In vitro 発熱性物質試験
目次
評価会議報告書
第三者評価報告書

ECVAM statement

ICCVAM

Test Method Evaluation Report

プロトコールAppendix B1

Cryopreserved Whole Blood (Cryo WB)/Interleukin-1β (IL-1β)
Test Method

Appendix B2

Mono Mac 6(MM6)/Interleukin-6 (IL-6) Test Method

Appendix B3

Blood Mononuclear Cell (PBMC) /Interleukin-6 (IL-6) Test Method

Appendix B4Whole Blood (WB)/Interleukin-1β (IL-1β) Test Method

Appendix B5Whole Blood (WB)/ Interleukin-6 (IL-6)

Test MethodIn vitro 発熱性物質試験の評価会議報告書

2010 年 9 月 6 日

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JaCVAM 評価会議
西川秋佳（国立医薬品食品衛生研究所 安全性生物試験研究センター）
田中憲穂（食品薬品安全センター 秦野研究所）
吉田武美（昭和大学薬学部）
横関博雄（東京医科歯科大学）
吉村 功（東京理科大学）
中村和市（日本製薬工業協会）
岡本裕子（日本化粧品工業連合会）
大島健幸（日本化学工業協会）
小野寺博志（医薬品医療機器総合機構）
小笠原弘道（医薬品医療機器総合機構）
吉田 緑 （国立医薬品食品衛生研究所 安全性生物試験研究センター 病理部）
五十嵐良明（国立医薬品食品衛生研究所 生活衛生化学部）

オブザーバー：
柴辻正喜（厚生労働省 医薬食品局 審査管理課 化学物質安全対策室）
実国慎一（経済産業省 製造産業局 化学物質安全対策室）
オブザーバー：JaCVAM 運営委員
大野泰雄（国立医薬品食品衛生研究所）
関野祐子（国立医薬品食品衛生研究所 安全性生物試験研究センター 薬理部）
増田光輝（国立医薬品食品衛生研究所 安全性生物試験研究センター 薬理部）
小島 肇（国立医薬品食品衛生研究所 安全性生物試験研究センター 薬理部）
秋田正治（日本動物実験代替法学会）

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In vitro 発熱性物質試験について、第三者評価委員会からの報告を受け、以下の 8 項目について審議した。本 2～8 項目は OECD ガイダンス文書 No．34 に示された検討項目である。
＜検討項目＞
１．検討対象の試験法は、日本のどの法規制やガイドラインに関係しているか。
２．検討対象の試験法とその妥当性を示すデータは、透明で独立な評価を受けているか。
３．当該試験法で得られるデータは、対象毒性を十分に評価あるいは予測できるものであるか。デー
タは、当該試験法と従来の試験法の、代替法としての繋がりを示しているか。あるいは（同時に）そのデータは、当該試験法と、対象としているあるいはモデルとしている動物種についての影響との繋がりを示しているか。
４．当該試験法は、ハザードあるいはリスク、あるいはその両方を評価するのに有用であるか。
５．当該試験法とその妥当性を示すデータは、その試験法で安全性を保証しようとする、行政上のプログラムあるいは関係官庁が対象としている化学物質や製品を、十分広く対象としたものとなっているか。当該試験法が適用できる条件及び適用できない条件が明確であるか。
６．当該試験法は、プロトコールの微細な変更に対して十分頑健で、適切な訓練経験を持つ担当者と適切な設備のある施設において、技術習得が容易なものであるか。
７．当該試験法は、時間的経費的に有用性があり、行政上で用いられやすいものであるか。
８．当該試験法は、従来の試験法と比べて、科学的・倫理的・経済的に、新しい試験法あるいは改訂
試験法であることが正当化されているか。
＜審議内容＞
１．

検討対象の試験法は、日本のどの法規制やガイドラインに関係しているか。

第十五改正日本薬局方（15 局，2006 年） 及び医療機器の生物学的安全性評価のための試験法 （医薬局審査管理課事務連絡，医療機器審査 No.36）が該当する。
・ 日本薬局方 15 局は、発熱性物質の検出法として「発熱性物質試験法」及び「エンドトキシン試
験法」 （リムルス試験）を収載している。
・ 日本薬局方 15 局の「製剤総則・注射剤」の項では「エンドトキシン試験法」が優先されているが、「エンドトキシン試験法の適用が困難な場合は，発熱性物質試験法を用いることができる」と規定されている。
・ 「輸液用ゴム栓試験法」（日本薬局方 15 局）及び「医療機器の生物学的安全性評価のための試験法」では「発熱性物質試験法」の実施が要求されている。
２．検討対象の試験法とその妥当性を示すデータは、透明で独立な評価を受けているか。

・ ICCVAM が、ECVAM の検討結果（EC STATEMENT ON THE VALIDITY OF IN-VITRO TESTS 及び ECVAM
Background Review Document (BRD)）を第 3 者評価し、2008 年 5 月に 2 つの報告書（ICCVAM
Test Method Evaluation Report 及び ICCVAM Background Review Document (BRD)）を公表した。これらの報告書について JaCVAM 発熱性物質試験評価委員会が評価した。
・ ICCVAM による妥当性評価では、OECD の指針書（GD34）に従って、要確認項目（Modular
approach）が精査された。
・ これらの報告書には評価した 5 つの試験のプロトコール及び被験物質が記載されている。
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総合評価の資料として取り入れなかった報告については概要を示し、ウサギ「発熱性物質試験法」との相関性については参考資料を挙げて比較したことが示されている。
・ 資料には、データ処理の仕方に若干の問題があったが、その結論に与える影響は小さく、
ICCVAM の総合評価は受け入れられるものである。
３．当該試験法で得られるデータは、対象毒性を十分に評価あるいは予測できるものであるか。デー
タは、当該試験法と従来の試験法の、代替法としての繋がりを示しているか。あるいは（同時に）そのデータは、当該試験法と、対象としているあるいはモデルとしている動物種についての影響との繋がりを示しているか。
・in vitro 発熱性物質試験法（in vitro PBMC 試験法）においては、発熱性物質の存在を評価するために、ヒト末梢血単核細胞あるいは株化ヒト単球様細胞から分泌される炎症性サイトカイン（インターロイキン（IL）-1β あるいは IL-6）を測定する。エンドトキシンを介した発熱の機序には即しており、科学的に妥当である。

・

試験法として標準的に参照されているウサギの体温上昇を測定する「発熱性物質試験法」、及び「エンドトキシン試験法」との比較を行い、結果が概ね一致していることを確かめている。

・

ヒト由来の細胞を用いることから、異なる動物種を用いる「発熱性物質試験法」や「エンドトキシン試験法」よりもヒトでの発熱性を正確に予測できる可能性はあるが、それは検証されていない。

・

評価書では、グラム陰性菌由来のエンドトキシンについてのみ検証されており、非エンドトキ
シンについては十分な検証がなされていない。

４．当該試験法は、ハザードあるいはリスク、あるいはその両方を評価するのに有用であるか。
当該試験法は、あるエンドトキシン投与量においてサイトカイン量がある値を越えているか
で陽性・陰性を評価する試験法であり、ハザードは評価できる。ウサギを用いた発熱性物質試
験法と同様、リスク評価を目的とするものではない。
５．当該試験法とその妥当性を示すデータは、その試験法で安全性を保証しようとする、行政上のプログラムあるいは関係官庁が対象としている化学物質や製品を、十分広く対象としたものとなっているか。当該試験法が適用できる条件及び適用できない条件が明確であるか。
・

当該試験法の正確性及び再現性は合計 13 物質のバリデーション研究によって評価されている。
いずれもエンドトキシンを添加した医薬品で、エンドトキシンを添加せずとも発熱性を示す物
質、生物学的製品及び医療機器などについては実施されていない。よって、非エンドトキシンの検出力が不明確である。当該試験法は非経口投与される医薬品のエンドトキシンによる発熱性の評価が可能であるが、医療機器や経口曝露される医薬品に対しては不十分であると考えられる。

・

参考資料として、臨床上で発熱の副作用報告のあったヒト血清アルブミン製剤、第Ⅷ因子製剤、及び血漿分画製剤にエンドトキシンを添加した試料について、ウサギ発熱性物質試験、エンド
トキシン試験、in vitro PBMC 試験での結果を比較した報告が上げられている。また、当該試験法を用いた各種グルカン、リポテタイコ酸、真菌胞子、各種医療材料、脂質等での結果も報告として示されており、他の試験法では適用困難なものについても検出することが可能であった
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としている。しかし、これらは検証的なデータとは言えない。
６．当該試験法は、プロトコールの微細な変更に対して十分頑健で、適切な訓練経験を持つ担当者と適切な設備のある施設において、技術習得が容易なものであるか。
・

採血後 4 時間以内のヒト全血、凍結保存血、全血から採取した末梢血単核細胞あるいは株化細胞を用い、IL-1β または IL-6 を測定する。組み合わせによって 5 つの試験法（全血（Whole
Blood： WB）/IL-1、保存血 CryoWB/IL-1、WB/IL-6、PBMC/IL-6、単球細胞株 MM6/IL-6）があり、それらの推奨プロトコールが ICCVAM により提案されている。発熱性の予測性能としては、どの試験法ともほぼ同じ一致率を示している。

・

PBMC/IL-6 及び WB/IL-1 については、変法が評価されている。しかし、その変法がどのような経緯で作られたか明らかではなく、頑健性については評価できない。

・

ELISA キットを用いてサイトカインを測定するので、技術移転の容易性については検討されていないが、経験を有する人が技術習得することは容易であると思われる。WB/IL-1 についての施設間再現性が悪いが、他の試験法の施設間再現性および施設内再現性は良好とされている。

７．当該試験法は、時間的経費的に有用性があり、行政上で用いられやすいものであるか。
・

多数の動物を用い、実施まで時間のかかるウサギを用いる「発熱性物質試験法」よりは時間的経費的に優位であると推察される。

・「エンドトキシン試験法」と比較したときの時間的経費的な有用性については明らかではない。

・

ヒト血液を用いる場合は、行政的な制約を伴う。

８．当該試験法は、従来の試験法と比べて、科学的・倫理的・経済的に、新しい試験法あるいは改訂
試験法であることが正当化されているか。
・

ウサギを用いる発熱性物質試験と比較した場合、当該試験法は新しい試験法であり、 3Rs 原則
を満たしており、経済的にも優位性がある。

・

エンドトキシン試験と比較した場合、作用機序の異なる新しい試験法ではあるが、倫理的・経済的には優位性が明らかでない。

以上の審議の結果、JaCVAM 評価会議は、In vitro 発熱性物質試験について以下のように結論した。
・ 当該試験法は、エンドトキシンによる発熱の機序をもとに考案された方法であり、原理面で科学的な妥当性はある。
・

バリデーション研究によって、ウサギ発熱性物質試験法との相関性も概ね高いと思われるが、これを代替法として採用するためには、化合物数を増やし、多施設バリデーションを実施することが必要である。

・

現時点でエンドトキシン試験にかわるものではない。本邦においては、日本薬局方 15 局でウサギを用いる発熱性物質試験からエンドトキシン試験への移行が進められている現状を踏まえると、利用価値は限定的と言える。

・

有望な試験法と考えられるが、現時点では、ただちに代替法として採用することは妥当でな
い。
5

参考文献
１．OECD (2005) OECD

Series on testing and assessment Number 34, Guidance document on thevalidation and international acceptance of new or updated test methods for hazard
assessment, ENJ/JM/MONO(2005) 14

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In vitro 発熱性物質試験の第三者評価報告書

平成 22 年（2010 年）8 月 18 日

発熱性物質試験評価委員会

1発熱性物質試験評価委員
中澤憲一（国立医薬品食品衛生研究所・薬理部）
篠田和俊（医薬品医療機器総合機構）
小島肇（国立医薬品食品衛生研究所・薬理部）
吉村功（東京理科大学）
西岡吾朗（扶桑薬品工業）
石井健（大阪大学）
オブザーバー
内藤 誠之郎（国立感染症研究所）

用語集
BET ：Bacterial Endotoxin Test：Limulus amebocyte Lysate(：LAL) TESTING（エンドトキシン試験）
BRD: Background Review Document
ECVAM: European Center for the Validation of Alternative methods
ELISA: Enzyme-Linked ImmunoSorbent Assay
GD: Guidance Document
ICCVAM: Interagency Coordinating Committee on the Validation of Alternative Methods
IL: Interleukin（インターロイキン）
IL-1β：Interleukin 1β
IL-6: Interleukin 6
IL-8: Interleukin 8
LAL: Limulus amebocyte Lysate（カブトガニ血球抽出物）
LPS：Lipopolysaccaride（リポ多糖）
LTA:Lipoteichoic acid (リポテイコ酸)
MM: Monocytoid Cell Line Nono Mac (単球細胞株)
OECD：Organization for Economic Co-operation and Development
PBMC：Peripheral Blood Mononuclear Cell（末梢血単核細胞）
RPT：Rabbit Pyrogen Test(ウサギ発熱性物質試験)
TNF-α：Tumor Necrosis Factor-α
WB: Whole Blood(全血)2要旨
当委員会は，製剤の発熱性物質（パイロジェン）の検査法である発熱性物質試験に関して提案されている新規試験法について，その妥当性を検討することを目的とする．この新規試験法は，従来の発熱性物質試験に代わるとされる in vitro 発熱性物質試験法であり，採取したヒト末梢血単核細胞（PBMC)から分泌されるサイトカイン（インターロイキン；IL）の量を指標とする．この試験法に対し，各委員が以下のような観点で考察を加え，その正当性を総合的に評価した．
1）試験法の科学的，規制の上での妥当性
従来の発熱性物質の検査法であるウサギを用いた発熱性物質試験法とカブトガニの血球抽出成分の凝固反応によるエンドトキシン試験法（リムルス試験），さらに，今回新たに提案された in vitro 発熱性物質試験法（以後、in vitro PBMC 試験法と記す）について，検出可能な発熱性物質の種類について考察を加えた．ウサギによる発熱性物質試験ではエンドトキシン，非エンドトキシンを問わず発熱性物質の検出が可能であるが，試験法が煩雑であり，また，検査に多数の動物を必要とする．これに対し，エンドトキシン試験は簡便であり，医薬品の品質管理上問題となるエンドトキシンに対し優れた検出力を有するが，非エンドトキシンの発熱性物質試験には無効である． in vitro PBMC 試験法は発熱の生化学的メカニズムに立脚しており，理論上はエンドトキシン，非エンドトキシンの両者に対応が可能であるが，これまでに提出されているデータは主としてエンドトキシンに関するもので，非エンドトキシンの発熱性物質検出に有効であるかどうかは明確には示されていない．エンドトキシンの検出には現在用いられているエンドト
キシン試験が有効であるため，動物実験代替法（以下、代替法と記す）としては非エンドトキシンをもカバーするウサギによる発熱性物質試験に代わる役割が期待されるが，個々の事例について検証する必要があると考えられる．また，ヒトの血液等を用いるので，ヒトへの影響を直接的に評価できるという利点も想定されるが，日本人の血液を用いる場合にはその検証が必要となろう．
2）プロトコールの妥当性およびバリデーション研究に用いた被験物質
in vitro PBMC 試験法の 5 つのプロトコールを詳細に検討し，例数およびそれに基づく解釈についての疑念に加えるとともに，日本における採血実施に伴うウイルス感染への危惧を指摘した．非エンドトキシ
ンの発熱性物質についての考慮不足が懸念された．
3）試験法の正確性・信頼性についての考察
文献 1．ICCVAM Test Method Evaluation Report，および，文献 2．ICCVAM Background Review Documentの 2 つのデータを精査し，その問題点を詳細に指摘している．文献 1 における ICCVAM の総合評価は，“5種の in vitro PBMC試験法は非経口投与によるエンドトキシンの発熱性物質試験として利用可能であるが，他の物質や器具の評価法としては不十分である”，というものであった．これは OECD の指針書 GuidanceDocument (GD)
34 に従ってなされている．データの質を吟味する上で，恣意的な選択がなされていないか，判定基準が後づけでないか，などの観点からの考慮が必要であり，質の良くないデータを削除した痕跡等の問題が文献
1 および2に散見された．
しかし，
文献のデータの吟味を行った13 名の委員からなる in vitro Pyrogenicity
Independent Peer Review Panel の中には統計に詳しいと思われる委員もおり，データの細部に性能を高めに評価する偏りが多少見受けられるものの，ICCVAM の総合評価の結果から in vitro PBMC 試験法をおおむね受け入れて差し支えないものと考えられた．
4）試験法の正確性を評価する物質の in vivo および参照データ
in vitro PBMC 試験法のバリデーションに用いられた 13 の物質に関し，比較のためのウサギを用いた発熱性物質試験は行われておらず，その代わりとして過去にドイツで行われたウサギによるデータとの比較がなされていることをまず指摘している．この比較はエンドトキシン試験の結果をも加えて行っている．
ここで，実際の臨床使用で発熱が報告された物質に対する応答性を比較したところでは，必ずしもウサギ
で発熱が認められるとは限らず，例えば，ヒト血清アルブミン製剤については，in vitro PBMC 試験法では 5 例で平均 5.52 倍の応答が認められたが，ウサギではすべて陰性であった．エンドトキシンをスパイクした試料でのウサギを用いた試験との比較においても，第 VIII 因子製剤あるいはヒト血清アルブミンの発熱性について，in vitro PBMC 試験法ははるかに優れた応答性を示した．また，発熱性を示したゼラ
チン含有輸液製剤 2 バッチを用いた比較では，in vitro PBMC 試験法はともに陽性を示したが，エンドトキシン試験はともに陰性であり，ウサギは一方が陽性，他方が陰性であった．以上のように，in vitro PBMC
試験法はウサギを用いる方法あるいはエンドトキシン試験よりもヒトの発熱性を的確に検出できる可能3

性があることが文献的に示された.
しかし，実際の製剤の原料や製造工程において，エンドトキシン以外の発熱性物質の汚染が考えられな
い場合には，現状のエンドトキシン試験で対応が可能である．また，ウサギによる発熱性物質試験のすべてを今回の新規試験法に切り替えるのが適当とは思われず，例えば，何らかの理由でエンドトキシン試験が適用できない場合に，ウサギによる発熱性物質試験の代替として in vitro PBMC 試験法を採用するといった限定的な使用にとどめるべきであるという考察がなされており，本委員会も同様に考える．
5）試験法の科学的根拠および有用性と限界
in vitro PBMC 試験法以外にも，in vitro 発熱性物質試験法は提案されており，発熱あるいはそれに類するバイオマーカーを指標とする多様な系が開発されつつある．しかしながら，in vitro PBMC 試験法については，ICCVAM の報告書にもあるように，ウサギ発熱性物質試験との相関についてはエンドトキシン試
験を引用しており，また，IL-1，IL-6 といったマーカーが発熱性について普遍性があるとは言えない．よって，in vitro PBMC 試験法をウサギ発熱性物質試験の代替法とすることには無理がある．発熱性の作用機序については，in vitro のみならず，in vivo のウサギにおいても，科学的に十分に解明されているとは言い難い．最新の知見を踏まえた上で現行の試験法の可否と新たな試験法の必要性を論じる必要がある，という考察がなされており，本委員会も同様に考える．
6)結論
上記の各考察より，今回提案された in vitro PBMC 試験法は，ウサギの使用数を削減可能とし，また，作用機序に立脚した発熱性物質試験の新たな方向性として評価でき，さらに，バリデーションでのデータの扱いに関しても納得のできる基準を満たしていると考えられる．しかし，ウサギを用いた発熱性物質試
験との互換性については不明な点が多く，この試験法の代替法とするには，問題がある．国内ではウサギ
発熱性物質試験からエンドトキシン試験への移行の問題も残されており，これに優先して独立した代替法とすることには無理がある．しかしながら，エンドトキシン試験が使用できないような特殊な状況やウサ
ギとヒトでの発熱性に関する種差が想定されるような状況においてウサギ発熱性物質試験と併用するな
ど，限定的な使用については推奨できよう．このような in vitro PBMC 試験法が科学的根拠に基づいた研究により，さらに広範囲な状況で使用可能となることが望まれる．

補）エンドトキシン試験と発熱性物質試験に関する実態調査結果報告
2009 年 1 月に日本製薬工業協会の協力を得て，アンケートを行い，36 社から回答を得た．エンドトキシ試験法が，日本薬局方で優先すべき試験法として推奨されていることもあり，最も広く用いられていた．
エンドトキシン試験法の原理は一連の酵素活性化反応であるため，これらの系に影響する反応干渉因子が存在する．しかし，実際には干渉のメカニズムが既知の物質については，製剤そのものが体温に影響する場合を含め，ほとんどが検証可能であった．今回提案されている新規試験法（in vitro PBMC 試験法）の実施経験の報告は皆無であった．また，ヒトの血液の入手あるいはバイオハザードの問題などがあり，動物愛護の観点等からウサギを用いた発熱性物質試験法から代替法へと切り替えるにしても，エンドトキシ
ン試験法への変更が妥当であると考えられた．上述したように，現状ではエンドトキシン試験法は日本薬局方においてその使用が推奨され最も汎用されている系ではあるが，推奨される以前にウサギによる発熱性物質試験のデータを用いて承認を受けた医薬品に関しては，動物愛護の観点を含めこれをエンドトキシ
ン試験法に変更することが望まれる．しかし，実行するとなると，両者の同等性を示すために多数のウサギを使用しなければならない，というジレンマが生じる．

4

０．評価資料
１）ICCVAM Test Method Evaluation Report
２）ICCVAM Background Review Document(BRD)
３）EC STATEMENT ON THE VALIDITY OF IN-VITRO PYROGEN TESTS
４）ECVAM BRD
１．試験法の科学的，規制の上での妥当性
発熱性物質（パイロジェン）は，体温調節中枢である視床下部に直接的あるいは間接的に作用して体温を上昇させる物質である． 主に，細菌やウィルス，菌類等の微生物に由来し，生体はこのような物質の暴露に反応して，
前炎症性サイトカインである IL-1 や IL-6，
TNF-α等の内因性発熱性物質を産生分泌し，これらがプロスタグランディンの合成を促進して中枢神経系に作用することにより発熱を生じる．発熱性物質のタイプや量，あるいは暴露を受けた個人の感受性によっては，サイトカインが過剰に放出され，シ
ョックや多臓器不全を発症して死に至る場合もあり，注射剤等の医薬品や医療用具の品質および安全性確保の観点から，発熱性物質の存在を適切かつ確実に評価できる試験法を確立することが重要である．
日本薬局方においては，2 種類の試験，すなわち，「発熱性物質試験法」および「エンドトキシン試験法」（リムルス試験）が収載されている．試料をウサギの静脈内に投与して体温上昇（発熱）を測定する「発熱性物質試験法」は，前述の微生物由来物質のほかに，化学的発熱性物質を含むさまざまなタイプの発熱性物質を検出できるが，実験操作が煩雑であることに加えて，多数の動物を使用することから，その動物実験代替法(以下、代替法と記す)として，カブトガニの血球抽出成分に対する凝固能を指標とした「エンドトキシン試験法」が開発された．
エンドトキシンは極めて活性の高い発熱性物質で，グラム陰性菌の細胞壁成分のリポ多糖であり，環境中に広く存在するとともに，その失活や除去が困難であることから，医薬品等の品質管理を行う上で特に重要である．2006 年に告示された第十五改正日本薬局方（15 局）の製剤総則・注射剤の項では，「エンドトキシン試験法の適用が困難な場合は，発熱性物質試験法を用いることができる」と規定され，発熱性物質の検出において，「エンドトキシン試験法」が優先され，「発熱性物質試験法」はその補助的な試験法として位置づけられた．しかし，「エンドトキシン試験法」はグラム陰性菌由来のエンドトキシンの検出において感度が高いものの，非エンドトキシン型の発熱性に対しては反応せず，また，ある種の物質（β-グルカン等）や試料によっては検出系が干渉され，適用が困難な場合もあり，さらに，「輸液用ゴム栓試験法」（15 局）や「医療機器の生物学的安全性評価のための試験法」（医薬局審査管理課事務連絡，医療機器審査 No.36）においては「発熱性物質試験法」の実施が要求されていることから，未だに多くのウサギが試験に用いられているのが現状である．また，ウサギには発熱性を示さないがヒトでは発熱性を示
す物質については，「発熱性物質試験法」では検出できない．従って，規制の上からも，発熱性物質の検出において，ウサギでの「発熱性物質試験法」に代わり得る，新規の in vitro 発熱性物質試験法の開発は有意義であると考えられる．
今回提案された新規の in vitro 発熱性物質試験法(in vitro PBMC 試験法)は，ウサギでの「発熱性物質試験法」が in vivo での体温上昇（発熱）を測定するのに対して，発熱反応における単球の役割に注目し，ヒト血液（新鮮血／凍結血）由来あるいは株化されたヒト単球様細胞から培養液中に分泌される前炎症性サイトカイン（インターロイキン（IL）-1βあるいは IL-6）を測定するものである．ウサギでの発熱性物
質投与前後のサイトカイン濃度の変動に関するデータは得られておらず，発熱とサイトカインとの関係を直接比較することはできないが，前述のように，発熱性物質による発熱は，主に前炎症性サイトカインの放出を引き金として誘発されることから，これらのサイトカイン濃度と発熱が相関するとの仮定は科学的に妥当と考えられる．さらに，検出系がヒトに由来することから，「エンドトキシン試験法」や「発熱性物質試験法」よりもヒトにおける発熱性を正確に予測できる可能性も示唆される（表１：Biotest 社資料より引用）．
しかし，今回 ECVAM （European Center for the Validation of Alternative methods）から提出された試験成績においては，グラム陰性菌由来のエンドトキシンについてのみ検証されており，エンドトキシ
ンに対する個々の試験法の反応性や適切性に関するデータは示されているものの，非エンドトキシン型の発熱性について十分な検証がなされていない．従って，現時点では，in vitro PBMC 試験法は，ウサギでの「発熱性物質試験法」の完全な代替法とはなりえず，試料の検出系に対する干渉や試料が非エンドトキ
シン型の発熱性物質を含む等の理由から「エンドトキシン試験法」の適用が困難であり，「発熱性物質試
験法」の実施が求められる個々の事例について，「発熱性物質試験法」と同等以上の検出感度を有することを検証した上で，ケースバイケースで適用することが妥当と考えられる．なお，これらの試験法の国内での実施にあたっては，ヒト試料を用いることよるバイオハザードの問題を解決するとともに，日本人の5

