. This is because it is not necessarily the case that positive chemicals at the promotion assay are carcinogens. Is the evaluation solely based on statistical significance, with no consideration of biological relevance? Paragraph 56 Add reference for positive and negative controls. NATIONAL POSIITON: Agreed Suggest deleting the sentence “Additionally, changes in laboratory personnel..” and moving up the sentence “before using this Test Guideline, it is recommended that…” If positive results are obtained in promotion assay, we conclude that the test chemical possess carcinogenic potential but not definite carcinogen, so it leave the sentences. Added the description on the biological relevance. Added reference no.13. Deleted. We added “Genotoxic carcinogen” for positive Table 2 It would be more appropriate to talk about carcinogens and non-carcinogens than chemicals for initiation assay, “Nongenotoxic positive and negative chemicals. carcinogens” for positive chemicals for promotion assay, “Noncarcinogens” for negative chemicals. “Before using this Test Guideline, it is recommended that personnel be trained in a laboratory experienced in this assay.” Harmonized with SHE cell CTA draft test guideline. Is this a practical recommendation? “Recommend” is a strong word in a test guideline, making it likely that regulatory agencies may want to see proof of such “seconded” training. While we agree that personnel should be trained adequately, we suggest the words “…personnel may find it helpful to be trained…”. Remote training or training by visiting scientists may also be helpful. Paragraph 57 Results should include observations of cytotoxicity, pH, and data presentation Added pH and data presentation instructions. instructions. Paragraph Paragraph 16 Appendix (3) 3 Outline of Cell Transformation Assay of Bhas 42 Cells (v-Ha-ras-Transfected BALB/c 3T3 Clone) to Detect Carcinogens Hatano Research Institute, Food and Drug Safety Center 14-16 January 2014 Contents 1. Bhas 42 cells 2. Methods of the transformation assay 3. In-house study for prediction of carcinogenic potential of 98 chemicals 4. Validation studies 5. What is the Bhas 42 cell transformation assay used for? Transformed Colony （Focus） in Bhas 42 Cells Transformed focus Basophilic Spindle-shaped Criss-cross Piling up Invasive Normal cells Monolayer Contact inhibition Two Stage Transformation in BALB/c 3T3 Cells DMSO + DMSO MCA 0.5 mg/ml + DMSO DMSO + TPA 100 ng/ml MCA 0.5 mg/ml + TPA 100 ng/ml MCA 3 mg/ml + DMSO Chemically Induced Initiation Can Be Replaced with v-Ha-ras transfection in BALB/c 3T3 Cells Plasmids –––––––––––––––––––––– v-Ha-ras Control Chemicals ––––––––––––––––––––– Control TPA This dish suggests that the presence of a model of initiated cells. Sasaki K et al. Jpn J Cancer Res. (1988) 79:921-930. Establishment of Bhas 42 Cells BALB/c 3T3 cells Transfection of v-Ha-ras Cloning of v-Ha-ras containing cells Screening of non-transformed cells which are morphologically changed by TPA Bhas 42 cells Sasaki K et al. Jpn J Cancer Res. (1988) 79:921-930. Bhas 42 Cells Can Be Transformed by TPA Alone Control Bhas 42 BALB/c 3T3 MCA TPA FISH Analysis of v-Ha-ras in Bhas 42 Cells Modal No. = 60 (nearly triploid) 100% cells contain v-Ha-ras on Ch No. 17 or 19. Average = 2.4 copies/nucleus Evidence That Transformation of Bhas 42 Cells Correlates with Carcinogenicity Transformed Bhas 42 cells Normal Bhas 42 cells 60 days after inoculation of 106 cells s.c. Tumors / 6 transplanted sites All Transformed Bhas 42 Clones Are Tumorigenic but Normal Bhas 42 Cells Are Not 6 5 4 Bhas 42 MCA-14 (106) Bhas 42 MCA-18 (106) Bhas 42 TPA-14 (106) Bhas 42 TPA-15 (106) Bhas 42 (106) BALB/c 3T3 (106) 3 2 1 0 0 10 20 30 40 50 60 70 Days ‧ 4 transformed