血液を用いる場合にはその検証を行う必要があろう．

表

２．プロトコールの妥当性
２－１．試験名
ボランティアから供与を受けた全血を用い，採血後 4 時間以内に実施すべき試験が 2 種，保存血（凍結保存）を用いる試験が 1 種，全血から取り出した PBMC(末梢血単核細胞)を用いる試験が 1 種，細胞株を用いる試験が 1 種記載されている．これらを総称して in vitro PBMC 試験法とした。
１）全血（Whole Blood： WB）/IL-1
２）保存血 CryoWB/IL-1
３）WB/IL-6
４）PBMC/IL-6５）単球細胞株 MM6/IL-6
２－２．試験法
試験法として提案されている 5 種の試験のプロトコールはいずれも明確である．試験法の共通事項を以下にまとめた．試験法の詳細は添付資料２にまとめた。
１）予備試験
エンドトキシンのスパイク回収率（50～200％）のために必要な最低希釈濃度を決めるための相互試験
２）使用限界相互試験の結果による非経口医薬品，生物学製品，医療機器
３）測定キット
ELISA(1 測定／n=4)４）陽性対照(PC)0.5EU/ｍL WHO-LPS94/580(E.coli0113:h10:K-)
陽性製品対照 (PPC)
0.5EU/ｍL の陽性対照を添加した製品
５）陰性対照(NC)
生理食塩水
陰性製品対照 (NPC)
生理食塩水を添加した製品
６）測定値
吸光度の平均値±標準偏差７）陽性基準セクション 4.2 で定められた発熱性閾値を基準にした発熱性様反応を示す>0.5EU/mＬ
これらの相違点のみを表 2-１にまとめた．
6

表 2-１．試験法の相違点一覧
試験法
WB/IL-1
起源
ヒト全血
培養液
生理食塩水

CryoWB/IL-1
ヒト全血
RPMI1640

測定指標
前処理

IL-1β
IL-1β
採血後，4 時間 Konstanz ま た
以内に使用
は PEI 法で凍結
保存
被験物質の 20μL サンプル Konstanz 法 20
適用法＋200μL 生理 μL サンプル＋
食 塩 水 ＋ 血 液 PRMI 200 μ L
20μL＋血液 20μL，PEI 法 20μL
サンプル＋RPMI
180μL ＋血液40μL
適用時間
10-24 時間
10-24 時間
ELISA 測定 10000g，2 分処 混合物
物
理後の上清
n
１
５(プール品)適合基準
陽性対照 OD1.6 陽性対照 OD1.6(陽性対照) 倍＞陰性対照， 倍＞陰性対照，
陽性製品対照 陽性製品対照
OD1.6 倍＞陰性 OD1.6 倍＞陰性
製品対照，陽性 製品対照，陽性
製品対照の OD 製品対照の OD
は陽性対照 OD は陽性対照 ODの 50-200％内の 50-200％内適合基準
OD≤0.1OD≤0.1(陰性対照)
その他

特許あり(US6696261 B2)

特許あり（USPTO
436518000）

WB/IL-6
ヒト全血
生理食塩水

50μL サンプル＋生理食塩水100 μ L ＋ 血 液
50μL

MM6/IL-6
MM 細胞バンク
10 または 2％非動化した牛胎児
血 清 を 含 む
RPMI1640
IL-6
IL-6
白 血 球 お よ び MM6 細胞
PBMC を遠心分 4×105 細胞/mL
離50μL サンプル 50μL サンプル
＋PRPMI-C100μ ＋PRPMI-C100μ
L＋PBMC 100μ L＋細胞 100μ
L
L100μL

16-24 時間混合物

16-24 時間
上清

16-24 時間上清３
陽性製品対照のOD は陽性対照
OD の 50-200％
内

３－４
陽性製品対照のOD は陽性対照
OD の 50-200％
内，
1EU/mL の ODが＞1000pg/mL IL-61
陽性対照 期待値（0.5EU/ｍL）
OD±20％，
陽性製品対照のOD は陽性対照
OD の 50-200％
内IL-6
採血後，4 時間
以内に使用

PBMC/IL-6
ヒト全血
RPMI1640

OD＜200pg/mL
IL-6

OD<0.15 および OD＜0.2
OD ＜ 500pg/mL
IL-6
特許あり
Prf.Ziegler-He(US6696261 B2)
itbrock（GermanCollection of
Microorganisms
and Cell bank）のライセンス

これに対して，ICCVAM が第三者評価を実施し，表 2-2 に示すような主に採用基準に関して推奨プロトコー
ルを示している．

7

表 2-2．ICCVAM が推奨するプロトコール
試験法
WB/IL-1
CryoWB/IL-1
WB/IL-6
PBMC/IL-6
MM6/IL-6
被験物質
最大有効希釈濃度を越えず，干渉のない原液または段階希釈
ドナー数
最小３（個々またはプールされたもの）
適用外
反応干渉の基準 陽性製品対照 陽 性製 品対照 陽性製品対照 陽性製品対照 陽性製品対照の 平 均 OD が の 平 均 OD が の 平 均 OD が の 平 均 OD が の 平 均 OD が
1.0EU/ｍL EC 0.5EU/ｍL EC 1.0EU/ｍL EC 0.25EU/ ｍ L 1.0EU/ｍL EC
で 50-200％で 50-200％で 50-200％
EC で 50-200％ で 50-200％
培養プレート
陰性対照（１）
エンドトキシンコントロール（５）被験物質（１4）
陽性製品対照（0）陰性製品対照（0）ELISA プレート IL-1βを 7 点およびブランク 2 IL-6 を 7 点およびブランク 2 点
点
アッセイ採用基 陰性対照の平均 OD≤0.15
準
IL-１β標準曲線が二次関数で r IL-6 標準曲線が二次関数で r≧0.95≧0.95
エンドトキシン標準曲線の OE 値がシグモイド濃度反応で上がる
適用外
適用外
高反応ドナー 高反応ドナー 適用外（ ＞ 200pg/mL （＞200 pg/mLIL-6）は除外
IL-6）または低反応ドナー（1EU/mL のエンド
トキシンコン
トロール＜
1000 pg/mL)は除外

発熱性陽性基準

Dioxin’s テストを用いる棄却検定
被験物質中のエンドトキシン濃度＞被験物質中のエンドトキシン測定限界濃度以下に評価委員会の意見を記す．
１）日本での実施においては，採血を伴う試験の普及度が気掛かりである．試験者が試験によって未知のウイルスによる感染する恐れも否定できず，可能であれば細胞株の利用を推し進めるべきであろう．
２）指標としては，インターロイキン 1β(IL-1β)が 2 種，または IL-6 が 3 種の試験で提案されており，
ELISA 法で簡便に測定できる．
３）ECVAM では，PBMC の場合，ドナーのバラツキを考慮して n=３～４，保存血の n=５と妥当な数を推奨している．これらの結果の多数決で判定される．WB／IL-6 が n=3 であるのに対し，WB／IL-1 の n が１であることには納得できていない．検体数は ICCVAM が推奨するように，最少でも 3 以上とすべきである．
４）陽性対照は，大腸菌の抽出液(WHO-リポポリサッカライド 94/580)という標準品（エンドトキシン）であり，0.25～5.0EU/mL で検量線を求め，その基準と合わせ妥当な選択がなされていると考える．
ECVAM では， 0.5 エンドトキシンユニット（EU）/mL を越えた場合を陽性とし，ICCVAM では試験法
によって，0.25～1.0（EU）/mL としている．ICCVAM が試験法毎に基準を変えている理由が明確でなく，判断できない．
５）エンドトキシンを添加していない対照品も試験されており，コントロールの設定は適切であると考えられる．
６）本研究は将来，n 数を増やし，エンドトキシンを添加せずとも発熱性物質試験で陽性となるような被
験物質を用いた追加バリデーション研究が必要であると考える．現状では，発熱性物質試験の完全な代替法とはいえず，補完試験の位置付けである．

8

３．バリデーション研究に用いた被験物質
表 3-1 および 3-2 に示すように，正確性の確認のために 10 物質，再現性確認のために 3 物質が実験に利用されている．
表 3-1．正確性を求めるためにバリデーション研究に用いられた 10 物質

表 3-2．再現性を確認するためにバリデーション研究に用いられた 3 物質

表 3-1 および 3-2 に示すような合計 13 物質を用いたバリデーション研究は少なすぎる．エンドトキシ
ンを製剤にスパイクしたものを被験物質として使用しており，この方式では被験物質を増やしたから適切であるとは言い切れない．選ばれている製品はコード化されたものが，医薬品しか実施されていない．生物製品，医療機器などの結果がない．
いずれもエンドキシンを製品に添加した被験物質を試験に用いているが，エンドトキシンをスパイクせずとも発熱試験で陽性となるような被験物質における結果が必要である．また，エンドトキシンを産生す
るグラム陰性細菌の評価は可能であるとしても，その他細菌による発熱作用は判別できているとはいえず，その使用は限定的であると考えられる．
４．試験法の正確性・信頼性を評価するデータについての考察
試験法の正確性や信頼性の評価に用いられているデータの質について述べる．検討したものは，次の二つの冊子の中で説明されているデータとそれを評価に利用したときの方法論である．
文献１． ICCVAM Test Method Evaluation Report
文献２． ICCVAM Background Review Document
ICCVAM のこの検討結果は，2008 年 5 月に公刊されたものであり，それより前の ECVAM の検討結果を利用している．従って，データを吟味するという面では，最新でかつ最も網羅的なものと考えられる．
実際，文献 2 の§9 では，ECVAM が 2005 年に「RPT, BET, in vitro pyrogen test について，実験デー
タを持っている場合は提供してほしいと呼びかけたが反応は皆無であった」と述べている．そして，文献検索で入手した報告において，試験プロトコールが異なるがゆえに総合評価に用いなかった 10 報告（§9.1）と，被験物質の関係で総合評価に取り入れなかった 9 報告（§9.2）について，概要を述べている．
以上の状況を考えると，5 つの in vitro PBMC 試験法の性能評価は，文献 2 に採録されている資料に基づいて行うのが妥当と考えられる．
9

文献 1 において ICCVAM は，検討した 5 試験について，「これらの 5 試験は経口でない投与におけるグ
ラム陰性エンドトキシンによる発熱性物質を評価・同定するための代替法として，完全ではないが利用可
能である（… can be considered for use to detect … in human parenteral drugs on a case-by-casebasis ）．しかし，他の物質や器具の評価法としては不十分である．」と総合評価している（p.xvi）．
この妥当性評価は，OECD の指針書（GD34）にしたがっている．そこでの要確認項目（Modular approach）
は次の通りである．
（i）当該試験法の定義（目的，必要性，科学的根拠を含む） Test definition (including purpose, needand scientific basis)（ii）施設内の同等性・再現性 intra-laboratory repeatability and reproducibility（iii）施設間の易移転性 inter-laboratory transferability（iv）施設間の再現性 inter-laboratory reproducibility（v）予測能（正確性） predictive capacity (accuracy)（vi）適用限界 applicability domain（vii）性能標準 performance standards
これらの各確認項目のうち，(i) 試験の標準手順とその正当化，（vi）適用限界，（vii）標準性能，については，被験物質や試験条件の技術的吟味が主要で，データの質について議論する事柄はほとんどない．
データの質について着目すべき吟味点は次の通りである．
(ii) 「施設内同等性・再現性」の根拠とされたデータの完全性（integrity）を確認するには，同じときに反復したとされる実験が，「・試料名をコード化してかつ適切に混ぜて行われたものかどうか，・どの程度の時間範囲で行われたか，・恣意的なデータ選択が行われなかったか，・判定基準が後知恵的でなかったか，」などを吟味することが必要である．
（iii）「施設間の易移転性」を確認する方法論は国際的にも，国内的にも確立していないので，これについてはどのような検討がされているかを吟味することが必要である．
（iv）「施設間の再現性」については，多くの被験物質を多くの施設で分業的に確認する，という方針を過去の日本でのバリデーション研究で取ってきたが，欧米ではそうではなく，少数の被験物質を多くの施設で共通に確かめることと，多くの被験物質を少数の施設で確かめること，の両方で良いとしてきた．この ECVAM と ICCVAM の方針に基づいて得られた結果が適切かどうかを，日本の方針に基づいて吟味することが必要がある．
（v）「予測能（正確性）」については，黄金標準となる真の評価が本当に「真」なものかどうかに疑問が生じることが稀でない．実際，人での結果と動物（in vivo）での結果の類似性（similarity）は，完全に一致するものではないことが知られている．これについて吟味することが必要である．
(v) で取り上げられている正確度について，文献 1 は 表 4-1 のデータを示している．ただしこの表では，英語で “accuracy” と書かれているものを一致度（concordance）と置き換えてある．概念としての正確性と，数値としての比率を区別するためである．
表 4-1
Test method
Cryo WB/IL-1
MM6/IL-6
PBMC/IL-6
PBMC/IL-6(Cryo)＊
WB/IL-6
WB/IL-1(Tube)
WB/IL-1(96-well plate)＊

Concordance
92% (110/120)
93% (138/148)
93% (140/150)
87% (130/150)
92% (136/138)
81% (119/147)
93% (129/139)

Sensitivity
97% (75/77)
96% (85/89)
92% (83/90)
93% (84/90)
89% (79/89)
73% (64/88)
99% (83/84)

Specificity
81% (35/43)
90% (53/59)
95% (57/60)
77% (46/60)
97% (57/59)
93% (55/59)
84% (46/55)

文献の 1, 2 ではこの種の表に，偽陰性率，偽陽性率等の数値も併記されているが，これらは感度と特異度から引き算で簡単に計算されるものであるから，データの質の吟味には不要である．
評価される試験法が 5 試験なのに，この表に 7 試験があるのは，＊をつけた試験がそのすぐ上の試験の変法として ECVAM のバリデーション研究で実験が行われ，データが評価に利用されたためである．
10

表 4-1 に示されている値は，ウサギを用いる発熱性物質試験を黄金標準として示されている．文献 2
ではこれについて，ウサギでの結果とヒトでの結果はおおむね類似している，と述べている．ただし，用量反応関係はウサギよりヒトの方が急峻になる傾向があることを注意している．これがどのように発熱性評価に影響するかは明らかでないが，代替法としての評価には影響しない程度と考えられる．
表 4-1 で示されている予測性能は，PBMC/IL-6 については原法，WB/IL-6 については変法の標準作業手
順（SOP）を用いることで，どの試験法も非常に良いことを示している．
問題としては，この変法がどのような経緯でバリデーション研究に加えられたかである．評価においては，SOP を固定して比較するのが原則だからである．
表 4-1 のデータについては，評価に採用されている実験データの量が，試験毎に異なっていることを注意しなければならない．
この理由について，
文献 1 は，§5.3 “Description of the Statistical Approaches
Used to Evaluate the Resulting Data” において次のように述べている．
「再現性解析においては，1 実験（run）が 4 反復（quadruplicate）の測定値からなっているので，その変動係数（coefficient of variation: CV）で反復におけるデータの精度を評価し，試験毎に多少異なる，CV<0.25 から CV<0.45 という基準でデータの採否を決めた．」
これは，バリデーション研究の実験データから，精度の悪さを反映しているデータを除去して計算したことを意味している．この基準をいつの時点（事前か事後か）どのような正当化に基づいて導入されたかは，明らかでない．
データの最大量が，10 物質ｘ5 施設ｘ3 実験，の 150 実験（runs）であるから，データ量が少ない試験は，それだけ試験法としての再現性が悪かったと評価すべきである．たとえば，Cryo WB/IL-1 や WB/IL-1(96-well plate) における高い感度は，陽性物質 90 実験の 14%, 7% を除外して評価した値であることに注意すべきである．極端な場合として，除外されたものがすべて「適正に評価されなかったもの」とすれ
ば，Cryo WB/IL- 1 の感度は，83%になる．
感度と特異度は，取引関係（trade-off relationship）を持っている．すなわち，判定限界値（cut-off
value）を変えれば，一方が大きくなることで他方が小さくなる，という関係を持っている．このデータに基づいて判定限界値を調整することで，感度や特異度は変更できる．しかしそうすると，バリデーショ
ン研究の正統的遂行がおかされる．このデータに基づいて，SOP の変更を行ったとすると，その妥当性を新たに検証しなければならない．
文献 2 の§5.5 によると，被験物質は基本的にはコードをつけて実験に供されたが，3 物質だけは例外であった．すなわち，「the identity of the three test substances was not blinded）とされている．
その理由が明らかでない．
ICCVAM は易移転性についての検討を全くしていない．これは，わずか 3 施設しかバリデーション研究に参加させていなかったためと考えられる．GD34 では，要確認項目としているのに，ECVAM も ICCVAM も，試験法の易移転性は重視していないようである．
実際に易移転性を評価するには，日本のバリデーション研究で行っているように，比較的広い範囲で一般を代表できる施設で実験を行うべきであるが，ECVAM にも ICCVAM にも，そのような視点が存在していないようである．これが問題であることは，日本から提起・発信していくべきではないだろうか．
実験の反復安定性（repeatability）と施設内再現性（intra-laboratory reproducibility）は，概念としては違うことが GD34 の用語集（Glossary）で示されている．しかし ECVAM のバリデーション研究では，この区別がされておらず，文献 2 では，§7.2.2 で両方を 3 実験（runs）での結果の対ごとの一致率で評価している．この評価法自体は悪くないが，その際に都合の悪いデータの除去が行われたようである．
これについては元データを吟味する必要があろう．
Lab 2 には問題があるようで，データが除外されているだけでなく，“not included” の欄が一つあって，”NI: Not included due to lack of sufficient data. The sensitivity criteria were not met
for 1 of 3 substances in run 2, and 1 of 3 substances in run 3” と注記されている．
数値としては悪くないが，Lab 2 での除外が何を意味するか，検討が必要である．
11

表 4-2Lab.

WB/IL-1
Lab1

Lab2

1vs
2

92%(1
1/12)

100%(
8/8)

1vs
3

83%(1
0/12)

88%(7
/8)

2vs
3

92%(1
1/12)

NI

All

83%

WB/IL-6
Lab3
100%(
12/12
)

Lab1

Lab2

75%(9
/12)

92%(1
1/12)

92%(1
1/12)

100%(
12/12
)

92%(1
1/12)

92%(1
1/12)

75%(9
/12)

92%(1
1/12)

92%

75%

92%

PBMC/IL-6
Lab3
100%(
12/12
)
100%(
12/12
)
100%(
12/12
)
100%

Lab1
92%(1
1/12)
100%(
12/12
)
92%(1
1/12)
92%

Lab2
100%(
12/12
)
100%(
12/12
)
100%(
12/12
)
100%

MM6/IL-6
Lab3
100%(
12/12
)
92%(1
1/12)
92%(1
1/12)
94%

Lab1
100%(
12/12
)
100%(
12/12
)
100%(
12/12
)
100%

Lab2
92%(1
1/12)
92%(1
1/12)
100%(
12/12
)
92%

Lab3
100%(
12/12
)
92%(1
1/12)
92%(1
1/12)
92%

施設間再現性（inter-laboratory reproducibility）も施設内再現性と同じように，一致率で検討がされている．結果は表 4-3 にまとめられている．
表 4-3
Lab.
1vs2
1vs3
2vs3
All3

WB/IL-1
92%(77/84)
77%(83/108)
68%(57/84)
58%(167/288)

Cryo WB/IL-1
92%(11/12)
92%(11/12)
92%(11/12)
92%(11/12)

Test Method
WB/IL-6
72%(78/108)
75%(81/108)
97%(105/108)
72%(234/324)

PBMC/IL-6
81%(87/108)
86%(93/108)
89%(96/108)
78%(252/324)

MM6/IL-6
97%(105/108)
89%(96/108)
86%(93/108)
86%(279/324)

特定の施設が他とかけ離れている，という現象は起こっていない．WB/IL-1β の施設間再現性が悪いようである．
データの吟味は，13 人の委員からなる，in vitro Pyrogenicity Independent Peer review Panel (Panel)で行われた．委員の中には統計に詳しいと思われる委員が一人，日本人が一人含まれている．
文献 1 の Executive Summary (p. xv) の中段に「The Panel … to review the ICCVAM draft BRD for errors
and omissions and to discuss the current validation status to the five in vitro test methods.」という記載がある．この中の，「errors and omissions」がデータのことを意味するのであれば，データの取捨選択が問題になったことになる．
文献 2 の§5.3 では，測定値の除外の方法についての約 1 ページにわたる記載があるが，最終的に定められた基準が，データを見てからの後付原則であるか，事前討議の結果であるか明らかでない．文脈をみると，前者のように感じられる．
以上，データの細部には，性能を高めに評価する偏りの存在が見受けられるが，その影響はそれほど大きいものではない．ICCVAM の総合評価の結果は，おおむね受け入れて差し支えないと考えられる．
試験法の正確性を評価する物質のin vivoおよび参照データ
・In vivo データについて
WB/IL-1,CRYO WB/IL-1,WB/IL-6,PBMC/IL-6,MM6/IL-6の5種類のin vitro PBMC試験法に関するECVAM BRD
March,2006 Section 3.3(ICCVAM BRD Appendix A1 ～ A5 May 2008 に 収 載 ) お よ び ICCVAM BRD May,
2008(Section 3.3)には，評価に使用した13種類の物質の一覧が記載されているが，ECVAM BRD March,2006
Section 4.1およびICCVAM BRD May 2008(Section 4.2)によれば，これらの物質でのバリデーションには，
倫理的理由からウサギを使用した発熱性物質試験は実施されていない．
これは，エンドトキシンをスパイクした13種類の物質についてのウサギ発熱性物質試験での発熱に関する干渉作用の有無は明らかにされなかったことを意味する．これと同様に，13種類の物質でのエンドトキシン以外の発熱物質についての反応に対する干渉作用の有無が明らかではない．
その代わりに他の研究成果によるデータが参考資料として使用されている．これは，ドイツの
12maximum rise in temperature

Paul-Ehrlich Instituteによって得られたデータで，エンドトキシンを用いた171羽のウサギ(Chinchilla
Bastards種)による発熱閾値の調査で，被験ウサギの50%が0.55℃の体温上昇を示す体重1kgあたりのエン
ドトキシン量は5EUであったとしている．

図4-1
ECVAM BRD March,2006 Section 4.1 Figure 4.1.1 "Dose-Temperature of standard endotoxin applied
to Chinchilla Bustards (n=171)"より
これは，ヒト男性ボランティアにエンドトキシンを投与した研究と同等の結果であり，エンドトキシン
に対するウサギとヒトの発熱反応が類似であるとしている．(ECVAM BRD March,2006 Section 4.5)
また，5種類のECVAM BRD March,2006 Section 9およびECVAM Response to ICCVAM Questions (ICCVAM BRD
Appendix Bに収載)にて， in vitro PBMC試験法，ウサギ発熱性物質試験，エンドトキシン試験についての過去の文献による比較が報告された．
ここでは，実際の臨床使用において発熱の副作用報告のあったヒト血清アルブミン製剤，第Ⅷ因子製剤，
および血漿分画製剤にエンドトキシンをスパイクした試料を用いて，ウサギ発熱性物質試験，エンドトキシン試験と，in vitro PBMC試験法の報告があり，非エンドトキシン性発熱物質やウサギで発熱を起こさないがヒトで発熱を起こす物質について，in vitro PBMC試験法により検出可能であったとしている．さらに，エンドトキシンと共に各種グルカンやリポテイコ酸(LTA)等での報告や，真菌胞子，各種医療材料，
脂質等での報告があり，ここでもin vitro PBMC試験法で検出が可能であったとしている．内容を以下にまとめた．
・発熱の副作用報告のあったヒト血清アルブミン製剤について，ウサギ発熱性物質試験およびエンドトキシン試験では全て陰性であったが，in vitro PBMC試験法のうち、WBを用いる試験法では，ドナー数5の平均値が対照ロットの平均値と5.52倍の差が見られた事が示された．
表4-4
Donor

Incriminated batch
Control batch
Quotient
IL-1 (pg/ml)
IL-1 (pg/ml)
incriminated/control
1
79.0
4.0
19.75
2
14.1
3.9
3.61
3
44.3
15.0
2.95
4
20.9
14.9
1.4
5
71.9
3.9
18.44
Mean
46.04
8.34
5.52
ECVAM Response to ICCVAM Questions 1.a "Fever reactions caused by a batch of Human Serum Albumin
Table: Incriminated Human Serum Albumin"(ICCVAM BRD Appendix B)より
13

・第Ⅷ因子製剤について，6製造業者の製剤各3ロットに，エンドトキシンをスパイクした時のウサギ発熱性物質試験およびWBを用いる試験法による比較では，ウサギの発熱閾値に相当する5 IU/kg体重のエン
ドトキシンを投与されたウサギでは明らかな陽性の結果が得られなかったが，WBを用いる試験法（WB/IL-1）では全て陽性の結果が示された．
表4-5
Endotoxin(WHO Standard)
RabbitIVPTIU LPS/kg（1mL/kg）

IU LPS/mL

Rabbit Pyrogen Test
3 animals per test
repetition
positive
possible> 2.65 ℃> 1,15 <2.65 ℃

WB
n = 4 donors eachnegative

positive

negative< 1.15 ℃

0
0
0
0
18
0(1)*
72(71)*
5
5
0
17
1
72
0
15
15
11
7
0
72
0
ECVAM Response to ICCVAM Questions 1.b "Coagulation Factor VIII Concentrates Table: Comparison
study Factor VIII (18 batches, 162 rabbits)"(ICCVAM BRD Appendix B)より
* testing the same sample, the blood of 3 donors remained negative, the blood of 1 donor reacted
slightly positive in the first experiment, the repetition was negative(* 初回の試験で1ドナーの血液のみ弱い陽性反応を示したため，同じ試料で再試験を実施した結果は陰性，他の3ドナーの血液も陰性．)
・発熱を起こした血漿分画製剤について，ウサギ発熱性物質試験で陽性，エンドトキシン試験で陰性であったと報告された．(図表なし：ECVAM Response to ICCVAM Questions 1.c "Pyrogenic batch of a plasma
derivative"(ICCVAM BRD Appendix B)より
・ヒト血清アルブミンについて，5製造業者の各種濃度製剤各3バッチ(合計29バッチ)に，エンドトキシンをスパイクした時のウサギ発熱性物質試験およびWBを用いる試験法による比較では，ウサギの発熱閾値に相当する5 IU/kg体重のエンドトキシンを投与されたウサギでは，29バッチの製剤中5バッチのみ陽性であったが，その他のバッチでは明らかな陽性の結果が得られなかった．一方，WBを用いる試験法では全て陽性の結果が示された．
表4-6
Endotoxin(WHO Standard)

Rabbit Pyrogen Test
WB
3 animals per test
repetition
Rabbit
IVPT
positive
negative
positive
negative
possible
IU LPS/kg IU LPS/mL> 1,15 <（1mL/kg）> 2.65 ℃
2.65 ℃< 1.15 ℃
0
0
0
0
29
0
29
5
5
5
23
1
29
0
15
15
21
8
0
29
0
ECVAM Response to ICCVAM Questions 1.e "Table: Comparison study Human Serum Albumin (29 batches,
261 rabbits)"(ICCVAM BRD Appendix B)より

・発熱の副作用報告のあったゼラチン含有輸液製剤について，副作用事例の生じていない対照バッチと共に，エンドトキシン試験，ウサギ発熱性物質試験およびWBを用いる試験法で比較を行った結果，WBでは
ヒトで発熱を起こした2つのバッチと陰性対照を識別できたのに対して，エンドトキシン試験では全て陰性，ウサギ発熱性試験ではヒトで発熱を起こした2バッチの内1バッチが陰性で，ヒトに発熱性を有す
るがウサギには発熱を起こさない事例が示された．

14

表 4-7batch

LAL test

rabbittest

fever inpatients

WB

IL-1
IL-6
TNF
(pg/mL)(pg/mL)(pg/mL)A
negative
negative
no
8.5
28.0
28.2
B
negative
positive
yes
142.6
654.4
67.6
C
negative
negative
yes
421.5
9444.0116.7
cut off: 32.6
127.6
43.6
ECVAM Response to ICCVAM Questions 1.f (ICCVAM BRD Appendix B) "Table 5: Incriminated infusion
solution containing gelatine"より
・11の物質について，それぞれの試験法で反応を示した最小濃度（ng/mL）が報告された．3種類のグルカ
ンは，エンドトキシン試験で偽陽性の結果となり，エンドトキシン試験で偽陽性のグルカンとカードランに反応せず，エンドトキシン試験で偽陰性のLTAに反応したと報告された．
表4-8
WBT/IL-1
WB/IL-6
PBMC/IL-6 MM6/IL-6 THP-1/Neo
Curdrlan
1000
1000
100
1000
1000
Glucan-Barley negative negative
negative
negative
negative
Glucan-Yeast
not done negative
negative
negative
negative
Zymosannegative
10000
10000
negative
10000
PHA-L
100
10000
100
100
1000
PHA-E
negative negative
negative
negative
negative
Lipid A
10000
1000
10000
1000
negative
Glucan STD
negative negative
negative
negative undiluted
Endotoxin-C
4
40
0.4
4
4
Endotoxin-G
0.4
40
4
4
4
LTA
1000
1000
100
1000
1000
ECVAM Response to ICCVAM Questions 2.(ICCVAM BRD Appendix B) "Table: Smallest concentration[ng/mL] or dilution of substances active in the respective method"より
・12人のドナー血液によるエンドトキシン(0.5EU/mL)とB.subtilis 由来LTAの反応性について，いずれも明らかな陽性反応を示したと報告された．

図4-2
ECVAM Response to ICCVAM Questions 2. "Fig. Reactivity of 12 donors towards LPS E. coli O113:
H10 (0.5 EU/mL) and LTA from B. subtilis"(ICCVAM BRD Appendix B)より
15

・真菌胞子によるヒト全血のIL-1β放出は，リポ多糖（LPS）阻害物質であるポリミキシンＢによって抑制されないことからエンドトキシンによる反応では無いことが示され(図4-3および4-4)，一方，E.coli
由来のLPSはポリミキシンＢによってIL-1β放出が抑制される．したがって，真菌由来の非エンドトキ
シン・発熱性に対しても活性を有することが示された．

IL-1β (OD450nm)(A)(図 4-3)IL-1β (OD450nm)(B)(図 4-4)

図4-3および4-4
ECVAM Response to ICCVAM Questions 2. "Figure 8: IL-1 release in human whole blood in response
to fungal spores (A) is not inhibited by 5 µg/ml polymyxin B, mean of 4 donors (±SEM), numbers
above the bars indicate the spore counts employed. In contrast (B), the response to LPS is
inhibited over a wide concentration range, mean of double values."(ICCVAM BRD Appendix B)
より・各種医療材料について，それら疑わしい物質の抽出液をウサギに移植することは，侵襲性により発熱性汚染と関連しない反応を引き起こす可能性があり，倫理面や科学的見地から問題があると述べている．
in vitro PBMC試験法は抽出液が必須ではなく，血液に直接接触させることで，エンドトキシンだけではなく全ての関連した発熱性を検出可能との報告が示された．
ECVAM Response to ICCVAM Questions 3. "Adaptation of the WB/IL-1 to biocompatible
materials"(ICCVAM BRD Appendix B)より
16

・透析装置および透析液について，米国ではダイアライザーを再処理して使用している透析施設は72%あり，また，透析施設で透析液の調製に使用する純水や透析液，フィルターや透析装置内配管のデッドス
ペース，等による細菌の増殖に起因する腎不全患者の透析終了後の発熱が問題となり，Association forthe
Advancement of Medical Instrumentation（AAMI）によって1993年に処理水と透析液の従属栄養細菌
に関する基準が設けられたが，1994年ドイツ，ギリシャ1998年，米国1990年，カナダ1995年の調査結果
では，このAAMIの緩い基準でさえ完全に達成できていないことが示された．多数のグラム陽性菌汚染の報告もあり，透析チューブ内のバイオフィルム片を調査したところ，in vitro PBMC試験法では，エンド
トキシン試験の20倍高い反応を示す調査結果も示された．エンドトキシンだけではなく菌体外毒素やペプチドグリカン等のサイトカイン誘導物質が透析膜を通過する可能性も指摘され，米国での1991年の調査では1年間に全透析施設の20%で複数の臨床的な発熱が報告されている．こうした慢性的に刺激を受けた患者の血液のサイトカインレベルは健常者のそれよりも高いとの報告が示された．(図表なし)
ECVAM Response to ICCVAM Questions 3. "Dialysis"(ICCVAM BRD Appendix B)より
・脂質製剤について，ビタミン剤やステロイド剤などの一部の製剤は非親水性の性状や筋注や皮下注等の投与経路の違いから，ウサギの静脈内に投与することが出来ず，また，エンドトキシン試験も水性試料を対象としているため試験が困難であるが，試験に対して非干渉性の油脂で希釈することにより，invitro PBMC試験法の適用が可能となることが示された．(図表なし)
ECVAM Response to ICCVAM Questions 3. "Lipidic formulations"(ICCVAM BRD Appendix B)より
・発熱性物質の標準品について
in vitro PBMC試験法に関するECVAM BRD March,2006 Section 2.1(ICCVAM BRD Appendix A1～A5 May
2008に収載)およびICCVAM BRD May 2008 Section 2.1によれば，発熱性物質の標準品として，WHO-LPS
エンドトキシン標準品[(94/580，O113:H10:K-)]が使用されており，エンドトキシンの由来となる大腸菌の株はU.S. Pharmacopoeia（USP）エンドトキシン標準品と同じものである．
発熱性物質の標準としてエンドトキシンを使用した理由は，エンドトキシンが安定的に入手可能で高い検出精度を有する標準化された発熱物質であるからとされている．これは，既に広く普及しているカ
ブトガニ血球成分から成るライセート試薬を用いたエンドトキシン試験において，このエンドトキシン
標準品が使用されているからであるが，この選択理由は妥当と考える．ここで，エンドトキシン標準品の表示単位がエンドトキシン試験で得られた値であることに留意する必要があるが，過去に得られたウサギの結果でエンドトキシン投与量と体温上昇の直線性が確認され，ここで得られた発熱閾値を利用して，in vitro PBMC試験法は「ヒトに発熱性」または「ヒトに非発熱性」を判定している(ECVAM BRD
March,2006 Section 2.2.13)ことから，エンドトキシン試験という異なる尺度での表示単位で表された標準品を使用しても問題ないと考える．
・考察

in vitro PBMC試験法で，過去の文献により非エンドトキシン性発熱性の検出が確認されている．よって，この試験法は他の発熱機序によるものは別として，ヒトに特異的な発熱物質の検出法の一つとして有用であると思われる．ただし，原料や製造工程でエンドトキシン以外の発熱物質の汚染が考えられない場
合には，エンドトキシン試験で代替可能と思われることから，現在，ウサギを用いて発熱性物質試験を行っている試験対象について，発熱性物質の検出の全てをin vitro PBMC試験法に代替を集約するのではな
く，何らかの理由からエンドトキシン試験が適用できず，ウサギによる試験でしか発熱性物質の検出が可能な試料にのみ，このin vitro PBMC試験法を適用すべきと考える．
５．他の科学的な報告
In vitro 発熱性物質試験に関してヒト末梢血由来細胞などを用いた IL-1β,IL-6 測定系以外にもウサ
ギ発熱性物質試験の代替として可能性のある試験法が提案されている．例を挙げると，末梢血前血を用いた IL-8 産生能に基づいた試験法は IL-1β，IL-6 より感受性が高いとされ，多くの発熱性の受容体である
Toll 様受容体（Toll-like receptor：TLR）の強制発現細胞株などはその特異性は非常に高いことが知られている．（例：Journal of Immunological Methods 336 (2008)45-55）
また，発熱，もしくはそれに類する毒性のバイオマーカーとして多様な評価系が提案，もしくは開発研究を開始している．そのなかでも，Embryonic Stem(ES)細胞や人工多能性幹細胞（Induced
pluripotent stem cells： iPS）細胞を利用した毒性試験やトキシコパノミクス（トランスクリプト17

ーム，プロテオーム，メタボロームを含めた網羅的な毒性解析）を利用した毒性試験などは in vitroと in vivo 試験それぞれの利点，欠点を補うものとしてその迅速な開発が期待されている．
６．３Rｓへの関与
ヒト血液を用いるあるいは細胞を用いる試験法は，動物を用いた発熱性物質試験に置き換わる方法として動物福祉面で貢献するものである．
７．本試験法の有用性と限界
in vitro PBMC 試験法のような人の白血球や細胞株によるサイトカインの産生量計測に基づく試験はウ
サギや人の発熱の誘導に相関はしているといえよう．しかしながら，ICCVAM の報告書にあるように，（ICCVAM (1.2.3 )) 「ウサギ発熱性物質試験は（多様な原因，メカニズムに基づいて誘導される全身の発熱を検出するものであるが，本代替法は培養細胞の１－２種類のサイトカインを計量しているに過ぎない．）現時点で，IL-1βおよび IL-6 が全身の発熱反応に必須であるという科学的根拠が欠落している．」とあり，「少なくともヒトの発熱反応とヒト由来細胞の培養における IL-1β,IL-6 の産生との相関に関する情報をさらに追加する必要がある．特に異なる内毒素(エンドトキシン)に対する反応の仕方の違い等に留意する必要がある」と述べており，本代替法には科学的根拠が少ない，もしくは十分でないとの認識が強い．
ICCVAM の報告書でも述べられているが，ウサギ発熱性物質試験と in vitro PBMC 試験法の相関性に加え，他の類似点，相違点に関し多くの文献にて精査されていることも事実である．なかでも報告書が注目し言及している点として，それらの文献の多くが，ウサギ発熱性物質試験の結果はヒトの発熱反応とよく相関している事実を再確認したことになっている点や，in vitro PBMC 試験法のウサギ発熱性物質試験との相関を示唆するデータはすべてグラム陰性細菌由来のエンドトキシンのみを引用していることがあげられる．言い換えれば代替法である in vitro PBMC 試験法があまりにかけ離れていてウサギ発熱性物質試
験の「代替法」と定義，解釈することに無理があると思われる．
in vitro PBMC 試験法が開発された時点，ECCVAM の報告書，さらには ICCVAM の報告がなされた後も，自然免疫研究成果が新たに集積している．しかしながら，そのような最新の知識をもってしても，IL-1
β,IL-6 の本代替法はおろか，ウサギ発熱性物質試験においても，どのような「発熱性」があることが知られていて，それらがどのような細胞の受容体，シグナル伝達経路，遺伝子発現制御，転写後制御，細胞間相互作用などを介して，全身の「発熱」という計測可能なパラメーターに影響を及ぼしているか，科学的に見て明らかになったとは到底言い難い．ただし，上記の一部，たとえばエンドトキシンの自然免疫受容体，シグナル経路などは明らかになってきており，本代替法を上回る感受性，特異性をもった検出方法も知られるようになった（上記参照）．
ECCVAM,ICCVAM の報告は発表された時点で，その効力が及ぶ地域の文化的な背景に基づけば，ある程度妥当な意見が述べられている．しかし科学的な観点，および倫理的な観点から鑑みた場合，また，時間的な経過による影響も加味した上で，（必ずしも in vitro 発熱性物質試験の必要性があると断定はせずに）
新たな発熱性テストの開発の可能性と，現行のテストの限界を独自に議論する必要があると考えられる．
８．結論
上記の各考察より，今回提案された in vitro PBMC 試験法は，ウサギの使用数を削減可能とし，また，作用機序に立脚した発熱性物質試験の新たな方向性として評価でき，さらに，バリデーションでのデータの扱いに関しても納得のできる基準を満たしていると考えられる．しかし，ウサギを用いた発熱性物質試
験との互換性については不明な点が多く，この試験法の代替法とするには，問題がある．国内ではウサギ
発熱性物質試験からエンドトキシン試験への移行の問題も残されており，これに優先して独立した代替法とすることには無理がある．しかしながら，エンドトキシン試験が使用できないような特殊な状況やウサ
ギとヒトでの発熱性に関する種差が想定されるような状況においてウサギ発熱性物質試験と併用するな
ど，限定的な使用については推奨できよう．このような in vitro PBMC 試験法が科学的根拠に基づいた研究により，さらに広範囲な状況で使用可能となることが望まれる．

18

添付資料１

エンドトキシン試験と発熱性物質試験に関する実態調査結果報告
日本薬局方に収載されているカブトガニ血球抽出物を用いたエンドトキシン試験法とウサギを用いた発熱性物質試験法，および in vitro 発熱性物質試験について，2009 年 1 月に日本製薬工業協会の協力を得て「エンドトキシン試験と発熱性物質試験に関する現状調査のためのアンケート」と題した実態調査を行い 36 社(1 社につき複数事業所含む)の回答を得たので，この調査結果を基に報告する．
一般にエンドトキシン試験での反応干渉因子には，ライセート試薬とエンドトキシンの一連の酵素活性化反応を阻害するものとして，酵素活性阻害剤，タンパク変性剤，キレート剤などがある．また，エンド
トキシンに作用して反応を阻害するものとして，界面活性剤や，鉄，アルミニウムなどの金属イオンがある．一方，ライセート試薬とエンドトキシンの一連の酵素活性化反応を促進させるものとして，セリンプ
ロテアーゼなどが挙げられる．その他に試料溶液の濁りや着色は光学的測定法に影響を及ぼすと言われている．また，リポソーム製剤なども反応を阻害する要因となる．実態調査の結果では，これらのエンドトキシン試験での反応干渉因子であっても，未確認の事例を除いて発熱性物質試験は実施可能であった．
日局発熱性物質試験の反応干渉因子として考えられるものは，製剤そのものが発熱または体温下降を起こす場合が考えられる．実態調査においてこのような反応干渉の報告があった 3 例は，未確認の事例を除いてエンドトキシン試験は実施可能であった．
In vitro 発熱性物質試験について，36 社中に試験の実施経験を有する回答は無く，実例による反応干渉因子の報告は得られなかった．
実態調査で今回の評価に際して自由記入の形式で意見を募ったところ，多くの意見が寄せられた．まず，発熱性物質試験については，感度その他の問題や動物愛護の観点から，エンドトキシン試験を含めた何らかの代替法に切り替えたいという意見が多かった．
ただし，今回のヒト血液を用いる in vitro 発熱性物質試験（in vitro PBMC 試験法）では，ヒト血液の入手方法やバイオハザードの問題，反応阻害や促進等の反応干渉因子試験の必要性を指摘する意見があった．
日本薬局方では発熱性物質試験法よりもエンドトキシン試験法を優先する規定があり，発熱性物質試験についてはエンドトキシン試験の適用が困難な品目に限り設定することとなっている．実際に新規製造品
目の試験規格設定時ではこの規定が適用されており，実態調査においても最終製品での実施例について発熱性物質試験よりもエンドトキシン試験の実施頻度が高い結果となっている．しかし，日本薬局方でエン
ドトキシン試験が優先する規定が出来る以前は，容量 10mL を超える注射剤には発熱性物質試験を適用する規定があったことから，この時期に製造(販売)承認を得た注射剤では発熱性物質試験が適用されているものが現在でも多く存在すると考えられる．これらの発熱性物質試験が適用された既承認医薬品に対しても，原料や直接容器等にエンドトキシン以外の発熱性が存在する可能性がある品目やエンドトキシン試験が困難な品目を除き，現行の日本薬局方に則ってエンドトキシン試験への変更を促進することは理にかな
うものと考えられる．しかし，これら発熱性物質試験が既に設定された既承認医薬品では，発熱性物質試
験をエンドトキシン試験に変更するためには，変更申請時にウサギに大量のエンドトキシンを投与して得られる発熱データの提出を求められ，ウサギの個体差による感度のバラつきや動物愛護の観点等の問題で，エンドトキシン試験へ変更する際の障害となる場合もある．
エンドトキシン以外の発熱性が存在する可能性がある品目やエンドトキシン試験が困難な品目について，現状ではウサギを用いた発熱性物質試験しか方法が無いため代替法の確立を望む意見が多く見受けられた．
現在行われている発熱性物質試験のすべてを今回のヒト血液を用いた代替法に切り替えるのではなく，
エンドトキシン試験で代替可能なものはエンドトキシン試験に変更することが妥当と考えられ，実態調査における最終製品でのウサギ使用数内訳から見ても，エンドトキシン試験への変更だけでも相当数の発熱性物質試験に使用するウサギを減少させることが可能と考える．

1

エンドトキシン試験と発熱性物質試験に関する実態調査結果
2009 年 1 月調査実施＊回答企業 36 社の形態：
国内企業(本調査に該当する海外向け製品あり)
国内企業(本調査に該当する海外向け製品なし)
海外企業(米国系)
海外企業(欧州系)

：10 社
：17 社
： 4社
： 5社

＊日局エンドトキシン試験および日局発熱性物質試験の実施経験について
36 社中(複数事業所を有する企業を含む)
エンドトキシン試験の実施経験あり ：32 社
エンドトキシン試験の実施経験なし ： 4 社
発熱性物質試験の実施経験あり：24 社
発熱性物質試験の実施経験なし：12 社
内訳：両試験共に経験あり:24 社
エンドトキシン試験のみ経験あり : 8 社
発熱性物質試験のみ経験あり: 0社
両試験共に経験なし: 4社＊日局エンドトキシン試験検体数内訳
36 社集計
ｹﾞﾙ化法
原料
2142
最終製品
13067
2006 年
製造工程管理
28872
合計
44081
原料
3024
最終製品
14465
2007 年
製造工程管理
34105
合計
51594
原料
3285
最終製品
14461
2008 年
製造工程管理
29408
合計
47154
＊日局発熱性物質試験検体数内訳
36 社集計
検体数
ゴム栓
561
原料
615
2006 年
最終製品
2519
合計
3695
ゴム栓
537
原料
495
2007 年
最終製品
2618
合計
3650
ゴム栓
608
原料
580
2008 年
最終製品
2483
合計
3671

比濁法
4952
14864
40966
60782
5912
14536
54875
75323
5821
13745
44310
63876

比色法
1357
1459
8486
11302
1244
1635
8428
11307
1189
1936
9351
12476

ウサギ使用数(羽)
1435
1570
5715
8720
1356
1406
6107
8869
1527
1442
7293
10262

2

合計
8451
29390
78324
116165
10180
30636
97408
138224
10295
30142
83069
123506

2006年 エンドトキシン試験検体数 32社集計
140000

合計:116165検体

12000011302

100000

合計:78324検体

80000

8486

60782

比濁法

60000
40000
20000
検
体
数

比色法

合計:8451検体
1357
4952
2142

0
原料

40966

合計:29390検体
1459
14864
13067

28872

最終製品

工程管理

ｹﾞﾙ化法
44081

合計

2006

2007年 エンドトキシン試験検体数 32社集計
合計:138224検体
14000011307

120000

合計:97408検体

100000

8428

80000

比色法
54875

60000

検
体
数

比濁法

合計:30636検体

40000
20000

75323

合計:10180検体
1244
5912
3024

0
原料

ｹﾞﾙ化法

1635
14536

34105

51594

14465

最終製品

工程管理

合計

2007

2008年 エンドトキシン試験検体数 32社集計
140000

合計:123506検体

120000
合計:83069検体

100000
80000

9351

60000

20000
検
体
数

合計:10295検体
1189
5821
3285

0
原料

63876

ｹﾞﾙ化法

1936
13745

29408

14461

最終製品

工程管理
2008

3

比色法

比濁法

44310

合計:30142検体

40000

12476

47154

合計

2006年 発熱性物質試験検体数 24社集計
12000
10000

合計:8720羽

8000

最終製品

6000

5715

原料
ゴム栓

合計:3695検体
4000
2000

0

2519

1570

615
561

1435

検体数

ウサギ使用数(羽)2007年 発熱性物質試験検体数 24社集計
12000

合計:8869羽

10000

6107

8000

最終製品

原料

6000

ゴム栓

合計:3650検体
4000
2618
2000

0

1406
495
537

1356

検体数

ウサギ使用数(羽)2008年 発熱性物質試験検体数 24社集計
12000

合計:10262羽

10000
8000
6000

最終製品

7293
合計:3671検体

ゴム栓

4000
2000

0

原料

2483

1442

580
608

1527

検体数

ウサギ使用数(羽)42006年

エンドトキシン試験
3法合計検体数

140000116165120000

ウサギ使用数(羽)100000
78324
80000

60000
40000
検
体
数

29390
8451

20000
0

1570

1435
ゴム栓

原料

8720

5715
最終製品

工程管理

2007年

合計

エンドトキシン試験
3法合計検体数

13822414000012000097408

ウサギ使用数(羽)100000
80000

60000
40000
検
体
数

30636
10180

20000
0

1406

1356
ゴム栓

原料

8869

6107
最終製品

工程管理

合計

2008年
140000

エンドトキシン試験
3法合計検体数123506120000100000

ウサギ使用数(羽)

83069

80000

60000
40000
検
体
数

30142
10295

20000
0

1527
ゴム栓

1442
原料

10262

7293
最終製品

5

工程管理

合計

＊日局エンドトキシン試験の実施が不可能であった事例について
経験あり： 8 社
経験なし：24 社＊日局エンドトキシン試験の実施が不可能であった事例の内訳
事例

要因

薬効分類等

事例を確認
した試験法

日局発熱性物質試験法の適用可否

反応阻害アルミニウムゲルのため

(非公開)

比色法

不明

反応阻害

リポソームのため

(非公開)

ゲル化法

試験可能

反応阻害

原薬の酵素阻害作用のため蛋白分解酵素 ゲル化法
阻害剤
比濁法

試験可能

反応阻害

エマルジョン製剤であり，水層と 中枢神経系用
ゲル化法
油層の分離が困難
薬

試験可能

反応阻害

原薬の酵素阻害作用のため反応阻害

リポソームのため

反応促進

タンパクのため血漿分画製剤

ゲル化法

試験可能

反応阻害

リポソームのため抗生物質製剤

不明

試験可能

ゲル化法
蛋白分解酵素
比濁法
阻害剤
比色法
ゲル化法
(非公開)
比濁法
比色法

試験可能

試験可能

＊日局発熱性物質試験の実施が不可能であった事例について
経験あり： 3 社
経験なし：21 社＊日局発熱性物質試験の実施が不可能であった事例の内訳
事例

薬効分類等

日局エンドトキシン試験法の適用可否

発熱作用あり制癌剤

試験可能

ウイルスの性状に起因した発熱作用

生物学的製剤

試験可能

抗発熱作用(阻害)