clones, Bhas 42 cells and BALB/c 3T3 cells ‧ 3 places s.c./mouse ‧ 2 mice/group Sasaki K et al. submitted. Replication of Cells Is Necessary for Genotoxic Carcinogens, While Cell-to-Cell Contact Is Necessary for Non-Genotoxic Carcinogens at Treatment with Chemicals MCA: positive, TPA: negative Treatment for 3 days at growth phase MCA: negative, TPA: positive for 10 days at stationary phase Sakai A. AATEX. (2008) 14:367-373. Schedule of Bhas 42 Cell Transformation Assay Days 0 4 7 11 14 21 Medium change Initiation assay Treat at growth phase Promotion assay Treat at stationary phase 6-well or 96-well plates are used. In parallel, the cells for cell growth assay are fixed on day 7. When either the initiation assay or promotion assay is positive, the chemical is positive. Expression of Transformation Frequency in 6-Well and 96-Well Methods Control MCA 1 mg/ml 6-well method Foci / Well e.g. 14 foci / Well 96-well method No. of wells with foci / 96 wells e.g. 52 wells with foci / 96 wells Arai S et al. AATEX. (2013) 18:1-19. 6-Well and 96-Well Methods Show Similar Results in the Treatment with MCA and TPA MCA 6-well method TPA 96-well method 6-well method 96-well method Initiation assay Promotion assay : transformation frequency, : relative cell growth. Arai S et al. AATEX. (2013) 18:1-19. Performances of Bhas 42 CTA for the Prediction of Carcinogenic Potential of Chemicals by 6-Well Method (In-House Study) –––––––––––––––––––––––––––––––––– Animals or Bhas 42 CTA –––––––––––––––––––– human Positive Negative –––––––––––––––––––––––––––––––––– Positive 38 14 Negative 6 31 –––––––––––––––––––––––––––––––––– 98 chemicals tested 89 chemicals have in vivo data 52 Carcinogens 37 Non-carcinogens 9 chemicals have no in vivo data Concordance 78% = (38 + 31) / (38 + 14 + 6 + 31) × 100 Sensitivity 73% = 38 / (34 + 14) ×100 Specificity 84% = 31 / (6 + 31) × 100 Positive predictivity 86% = 38 / (38 + 6) × 100 Negative predictivity 69% = 31 / (14 + 31) × 100 False positive 16% = 6 / (6 + 31) × 100 False negative 27% = 14 / (38 + 14) × 100 Sakai A et al. Mutat Res. (2010) 702:100-122. Comparison of the Results in Bhas 42 CTA with Those in Ames Assay Carcinogens (52 chemicals) Chemicals 52 Carcinogens) Bhas 42 CTA Initiation Promotion Ames MN Described in DRP31 2-Acetylaminofluorene + + + + + Benz[a]anthracene + + +,+,+ + + 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) 5-Azacytidine Barium chromate Benzo[a]pyrene Cyclophosphamide Cyclosporin A 2,4-Diaminotoluene Dibenz[a,h]anthracene Melphalan 3-Methylcholanthrene Mitomycin C MNNG Sterigmatocystin Thio-TEPA Cadmium chloride Chenodeoxycholic acid 4-Chloro-o-toluidine HCl Cholic acid Deoxycholic acid Dichlorvos Epichlorohydrin D-Limonene Lithocholic acid Methapyrilene HCl + - + - + + + + + + + + + + + + + + - + + + + + + + + + + +,+/+,+ +,+/-,+/+,+ +,+/-,+ +,+,+,+/+,+ + +,+ + + -,+/+/+/+,+ +,+,+/-,-,- + +,+,+ - + +,+,+ + + + - + + + + + + + + + + + + + + + - + + + + 18 of 38 carcinogens: Positive in the Bhas 42 CTA but negative or discordant in the Ames assay. These chemicals are detected mainly by the promotion assay. 52 Carcinogens (continued) Chemicals Bhas 42 CTA Initiation Promotion Ames MN Methylarsonic acid Mezerein 2-Naphthylamine Phorbol 12,13-didecanoate Quercetin Sodium arsenate Sodium arsenite Sodium saccharin Styrene oxide TPA o-Toluidine Zinc chloride o-Anisidine Benzene Cobalt sulfate heptahydrate Diethylstilbestrol Dimethylarsinic acid 1,4-Dioxane Ethyl carbamate (Urethane) Formaldehyde Furylfuramide (AF-2) Methyl carbamate Nickel (II) chloride Nickel monooxide Phenobarbital sodium p-Toluidine +/+ +/+/- -,+,+,+/-,+,+/- - +,+,+/-,+/-,+,+,+/-,+/+,+ + + -,-,-,-,+/-,-,+,+/-,+/+,+,+ -,-,-,+/- +,+ + + + + + + + + + + + + + - Described in DRP31 + -,+,-,+ +/-,- +,+ +/-,+,+/+,+ +/- + + + + + + + + + + + + + + + + + + + + + + 18 of 38 carcinogens: Positive in the Bhas 42 CTA but negative or discordant in the Ames assay. These chemicals are detected mainly by the promotion assay. 