アミノ酸

不明

＊in vitro 発熱性物質試験(エンドトキシン試験以外)の実施経験について経験あり： 0 社
経験なし：36 社＊エンドトキシン試験，発熱性物質試験，in vitro 発熱性物質試験についての意見
意見あり：17 社
意見なし：19 社＊エンドトキシン試験，発熱性物質試験，in vitro 発熱性物質試験についての意見
6

・ウサギを使った眼刺激性試験と同様，可能であれば in vitro 試験に変更すべき．
・局方で発熱性物質試験を規定されていない限りは，(新たな開発品に対して)発熱性物質試験規格は設
定せず，エンドトキシン試験規格を設定する方針．
・実施経験がないが，今後必要となった場合には in vitro 試験での評価系が確立されていれば有用と考える．
・in vitro 発熱性物質試験について検討を考えたい．具体的な手順等を教えて頂ければありがたい．
・輸液用ゴム栓以外の発熱性評価は，エンドトキシン試験で十分評価できると考える．
また，動物愛護の観点から動物の使用を削減し代替法の導入が推奨されている．従って，輸液用ゴム
栓以外の発熱性物質試験は必要ないと考える．
・発熱性物質試験はゴム栓で外部へ試験委託を行ってきたが，ここ 3 年間は行っていない．
・日局一般試験法，輸液用ゴム栓試験法および医薬品各条の一部に規定されている発熱性物質試験について，エンドトキシン試験への置き換えを要望する．
・今回の趣旨とは異なるが，日・ＥＵ相互承認協定（ＭＲＡ）締結内容に早期の無菌医薬品および生物学的製剤の追加を望む．
・動物愛護と試験の効率化の観点から，発熱性物質試験からエンドトキシン試験への変更や，新規品目については開発当初からエンドトキシン試験を設定する事例が多いと考える．エンドトキシンは最も強力な発熱性物質に相違ないが，エンドトキシン試験では検出できない発熱性物質の存在を否定するために，エンドトキシン試験導入前に，発熱性物質試験においても問題ない結果が得られることを確認しておくことは重要と考える．
・発熱性物質試験の早期国際調和(EP,USP,JP)を希望する．
・エンドトキシン試験は，エンドトキシン以外の発熱性物質を検出できない課題がある一方，発熱性物
質試験は感度が低く，動物愛護の面での問題も多い．よって，in vitro 発熱性物質試験 が，将来にこれらの問題を解決する有力な手段であるならば，業界として積極的に導入することを期待する．
・発熱性物質試験について，当施設では，日局準拠ではないが，ほぼ同等の手順による「医療機器の生
物学的安全性試験の基本的考え方に関する参考資料について(事務連絡 医療機器審査 No.36)」に基
づき，医療機器の中間・最終製品もしくは原料・部材の安全性を評価する目的でウサギを用いた発熱性物質試験を実施している．医療機器の安全性評価の一環として実施する発熱性物質試験は，医療機器または原材料から抽出した抽出液中に，エンドトキシンおよび非エンドトキシン性発熱性物質が存在しないことを確認することを目的としており，特に，ウサギを用いた試験は化学物質等による「非エンドトキシン性発熱性物質」を検出する方法として，医療機器の承認・認証申請において審査当局
から試験実施が規定されている．現在，公知の代替方法がないために，当施設では医療機器の安全性評価においてウサギを使用しており，動物愛後の面からも in vitro 発熱性物質試験のような動物を使用しない代替方法が受け入れられ，選択肢の１つとして拡充することに期待する．
・エンドトキシン試験は，比濁時間分析法で定量的にエンドトキシン濃度が判明するので，工程におけるエンドトキシン汚染の迅速管理に有効であることと，動物愛護的観点と動物飼育経費の削減に寄与してきた．また，発熱性物質試験は発熱増強活性*1 がない製剤に関しては，エンドトキシン試験に移行している．
(*1：発熱性物質試験を実施したとき，製剤に 0.5 EU/mL になるようにエンドトキシンを加えた液が，生理食塩液に 0.5 EU/mL になるようにエンドトキシンを加えた対照液に比べて有意に発熱性を認めた場合，発熱増強活性があるものと判断．)
in vitro 発熱性物質試験の話題に変えると，ヒト単球を用いていることから，糖濃度が高いと，末梢血単球からのインターロイキン 8（好中球,T リンパ球に選択時に働く走化性因子である）の分泌が低下し，白血球の遊走能が低下することが知られている．このことから，製剤の成分が単球のサイ
トカインの分泌制御に影響し，in vitro 発熱性物質試験にも反応阻害などに対する反応干渉因子試
験は必要なものと考える．
また，in vitro 発熱性物質試験は，生きた単球の安定供給，バイオハザード的観点にも問題があり，工程管理のためのエンドトキシン試験も含めてすべての試験を in vitro 発熱性物質試験に切り替えるのは疑問である．
医療用具および医用材料の基礎的な生物学的試験のガイドラインに記載されている発熱性物質試
験は，医療用具又は医用材料から抽出した抽出液中に，材料に由来するエンドトキシン以外の発熱性物質が存在しないことを確認する試験と記載されている．（エンドトキシンを確認するには，エンド
トキシン試験が別途必要）．この観点からすると，医療機器の発熱性物質試験をエンドトキシン試験に置き換えるには，生産している製品材料に文献的に既知の発熱性化学物質（例えば，ゴムの老化防7

止剤 n-phenyl－β-naphthylamine, aldol-α-naphthylamine（現在は使用禁止）など）が含まれないことを確認して，試験法をエンドトキシン試験法に変更していく必要があると考える．よって，invitro 発熱性物質試験が化学物質の発熱性を確認する手段となれば，医療用具の(材料に由来するエンドトキシン以外の発熱性物質が存在しないことの確認を目的とした)発熱性物質試験の有効な代替法になるものと考えます．
・有限かつ高価な天然物試薬であるライセート試薬の低減化として，日局規定の各種試験の試験回数規定を試験の信頼性を欠くことなく最小限での試験を認めて欲しい．
（例えば）（ゲル化法予備試験のライセート試薬の表示感度試験）
エンドトキシン標準原液を現行 4 濃度から 3 濃度以上とし，繰返し 4 回を 3 回以上とする．
（ゲル化法予備試験の反応干渉因子試験）
添加エンドトキシン濃度を現行 4 濃度から 3 濃度以上とし，繰返し回数を現行 2 および 4 回をすべて2 回以上とする．
（ゲル化法限度試験法）エンドトキシン無添加の陰性試験を現行 2 回から 1 回以上とする．
（ゲル化法定量試験法）
現行エンドトキシン希釈倍数を 4 条件から 3 条件以上とする．エンドトキシン無添加の陰性試験を現
行 2 回から 1 回以上とする．
（比濁法の予備試験の反応干渉因子試験）
エンドトキシン添加濃度を現行 3 濃度から 2 濃度以上とする．
・日局では発熱性物質試験よりもエンドトキシン試験が優先される規定になっているが，このような規定が出来る以前に製造販売承認を取得した注射剤には，未だに発熱性物質試験が設定された製品が多い．一般の注射剤等で考えられる微生物汚染は，製造工程での水棲菌(主にグラム陰性菌)によるものが殆どと考えられるため，原料等がエンドトキシン以外の発熱性物質で汚染される可能性があるもの
以外は，速やかにエンドトキシン試験へ変更できるよう(変更申請時に必要とされるデータの簡略化を)考慮すべきと考える．発熱性物質試験も動物を使用しない方法が良いと思うが，日常試験として実施するには，今回のようなヒト血液を用いる方法では，その入手方法やバイオハザード対策等で不安がある．動物を使用せずに代替するのであれば，輸液用ゴム栓などでは(輸液用プラスチック容器
試験法からプラスチック製医薬品容器試験法への改正時のように)発熱だけに絞ったものでなく，既に日局にも収載されている細胞毒性試験などで代替することは出来ないか．
・発熱性物質試験からエンドトキシン試験へ試験法を代替する場合に過剰な実験動物データを必要とされるため代替が困難である．現在検討されている in vitro 発熱性物質試験には，どのようなものがあるかご教示ください．
・ライセート試薬を使用しているが，高価な試薬なので安価になれば良いと思う．比濁法で実施しているが，試験管も使い捨てのため Reuse できる洗浄方法などがあれば良いと思う．
・輸液用ゴム栓試験での発熱性物質試験はエンドトキシン試験への代替が可能なのか？

以上

8

EUROPEAN COMMISSION
DIRECTORATE GENERAL JRC
JOINT RESEARCH CENTRE
Institute for Health and Consumer Protection
European Centre for the Validation of Alternative Methods (ECVAM)

STATEMENT ON THE VALIDITY OF IN-VITRO PYROGEN TESTSAt its 24th meeting, held on 20-21 March 2006 at the European Centre for thevalidation of alternative methods (ECVAM), Ispra, Italy, the non-Commission
members of the ECVAM Scientific Advisory Committee (ESAC)1 unanimously
endorsed the following statement:
Following a review of scientific reports and peer reviewed publications on thefollowing range of in-vitro pyrogen tests:
1.
2.
3.
4.
5.

Human Whole Blood IL-1,
Human Whole Blood IL-6,
PBMC IL-6,
MM6 IL-6, andHuman Cryopreserved Whole Blood IL-1,

it is concluded that these tests have been scientifically validated for the detection ofpyrogenicity mediated by Gram-negative endotoxins, and quantification of thispyrogen, in materials currently evaluated and characterized by rabbit pyrogen tests.
These methods have the potential to satisfy regulatory requirements for the detection
and quantification of these pyrogens in these materials subject to product-specificvalidation.
The test methods have the capacity of detecting pyrogenicity produced by a wider
range of pyrogens, but the evidence compiled for, and considered within this peer
review and validation process, is not sufficient to state that full scientific validation ofthis wider domain of applicability has been demonstrated and confirmed.
Thus, the above test methods can currently be considered as full replacements for theevaluation of materials or products where the objective is to identify and evaluate
pyrogenicity produced by Gram-negative endotoxins, but not for other pyrogens.
This endorsement takes account of the dossiers prepared for peer review; the views of
independent experts who evaluated the dossiers against defined validation criteria;
supplementary submissions made by the Management Team; and the considered viewof the Peer Review Panel appointed to oversee the process.
Thomas Hartung
Head of Unit
ECVAM
Institute for Health & Consumer Protection
Joint Research Centre
European Commission
Ispra
21 March 2006

1. The ESAC was established by the European Commission, and is composed ofnominees from the EU Members States, industry, academia and animal
welfare, together with representatives of the relevant Commission services.
This statement was endorsed by the following Members of the ESAC:
Prof Helmut Tritthart (Austria)
Dr Dagmar Jírová (Czech Republic)
Prof Elisabeth Knudsen (Denmark)Dr Timo Ylikomi (Finland)Prof André Guillouzo (France)
Dr Manfred Liebsch (Germany)
Dr Efstathios Nikolaidis (Greece)
Dr Katalin Horvath (Hungary)Prof Michael Ryan (Ireland)
Dr Annalaura Stammati (Italy)
Dr Mykolas Maurica (Lithuania)
Prof Eric Tschirhart (Luxembourg)
Dr Jan van der Valk (The Netherlands)
Dr Dariusz Sladowski (Poland)Prof Milan Pogačnik (Slovenia)
Dr Argelia Castaño (Spain)
Dr Patric Amcoff (Sweden)
Dr Jon Richmond (UK)Dr Odile de Silva (COLIPA)
Dr Julia Fentem (ECETOC)Dr Nathalie Alépée (EFPIA)Prof Robert Combes (ESTIV)Dr Maggy Jennings (Eurogroup for Animal Welfare)
Mr Roman Kolar (Eurogroup for Animal Welfare)
The following Commission Services and Observer Organisations wereinvolved in the consultation process, but not in the endorsement process itself.
Mr Thomas Hartung (ECVAM; chairman)
Mr Jens Linge (ECVAM; ESAC secretary)
Mr Juan Riego Sintes (ECB)
Ms Beatrice Lucaroni (DG Research, Unit F.5)
Mr Sylvain Bintein (DG Environment, Unit C.3)
Mr Sigfried Breier (DG Enterprise, Unit F.3)
Prof Dr Constantin Mircioiu (Romania)
Dr William Stokes (NICEATM, USA)
Prof Dr Vera Rogiers (ECOPA)

Annex
The novel pyrogen tests are based on the human fever reaction. Monocytoid cells,
either primary from human blood or as propagated cell lines, detect pyrogens ofdifferent chemical nature and respond by the release of inflammatory mediators suchas cytokines. Since lipopolysaccharides from Gram-negative bacteria are the only
type of proven pyrogen, for which an International reference material is available, thetests were standardised to detect the presence of significantly less than 0.5 Endotoxin
Units of this preparation, which is considered to be the threshold level for fever
induction in the most sensitive rabbit species according to pharmacopoeia test
procedures.
The five tests which were sufficiently reproducible and exceeded the rabbit test withregard to sensitivity and specificity for the detection of lipopolysaccharide spiked
samples, differ with regard to cell source and preparation, cryopreservation andcytokine measured. The tests have been described elsewhere (1-4). The concept ofthe validation study (5) and the international validation studies are available (6-7).
1. Poole, S., Thorpe, R., Meager, A., Hubbard, A.R., Gearing, A.J. (1988) Detectionof pyrogen by cytokine release. Lancet 8577, 130.
2. Taktak, Y.S., Selkirk, S., Bristow, A.F., Carpenter, A., Ball, C., Rafferty, B., Poole,
S. (1991) Assay of pyrogens by interleukin-6 release from monocytic cell lines. J.
Pharm. Pharmacol. 43, 578.
3. Hartung, T., Wendel, A. (1996) Detection of pyrogens using human whole blood.
In Vitro Toxicol. 9, 353.
4. Schindler S, Asmus S, von Aulock S, Wendel A, Hartung T and Fennrich S. (2004)
Cryopreservation of human whole blood for pyrogenicity testing. J. Immunol. Meth.
294, 89-100.
5. Hartung, T., Aaberge, I., Berthold, S., Carlin, G., Charton, E., Coecke, S., Fennrich,S., Fischer, M., Gommer, M., Halder, M., Haslov, K., Jahnke, M., Montag-Lessing, T.,
Poole, S., Schechtman, L., Wendel, A., Werner-Felmayer, G. (2001) Novel pyrogentests based on the human fever reaction. The report and recommendations of ECVAM
Workshop 43. European Centre for the Validation of Alternative Methods. Altern.
Lab. Anim. 29, 99.
6. Hoffmann S, Peterbauer A, Schindler S, Fennrich S, Poole S. Mistry Y, MontagLessing T, Spreitzer I, Loschner B, vam Aalderen M, Bos R, Gommer M, Nibbeling
R, Werner-Felmayer G, Loitzl P, Jungi T, Brcic M, Brugger P, Frey E, Bowe G,
Casado J, Coecke S, de Lange J, Mogster B, Naess LM, Aaberge IS, Wendel A and
Hartung T. (2005) International validation of novel pyrogen tests based on the humanfever reaction. J. Immunol. Meth. 298, 161-173.
7. Schindler S, Spreitzer I, Loschner, Hoffmann S, Hennes K, Halder M, Brügger P,
Frey E, Hartung T and Montag T. (2006) International validation of pyrogen tests
based on cryopreserved human primary blood cells. J. Immunol. Meth. 316, 42-51.

NIH Publication Number 08-6392

ICCVAM TEST METHOD EVALUATION REPORT
Validation Status of Five In Vitro Test Methods Proposed
for Assessing Potential Pyrogenicity of Pharmaceuticals
and Other ProductsInteragency Coordinating Committee on the Validation of Alternative Methods(ICCVAM)
National Toxicology Program (NTP) Interagency Center for the Evaluation of
Alternative Toxicological Methods (NICEATM)
National Institute of Environmental Health Sciences
National Institutes of Health
U. S. Public Health Service
Department of Health and Human Services

About the Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM)andThe National Toxicology Program Interagency Center for the Evaluation of Alternative
Toxicological Methods (NICEATM)
In 1997, the National Institute of Environmental Health Sciences (NIEHS), one of the National
Institutes of Health (NIH), established ICCVAM to:

Coordinate interagency technical reviews of new and revised toxicological test methods,
including alternative test methods that reduce, refine, or replace the use of animals
Coordinate cross-agency issues relating to validation, acceptance, and national andinternational harmonization of new, modified, and alternative toxicological test methods

On December 19, 2000, the ICCVAM Authorization Act (Public Law 106-545, 42 U.S.C. 2851-3)
established ICCVAM as a permanent interagency committee of NIEHS under NICEATM.
ICCVAM is comprised of representatives from 15 U.S. Federal regulatory and research agencies thatuse, generate, or disseminate toxicological information. ICCVAM conducts technical evaluations ofnew, revised, and alternative methods with regulatory applicability. ICCVAM promotes the scientificvalidation and regulatory acceptance of toxicological test methods that more accurately assess thesafety or hazards of chemicals and products and that refine (decrease or eliminate pain and distress),
reduce, and/or replace animal use. NICEATM administers ICCVAM and provides scientific andoperational support for ICCVAM-related activities. More information about ICCVAM and
NICEATM can be found on the NICEATM-ICCVAM web site (http://iccvam.niehs.nih.gov) or
obtained by contacting NICEATM (telephone: [919] 541-2384, e-mail: niceatm@niehs.nih.gov).
The following Federal regulatory and research agencies are ICCVAM members:

•
•
•
•
•

•
•
•
•

Consumer Product Safety Commission
Department of Agriculture
Department of Defense
Department of Energy
Department of Health and Human Services- Centers for Disease Control and Prevention

Agency for Toxic Substances and Disease Registry
National Institute of Occupational Safety and Health- Food and Drug Administration- National Institutes of Health
Office of the Director
National Cancer Institute
National Institute of Environmental Health Sciences
National Library of Medicine
Department of the Interior
Department of Labor- Occupational Safety and Health Administration
Department of Transportation
Environmental Protection Agency
The NICEATM-ICCVAM graphic symbolizes the important role of new andalternative toxicological methods in protecting and advancing the health of people,
animals, and our environment.

ICCVAM Test Method Evaluation Report:
Validation Status of Five In Vitro Test Methods Proposed forAssessing Potential Pyrogenicity of Pharmaceuticals and Other
Products

Interagency Coordinating Committee on the Validation of Alternative
Methods (ICCVAM)

National Toxicology Program (NTP) Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM)
National Institute of Environmental Health Sciences
National Institutes of Health
U.S. Public Health Service
Department of Health and Human Services

May 2008
NIH Publication No. 08-6392

National Toxicology Program
P.O. Box 12233
Research Triangle Park, NC 27709

This document is available electronically at:
http://iccvam.niehs.nih.gov/methods/pyrogen/pyrogen.htm

ICCVAM Test Method Evaluation Report: Table of Contents

May 2008

Table of Contents
Page Number
List of Tables ......................................................................................................................iv
List of Abbreviations and Acronyms..................................................................................v
Interagency Coordinating Committee on the Validation of Alternative
Methods: Agency Representatives ........................................................................vii
Acknowledgements.......................................................................................................... viii
Preface ................................................................................................................................xi
Executive Summary ..........................................................................................................xv
1.0
Introduction .............................................................................................................1
2.0
ICCVAM Recommendations for In Vitro Pyrogen Test Methods .........................3
2.1
ICCVAM Recommendations: Test Method Uses and Limitations ..................3
2.2
ICCVAM Recommendations: Test Method Protocols ....................................4
2.3
ICCVAM Recommendations: Future Studies .................................................6
2.4
ICCVAM Recommendations: Performance Standards....................................7
3.0
Validation Status of In Vitro Pyrogen Test Methods..............................................9
3.1
Test Method Description ................................................................................9
3.2
Validation Database .....................................................................................10
3.3
Reference Test Method Data ........................................................................11
3.4
Test Method Accuracy .................................................................................11
3.5
Test Method Reliability................................................................................12
3.6
Animal Welfare Considerations: Reduction, Refinement,
and Replacement ..........................................................................................18
4.0
ICCVAM Consideration of Public and SACATM Comments ............................19
4.1
Public Comments in Response to FR Notice (70FR74833, December
16, 2005): Peer Panel Evaluation of In Vitro Pyrogenicity Testing
Methods: Request for Comments, Nominations of Experts, andSubmission of In Vivo and In Vitro Data ......................................................19
4.2
Public Comments in Response to FR Notice (71FR74533,
December 12, 2006): Announcement of an Independent ScientificPeer Review Meeting on the Use of In Vitro Pyrogenicity Testing
Methods; Request for Comments..................................................................19
4.3
Public Comments in Response to FR Notice (72FR26395, May 9,2007): Peer Review Panel Report on Five In Vitro Pyrogen Test
Methods: Availability and Request for Public Comments.............................20
4.4
Public and SACATM Comments: SACATM Meeting on June 12,2007.............................................................................................................21
5.0
References ..............................................................................................................23
Appendix A Independent Scientific Peer Review Panel Assessment ..........................A-1
A1
Peer Review Panel Report: The Use of Five In Vitro Test Methods
Proposed for Assessing Potential Pyrogenicity of Pharmaceuticals
and Other Products.....................................................................................A-3
A2
Summary Minutes from Peer Review Panel Meeting on
February 6, 2007 ......................................................................................A-53
A3
Pyrogenicity Peer Review Panel Biographies ...........................................A-65

i

ICCVAM Test Method Evaluation Report: Table of Contents

May 2008

Appendix B Relevant Federal Pyrogenicity Regulations and Testing
Guidelines................................................................................................. B-1
B1
U.S. Code of Federal Regulations (CFR).................................................... B-3
B1-1 21 CFR 211.167 - Current Good Manufacturing Practice for
Finished Pharmaceuticals: Special Testing Requirements(April 1, 2007)................................................................................ B-5
B1-2 21 CFR 314.50(d)(1)(ii)(a) - Applications: Content andFormat of an Application (April 1, 2007)........................................ B-6
B1-3 21 CFR 610.9 - General Provisions: Equivalent Methods andProcesses (April 1, 2007).............................................................. B-17
B1-4 21 CFR 610.13 - General Biological Products Standards:
Purity (April 1, 2007) ................................................................... B-19
B2
International Organization for Standardization - ISO 10993-11 Biological Evaluation of Medical Devices - Part 11: Tests for
Systemic Toxicity (First Edition 1993-12-15) .......................................... B-21
B3
U.S. Pharmacopeia (USP) 30-NF25 ......................................................... B-23
B3-1 (85) - Bacterial Endotoxins Test ................................................... B-23
B3-2 (151) - Pyrogen Test..................................................................... B-23
B3-3 (1041) - Biologics ........................................................................ B-23
B4
European Pharmacopoeia 5.0 ................................................................... B-25
B4-1 2.6.8. Pyrogens............................................................................. B-25
B4-2 2.6.14. Bacterial Endotoxins......................................................... B-25
B5
FDA Guideline on Validation of the Limulus Amebocyte Lysate Test
as an End-Product Endotoxin Test for Human and Animal
Parenteral Drugs, Biological Products, and Medical Devices(December 1987) ..................................................................................... B-27
Appendix C ICCVAM Recommended Test Method Protocols................................... C-1
C1
The Human Whole Blood (WB)/Interleukin (IL)-1β
In Vitro Pyrogen Test................................................................................. C-3
C2
The Human WB/IL-1β In Vitro Pyrogen Test: Application of
Cryopreserved (Cryo) Human WB........................................................... C-25
C3
The Human WB/IL-6 In Vitro Pyrogen Test............................................. C-47
C4
The Human Peripheral Blood Mononuclear Cell (PBMC)/IL-6
In Vitro Pyrogen Test............................................................................... C-71
C5
The Monocytoid Cell Line Mono Mac 6 (MM6)/IL-6
In Vitro Pyrogen Test............................................................................... C-95
Appendix D Federal Register Notices, Public Comments, and Relevant
SACATM Meeting Minutes..................................................................... D-1
D1
Federal Register Notices............................................................................ D-3
D1-1 Vol. 70, No. 241, pp. 74833-74834, December 16, 2005:
Peer Panel Evaluation of In Vitro Pyrogenicity Testing Methods:
Request for Comments, Nominations of Experts, and Submissionof In Vivo and In Vitro Data ....................................................................... D-5ii

ICCVAM Test Method Evaluation Report: Table of Contents

D2
D3

May 2008

D1-2 Vol. 71, No. 238, pp. 74533-74534, December 12, 2006:
Announcement of an Independent Scientific Peer Review Meetingon the Use of In Vitro Pyrogenicity Testing Methods; Request forComments..................................................................................................D-7
D1-3 Vol. 72, No. 89, pp. 26395-26396, May 9, 2007: Peer
Review Panel Report on Five In Vitro Pyrogen Test Methods:
Availability and Request for Public Comments ..........................................D-9
Public Comments Received in Response toFederal Register Notices..........................................................................D-11
SACATM Comments: ICCVAM Evaluation of In Vitro Pyrogen Test
Methods ...................................................................................................D-43

Appendix E ESAC Statement on the Validity of In Vitro Pyrogen Tests ................... E-1

iii

ICCVAM Test Method Evaluation Report: List of Tables

May 2008

List of Tables
Page Number
Table 1*
Table 2-1
Table 3-1
Table 3-2
Table 3-3*
Table 3-4
Table 3-5
Table 3-6

Accuracy of In Vitro Pyrogen Test Methods............................................... xvii
Summary of ICCVAM Recommended In Vitro Pyrogen Test Method
Protocols........................................................................................................ 5
Parenteral Drugs Used in the Validation Studies for Determining
Test Method Accuracy ................................................................................. 10
Parenteral Drugs Used in the Validation Studies for Determining
Test Method Reproducibility........................................................................ 11
Accuracy of In Vitro Pyrogen Test Methods................................................. 12
Intralaboratory Reproducibility of In Vitro Pyrogen Test Methods ............... 15
Interlaboratory Reproducibility of In Vitro Pyrogen Test
Methods: Study One .................................................................................... 17
Interlaboratory Reproducibility of In Vitro Pyrogen Test
Methods: Study Two.................................................................................... 18*

Tables 1 and 3-3 are identical. Table 1 provides supporting information for the Executive Summary, while
Table 3-3 provides the information in Section 3.2, where test method accuracy is discussed in greater detail.

iv

ICCVAM Test Method Evaluation Report: List of Abbreviations and Acronyms

May 2008

List of Abbreviations and Acronyms
APHIS
BET
BRD
CBER
CFR
Cryo
CV
EC
ECVAM
ELC
ELISA
EP
ESAC
EU
EU/mL
FDA
FR
ICCVAM
IL
ILS
ISO
LAL
LLNA
LPS
MM6
MVD
MRC
NA
NC
ND
NI
NICEATM
NIEHS
NPC
NSC
NTP
OECD
OD
PBMC
PEI
PPC

Animal and Plant Health Inspection Service (USDA)
Bacterial Endotoxin Test
Background Review Document
Center for Biologics Evaluation and Research (FDA)
Code of Federal Regulations
Cryopreserved
Coefficient of variation
Endotoxin control
European Centre for the Validation of Alternative Methods
Endotoxin limit concentration
Enzyme-linked immunosorbent assay
European Pharmacopeia
ECVAM Scientific Advisory Committee
European Union
Endotoxin Units per milliliter
U.S. Food and Drug Administration
Federal Register
Interagency Coordinating Committee on the Validation of
Alternative Methods
Interleukin
Integrated Laboratory Systems
International Organization for Standardization
Limulus Amebocyte Lysate test method
Murine local lymph node assay
Lipopolysaccharide
Mono Mac 6
Maximum valid dilution
Medical Research Council (U.K.)
Not applicableNot calculated
Not done
Not included
National Toxicology Program (NTP) Interagency Center for
the Evaluation of Alternative Toxicological Methods
National Institute of Environmental Health Sciences
Negative product control
Negative saline control
National Toxicology Program
Organization for Economic Co-operation and Development
Optical density
Peripheral blood mononuclear cellPaul Ehrlich Institut
Positive product controlv

ICCVAM Test Method Evaluation Report: List of Abbreviations and Acronyms

PWG
RPT
RSE
SACATM
SC
SOP
TS
TLR
U.S.
U.K.
USP
WB
WHO

May 2008

Pyrogenicity Working Group
Rabbit pyrogen test
Reference standard endotoxin
Scientific Advisory Committee on Alternative Toxicological
Methods
Standard curveStandard operating procedure
Test substanceToll-like receptors
United States
United Kingdom
U.S. Pharmacopeia
Whole blood
World Health Organizationvi

ICCVAM Test Method Evaluation Report: ICCVAM Representatives

May 2008

Interagency Coordinating Committee on the Validation of Alternative
Methods: Agency Representatives
Agency for Toxic Substances and Disease Registry★Moiz Mumtaz, Ph.D.

Department of Energy★ Michael Kuperberg, Ph.D.
◊ Marvin Stodolsky, Ph.D.

Food and Drug Administration
Office of Science★ Suzanne Fitzpatrick, Ph.D., D.A.B.T.
Center for Drug Evaluation and Research◊ Abigail C. Jacobs, Ph.D.
Paul C. Brown, Ph.D.
Center for Devices and Radiological Health
Melvin E. Stratmeyer, Ph.D.
Center for Biologics Evaluation and Research
Richard McFarland, Ph.D., M.D.
Ying Huang, Ph.D.
Center for Food Safety and Nutrition
David G. Hattan, Ph.D.
Robert L. Bronaugh, Ph.D.
Center for Veterinary Medicine
Devaraya Jagannath, Ph.D.
M. Cecilia Aguila, D.V.M.
National Center for Toxicological ResearchWilliam T. Allaben, Ph.D.
Paul Howard, Ph.D.
Office of Regulatory Affairs
Lawrence A. D’Hoostelaere, Ph.D.

Department of the Interior★ Barnett A. Rattner, Ph.D.
◊ Sarah Gerould, Ph.D.

National Cancer Institute★ Alan Poland, M.D.
◊ T. Kevin Howcroft, Ph.D.

Department of Transportation★ George Cushmac, Ph.D.
◊ Steve Hwang, Ph.D.

National Institute of Environmental Health Sciences★ William S. Stokes, D.V.M., D.A.C.L.A.M.
◊ Raymond R. Tice, Ph.D.
Rajendra S. Chhabra, Ph.D., D.A.B.T
Jerrold J. Heindel, Ph.D.

Consumer Product Safety Commission★ Marilyn L. Wind, Ph.D. (Chair)◊ Kristina Hatlelid, Ph.D.
Joanna Matheson, Ph.D.
Department of Agriculture★ Jodie Kulpa-Eddy, D.V.M. (Vice-Chair)◊ Elizabeth Goldentyer, D.V.M.
Department of Defense★ Robert E. Foster, Ph.D.
◊ Patty Decot
Peter J. Schultheiss, D.V.M., D.A.C.L.A.M.
Harry Salem, Ph.D.

Environmental Protection Agency
Office of Science Coordination and Policy★ Karen Hamernik, Ph.D.
Office of Research and Development◊ Julian Preston, Ph.D.
Suzanne McMaster, Ph.D.
Office of Pesticides Programs
Amy Rispin, Ph.D.
Deborah McCall
OECD Test Guidelines Program
Jerry Smrchek, Ph.D.

National Institute for Occupational Safety and
Health★ Paul Nicolaysen, V.M.D.
◊ K. Murali Rao, MD, Ph.D.
National Institutes of Health★ Margaret D. Snyder, Ph.D.
National Library of Medicine◊ Jeanne Goshorn, M.S.
Occupational Safety and Health Administration★ Surender Ahir, Ph.D

★ Principal Agency Representative◊ Alternate Principal Agency Representativevii

ICCVAM Test Method Evaluation Report: Acknowledgements

May 2008

Acknowledgements
The following individuals are acknowledged for their contributions to the in vitro pyrogentest method review process.
Pyrogenicity Working Group (PWG)
Interagency Coordinating Committee on the Validation of
Alternative Methods (ICCVAM)

U.S. Food and Drug Administration
Center for Biologics Evaluation and ResearchMustafa Akkoyunlu, M.D., Ph.D.
Pankaj Amin
Christine Anderson
Kimberly Benton, Ph.D.
Joseph George, Ph.D.
Christopher Joneckis, Ph.D.
Richard McFarland, Ph.D., M.D. (Chair)Center for Drug Evaluation and Research
David Hussong, Ph.D.
Abigail Jacobs, Ph.D.
Robert Mello, Ph.D.
Amy Rosenberg, M.D.
Daniela Verthelyi, M.D., Ph.D.
Jiaqin Yao, Ph.D.
Center for Devices and Radiological Health
Raju Kammula, D.V.M., Ph.D., D.A.B.T.
Ramesh Panguluri
Center for Food Safety and Nutrition
Penelope Rice, Ph.D.

*

U.S. Environmental Protection Agency
Office of Science Coordination and Policy
Karen Hamernik, Ph.D.
National Institute of Environmental
Health Sciences
William Stokes, D.V.M., D.A.C.L.A.M.
Raymond Tice, Ph.D.
U.S. Department of Agriculture
Jodie Kulpa-Eddy, D.V.M.
Liaison, European Centre for the
Validation of Alternative Methods
Marlies Halder, Ph.D.*

Dr. Halder was responsible for the trial plan of the catch-up validation study, but she was not involved in theevaluation of the data.

viii

ICCVAM Test Method Evaluation Report: Acknowledgements

May 2008

The following scientists are acknowledged for their independent evaluation of the five in
vitro pyrogen tests as members of the Peer Review Panel.
In Vitro Pyrogenicity Independent Peer Review Panel
Karen Brown, Ph.D., (Panel Chair), DRL Pharma and Pair O’Doc’s Enterprises, Parkville,
Missouri
Brian Crowe, Ph.D., Baxter Vaccine AG, Orth an der Donau, Austria
Nancy Flournoy, Ph.D., University of Missouri-Columbia, Columbia, Missouri
Ihsan Gursel, Ph.D., Bilkent University, Bilkent, Ankara, Turkey
Ken Ishii, M.D., Ph.D., ERATO, Japan Science and Technology Agency, Osaka University,
Osaka, JapanJack Levin, M.D., University of California-San Francisco, San Francisco, California
Albert Li, Ph.D., In Vitro ADMET Laboratories, Rockville, Maryland
David Lovell, Ph.D., University of Surrey, Guilford, United Kingdom
Melvyn Lynn, Ph.D., Eisai Medical Research, Inc., Ridgefield Park, New Jersey
Anthony Mire-Sluis, Ph.D., AMGEN, Inc., Thousand Oaks, California
Jon Richmond, M.D., Home Office, Tayside, United Kingdom
Peter Theran, V.M.D., Massachusetts Society for the Prevention of Cruelty to Animals,
Novato, California
Kevin Williams, Eli Lilly, Indianapolis, Indianaix

ICCVAM Test Method Evaluation Report: Acknowledgements

May 2008

National Toxicology Program (NTP) Interagency Center for the Evaluation of
Alternative Toxicological Methods (NICEATM)
National Institute of Environmental Health Sciences
William Stokes, D.V.M., D.A.C.L.A.M.
Director; Project Officer
Raymond Tice, Ph.D.
Deputy Director
Deborah McCarley
Special Assistant; Asst. Project Officer
NICEATM Support Contract Staff (Integrated Laboratory Systems [ILS], Inc.)David Allen, Ph.D.
Principal Investigator
Douglas Winters, M.S.
Project Manager
Neepa Choksi, Ph.D.
Sr. Staff Toxicologist
Judy Strickland, Ph.D., D.A.B.T.
Sr. Staff Toxicologist
Frank Deal, M.S.
Staff Toxicologist
Elizabeth Lipscomb, Ph.D.
Staff Toxicologist
Thomas Burns, M.S.
Sr. Project Coordinator/Technical Writer
Michael Paris
Sr. Project Coordinator/Technical Writer
Patricia Ceger, M.S.
Project Coordinator/Technical Writer
James Truax, M.A.
Project Coordinator/Technical Writer
Catherine Sprankle
Communications Specialist/Web Developer
Linda Litchfield
Meeting Planner and Coordinator
Consultants for ILS, Inc.
Joe Haseman, Ph.D.
Errol Zeiger, Ph.D., J.D.

Statistical Consultant
Editorial Consultantx

ICCVAM Test Method Evaluation Report: Preface

May 2008

Preface
The Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM) is charged by the ICCVAM Authorization Act of 2000 with evaluating thescientific validity of new, revised, and alternative toxicological test methods applicable to
U.S. Federal agency safety testing requirements (ICCVAM 2000). ICCVAM is required toprovide recommendations to U.S. Federal agencies regarding the usefulness and limitationsof test methods based on this scientific evaluation. This Test Method Evaluation Reportprovides ICCVAM recommendations for five in vitro test methods proposed for assessing thepotential pyrogenicity of pharmaceuticals and other products. These recommendations are
based on a comprehensive evaluation of the current validation status of these test methods.
In March 2005, the European Centre for the Validation of Alternative Methods (ECVAM), a
unit of the Institute for Health and Consumer Protection at the European Commission’s Joint
Research Centre, submitted background review documents (BRDs) to ICCVAM for five in
vitro test methods, which were proposed as replacements for the rabbit pyrogen test. The
information in the BRDs was based on validation studies financed by the European
Commission within the 5th Framework Programme of Directorate General Research, theresults of which were recently published (Hoffmann et al. 2005a; Schindler et al. 2006). The
five test methods are:
•
•
•
•
•

The Human Whole Blood (WB)/Interleukin (IL)-1β In Vitro Pyrogen Test
The Human WB/IL-1β In Vitro Pyrogen Test: Application of Cryopreserved
Human WB
The Human WB/IL-6 In Vitro Pyrogen Test
The Human Peripheral Blood Mononuclear Cell/IL-6 In Vitro Pyrogen Test
The Monocytoid Cell Line Mono Mac 6/IL-6 In Vitro Pyrogen Test

In June 2005, ICCVAM initiated evaluation of the validation status of these five test
methods. An ICCVAM Pyrogenicity Working Group (PWG) was established to work with
the National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative
Toxicological Methods (NICEATM) to carry out this review. Dr. Marlies Halder was
designated by ECVAM as their liaison to the PWG. Following a NICEATM pre-screen
evaluation of the ECVAM BRDs, NICEATM, ICCVAM and the ICCVAM PWG requested
additional information and clarification from ECVAM on a number of issues. In March 2006,
ECVAM provided revised BRDs and responses addressing these issues.
NICEATM, in conjunction with the PWG, prepared a comprehensive BRD to combine theavailable data and information for each of the five in vitro test methods into one document.
The ICCVAM BRD describes the current validation status of these test methods, including
what is known about their reliability and accuracy, the scope of the substances tested, and theavailability of standardized protocols for each test method. The ICCVAM BRD was based on
the ECVAM BRDs, but also includes other relevant data and analyses, including data andinformation submitted to NICEATM in response to a Federal Register (FR) Notice (Vol. 70,
No. 241, pp. 74833-74834, December 16, 2005). The ICCVAM draft BRD was madeavailable to the public on December 12, 2006 (announced in FR Vol. 71, No. 238, pp. 74533-

xi

ICCVAM Test Method Evaluation Report: Preface

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74534, December 12, 2006) for comment and a public peer review panel meeting on
February 6, 2007 was announced.
The independent scientific peer review panel (Panel) met in public session on February 6,
2007 at the National Institutes of Health in Bethesda, Maryland. The Panel first reviewed the
ICCVAM draft BRD for errors and omissions and then discussed the current validation statusof the five in vitro test methods. The Panel also reviewed the extent that the information in
the ICCVAM BRD supported the ICCVAM draft test method recommendations for proposed
test method uses, standardized protocols, test method performance standards, and futurestudies. Throughout the review process, interested stakeholders from the public were
provided opportunities to provide comments including oral comments at the Panel meeting.
The Panel considered these comments as well as public comments submitted in advance ofthe meeting before concluding their deliberations. The final independent Panel report wasmade available to the public (http://iccvam.niehs.nih.gov/docs/pyrogen/PrRevPanFinRpt.pdf)
for review and comment on May 9, 2007 (announced in FR Vol. 72, No. 89, pp. 2639526396).
The ICCVAM draft BRD and draft recommendations, the Panel report, and all publiccomments were made available to ICCVAM’s advisory committee, the Scientific Advisory
Committee on Alternative Toxicological Methods (SACATM), and comments were provided
at their meeting on June 12, 2007.
ICCVAM and the PWG considered the Panel report, all public comments, and the commentsof SACATM in preparing the final ICCVAM test method recommendations provided in thisreport. This report will be made available to the public and provided to U.S. Federal agencies
for consideration, in accordance with the ICCVAM Authorization Action of 2000 (ICCVAM
2000). Agencies must respond to ICCVAM within 180 days after receiving an ICCVAM test
method recommendation. These responses will be made available to the public on the
NICEATM/ICCVAM website (http://iccvam.niehs.nih.gov) as they are received.
The efforts of the many individuals who contributed to the preparation, review and revisionof this report are gratefully acknowledged. We greatly appreciate the careful preparation of
the BRDs by ECVAM and their prompt response to requests for additional information. We
especially recognize all of the Panel members for their thoughtful evaluations and generous
contributions of time and effort. Special thanks are extended to Dr. Karen Brown for serving
as the Panel Chair and to Drs. Jack Levin, Melvyn Lynn, Anthony Mire-Sluis, and Jon
Richmond for their service as Evaluation Group Chairs. The efforts of the PWG wereinvaluable for assuring a meaningful and comprehensive review. We especially thank the
Chair of the PWG, Dr. Richard McFarland (FDA, Center for Biologics Evaluation andResearch) for his effective leadership. The efforts of the NICEATM staff and support
contractor in preparing the BRD, organizing the Panel meeting, and preparing this final
report are greatly appreciated. We acknowledge Drs. David Allen and Elizabeth Lipscomb,
Catherine Sprankle, James Truax, and Doug Winters of Integrated Laboratory Systems, Inc.,
the NICEATM support contractor, for their assistance. We also thank Dr. Raymond Tice,
Deputy Director of NICEATM, for his efforts on this project.

xii

ICCVAM Test Method Evaluation Report: Preface

May 2008

This comprehensive ICCVAM evaluation of the validation status of these five test methods
and the accompanying recommendations should aid agencies in providing guidance on their
future use for regulatory safety testing. The ICCVAM recommendations for future studies
are expected to advance broader applicability of these methods, which may further reduceanimal use while ensuring continued or better protection of human health.

William S. Stokes, D.V.M., D.A.C.L.A.M
Rear Admiral, U.S. Public Health Service
Director, NICEATM
Executive Director, ICCVAM
Marilyn Wind, Ph.D.
U.S. Consumer Product Safety Commission
Chair, ICCVAM

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ICCVAM Test Method Evaluation Report: Preface

May 2008[This Page Intentionally Left Blank]xiv

ICCVAM Test Method Evaluation Report: Executive Summary

May 2008

Executive Summary
This Test Method Evaluation Report, prepared by the Interagency Coordinating Committee
on the Validation of Alternative Methods (ICCVAM), summarizes ICCVAM’s evaluation of
the validation status of five in vitro test methods proposed for assessing the potential
pyrogenicity of pharmaceuticals and other products, as potential replacements for the in vivo
rabbit pyrogen test (RPT). The five test methods are:•

The Human Whole Blood (WB)/Interleukin (IL)-1β In Vitro Pyrogen Test•

The Human WB/IL-1β In Vitro Pyrogen Test: Application of Cryopreserved(Cryo) Human WB•

The Human WB/IL-6 In Vitro Pyrogen Test•

The Human Peripheral Blood Mononuclear Cell (PBMC)/IL-6 In Vitro
Pyrogen Test•

The Monocytoid Cell Line Mono Mac 6 (MM6)/IL-6 In Vitro Pyrogen Test

This report also provides ICCVAM's recommendations for current uses and limitations foreach test method, as well as recommendations for standardized protocols, future studies, andperformance standards. In support of this evaluation, ICCVAM prepared a draft Background
Review Document (BRD) and ICCVAM draft test method recommendations, which were
provided to an independent scientific peer review panel (Panel) and the public forconsideration and comment. The ICCVAM draft BRD was prepared using data fromvalidation studies that had been conducted by the European Centre for the Validation of
Alternative Methods (ECVAM), a unit of the Institute for Health and Consumer Protection at
the European Commission’s Joint Research Centre. The ECVAM submission, preparedaccording to the ICCVAM submission guidelines (ICCVAM 2003), included five individual
BRDs (i.e., one BRD for each test method), which summarized the validation studies foreach of the five in vitro test methods.
The Panel met on February 6, 2007 to review the ICCVAM draft BRD for errors andomissions and to discuss the current validation status of the five in vitro test methods. The
Panel also reviewed the extent that the information contained in the ICCVAM draft BRD
supported the ICCVAM draft test method recommendations. In finalizing the test method
recommendations presented here, ICCVAM considered the conclusions andrecommendations of the Panel as well as comments from the public and its Scientific
Advisory Committee on Alternative Toxicological Methods.
ICCVAM Recommendations: Test Method Uses and Limitations
Based on this evaluation, ICCVAM recommends that, although none of these test methods
can be considered a complete replacement for the RPT for all testing situations for thedetection of Gram-negative endotoxin, they can be considered for use to detect
Gram-negative endotoxin in human parenteral drugs on a case-by-case basis, subject tovalidation for each specific product to demonstrate equivalence to the RPT, in accordancexv

ICCVAM Test Method Evaluation Report: Executive Summary

May 2008

with applicable U.S. Federal regulations (e.g., U.S. Food and Drug Administration [FDA] *)†.
When used in this manner, these methods should be able to reduce the number of animals
needed for pyrogenicity testing. Pyrogenicity testing may involve more than slight or
momentary pain or distress when a pyrogenic response occurs. Accordingly, alternative test
methods must be considered prior to the use of animals for such testing, as required by U.S.
Federal animal welfare regulations and policies. Therefore, these and other in vitro
alternative test methods should be considered prior to the use of animals in pyrogenicity
testing and should be used where determined appropriate for a specific testing situation. Useof these methods, once appropriately validated, will support improved animal welfare while
ensuring the continued protection of human health.
ICCVAM developed a recommended standardized protocol for each test method based
primarily on ECVAM standard operating procedures (SOPs). ICCVAM also provided
recommendations for further research and development, optimization, and validation efforts.
These recommendations should be helpful to various stakeholders (e.g., applicable U.S.
Federal regulatory agencies, the international regulatory community, the pharmaceuticalindustry) for determining when these test methods might be useful.
The Panel concluded that the validation criteria were adequately addressed in the ICCVAM
BRD to determine the usefulness and limitations of these test methods to serve as a substitutefor the RPT to identify Gram-negative endotoxin on a case-by-case basis, subject tovalidation for that specific product. However, the Panel stated the performance of these test
methods in terms of their reliability and relevance did not support this proposed use.
In March 2006, the ECVAM Scientific Advisory Committee (ESAC) endorsed a statement ofvalidity for these five in vitro pyrogen test methods (see Appendix E). Like ICCVAM,ESAC concluded that these five methods can detect Gram-negative endotoxin in materials
currently tested with the RPT, and, therefore, may be useful for regulatory decisions, subject
to validation for that specific product. Both ICCVAM and ESAC also concluded that thecurrently available database does not support the use of these test methods to detect a wider
range of pyrogens, as suggested in the original ECVAM submission. However, ESAC
concluded that these tests "can currently be considered as full replacements for the evaluationof materials or products where the objective is to identify and evaluate pyrogenicity producedby Gram-negative endotoxins, but not for other pyrogens." ICCVAM has concluded that thecurrent validation database for these test methods is inadequate to support such a definitive
statement based on the ECVAM validation study design, which did not include biologics ormedical devices and evaluated only a limited range and number of pharmaceutical products.
Additionally, no RPT data were generated with the same test samples used in the in vitro test
methods (i.e., parallel testing).

*

Mechanisms exist for test method developers to qualify their method on a case-by-case basis. The use of any
recommended method will be subject to product-specific validation to demonstrate equivalence asrecommended by the FDA (e.g., U.S. Code of Federal Regulations (CFR) 21 CFR 610.9 and 21 CFR
314.50(d)(1)(ii)(a)).
†
Substances other than endotoxin may induce the cellular release of IL-1β and/or IL-6. For this reason, users ofthese test methods should be aware that the presence of other materials might erroneously suggest the presenceof endotoxin and lead to a false positive result.
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ICCVAM Test Method Evaluation Report: Executive Summary

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Accuracy and Reliability
The accuracy of in vitro pyrogen test methods for detecting Gram-negative endotoxin was
based on the results for 10 parenteral pharmaceuticals, each spiked with four concentrationsof endotoxin (0, 0.25, 0.5, or 1.0 Endotoxin Units [EU]/mL, with 0.5 EU/mL tested in
duplicate). As shown in Table 1, accuracy among the test methods ranged from 81% to 93%,
sensitivity ranged from 73% to 99%, specificity ranged from 77% to 97%, false negative
rates ranged from 1% to 27%, and false positive rates ranged from 3% to 23%.
Table 1

Accuracy of In Vitro Pyrogen Test Methods1

Test
MethodCryo
WB/IL-1βAccuracy2

Sensitivity3

Specificity492%(110/120)93%(138/148)93%(140/150)87%(130/150)92%(136/148)
81%(119/147)

97%(75/77)96%(85/89)92%(83/90)93%(84/90)89%(79/89)73%(64/88)93%(129/139)99%(83/84)

MM6/IL-6
PBMC/IL-6
PBMC/IL-6(Cryo)7
WB/IL-6
WB/IL-1β(Tube)
WB/IL-1β(96-wellplate)881%(35/43)90%(53/59)95%(57/60)77%(46/60)97%(57/59)93%(55/59)

False Negative
Rate5
3%(2/77)
5%(4/89)
8%(7/90)
7%(6/90)
11%(10/89)27%(24/88)

False Positive
Rate6
19%
(8/43)
10%(6/59)
5%(3/60)
23%(14/60)3%(2/59)
7%(4/59)

84%(46/55)1%(1/84)

16%(9/55)

Abbreviations: Cryo = Cryopreserved; EU/mL = Endotoxin units per milliliter; IL = Interleukin; MM6 = Mono Mac 6;
PBMC = Peripheral blood mononuclear cells; WB = Whole blood
1
Data shown as a percentage (number of correct runs/total number of runs), based on results of 10 parenteral drugs tested ineach of three different laboratories. Samples of each drug were tested with or without being spiked with a Gram-negative
endotoxin standard (0, 0.25, 0.5, or 1.0 EU/mL, with 0.5 EU/mL tested in duplicate).
2
Accuracy = the proportion of correct outcomes (positive and negative) of a test method.
3
Sensitivity = the proportion of all positive substances that are classified as positive.
4
Specificity = the proportion of all negative substances that are classified as negative.
5
False negative rate = the proportion of all positive substances that are falsely identified as negative.
6
False positive rate = the proportion of all negative substances that are falsely identified as positive.
7
A modification of the PBMC/IL-6 test method that uses Cryo PBMCs.
8
A modification of the WB/IL-1β test method that uses 96-well plates instead of tubes for the test substance incubation.