37 Non-carcinogens 8 of 31 non-carcinogens: Negative in the Bhas 42 CTA but positive or Discordant in the Ames assay. Chemicals Bhas 42 CTA Initiation Promotion Ames 4-Acetylaminofluorene Acid red 14 Ampicillin sodium salt Anthracene L-Ascorbic acid Aspartame Benzoin Caffeine Caprolactam 2-Chloroethanol Chromium (Ⅲ) Chloride, anhydrous 2,6-Diaminotoluene Diazepam N,N-Dimethylformamide Eugenol HC Blue 2 Hydrocortisone D-Mannitol Methotrexate 1-Naphthylamine Phenanthrene Phenol p-Phenylenediamine 2HCl Phthalic anhydride Rotenone Sodium chloride Sodium nitrite Sunset yellow FCF Thiabendazole m-Toluidine Triphenyltin hydroxide Barium chloride dihydrate tert-Butylhydroquinone 8-Hydroxyquinoline Propyl gallate Sodium fluoride Tetracycline HCl + - + -,-,+, -,+/+/-,+/-,+/-,-,-,+,+,+/+ -,-,+ + + + + + + -,+,+,+/+,-,+/+ -,+,+ + -,-,-,+,+ -,-,-,- MN - + + -,- -,+ + +,+ + + -,+,+ + +/-,+ Described in DRP31 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 9 chemicals of unknown carcinogenicity Chemicals Bhas 42 CTA Initiation Promotion Sodium valproate + + Valproic acid + + 2,3-Diaminotoluene - + 2,5-Diaminotoluene 2HCl - + 3,4-Diaminotoluene - + Sodium orthovanadate - Capsaicin Ames MN Described in DRP31 + - + - - - Ethidium bromide - - - Salicylic acid - - + + Sakai A et al. Mutat Res. (2010) 702:100-122. Bhas 42 CTA Can Detect Ames-Negative Carcinogens Bhas 42 Ames False negative 52 carcinogens Some carcinogens are detected. e.g. methapyrilene HCl sodium arsenate TPA 37 non-carcinogens False negative False positive False positive ▆: positive, ▛: negative Sakai A et al. Mutat Res. (2010) 702:100-122. Performances of Various Assays for the Prediction of Carcinogenic Potential of Chemicals (All Data Are Obtained from DRP 31 Except Bhas 42) 100 100 100 50 50 50 0 0 0 Low Concordance Bhas 42 100 Sensitivity BALB/c 3T3 Ames + Predictivity 50 0 Specificity 100 100 50 50 0 0 100 50 0 - Predictivity False Negative False Positive Performance SHE pH 6.7 SHE pH > 7.0 C3H10T1/2 100 100 100 50 50 50 0 0 0 High CA ML HGPRT Conclusions from the In-House Study on 98 Chemicals 1. Bhas 42 CTA could detect a considerable number of Ames-negative and Ames-discordant carcinogens. 2. Those Ames-negative and Ames-discordant carcinogens were detected mainly by the promotion assay. 3. The performances of Bhas 42 CTA are well-balanced compared with other short-term assays. Overview of Validation Studies –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Study No. Plates No.of chemicals / No. of No. of chemicals / Study labs Lab –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Pre-validation (for refinement, transferability and performance of the protocol) 1 6-well 9 6 3 Validation (to evaluate reproducibility and relevance of the assay) 2 6-well 12 6 3 3 (Phase I) 96-well 7 4 4 3 (Phase II) 96-well 16 3 2 –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Total 23 chemicals (excluding overlapping) Results of Study No. 3 - Phase I Labs 2AAF I P Anthracene I P B[a]P MCA I I P P Phenanthrene I P O-Toluidine I P TPA I P I + + - - + - + - - - - - - + II + - + - + - + - - - - - - + III + + - - + - + +/- - - - - - + IV + + - - + - + - - - - - - + All over judgment + + - - + - + - - - - - - + In vivo + - + + - + + I: initiation assay, P: promotion assay Evaluation of Each Chemical from Data of Study No. 1-3 ------------------------------------------------------------------------------------------------------------------------------Chemicals Study 1 Ini Pro Study 2 Ini