Repeatability within individual laboratories was determined for each in vitro test method,using saline and various endotoxin spikes to evaluate the closeness of agreement among
optical density (OD) readings for cytokine measurements at each concentration. The results
indicated that the variability in OD measurements increased with increasing endotoxin
concentration. However, the variability was low enough that the threshold for pyrogenicity
could still be detected (i.e., the 0.5 EU/mL spike concentration could still be distinguishedfrom the lower concentrations).
Reproducibility within individual laboratories was evaluated using three marketed
pharmaceuticals spiked with various concentrations of endotoxin. Three identical,
independent runs were conducted in each of the three testing laboratories, with the exceptionxvii

ICCVAM Test Method Evaluation Report: Executive Summary

May 2008

of the Cryo WB/IL-1β test method‡. The correlations (expressed as percentage of agreement)
between pairs of the independent runs (i.e., run 1 vs. run 2; run 1 vs. run 3; run 2 vs. run 3)
were determined, and the mean of these three values was calculated. Agreement across threeruns within a single laboratory ranged from 75% to 100%.
Reproducibility across all laboratories was evaluated in two different studies in which eachrun from one laboratory was compared to all other runs of another laboratory. The proportionof equally qualified samples provided a measure of reproducibility. In the first
reproducibility study, three marketed pharmaceutical products were spiked with either saline
control or various concentrations of endotoxin, and each sample was tested in triplicate ineach of three different laboratories, except for the Cryo WB/IL-1β. In the second study,
reproducibility was determined using the results from the 10 substances used in the accuracy
analysis. Each drug was spiked with four concentrations of endotoxin and tested once in eachof three laboratories. The extent and order of agreement among laboratories were similar inboth studies: the WB/IL-1β test method showed the least agreement (57% to 58%), and theCryo WB/IL-1β test method showed the most (88% to 92%).
ICCVAM Recommendations: Test Method Protocols
ICCVAM recommends standardized protocols for each test method that should be used forvalidation of specific products on a case-by-case basis for U.S. regulatory consideration.
These recommended protocols, provided in Appendix C, are primarily based on ECVAM
SOPs for each test method. ICCVAM has updated these protocols to address inadequacies
identified by the Panel, including modifications to standardize essential test method
components across the five in vitro test methods. These modifications are not expected toreduce or otherwise impact test method accuracy and reliability.
The Panel concluded that the information provided in the ICCVAM draft BRD supported the
ICCVAM draft recommended protocols for these five in vitro test methods, as long as
inadequacies identified by the Panel with respect to reliability and relevance are fully
addressed.
ICCVAM Recommendations: Future Studies
ICCVAM recognizes that these test methods could be applicable for detection of a wider
range of pyrogens (i.e., endotoxin and pyrogens other than endotoxin) and test materials,
provided that they are adequately validated for such uses. Test materials that have been
identified clinically as pyrogenic might be invaluable for use in future validation studies andmight allow such studies to be conducted without the use of animals. Wherever possible,
historical data generated with the same test samples in both in vitro and in vivo studies (i.e.,parallel testing data) should be retrospectively evaluated, or in vitro testing should be
performed in parallel with RPT and/or bacterial endotoxin tests (BET) conducted for‡

The ECVAM Cryo WB/IL-1β test method BRD stated that there was no direct assessment of intralaboratory
reproducibility because such an evaluation was performed in the WB/IL-1 test method, and the authors assumed
that variability would not be affected by the use of cryopreserved blood.

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ICCVAM Test Method Evaluation Report: Executive Summary

May 2008

regulatory purposes§. Future validation studies should include the following considerations:
1.

Both endotoxin-spiked and non-endotoxin spiked samples should be included.
Non-endotoxin pyrogen standards should be characterized prior to their use inany study, if possible.

2.

All aspects of the studies should comply with Good Laboratory Practices.

3.

Future studies should include products that have intrinsic pro-inflammatory
properties in order to determine if these tests can be used for such substances.

4.

Optimally, a study that includes three-way parallel testing, with the in vitro
assays being compared to the RPT and the BET, should be conducted tocomprehensively evaluate the relevance and comparative performance of thesetest methods. These studies may be conducted with historical RPT data
provided that the same substances (i.e., same lot) are tested in each method.
Based on ethical and scientific rationale, any in vivo testing should be limited
to those studies that will fill existing data gaps.

5.

Test substances that better represent all categories of sample types (e.g.,
pharmaceuticals, biologicals, and medical devices) intended for testing by themethods should be included.

6.

The hazards associated with human blood products should be carefully
considered, and all technical staff should be adequately trained to observe allnecessary safety precautions.

7.

Formal sample size calculations should be made to determine the required
number of replicates needed to reject the null hypothesis at a given level ofsignificance and power. For reliability assessments, formal hypothesis testingis essential with the alternative hypothesis being no difference between
groups.

The Panel agreed with ICCVAM that any future studies should be performed using the
ICCVAM recommended test method protocols. The Panel also provided other suggestions
and recommendations for future studies (see Appendix A). Like ICCVAM, the Panel also
recognized that these test methods could be applicable to a wider range of pyrogens and test
materials, provided that they are adequately validated for such uses.
ICCVAM Recommendations: Performance Standards
As indicated above, these test methods have not yet been adequately evaluated for theirability to detect Gram-negative endotoxin in parenteral pharmaceuticals, biological products,
and medical devices compared to the RPT or the BET. For this reason, ICCVAM does notconsider it appropriate at this time to develop performance standards that can be used toevaluate the performance of other test methods that are structurally and functionally similar.
§

In order to demonstrate the utility of these test methods for the detection of non-endotoxin pyrogens, either aninternational reference standard is needed (as is available for endotoxin [i.e., WHO-LPS 94/580 E. coli
O113:H10:K-]) or, when a positive non-endotoxin-mediated RPT result is encountered, this same sample
should be subsequently tested in vitro.
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May 2008

ICCVAM Test Method Evaluation Report: Section 1.0

1.0

May 2008

Introduction

In June 2005, the Interagency Coordinating Committee on the Validation of Alternative
Methods (ICCVAM) initiated a review of the validation status of five in vitro pyrogen test
methods proposed as replacements for the rabbit pyrogen test (RPT). The test methods were
submitted by the European Centre for the Validation of Alternative Methods (ECVAM), a
unit of the Institute for Health and Consumer Protection at the European Commission’s Joint
Research Centre. This submission was based on a validation study financed by the European
Commission within the 5th Framework Programme of Directorate General Research and wasrecently published (Hoffmann et al. 2005a; Schindler et al. 2006). The proposed test methodsare:•

The Human Whole Blood (WB)/Interleukin (IL)-1β In Vitro Pyrogen Test•

The Human WB/IL-1β In Vitro Pyrogen Test: Application of Cryopreserved(Cryo) Human WB•

The Human WB/IL-6 In Vitro Pyrogen Test•

The Human Peripheral Blood Mononuclear Cell (PBMC)/IL-6 In Vitro
Pyrogen Test•

The Monocytoid Cell Line Mono Mac 6 (MM6)/IL-6 In Vitro Pyrogen Test

For simplicity, the submitted studies are referred to collectively as the ECVAM validation
study in this document.
ICCVAM, which is charged with coordinating the technical evaluations of new, revised, andalternative test methods with regulatory applicability (ICCVAM 2000), unanimously agreed
that the five submitted in vitro test methods should have a high priority for evaluation. An
ICCVAM Pyrogenicity Working Group (PWG) was established to work with the National
Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative
Toxicological Methods (NICEATM) to carry out these evaluations; Dr. Marlies Halder was
the ECVAM liaison to the PWG. Following a NICEATM pre-screen evaluation of thecomprehensive background review documents (BRDs) submitted by ECVAM, NICEATM,ICCVAM and the ICCVAM PWG requested additional information and clarification fromECVAM on a number of issues. In March 2006, in response to this request, ECVAM
submitted revised BRDs and a list of responses to address these issues.
NICEATM, which administers ICCVAM and provides scientific support for ICCVAM
activities, subsequently prepared a comprehensive draft BRD that provided information anddata from the validation studies and scientific literature to enable a peer review of thevalidation status of each of the five in vitro test methods. A request for any other data andinformation on these test methods and for nominations to serve on an independent, scientificpyrogenicity review panel (Panel) was made through a 2005 Federal Register (FR) notice(Vol. 70, No. 241, pp. 74833-74834, December 16, 2005, available at
http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/FR/FR_E5_7410.pdf), through the ICCVAM
electronic mailing list, and through direct requests to over 100 stakeholders. Panelnominations were received, but no additional data or information was submitted in responseto this request.
1

ICCVAM Test Method Evaluation Report: Section 1.0

May 2008

Announcement of a public Panel meeting to review the validation status of the five in vitro
pyrogen test methods and availability of the ICCVAM BRD was made through a 2006 FR
notice (Vol. 71, No. 238, pp. 74533-74534, December 12, 2006, available at
http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/FR/FR_E6_21038.pdf). The draft BRD wasmade publicly available on the NICEATM/ICCVAM website (http://iccvam.niehs.nih.gov).
Additional information provided by ECVAM in response to a request from Panel wasappended to this BRD. All of the information provided to the Panel was also made publicly
available. Comments from the public and scientific community are available on the
NICEATM/ICCVAM website.
The adequacy of the data and information contained in the ICCVAM BRD to support the
ICCVAM draft test method recommendations were discussed by the Panel in a public
meeting on February 6, 2007 at the National Institutes of Health campus in Bethesda, MD. A
report of the Panel's recommendations (see Appendix A; Panel Report, available at
http://iccvam.niehs.nih.gov/docs/pyrogen/PrRevPanFinRpt.pdf) was made available forpublic comment on the NICEATM/ICCVAM website (see FR notice [Vol. 72, No. 89, pp.
26395-26396, May 9, 2007], available at
http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/FR/FR_E7_8896.pdf).
The ICCVAM draft BRD, the Panel report, and all public comments were made available to
ICCVAM’s advisory committee, the Scientific Advisory Committee on Alternative
Toxicological Methods (SACATM), for review and comment at their meeting on June 12,
2007.
ICCVAM and the PWG then considered the Panel report, all public comments, and thecomments of SACATM in preparing the final BRD and the final test method
recommendations that are provided in this ICCVAM Test Method Evaluation Report. This
report will be made available to the public and provided to U.S. Federal agencies forconsideration (ICCVAM 2000). The ICCVAM final BRD, revised in response to the Paneland PWG comments, will also be provided as background information and technical support
for this report. Agencies with applicable testing regulations and guidelines (see Appendix B)
are required by law to respond to ICCVAM within 180 days of receiving an ICCVAM test
method recommendation. These responses will be made available to the public on the
NICEATM/ICCVAM website (http://iccvam.niehs.nih.gov) as they are received.

2

ICCVAM Test Method Evaluation Report: Section 2.0

2.0

May 2008

ICCVAM Recommendations for In Vitro Pyrogen Test Methods

ICCVAM evaluated the validation status of the five in vitro pyrogen test methods as potential
replacements for the RPT. ICCVAM was unable to evaluate these tests as possible
replacements for the Bacterial Endotoxin Test (BET) because the validation studies were notdesigned for this purpose.
2.1

ICCVAM Recommendations: Test Method Uses and Limitations

The ability of the WB/IL-1β, Cryo WB/IL-1β, WB/IL-6, PBMC/IL-6, and MM6/IL-6 test
methods to detect Gram-negative endotoxin in a limited number of human parenteral drugs
have been tested in recent validation studies. The performance assessment for these five test
methods, and the drugs included in the associated validation studies are detailed in Section
3.0. Based on a review of the available data, these test methods have not been adequately
evaluated for their ability to detect Gram-negative endotoxin in parenteral pharmaceuticals,
biological products, and medical devices compared to the RPT or the BET. This is based on
the fact that the validation study only evaluated a limited range and number ofpharmaceutical products and did not evaluate the potential to detect endotoxin in biologics ormedical devices. Therefore, none of the test methods should be considered as a complete
replacement for the RPT or the BET for the detection of Gram-negative endotoxin. However,these test methods can be considered for use to detect Gram-negative endotoxin in humanparenteral drugs on a case-by-case basis, subject to product-specific validation todemonstrate equivalence to accepted pyrogen tests in accordance with applicable U.S.
Federal regulations (e.g., U.S. Food and Drug Administration [FDA] *)†. Potential users
should consider the false negative/false positive rates as well as ease of use in selecting any
test method for possible use. In addition, while the scientific basis of these test methods
suggests that they have the capability to detect pyrogenicity mediated by non-endotoxin
sources, there is insufficient data to support this broader application. Users should be awarethat the performance characteristics for these in vitro pyrogen test methods might be revised
based on additional data. Therefore, ICCVAM recommends that test method users routinely
consult the NICEATM/ICCVAM website (http://iccvam.niehs.nih.gov/) and otherappropriate sources to ensure that the most current information is considered.
2.1.1
Independent Peer Review Panel Conclusions and Recommendations
The Panel agreed that the applicable validation criteria have been adequately addressed in the
ICCVAM draft BRD in order to determine the usefulness and limitations of these test
methods to serve as a substitute for the RPT, for the identification of Gram-negative
endotoxin on a case-by-case basis, subject to product-specific validation. However, the Panelgenerally agreed that the performance of these test methods in terms of their reliability andrelevance did not support this proposed use (see Appendix A).
*

Mechanisms exist for test method developers to qualify their method on a case-by-case basis. The use of any
recommended method will be subject to product-specific validation to demonstrate equivalence asrecommended by the FDA (e.g., 21 CFR 610.9 and 21 CFR 314.50(d)(1)(ii)(a)).
†
Substances other than endotoxin may induce the cellular release of IL-1β and/or IL-6. For this reason, users ofthese test methods should be aware that the presence of other materials might erroneously suggest the presenceof endotoxin and lead to a false positive result.
3

ICCVAM Test Method Evaluation Report: Section 2.0

May 2008

While ICCVAM agreed with the Panel that these test methods cannot be considered
complete replacements for the RPT, they did recommend their use to detect Gram-negative
endotoxin in human parenteral drugs on a case-by-case basis, subject to product-specificvalidation to demonstrate equivalence to the RPT.
2.1.2
ECVAM Scientific Advisory Committee (ESAC) Statement of Validity
In March 2006, the ESAC unanimously endorsed a statement of validity for these five in
vitro pyrogen test methods, which describes their recommendations on test method uses (seeAppendix E). Like ICCVAM, ESAC concluded that these five methods can detect
pyrogenicity mediated by Gram-negative endotoxin in materials currently tested in the RPT,
and that they may be useful for regulatory decisions, subject to product-specific validation.
Both ICCVAM and ESAC also concluded that the currently available database does notsupport their use to detect a wider range of pyrogens, as was suggested in the original
ECVAM submission.
However, ESAC concluded that these tests have been scientifically validated for thedetection of pyrogenicity mediated by Gram-negative endotoxins, and quantification of thispyrogen, in materials currently evaluated and characterized by rabbit pyrogen tests. In
contrast, as described in Section 2.1, ICCVAM has concluded that the current validation
database for these test methods is inadequate to support such a definitive statement based on
the ECVAM validation study design, which did not include biologics or medical devices andevaluated only a limited range and number of pharmaceutical products and additionally did
not include parallel testing with the RPT.
2.2

ICCVAM Recommendations: Test Method Protocols

ICCVAM recommends that when testing is conducted, the in vitro pyrogen test method
protocols should be based on the standardized test method protocols provided in Appendix
C. These ICCVAM recommended protocols, summarized in Table 2-1, are based primarilyon ECVAM Standard Operating Procedures (SOPs) for each test method, with modifications
made by NICEATM and ICCVAM in an effort to standardize essential test method
components across protocols where possible. These modifications are not expected to reduce
test method performance. A table summarizing the differences between the ICCVAM
recommended protocol and the relevant ECVAM protocol/SOP is provided as an
introduction to each protocol included in Appendix C.
By comparison, the Panel concluded that the information provided in the ICCVAM draft
BRD supported the ICCVAM draft recommended protocols for these five in vitro test
methods, providing that the list of inadequacies identified by the Panel with respect toreliability and relevance are fully addressed. The revised ICCVAM recommended protocols(see Appendix C) have been updated to address many of the Panel's concerns.
Using these recommended standardized protocols will facilitate collection of consistent dataand expand the current validation database. Exceptions and/or changes to the recommended
standardized test method protocols should be accompanied by a scientific rationale. Users
should be aware that the test method protocols could be revised based on future optimization
and/or validation studies. Therefore, test method users should consult the
NICEATM/ICCVAM website (http://iccvam.niehs.nih.gov) or other appropriate sources toensure use of the most current recommended test method protocol.
4

ICCVAM Test Method Evaluation Report: Section 2.0

Table 2-1
ProtocolComponent
Test Substance
Number of
Blood Donors
Decision
Criteria forInterference

May 2008

Summary of ICCVAM Recommended In Vitro Pyrogen Test Method
Protocols
ICCVAM Recommended In Vitro Pyrogen Protocols
Cryo
WB/IL-1β
WB/IL-6
PBMC/IL-6
MM6/IL-6
WB/IL-1βTest neat or in serial dilutions that produce no interference, not to exceed the MVD
Minimum of 3 (independent or pooled)
1

Mean OD of
PPC is 50% to200% of 1.0
EU/mL EC

Mean OD of
PPC is 50% to200% of 0.5
EU/mL EC

Mean OD of
PPC is 50% to200% of 1.0
EU/mL EC
NSC (1)EC (5)
TS (14)
PPC (0)NPC (0)

Incubation
Plate(The number ofsamples or controls
measured inquadruplicate)

ELISA Plate

Assay
Acceptability
Criteria

Decision
Criteria for
Pyrogenicity

PPC2 (0)PPC (0)
NPC2 (0)NPC (0)Includes seven point IL-1β SC
and blank in duplicate

NA
Mean OD of
PPC is 50% to200% of 0.25
EU/mL EC

Mean OD of
PPC is 50% to200% of 1.0
EU/mL EC

PPC (0)NPC (0)

PPC3 (0)NPC (0)Includes seven point IL-6 SC and blank in duplicate

Mean OD of NSC ≤0.15
Quadratic function of IL-1β SC
Quadratic function of IL-6 SC
r ≥0.95
r ≥0.953
EC SC produces OD values that ascend in a sigmoidal concentration responseHigh responder
blood donors(i.e., > 200
pg/mL IL-6) orHigh responder
low responder
blood donors
blood donors(i.e., >200
NANA
NA
(i.e., Mean OD
pg/mL IL-6)
of 1EU/mL EC
may beis significantly
excludedless than that of1000 pg/mL IL6) may be
excluded
Outliers rejected using Dixon's test4
Endotoxin concentration TS > ELC5 TS

Abbreviations: Cryo = Cryopreserved; EC = Endotoxin control; ELC = Endotoxin Limit Concentration; ELISA = Enzymelinked immunosorbent assay; EU = Endotoxin units; IL= Interleukin; MM6 = Mono Mac 6; MVD = Maximum valid
dilution; NA = Not applicable; NPC = Negative product control; NSC = Negative saline control; OD = Optical density;
PBMC = Peripheral blood mononuclear cell; PPC = Positive product control; SC = Standard curve; TS = Test substance;
WB = Whole blood
1
In WB/IL-1β and MM6/IL-6 test methods, the mean OD values are corrected (i.e., reference filter reading, if applicable,
and NSC are subtracted).
2
In the ICCVAM protocols (see Appendix C), PPC and NPC are assessed in the interference test described in Section 4.2,
which is performed prior to the ELISA.
3
Correlation coefficient (r), an estimate of the correlation of x and y values in a series of n measurements.
4
Dixon 1950.
5
Where unknown, the ELC is calculated (see Appendix C).

5

ICCVAM Test Method Evaluation Report: Section 2.0

2.3

May 2008

ICCVAM Recommendations: Future Studies

ICCVAM recognizes that these test methods could be applicable for the detection of a wider
range of pyrogens (i.e., endotoxin and non-endotoxin) and test materials, provided that they
are adequately validated for such uses. Test materials identified clinically as pyrogenic might
be invaluable for use in future validation studies and might allow such studies to be
conducted without the use of animals. Wherever possible, historical data from parallel invivo/in vitro studies should be retrospectively evaluated, or parallel in vitro testing should be
conducted with RPT and/or BET tests that are performed for regulatory purposes‡. Future
validation studies should include the following considerations:
1.

Both endotoxin-spiked and non-endotoxin spiked samples should be included.
Non-endotoxin standards should be characterized prior to their use in any
study, if possible.

2.

All aspects of the studies should be compliant with Good Laboratory Practice.

3.

Future studies should include products that have intrinsic pro-inflammatory
properties in order to determine if such substances are amenable to these tests.

4.

Optimally, a study that includes 3-way parallel testing, with the in vitro assays
being compared to the RPT and the BET, should be conducted to allow for acomprehensive evaluation of the relevance and comparative performance of
these test methods. These studies may be conducted with historical RPT data
provided that the same substances (i.e., same lot) are tested in each method.
Based on ethical and scientific rationale, any in vivo testing should be limited
to those studies that will fill existing data gaps.

5.

Test substances that better represent all categories of sample types (e.g.,
pharmaceuticals, biologicals, and medical devices) intended for testing by themethods should be included.

6.

The hazards associated with human blood products should be carefully
considered, and all technical staff should be adequately trained to observe allnecessary safety precautions.

7.

Formal sample size calculations should be made to determine the required
number of replicates needed to reject the null hypothesis at a given level ofsignificance and power. For reliability assessments, formal hypothesis testingis essential with the alternative hypothesis being no difference between
groups.

The Panel agreed that any future studies should be performed using the ICCVAM proposed
protocols. Like ICCVAM, the Panel also recognized that these test methods could beapplicable to a wider range of pyrogens and test materials, provided that they are adequately
validated for such uses.
‡

In order to demonstrate the utility of these test methods for the detection of non-endotoxin pyrogens, either aninternational reference standard is needed (as is available for endotoxin [i.e., WHO-LPS 94/580 E. coli
O113:H10:K-]) or, when a positive non-endotoxin-mediated RPT result is encountered, this same sample
should be subsequently tested in vitro.
6

ICCVAM Test Method Evaluation Report: Section 2.0

May 2008

The Panel also recommended other studies for consideration:
1.

A proposed strategy for the Cryo WB/IL-1β test method is to retest if a test
fails because of too much variability. The statistical properties of thismultistage procedure should be characterized.
•

2.

The effects of direct administration of IL-1β and IL-6 to rabbits and thecomparison of the resulting pyrogenic response with endotoxin-mediatedpyrogenicity should be evaluated. In addition, the correlation of IL-1β andIL-6 levels in the in vitro tests with levels produced in rabbits using similardoses of endotoxin should be evaluated.
•

3.

ICCVAM note: ECVAM has previously commented that, "The study
design, using borderline spikes, aimed to profile differences in pyrogentests (i.e., RPT, BET, and in vitro tests), but does not reflect routine test
situations. Furthermore, the threshold chosen represents the endotoxin
limit, where 50% of the rabbits using the most sensitive rabbit strain reactwith fever." Therefore, the validation study was designed to maximallychallenge the sensitivity of the in vitro pyrogen tests. For this reason, andbecause the in vitro test methods are being recommended forconsideration on a case-by-case basis, subject to product-specificvalidation, ICCVAM has not included this specific recommendation.

A 'limit' test design protocol and a 'benchmark reference lot comparison' test
design protocol for each assay should be included.

•

2.4

ICCVAM note: This information would certainly be interesting andpossibly useful in the comparison of the responses of the in vitro humancells to that of the in vivo rabbit. However, ICCVAM did not consider thatthe information gained could justify the additional resources and animals
that would be required to perform such studies, and therefore, ICCVAM
has not included this specific recommendation.

The endotoxin-spike concentrations used for the performance assessment
studies should not be so close to the positive test concentration limit,
especially considering the relatively large enhancement and inhibition range
permitted in the sample specific qualification investigations.
•

4.

ICCVAM note: This comment, which pertains to the ECVAM Catch-Up
Validation SOP for the Cryo WB/IL-1β pyrogen test, is not relevant to the
ICCVAM recommended protocol.

ICCVAM note: Because these in vitro test methods are being
recommended for consideration on a case-by-case basis, subject toproduct-specific validation, ICCVAM did not consider the additional
resources required to perform both study designs practical.

ICCVAM Recommendations: Performance Standards

As indicated above, these five in vitro test methods have not been adequately evaluated fortheir ability to detect Gram-negative endotoxin compared to the RPT or the BET in a
7

ICCVAM Test Method Evaluation Report: Section 2.0

May 2008

sufficient number and range of parenteral pharmaceuticals, and in no biological products andmedical devices. For this reason, it is not feasible at this time to develop performancestandards that can be used to evaluate the performance of other test methods that are
structurally and functionally similar.

8

ICCVAM Test Method Evaluation Report: Section 3.0

3.0

May 2008

Validation Status of In Vitro Pyrogen Test Methods

The following is a synopsis of the information in the ICCVAM BRD, which reviews theavailable data and information for each of the five test methods. The ICCVAM BRD
describes the current validation status of the five in vitro pyrogen test methods, including
what is known about their reliability and accuracy, the scope of the substances tested, andstandardized protocols used for the validation study. The ICCVAM BRD may be obtained
electronically from the NICEATM/ICCVAM website (http://iccvam.niehs.nih.gov/) or bycontacting NICEATM via email at niceatm@niehs.nih.gov. A hard copy of the ICCVAM
BRD may be requested by email or by mail to NICEATM, NIEHS, P.O. Box 12233, Mail
Drop EC-17, Research Triangle Park, NC 27709.
3.1

Test Method Description

According to the ECVAM submission, these in vitro pyrogen test methods are intended forthe detection of Gram-negative endotoxin contained in substances intended for parenteral use(e.g., pharmaceuticals, biologics, medical devices). These methods are based on the detectionof the release of proinflammatory cytokines (i.e., IL-1β or IL-6) from human monocytes ormonocytoid cells induced by exposure to a product contaminated with Gram-negative
endotoxin.
3.1.1
General Test Method Procedures
The in vitro pyrogen test methods measure cytokine release from monocytes or monocytoid
cells (i.e., WB, PBMCs, or the MM6 cell line) by using an enzyme-linked immunosorbent
assay (ELISA) that includes monoclonal or polyclonal antibodies specific for either IL-1β or
IL-6. The amount of endotoxin present is determined by comparing the values of endotoxin
equivalents produced by WB cells exposed to the test substance to those exposed to aninternationally harmonized Reference Standard Endotoxin (RSE)4 or an equivalent standard
expressed in Endotoxin Units (EU)/mL. A product is considered to be pyrogenic if theendotoxin concentration exceeds the Endotoxin Limit Concentration (ELC) for the test
substance.
3.1.2
Protocol Similarities and Differences
Although there are differences among the five in vitro pyrogen test methods, the basic
procedural steps are consistent across all test methods:•

The test substance is mixed with a suspension of human-derived cells.

•

The mix of cells and test product is incubated for a specific time.

•

The concentration of pro-inflammatory cytokines (e.g., IL-1β, IL-6) ismeasured with an ELISA by comparison to a standard curve.

4

RSEs are internationally harmonized reference standards (e.g., WHO-lipopolysaccharide [LPS] 94/580
Escherichia coli [E. coli] O113:H10:K-; U.S. Pharmacopeia [USP] RSE E. coli LPS Lot G3E069; USP RSE E.
coli Lot G; FDA E. coli Lot EC6). Equivalent endotoxins include commercially available E. coli-derived LPS
Control Standard Endotoxin or other E. coli LPS preparations that have been calibrated with an appropriate
RSE.
9ICCVAM Test Method Evaluation Report: Section 3.0

May 2008•

The endotoxin content is calculated by comparing the measured concentrationof pro-inflammatory cytokines to an endotoxin standard curve.

•

A test substance is considered pyrogenic if the estimated endotoxin
concentration of the test substance exceeds the ELC for the test substance.

3.2

Validation Database

The test substances selected for use in the validation studies were marketed parenteralpharmaceuticals. No biological or medical device products were included in the validation
study. A total of 13 test substances were included in the performance analysis of each of thefive in vitro test methods. Ten substances (Table 3-1), each spiked with four concentrationsof endotoxin (0, 0.25, 0.5, and 1.0 EU/mL, with 0.5 EU/mL tested in duplicate), were used toevaluate accuracy. Three substances (Table 3-2), each spiked with three concentrations ofendotoxin (0, 0.5, and 1.0 EU/mL, with 0 EU/mL tested in duplicate), were used to assess
intralaboratory reproducibility. Interlaboratory reproducibility was evaluated in two different
studies. The first study tested the substances listed in Table 3-2 in triplicate in each of threelaboratories. In the second study, interlaboratory reproducibility was tested using thesubstances in Table 3-1, which were tested once in each of three laboratories.
Table 3-1

Parenteral Drugs Used in the Validation Studies for Determining Test
Method Accuracy1
ActiveIngredient
Metoprolol
tartrate
Ampicillin
Ethanol
Dimetindenmale
at

Astra
ZenecaGrünenthal
B. Braun

GlucoseEifelfango

Metoclopramid

Hexal

117EL2
2465Z01
21402
268033
1162
31323
21JX22Oxytocin

Hoechst

W015

Sostril®

Ranitidine

Glaxo
Wellcome

1L585B
3H01N3

Syntocinon®

Oxytocin

NovartisS00400Drug A - 0.9% NaCl
Drug B - 0.9% NaCl

0.9% NaCl
0.9% NaCl--

Test Substance2
Beloc®
Binotal®
Ethanol 95%
Fenistil®
Glucose 5%
MCP®
Orasthin®

SourceNovartis

Lot
Number(s)DA419A1

IndicationHeart
dysfunction
Antibiotic
Diluent

MVD(-fold)14014035Antiallergic

175

Nutrition

70

Antiemetic
Initiation ofdelivery

350

Antiacidic

140

Initiation ofdelivery
-

700

35
70

Abbreviations: MVD = Maximum valid dilution
1
Each substance was tested in all five in vitro pyrogen test methods.
2Each test substance was spiked with 0, 0.25, 0.5, or 1.0 Endotoxin Units (EU)/mL of endotoxin (WHO-LPS 94/580 [E. coli
O113:H10:K-]), with 0.5 EU/mL tested in duplicate. Each sample contained the appropriate spike concentration when tested
at its MVD.
3
Indicates the lot numbers used in the catch-up validation study for the Cryopreserved whole blood/Interleukin-1β test
method.

10

ICCVAM Test Method Evaluation Report: Section 3.0

Table 3-2

May 2008

Parenteral Drugs Used in the Validation Studies for Determining Test
Method Reproducibility1

Test Substance2
Gelafundin®
Haemate®
Jonosteril®

SourceBraun Melsungen
AventisFresenius

Agent
Gelatin
Factor VIIIElectrolytes

Indication
Transfusion
Hemophilia
Infusion

1

Each substance was tested in all five in vitro pyrogen test methods.
Each test substance was spiked with 0, 0.5, or 1.0 Endotoxin Units (EU)/mL of endotoxin (WHO-LPS 94/580 [E. coli
O113:H10:K-]), with 0 EU/mL tested in duplicate. Each sample contained the appropriate spike concentration when tested
at its maximum valid dilution.
2

3.3

Reference Test Method Data

The historical RPT studies were conducted at the Paul Ehrlich Institut (PEI), which supports
regional German regulatory authorities, provides marketing approval of certain marketedbiological products (e.g., sera, vaccines, test allergens), and functions as a World HealthOrganization (WHO) collaborating center for quality assurance of blood products and in vitro
diagnostics. The unit for pyrogen and endotoxin testing of the PEI is accredited following theInternational Organization for Standardization (ISO) and the International ElectrotechnicalCommission 17025 (ISO 2005). In a request for additional information from ECVAM, it wasstated that the RPT data was generated according to the European Pharmacopeia (EP)monograph, but the detailed protocol used by this laboratory was not provided.
These data were generated for internal quality control studies from 171 rabbits (Chinchilla
Bastards). Chinchilla Bastards are reported to be a more sensitive strain than the New
Zealand White rabbit strain for pyrogenicity testing (Hoffmann et al. 2005b). However, the
USP (USP 2007) and the EP (EP 2005) do not prescribe a specific rabbit strain for the RPT.
3.4

Test Method Accuracy

The ability of the in vitro pyrogen test methods to correctly identify the presence of Gramnegative endotoxin was evaluated using parenteral pharmaceuticals spiked with endotoxin(WHO-LPS 94/580 [E. coli O113:H10:K-]). As described in Section 3.2, 10 substances (seeTable 3-1) spiked with four concentrations of endotoxin (i.e., 0, 0.25, 0.5, or 1.0 EU/mL,
with 0.5 EU/mL tested in duplicate) were used for the evaluation. The individual spike
concentrations in each substance were tested once, using each test method, in three different
laboratories, providing a total of 150 runs (i.e., 10 substances x 5 spike solutions x 3laboratories = 150). Outliers were identified using Dixon's test (i.e., significance level of α =0.01) and subsequently excluded from the evaluation, which resulted in fewer than a total of
150 runs per evaluation (Dixon 1950; Barnett et al. 1984). A comparison of the results for thein vitro test methods indicates that the number of runs excluded was greatest for the Cryo
WB/IL-1β and WB/IL-1β (plate method) test methods, which had 30 and 11 runs excluded,
respectively. No other test method had more than three runs excluded.
As described in Section 3.3, no RPTs were conducted in parallel with the in vitro pyrogentest methods during the ECVAM validation studies. Instead, historical RPT data from rabbits
tested with endotoxin were used to establish a threshold pyrogen dose (i.e., the endotoxin
11

ICCVAM Test Method Evaluation Report: Section 3.0

May 2008

dose at which fever was induced in 50% of the rabbits). This historical data were
subsequently used to establish the limit of detection (i.e., 0.5 EU/mL) that the in vitro test
methods being validated must meet. Accordingly, the in vitro call was compared to the "truestatus" (based on the known endotoxin spike concentration) of the sample. The resulting calls
were used to construct 2x2 contingency tables, which were used to calculate the resulting test
method performance values.
The accuracy of each in vitro pyrogen test method for correctly identifying samples spiked
with 0.5 or 1.0 EU/mL endotoxin as positive and samples spiked with 0 or 0.25 EU/mL
endotoxin as negative was evaluated. As provided in Table 3-3, accuracy ranged from 81%
to 93%, sensitivity ranged from 73% to 99%, specificity ranged from 77% to 97%, false
negative rates ranged from 1% to 27%, and false positive rates ranged from 3% to 23%.
Table 3-3

Accuracy of In Vitro Pyrogen Test Methods1

Test
MethodCryo
WB/IL-1βAccuracy2

Sensitivity3

Specificity492%(110/120)93%(138/148)93%(140/150)87%(130/150)92%(136/148)
81%(119/147)

97%(75/77)96%(85/89)92%(83/90)93%(84/90)89%(79/89)73%(64/88)93%(129/139)99%(83/84)

MM6/IL-6
PBMC/IL-6
PBMC/IL-6(Cryo)7
WB/IL-6
WB/IL-1β(Tube)
WB/IL-1β(96-wellplate)881%(35/43)90%(53/59)95%(57/60)77%(46/60)97%(57/59)93%(55/59)

False Negative
Rate5
3%(2/77)
5%(4/89)
8%(7/90)
7%(6/90)
11%(10/89)27%(24/88)

False Positive
Rate6
19%
(8/43)
10%(6/59)
5%(3/60)
23%(14/60)3%(2/59)
7%(4/59)

84%(46/55)1%(1/84)

16%(9/55)

Abbreviations: Cryo = Cryopreserved; EU/mL = Endotoxin units per milliliter; IL = Interleukin; MM6 = Mono Mac 6;
PBMC = Peripheral blood mononuclear cells; WB = Whole blood
1
Data shown as a percentage (number of correct runs/total number of runs), based on results of 10 parenteral drugs tested ineach of three different laboratories. Samples of each drug were tested with or without being spiked with a Gram-negative
endotoxin standard (0, 0.25, 0.5, or 1.0 EU/mL, with 0.5 EU/mL tested in duplicate).
2
Accuracy = the proportion of correct outcomes (positive and negative) of a test method.
3
Sensitivity = the proportion of all positive substances that are classified as positive.
4
Specificity = the proportion of all negative substances that are classified as negative.
5
False negative rate = the proportion of all positive substances that are falsely identified as negative.
6
False positive rate = the proportion of all negative substances that are falsely identified as positive.
7
A modification of the PBMC/IL-6 test method that uses Cryo PBMCs.
8
A modification of the WB/IL-1β test method that uses 96-well plates instead of tubes for the test substance incubation.

3.5

Test Method Reliability

Intralaboratory repeatability was evaluated by testing saline spiked with various
concentrations of endotoxin (0, 0.06, 0.125, 0.25, 0.5, and 1.0 EU/mL) and then evaluating
the closeness of agreement among OD readings for cytokine measurements at eachconcentration. For each test method, each experiment was conducted up to three times. From
5 to 32 replicates per concentration were tested and results indicated that variability in OD
measurements increased with increasing endotoxin concentration. However, the variability
12

ICCVAM Test Method Evaluation Report: Section 3.0

May 2008

did not interfere with distinguishing the 0.5 EU/mL spike concentration (i.e., the thresholdfor pyrogenicity) from the lower concentrations.
Intralaboratory reproducibility was evaluated using three marketed pharmaceuticals spiked
with three concentrations of endotoxin (i.e., 0, 0.5, and 1.0 EU/mL, with 0 EU/mL tested in
duplicate). Three identical, independent runs were conducted in each of the three testinglaboratories, with the exception of the Cryo WB/IL-1β test method5. The correlations(expressed as a percentage of agreement) between pairs of the independent runs (i.e., run 1
vs. run 2; run 1 vs. run 3; run 2 vs. run 3) were determined and the mean of these three values
was calculated. In all reproducibility analyses, a single run consisted of each of the products
assayed in quadruplicate. Acceptability criteria for each run included a Coefficient of
Variation (CV) analysis to remove highly variable responses from the analyses. The criterion
used to identify outliers ranged from CV <0.25 to CV <0.45, depending on the method being
considered, and was arbitrarily set based on results using saline spiked with endotoxin. As an
example, for the MM6/IL-6 test method, the CV for any single spike concentration was≤0.12, and therefore, the outlier criterion was set at 0.25. Agreement between different runs
was determined for each substance in three laboratories. As shown in Table 3-4, theagreement across three runs in an individual lab ranged from 75% to 100%.
Interlaboratory reproducibility was evaluated in two different studies. In both studies, eachrun from one laboratory was compared with all runs of another laboratory. The proportionsof similarly classified samples provide a measure of reproducibility. In the first study, theinterlaboratory reproducibility was evaluated using results from three marketed
pharmaceuticals spiked with endotoxin and tested in triplicate in each of the threelaboratories. As shown in Table 3-5, the agreement across three laboratories for each test
method, where three runs per laboratory were conducted, ranged from 58% to 86%,
depending on the test method considered (excludes the Cryo WB/IL-1β test method, whichused only one run per laboratory). However, if the WB/IL-1β tube method is excluded, therange of agreement across laboratories is 72% to 86%. In comparison, the agreement acrossthree laboratories for the Cryo WB/IL-1β test method, for which only one run per laboratory
was conducted, was 92%.
The ECVAM Cryo WB/IL-1β test method BRD stated that there was no direct assessment of intralaboratory
reproducibility because such an evaluation was performed in the WB/IL-1β test method, and the authors
assumed that variability would not be affected by the use of cryopreserved blood.
5

13

ICCVAM Test Method Evaluation Report: Section 3.0

May 2008[This Page Intentionally Left Blank]

14

ICCVAM Test Method Evaluation Report: Section 3.0

Table 3-4
Run
Comparison1
1 vs 2
1 vs 3
2 vs 3
Mean
Agreement2
across 3 runs

May 2008

Intralaboratory Reproducibility of In Vitro Pyrogen Test Methods
WB/IL-1βCryo WB/IL-1β

Lab 1

Lab 2

Lab 3

Lab 1

Lab 2

Lab 3

92%
(11/12)
83%(10/12)92%(11/12)
89%

100%
(8/8)
88%
(7/8)

ND3

ND

ND

ND

ND

ND

ND

ND

ND

NC

100%
(12/12)92%(11/12)92%(11/12)
95%

ND

ND

83%

NC

92%

ND

ND

NI4

WB/IL-6

PBMC/IL-6

MM6/IL-6

Lab 1

Lab 2

Lab 3

Lab 1

Lab 2

Lab 3

Lab 1

Lab 2

Lab 3

ND

75%
(9/12)
100%(12/12)75%(9/12)
83%

92%(11/12)92%(11/12)92%(11/12)92%

100%(12/12)100%(12/12)100%(12/12)100%

92%(11/12)100%(12/12)92%(11/12)95%

100%(12/12)100%(12/12)100%(12/12)100%

100%(12/12)92%(11/12)92%(11/12)95%

100%(12/12)100%(12/12)100%(12/12)100%

92%(11/12)92%(11/12)100%(12/12)95%

100%(12/12)92%(11/12)92%(11/12)
95%

ND

75%

92%

100%

92%

100%

92%

100%

92%

92%

Abbreviations: Cryo = Cryopreserved; IL = Interleukin; MM6 = Mono Mac 6; NC = Not calculated; ND = Not done; NI = Not included; PBMC = Peripheral blood mononuclear
cells; WB = Whole blood
1Comparison among 3 individual runs within each laboratory.
2
All possible combinations of runs among the 3 laboratories were compared.
3
Not done. The ECVAM Cryo WB/IL-1β BRD states that an assessment of intralaboratory reproducibility was performed using the WB IL-1β (fresh blood) test method, and it was
assumed that intralaboratory variability would not be affected by the change to cryopreserved blood assayed in 96-well plates.
4
Not included due to lack of sufficient data. The sensitivity criteria were not met for 1 of 3 substances in run 2, and 1 of 3 substances in run 3.

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16

ICCVAM Test Method Evaluation Report: Section 3.0

Table 3-5
Lab
Comparison1
1 vs 2
1 vs 3
2 vs 3
Mean
Agreement
across 3 labs4

May 2008

Interlaboratory Reproducibility of In Vitro Pyrogen Test Methods:
Study One
WB/IL-1β(Tube)
92%(77/84)2
77%(83/108)68%(57/84)2
79%
58%(167/288)2

Agreement Between Laboratories1
Cryo
WB/IL-6
PBMC/IL-6
WB/IL-1β
92%
72%
81%
(11/12)3(78/108)(87/108)
92%
75%
86%
(11/12)3(81/108)(93/108)
92%
97%
89%
(11/12)3(105/108)(96/108)
92%
81%
85%
92%
72%
78%
(11/12)3(234/324)(252/324)

MM6/IL-6
97%(105/108)89%(96/108)86%(93/108)90%86%(279/324)

Abbreviations: Cryo = Cryopreserved; IL = Interleukin; MM6 = Mono Mac 6; PBMC = Peripheral blood mononuclear
cells; WB = Whole blood
1
Data from three substances (see Table 3-2) spiked with endotoxin (WHO-LPS 94/580 [E. coli O113:H10:K-]) at 0, 0.5 and1.0 EU/mL, with 0 EU/mL spiked in duplicate, were tested three times in three different laboratories, with the exception of
Cryo WB/IL-1β (only the preliminary run from each laboratory used for analysis).
2
Some of the runs did not meet the assay acceptance criteria and therefore were excluded from the analysis.
3
For the Cryo WB/IL-1β test method, each substance tested only once in each laboratory.
4
All possible combinations of runs among the 3 laboratories were compared (with the exception of Cryo WB/IL-1β, which
was only tested once in each laboratory, resulting in only one possible combination per substance).

In the second study, interlaboratory reproducibility was evaluated with the same 10
substances used for evaluating accuracy. In this study, each of the substances was spiked
with four concentrations of endotoxin (0, 0.25, 0.5, and 1.0 EU/mL, with 0.5 EU/mL spiked
in duplicate) and tested once in each of three laboratories. As shown in Table 3-6, theagreement across three laboratories for each test method ranged from 57% to 88%,
depending on the test method considered. The extent and order of agreement amonglaboratories was the same for both studies; the WB/IL-1β test method showed the leastagreement (57-58%) and the Cryo WB/IL-1β test method showed the most (88-92%).

17

ICCVAM Test Method Evaluation Report: Section 3.0

Table 3-6

May 2008

Interlaboratory Reproducibility of In Vitro Pyrogen Test Methods:
Study Two

Mean

WB/IL1β(Tube)
73%(35/48)82%(40/49)70%(33/47)75%

WB/IL1β(Plate)88%(37/42)90%(35/39)92%(43/47)90%

Agreement Between Laboratories1
Cryo
WB/ILWB/IL-6 PBMC/IL-6
1β
84%
85%
84%
(38/45)(41/48)(42/50)88%
85%
86%(21/24)(41/48)(43/50)
100%
88%
90%
(25/25)(44/50)(45/50)
91%
86%
87%

Agreement
across 3 labs

57%
(27/47)85%(33/39)88%(21/24)Lab
Comparison1
1 vs 2
1 vs 3
2 vs 379%(38/48)80%(40/50)

PBMC/IL6(Cryo)
96%(48/50)76%(38/50)80%(40/50)84%
76%(38/50)MM6/IL-6
90%(45/50)90%(43/48)83%(40/48)88%
81%(39/48)Abbreviations: Cryo = Cryopreserved; IL = Interleukin; MM6 = Mono Mac 6; PBMC = Peripheral blood mononuclear cells;
WB = Whole blood
1
Data from 10 substances spiked with endotoxin (WHO-LPS 94/580 [E. coli O113:H10:K-]) at 0, 0.25, 0.5, and 1.0 EU/mL,
with 0.5 EU/mL spiked in duplicate, were tested once in three different laboratories.

3.6

Animal Welfare Considerations: Reduction, Refinement, and Replacement

The currently accepted pyrogen test methods require the use of rabbits or horseshoe crab
hemolymph. The proposed in vitro pyrogen test methods use monocytoid cells of humanorigin, obtained either from WB donations or from an immortalized cell line. The capabilityof these five in vitro assays to detect Gram-negative endotoxin suggests that they may reduceor eventually replace the use of rabbits and/or horseshoe crab hemolymph for pyrogentesting. However, at the present time, the RPT detects classes of pyrogens that have neither
been examined nor validated with the in vitro pyrogen test methods and thus, the RPT will
still be required for most test substances.
Human blood donations are required for four of the five in vitro test methods (WB/IL-1β,
WB/IL-6, Cryo WB/IL-1β, and PBMC/IL-6) proposed as replacements for the RPT, and assuch, no animals will be used when these assays are appropriate for use. While the collectionof human blood is a common medical procedure, the many aspects of human blood collection
must be considered to ensure that human donors are treated appropriately, and that suchcollection and use is in accordance with all applicable regulations, policies, and guidelines.

18

ICCVAM Test Method Evaluation Report: Section 4.0

4.0

May 2008

ICCVAM Consideration of Public and SACATM Comments

In response to three FR notices that were released between December 2005 and May 2007,
eight public comments were received (see Appendix D). Comments received in response toor related to the FR notices are also available on the NICEATM/ICCVAM website(http://ntp-apps.niehs.nih.gov/iccvampb/searchPubCom.cfm). The following sections,delineated by FR notice, provide a brief discussion of the public comments received.
4.1

Public Comments in Response to FR Notice (70FR74833, December 16, 2005):
Peer Panel Evaluation of In Vitro Pyrogenicity Testing Methods: Request forComments, Nominations of Experts, and Submission of In Vivo and In Vitro
Data

NICEATM, in an FR notice (Vol. 70, No. 241, pp. 74833-74834, December 16, 2005),
requested (1) public comments on the appropriateness and relative priority of convening an
independent peer review panel (Panel) to evaluate the validation status of five in vitro
pyrogen test methods, (2) the nomination of scientists with relevant knowledge andexperience to potentially serve on the Panel should it be convened, and (3) submission ofdata from the RPT, the BET, and in vitro pyrogenicity testing using any of the five in vitro
pyrogen test methods under consideration by NICEATM.
In response to this FR notice, NICEATM received two comments. No additional data or
information was submitted in response to this request. One nomination requested
consideration of three potential panelists.
One commenter provided a reference for an in vitro pyrogen test method that measured TNFα (Martinez et al. 2004). The comment and article were provided to the Panel. However, thereference was not included in the ICCVAM BRD because the in vitro pyrogen methods being
evaluated by NICEATM measured only IL-1β and IL-6.
A second commenter requested an expeditious review of the in vitro pyrogen test methods
and described limitations of the currently used in vivo pyrogen test methods (i.e., the RPT
and the BET). This commenter also stated that the peer review of the in vitro test methods isappropriate, necessary, and should be given extremely high priority.
4.2

Public Comments in Response to FR Notice (71FR74533, December 12, 2006):
Announcement of an Independent Scientific Peer Review Meeting on the Useof In Vitro Pyrogenicity Testing Methods; Request for Comments

NICEATM, in an FR notice (Vol. 71, No. 238, pp. 74533-4, December 12, 2006), announced(1) an independent scientific peer review meeting to evaluate the validation status of five in
vitro pyrogen test methods proposed as replacements for the RPT, and (2) the availability ofan ICCVAM draft BRD on five in vitro pyrogen test methods, which describes the current
validation status of these methods and contains all of the data and analyses supporting theircurrent validation status, and ICCVAM draft recommendations on the proposed use of thesetest methods, draft test method protocols, and draft performance standards. NICEATM
invited the submission of written comments on the ICCVAM draft BRD and on the
ICCVAM draft test method recommendations. In response to this FR notice, NICEATM
received four comments.
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One commenter expressed that it was not clear why ICCVAM was neither considering the invitro pyrogen test methods for detection of non-endotoxin pyrogens nor for replacement ofboth the RPT and the BET. The commenter suggested that exclusion of these broader uses
would minimize the impact of these test methods on reduction in animal use and urged
ICCVAM "to significantly revise its recommendations and BRD to more accurately reflect
the potential use of these methods as full replacements for both the {BET} and RPT."
Furthermore, they "strongly encouraged ICCVAM to delete the recommendation regarding
the conduct of de novo RPTs to further demonstrate in vivo/in vitro concordance." ICCVAM
appreciates the concern for the proposed limited use of these test methods. However, neither
data comparing the in vitro test methods to the BET nor data directly comparing nonendotoxin pyrogens to the BET or the RPT were included in the validation studies submitted
by ECVAM. Therefore, ICCVAM was unable to consider the in vitro test methods as
replacements for the BET or to propose the use of these test methods for non-endotoxin
pyrogens. However, ICCVAM did identify and recommend future studies that could fill thesedata gaps and in turn, potentially broaden the applicability of these test methods to thatsuggested by the commenter.
Several commenters argued that the scope of the test substances was limited and the data
provided were inadequate to support the intended use of the in vitro test methods (i.e., as acomplete replacement for the RPT). These commenters emphasized that additional testing isneeded before these test methods can be recommended for this broader application.
ICCVAM agreed with these comments, which are reflected in the ICCVAM recommended
future studies.
One commenter provided data on an alternative in vitro pyrogen test method that is based on
the measurement of reactive oxygen species from the human HL-60 promyelocytic leukemia
cell line (Blatteis 2006; Timm et al. 2006). The comment and articles were provided to the
Panel. However, these data were not included in the ICCVAM BRD because the in vitro
pyrogen methods being evaluated by NICEATM measured only IL-1β and IL-6.
4.3