Pro Study 3 Ini Pro All over In vivo judgment ------------------------------------------------------------------------------------------------------------------------------Alkylating agent MNNG -,+,+ -,-,- +,+ -,- + + Aromatic amine 2-Acetylaminofluorene +,+,+ +,+,+ +,+,+,+ -,+,+,+ + + o-Toluidine -,+,+ -,+,+ -,-,-,- -,-,-,- +/- + -,- -,- - - -,-,-,- +,+,+,+ + + -,- +,+ + + Phorbol Phorbol -,-,- -,-,+/- TPA Steroid Lithocholic acid -,-,- +,+,+ -,-,- +,+,+ ------------------------------------------------------------------------------------------------------------------------------Each symbol represents a result of one lab, Ini: initiation assay, Pro: promotion assay. (Continued) ------------------------------------------------------------------------------------------------------------------------------Chemicals Study 1 Ini Pro Study 2 Ini Pro Study 3 Ini Pro All over In vivo judgment ------------------------------------------------------------------------------------------------------------------------------Aromatic hydrocarbon Anthracene -,-,- -,-,- -,-,- -,-,+ Phenanthrene Benz[a]anthracene +,+,+ -,+,+ Benzo[a]pyrene -,-,- +,+,+ Dibenzo[a,h]anthracene +,+,+ -,-,-,- - - -,-,-,- -,-,-,- - - + + + + +,+,+,+,+,+ -,-,-,-,-,+,+,+ -,-,- 3-Methylcholanthrene Pyrene -,-,-,+ -,+,+ +,+ -,- + + +,+,+,+ -,-,-,- + + +,+ +,+ + - -,- -,- - - -,- -,- - - Antibiotic Ampicillin sodium Alkaloid Caffeine -,- -,-,- ------------------------------------------------------------------------------------------------------------------------------- (Continued) ------------------------------------------------------------------------------------------------------------------------------Chemicals Study 1 Ini Pro Study 2 Ini Study 3 Pro Ini Pro All over In vivo judgment ------------------------------------------------------------------------------------------------------------------------------Inorganic compound Cadmium chloride -,-,- +,+,+ -,- +,+ + + Sodium arsenite -,+ +,+,+ -,- -,- +/- + -,-,- -,-,- -,- -,- - - -,- -,- - - Others L(+)-Ascorbic acid Eugenol D(-)-Mannitol -,-,- -,-,- -,- -,- - - -,-,- Methapyrilene HCl -,-,+ +,+,+ +,+,+ -,- +,+ + + Mezerein -,-,- +,+,+ -,+/-,+/- +,+,+ +,+ +,+ + + ε-Caprolactam (< 5 mM) -,- -,- - - ε-Caprolactam (> 10 mM) +,+ -,- + - ------------------------------------------------------------------------------------------------------------------------------Tanaka N et al. AATEX. (2009) 14:831-848. Sakai A et al. Mutat Res. (2011) 725:57-77. Results of Dibenz[a,h]anthracene in Study No. 2 Positive results Negative results Sakai A et al. Mutat Res. (2011) 725:57-77. Results of Lithocholic Acid in Study No. 2 Negative results Positive results Sakai A et al. Mutat Res. (2011) 725:57-77. Advantages of Using Bhas 42 Cell Transformation Assay to Detect Carcinogens (1) Low cost and short-term assay without animals ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Animals (rats, mice) Bhas 42 cells ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– No. of animals Test period Cost 150-300 0 2-3 years (whole life study) < 1 month > Two million $ < Ten thousand $ ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Advantages of Using Bhas 42 Cell Transformation Assay to Detect Carcinogens (2) 1. Either of 6-well and 96-well plates can be used. 2. Bhas 42 CTA can detect both genotoxic and nongenotoxic carcinogens depending on a change between two protocols. What Is the Bhas 42 Cell Transformation Assay Used for? Screening of carcinogens presumed by some information (e.g. structure) but Screening of nongenotoxic carcinogens but Screening of genotoxic noncarcinogens TPA + chemical X Screening of anti-carcinogens Mechanistic research
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