Public Comments in Response to FR Notice (72FR26395, May 9, 2007): Peer
Review Panel Report on Five In Vitro Pyrogen Test Methods: Availability andRequest for Public Comments

NICEATM, in an FR notice (Vol. 72, No. 89, pp. 26395-26396, May 9, 2007), announced
the availability of the Panel report and invited the submission of written comments on thereport. In response to this FR notice, NICEATM received two comments.
One commenter indicated that several of the Panel's observations and recommendations were"nonsensical, irrelevant, or inappropriate." This commenter also expressed concern about the“random” selection of Panel members and recommended both simplification of the questions
posed to the Panel and an orientation meeting to provide the panelists with backgroundinformation and focus. It was recommended that "ICCVAM coordinate with thepharmaceutical and medical devices industry to conduct product-specific validation on a setof pre-selected products and devices to serve as further validation work." ICCVAM
appreciates comments related to the evaluation process of new alternative test methods.
ICCVAM notes that Panel members were selected from nominations received in response to
an FR notice (Vol. 70, No. 241, pp. 74833-74834, December 16, 2005), in conjunction withrecommendations from the ICCVAM PWG, which includes a liaison from ECVAM.
20ICCVAM Test Method Evaluation Report: Section 4.0

May 2008

Additionally, orientation sessions are routinely convened for the Panel to provide backgroundinformation on the ICCVAM test method evaluation process.
A second commenter outlined responses to specific comments and/or recommendations madein the Panel report. These comments provided rationale for the design of the ECVAM
validation study and summarized existing data to address many of the Panel's concerns.
ICCVAM appreciates these written responses and clarifications to specific Panel comments.
ICCVAM considered all comments prior to finalization of the ICCVAM BRD and in
preparation of the ICCVAM test method evaluation report.
4.4

Public and SACATM Comments: SACATM Meeting on June 12, 2007

The June 12, 2007 SACATM Meeting included a discussion of the ICCVAM review of thein vitro pyrogen test methods. At this meeting, three public comments and four SACATM
comments were presented.
One public commenter reiterated the written comments submitted in response to the FR
notice announcing the availability of the Panel report (see Section 4.3, first commenter).
A second public commenter (who was also the Chair of the ICCVAM peer review panel)stated that, "given more time to discuss these methods, the Panel might have been able toprovide a stronger recommendation for one or more of the assays." ICCVAM appreciates
comments related to the evaluation process and now intends to extend the time allocated forPanel meetings to ensure that sufficient time is allotted.
A third public commenter noted that the long list of future studies recommended by the Panel
were impractical and not feasible to complete, particularly considering the expense that had
already been invested in the validation effort. This commenter also provided additionalcomments relevant to the criticisms of these in vitro test methods made by the Panel (e.g., thelimitations of the in vitro methods were not fairly compared to the limitations of the RPT and
BET; only endotoxin was included in the validation study because no non-endotoxinreference standard is available; and false positives were recorded because the assays are too
sensitive). ICCVAM considered many of these comments in the revisions of the ICCVAM
BRD and in the preparation of the ICCVAM test method evaluation report.
One SACATM member expressed concern with the high false negative rates reported forsome of the assays, the proprietary issues associated with using the Novartis IL-6 ELISA, thelack of concordance assessment between the RPT and the in vitro data, and the range ofsubstances included in the validation studies. A second SACATM member provided
comments on the statistical analyses used to assess the in vitro data. ICCVAM agrees withmany of these concerns, which are reflected in the ICCVAM test method recommendations.
A third SACATM member recommended that multiple test methods not be reviewed
simultaneously. As stated above, ICCVAM plans to allocate additional time for deliberation
at Panel meetings.
A fourth SACATM member suggested the concept of "core panelists" who areknowledgeable about the ICCVAM evaluation process for ICCVAM reviews with theaddition of ad hoc experts for specific methods. ICCVAM also appreciates this suggestion
and makes every effort to include in each panel individuals with direct experience with the
ICCVAM evaluation process as well as experts in the subject matter being evaluated.
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ICCVAM Test Method Evaluation Report: Section 5.0

5.0

May 2008

References

Barnett V, Lewis T. 1984. Outliers in statistical data. In: Wiley Series in Probability andMathematical Statistics. (Barnett V, Lewis T, eds). 3rd ed. New York: John Wiley & Sons.
Blatteis CM. 2006. Endotoxic fever: new concepts of its regulation suggest new approaches
to its management. Pharmacol Ther 111:194-223.
Dixon WJ. 1950. Analysis of extreme values. Ann Math Stat 21:488-506.
EP. 2005. Biological Tests. Supplement 5.2. 2.6.8 Pyrogens. In: European Pharmacopoeia(EP). 5th ed. Strasbourg, France: Council of Europe - European Directorate for the Quality of
Medicines.
Hoffmann S, Peterbauer A, Schindler S, Fennrich S, Poole S, Mistry Y, et al. 2005a.
International validation of novel pyrogen tests based on human monocytoid cells. J Immunol
Methods 298:161-173.
Hoffmann S, Lüderitz-Püchel U, Montag T, Hartung T. 2005b. Optimisation of pyrogentesting in parenterals according to different pharmacopoeias by probabilistic modelling. J
Endo Res 11:25-31.
ICCVAM. 2000. ICCVAM Authorization Act of 2000. Public Law 106-545 (42 U.S.C. 285).
ICCVAM. 2003. ICCVAM Guidelines for the Nomination and Submission of New, Revised,
and Alternative Test Methods. NIH Publication No: 03-4508. Research Triangle Park,
NC:National Toxicology Program.
ISO. 2005. ISO/IEC 17025. General Requirements for the Competence of Testing andCalibration Laboratories. 2nd ed. Geneva:International Organization for Standardization(ISO).
Martinez V, Mitjans M, Vinardell MP. 2004. TNFα measurement in rat and human whole
blood as an in vitro method to assay pyrogens and its inhibition by dexamethasone anderythromycin. J Pharm Sci 93:2718-2723.
Schindler S, Spreitzer I, Löschner B, Hoffmann S, Hennes K, Halder M, et al. 2006.
International validation of pyrogen tests based on cyropreserved human primary blood cells.
J Immunol Methods 316:42-51.
Timm M, Hansen EW, Moesby L, Christensen JD. 2006. Utilization of the human cell line
HL-60 for chemiluminescence based detection of microorganisms and related substances.
Eur J Pharm Sci 27:252-258.
USP. 2007. Biological Tests. USP30 NF25<151> Pyrogen Test. The U.S. Pharmacopeia(USP). Rockville, MD:The U.S. Pharmacopeial Convention.

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ICCVAM Test Method Evaluation Report: Appendix A

May 2008

Appendix A
Independent Scientific Peer Review Panel Assessment
A1
A2

Peer Review Panel Report: The Use of Five In Vitro Test Methods
Proposed for Assessing Potential Pyrogenicity of Pharmaceuticals
and Other Products .............................................................................................A-3
Summary Minutes from Peer Review Panel Meeting on
February 6, 2007................................................................................................A-53

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A-2

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ICCVAM Test Method Evaluation Report: Appendix A1

May 2008

Appendix A1Peer Review Panel Report: The Use of Five In Vitro Test Methods Proposed forAssessing Potential Pyrogenicity of Pharmaceuticals and Other Products

A-3

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A-4

May 2008

ICCVAM Test Method Evaluation Report: Appendix A1

May 2008

Independent Peer Review Panel Report:
Five In Vitro Test Methods Proposed for Assessing Potential Pyrogenicityof Pharmaceuticals and Other Products

April 2007

Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM)
National Toxicology Program (NTP) Interagency Center for the Evaluation of
Alternative Toxicological Methods (NICEATM)
National Institute of Environmental Health Sciences
National Institutes of Health
U.S. Public Health Services
Department of Health and Human Services

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ICCVAM Test Method Evaluation Report: Appendix A1

May 2008

This document is available electronically at:
http://iccvam.niehs.nih.gov/docs/pyrogen/PrRevPanFinRpt.pdf

The findings and conclusions of this report are those of the Independent ScientificPeer Review Panel and should not be construed to represent the official views of
ICCVAM or its member agencies.

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ICCVAM Test Method Evaluation Report: Appendix A1

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TABLE OF CONTENTS
IN VITRO PYROGENICITY INDEPENDENT PEER REVIEW PANEL ............... iv
PREFACE ..................................................................................................................... v
EXECUTIVE SUMMARY ......................................................................................... vii
A.

REVIEW OF THE VALIDATION STATUS OF IN VITROPYROGEN TEST METHODS ......................................................................... 1

1.0

Introduction and Rationale for the Proposed Use of In Vitro Pyrogen
Test Methods.................................................................................................... 19
1.1 Introduction ............................................................................................ 19
1.2 Regulatory Rationale and Applicability................................................. 20
1.3 Scientific Basis for the In Vitro Pyrogen Test Methods ......................... 22

2.0

In Vitro Pyrogen Test Method Protocol Components .................................... 24
2.1 Overview of How the In Vitro Pyrogen Test Methods are
Conducted............................................................................................... 24
2.2 Description and Rationale for the Test Method Components for
Proposed Standardized Protocols .......................................................... 24
2.3 Basis for Selection of Test Method Systems ........................................... 26
2.4 Proprietary Components ........................................................................ 26
2.5 Number of Replicates.............................................................................. 26
2.6 Modifications to the Test Method Protocols Based on ECVAM
Validation Study Results........................................................................ 27

3.0

Substances Used for the Validation of In Vitro Pyrogen Test Methods......... 27
3.1 Rationale for the Substances or Products Selected for Testing............. 27
3.2 Number of Substances ............................................................................ 27
3.3 Identification and Description of Substances Tested............................. 28
3.4 Sample Coding Procedure ...................................................................... 28

4.0

In Vivo Reference Data for the Assessment of Test Method Accuracy.......... 28
4.1 Description of the Protocol Used to Generate In Vivo Data .................. 28
4.2 Reference Data Used to Assess In Vitro Test Method Accuracy ........... 29
4.3 Availability of Original Records for the In Vivo Reference Data.......... 29
4.4 In Vivo Data Quality ............................................................................... 29
4.5 Availability and Use of Toxicity Information from the Species ofInterest .................................................................................................... 29
4.6 Information on the Relevance and Reliability of the In Vivo Test
Methods .................................................................................................. 30

5.0

Test Method Data and Results ........................................................................ 12
5.1 Test Method Protocol.............................................................................. 30
5.2 Availability of Copies of Original Data Used to Evaluate Test
Method Performance.............................................................................. 31
5.3 Description of the Statistical Approaches Used to Evaluate theResulting Data ........................................................................................ 31
5.4 Summary of Results................................................................................ 32
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ICCVAM Test Method Evaluation Report: Appendix A1

5.5
5.6

May 2008

Use of Coded Chemicals and Compliance with GLP Guidelines.......... 33
Lot-to-Lot Consistency of Test Substances............................................ 33

6.0

Relevance of the In Vitro Pyrogen Test Methods ........................................... 34
6.1 In Vitro Pyrogen Test Method Relevance .............................................. 34
6.2 Summary of the Performance Statistics for In Vitro Pyrogen Test
Methods .................................................................................................. 34

7.0

Reliability of the In Vitro Pyrogen Test Methods........................................... 35
7.1 Selection Rationale for the Substances Used to Evaluate the
Reliability of In Vitro Pyrogen Test Methods........................................ 35
7.2 Analysis of Repeatability and Reproducibility ...................................... 35
7.3 Historical Positive and Negative Control Data ...................................... 37

8.0

Test Method Data Quality............................................................................... 37
8.1 Adherence to National and International GLP Guidelines................... 37
8.2 Data Quality Audits................................................................................ 37
8.3 Impact of Deviations from GLP Guidelines .......................................... 37
8.4 Availability of Laboratory Notebooks or Other Records...................... 38

9.0

Other Scientific Reports and Reviews ............................................................ 38
9.1 Have Relevant Data Identified in Other Published or
Unpublished Studies Conducted Using the In Vitro Test
Methods Been Adequately Considered?................................................ 38
9.2 Are the Conclusions Published in Independent Peer-Reviewed
Reports or Other Independent Scientific Reviews of the In Vitro
Test Methods Adequately Discussed and Compared?.......................... 38
9.3 Are There Other Comparative In Vitro Test Method and RPT
Data That Were Not Considered in the ICCVAM Draft BRD,
But are Available for Consideration?.................................................... 38

10.0

Animal Welfare Considerations (Refinement, Reduction, andReplacement) ................................................................................................... 39
10.1 How the Five In Vitro Pyrogen Test Methods Will Refine,
Reduce, or Replace Animal Use............................................................. 39
10.2 Requirement for the Use of Animals ..................................................... 39

11.0

Practical Considerations ................................................................................. 39
11.1 Transferability of the In Vitro Pyrogen Test Methods.......................... 39
11.2 Personnel Training Considerations ....................................................... 40
11.3 Cost Considerations ............................................................................... 40
11.4 Time Considerations .............................................................................. 40

12.0 Recommended Additional References ............................................................. 23
13.0

Summary of Validation Status of the In Vitro Pyrogen Test Methods .......... 25

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B.

REVIEW OF ICCVAM DRAFT TEST METHODRECOMMENDATIONS................................................................................. 26
1.0
Proposed Test Method Usefulness and Limitations............................ 26
2.0
Proposed Test Method Standardized Protocols.................................. 26
3.0
Proposed Test Method Performance Standards ................................. 29
4.0
Proposed Additional Studies................................................................ 30

C.

OVERALL PEER REVIEW OUTCOMES .................................................... 33

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ICCVAM Test Method Evaluation Report: Appendix A1

May 2008

IN VITRO PYROGENICITY INDEPENDENT PEER REVIEW PANEL
Karen Brown, Ph.D., (Panel Chair), DRL Pharma and Pair O’Doc’s Enterprises, Parkville,
Missouri
Brian Crowe, Ph.D., Baxter Vaccine AG, Orth an der Donau, Austria
Nancy Flournoy, Ph.D., University of Missouri-Columbia, Columbia, Missouri
Ihsan Gursel, Ph.D., Bilkent University, Bilkent, Ankara, Turkey
Ken Ishii, MD, Ph.D., ERATO, Japan Science and Technology Agency, Osaka University,
Osaka, JapanJack Levin, MD, University of California-San Francisco, San Francisco, California
Albert Li, Ph.D., In Vitro ADMET Laboratories, Rockville, Maryland
David Lovell, Ph.D., University of Surrey, Guilford, United Kingdom
Melvyn Lynn, Ph.D., Eisai Medical Research, Inc., Ridgefield Park, New Jersey
Anthony Mire-Sluis, Ph.D., AMGEN, Inc., Thousand Oaks, California
Jon Richmond, MD, Home Office, Tayside, United Kingdom
Peter Theran, V.M.D., Massachusetts Society for the Prevention of Cruelty to Animals,
Novato, California
Kevin Williams, Eli Lilly, Indianapolis, Indiana

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ICCVAM Test Method Evaluation Report: Appendix A1

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PREFACEThis document is an independent report of the In Vitro Pyrogenicity Peer Review Panel('Panel') evaluation of the validation status of five in vitro test methods for pyrogenicity
testing. The Panel was convened as a National Institutes of Health (NIH) Special Emphasis
Panel by the National Toxicology Program (NTP) Interagency Center for the Evaluation of
Alternative Toxicological Methods (NICEATM) to provide advice to the Interagency
Coordinating Committee on the Validation of Alternative Methods (ICCVAM). This reportsummarizes the discussions, conclusions, and recommendations of the Panel’s public
meeting convened at the NIH in Bethesda, MD on February 6, 2007. ICCVAM and the
ICCVAM Pyrogenicity Working Group (PWG) will consider the Panel report, along withcomments from the public and the Scientific Advisory Committee on Alternative
Toxicological Methods (SACATM), and prepare final test method recommendations for U.S.
Federal agencies. ICCVAM test method recommendations will be forwarded to U.S. Federal
agencies for consideration and action, in accordance with the ICCVAM Authorization Act of2000 (42 U.S.C. 285l-3, available at
http://iccvam.niehs.nih.gov/docs/about_docs/PL106545.pdf).
The Panel considered five in vitro test methods submitted to ICCVAM by the European
Centre for the Validation of Alternative Methods (ECVAM), a unit of the Institute for Healthand Consumer Protection (IHCP) at the European Commission’s Joint Research Centre.
ECVAM submitted background review documents (BRDs) for these test methods to
ICCVAM for consideration as replacements for the rabbit pyrogen test (RPT) in June 2005.
The proposed test methods are:•

The Human Whole Blood (WB)/IL-1 In Vitro Pyrogen Test•

The Human WB/IL-1 In Vitro Pyrogen Test: Application of Cryopreserved
Human WB•

The Human WB/IL-6 In Vitro Pyrogen Test•

The Human Peripheral Blood Mononuclear Cell (PBMC)/IL-6 In Vitro
Pyrogen Test•

An Alternative In Vitro Pyrogen Test Using the Monocytoid Cell Line Mono
Mac 6 (MM6)/IL-6

ICCVAM established an ICCVAM PWG to work with NICEATM to carry out theevaluation of these test methods. The ICCVAM PWG developed draft test method
recommendations and questions for consideration by the Panel. The ICCVAM PWG alsocollaborated closely with ECVAM throughout the evaluation process to obtain additional
information for consideration by the Panel and ICCVAM.
The Panel was provided a comprehensive draft BRD prepared by NICEATM in conjunction
with the PWG and ICCVAM. The draft BRD provided all available data and information
related to the five in vitro pyrogen test methods. The five ECVAM submitted BRDs (one foreach test method), the ECVAM response to PWG questions, and other supplemental
information (i.e., key references and testing guidelines/regulations for pyrogenicity testing)were appended to the draft BRD. All of the information provided to the Panel was also madeA-11

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publicly available, and public comments were requested via a Federal Register (FR) notice(Vol. 71, No. 238, pp. 74533-74534, 12/12/06). The FR notice also announced the public
ICCVAM independent peer Panel review meeting scheduled for February 6, 2007.
The Panel was charged with:•

Reviewing the ICCVAM draft BRD for completeness and to identify any
errors or omissions in the draft BRD•

Evaluating the information in the draft BRD to determine the extent to which
each of the applicable criteria for validation and acceptance of toxicologicaltest methods (ICCVAM 20031) have been appropriately addressed•

Considering the ICCVAM draft test method recommendations for thefollowing and commenting on the extent to which they are supported by the
information in the draft BRD:o proposed test method uses
o proposed recommended standardized protocols
o proposed test method performance standards
o proposed additional studies

At the Panel’s public meeting on February 6, 2007, the Panel made recommendations forcorrections and additions to the draft BRD and then discussed the current validation status ofthese five in vitro test methods. The Panel also commented on the ICCVAM draft test
method recommendations for proposed test method uses, recommended standardized
protocols, test method performance standards, and additional studies. The public wasprovided the opportunity to comment several times during the meeting. The Panel consideredthese comments as well as public comments submitted in advance of the meeting before
concluding their deliberations.
The Panel gratefully acknowledges the efforts of NICEATM staff in coordinating thelogistics of the peer review Panel meeting and in preparing materials for the review. The
Panel also thanks Dr. Thomas Hartung (Head of ECVAM) for providing an overview of thetest methods and for additional clarifications at the meeting. Finally, as Panel Chair, I want tothank each Panel member for their thoughtful and objective review of these test methods.

Karen Brown, Ph.D.
Chair, In Vitro Pyrogenicity Peer Review Panel
April 2007

1

ICCVAM. 2003. ICCVAM Guidelines for the Nomination and Submission of New, Revised, and Alternative
Test Methods. NIH Publication No. 03-4508. Research Triangle Park, NC. NIEHS. The guidelines can beobtained at http://iccvam.niehs.nih.gov/SuppDocs/SubGuidelines/SD_subg034508.htm.
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EXECUTIVE SUMMARY
This report describes the conclusions and recommendations of the In Vitro Pyrogenicity Peer
Panel ('Panel') regarding the validation status of five in vitro pyrogen test methods1, and theability of these test methods to individually serve as a substitute for the Rabbit Pyrogen Test
(RPT) for the identification of Gram-negative endotoxin on a case-by-case basis, subject toproduct specific validation. The test methods are:•

The Human Whole Blood (WB)/IL-1 In Vitro Pyrogen Test•

The Human WB/IL-1 In Vitro Pyrogen Test: Application of Cryopreserved
Human WB•

The Human WB/IL-6 In Vitro Pyrogen Test•

The Human Peripheral Blood Mononuclear Cell (PBMC)/IL-6 In Vitro
Pyrogen Test•

An Alternative In Vitro Pyrogen Test Using the Monocytoid Cell Line Mono
Mac 6 (MM6)/IL-6

Panel Recommendations for the ICCVAM Background Review Document
The Panel stated that, in general, the information presented in the ICCVAM draft
Background Review Document (BRD) was sufficient for its purpose. Exceptions areincluded within the body of the Panel report. The Panel identified a number of sections whereclarification or a more comprehensive explanation would improve the ICCVAM draft BRD.
For example, the extent to which the RPT is currently performed when risk assessments andregulatory decisions are concerned only with the presence of endotoxin should be provided.
Likewise, a more detailed review of the various mechanisms and processes thought to be
involved in the actual induction of fever itself, and a more detailed description of thestatistical approaches used to evaluate the resulting data would be helpful. The Panel statedthat the rationale for the selected test substances was neither appropriate nor acceptable andthey recommended the inclusion of non-endotoxin pyrogens, protein- and lipid-containing
materials that are used parenterally, and 'classical' examples of biological products andmedical devices. The Panel also requested that the formal validation statement from the
ECVAM Scientific Advisory Committee (ESAC) (and the supporting documents) beappended to the ICCVAM BRD. The Panel agreed that a comprehensive summary offindings on overall conclusions about the usefulness and limitations of each of the in vitro
pyrogen tests compared to the Bacterial Endotoxin Test (BET) or the RPT should be includedin the ICCVAM final BRD.
With regard to animal welfare, the Panel suggested that the ICCVAM final BRD provide
information on the number of rabbits used for pyrogenicity testing to permit an accurate
assessment of the actual impact on animal use. The Panel recommended that the ICCVAM
1

These test methods are referred to in this report as in vitro pyrogen tests in order to maintain consistency withthe designation provided by the test methods' submitter (ECVAM). However, the Panel noted that thisdesignation may be inappropriate because the usefulness and limitations for these test methods have been
defined only for their ability to detect bacterial endotoxin and not other pyrogens.
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final BRD discuss the practice of, and the U.S. Federal restrictions on, the reuse of rabbits inpyrogenicity testing, as well as the availability and use of the recombinant clotting factor C(rFC) that could replace the need for horseshoe crab hemolymph. The Panel also felt that thelack of direct parallel testing in rabbits with the products tested in the validation study was asignificant limitation to the study design.
The Panel concluded that the cost and logistical considerations involved in conducting a
study using the in vitro test methods were incompletely stated. The Panel recommended thata more detailed cost comparison for conducting the RPT and the in vitro test methods be
performed. The Panel also commented that both the cost and logistical problems associatedwith the need to harvest and use human blood in four of the test methods were understated.
Validation Status of the In Vitro Pyrogen Test Methods
The Panel agreed that the applicable validation criteria have been adequately addressed in the
ICCVAM draft BRD in order to determine the usefulness and limitations of these test
methods to serve as a substitute for the RPT, for the identification of Gram-negative
endotoxin on a case-by-case basis, subject to product specific validation. However, the Panelgenerally agreed that the performance of these test methods in terms of their reliability andrelevance did not support this proposed use. A minority opinion (Dr. Peter Theran) suggested
that the qualification in the above statement (i.e., that uses were subject to product specificvalidation) should allow for these test methods to be used for the specified purpose. A second
minority opinion (Drs. Karen Brown, Albert Li, and Jon Richmond) expressed concern that it
is not clear that the qualification included in the above statement would preclude the use of
the in vitro test methods as replacements for the RPT in those circumstances where the BET
is currently serving to replace the RPT.
Review of the Interagency Coordinating Committee on the Validation of Alternative
Methods (ICCVAM) Draft Recommendations for Test Method Usefulness andLimitations
The Panel concluded that the available data and demonstrated performance of these five in
vitro test methods, in terms of their relevance2 and reliability3, did not support the ICCVAM
draft recommendations in terms of their usefulness and limitations. The Panel felt that theusefulness of these test methods for detecting Gram-negative endotoxin has not been
properly assessed for concordance with the RPT or for relevance in comparison to the BET,
and therefore, it was not possible to truly assess their usefulness and limitations.
One minority opinion stated (Dr. Peter Theran): This Panel has considered the failure toundertake additional RPTs a significant flaw in this validation study and therefore proposed
that, in the future, similar validation studies should use the RPT to provide concordance data.
I have no objection to the performance of in vitro tests in parallel with rabbit tests, which are
already scheduled to be performed, in order to achieve concordance data. But, it is my2

The extent to which a test method correctly predicts or measures the biological effect of interest in humans oranother species of interest. Relevance incorporates consideration of the "accuracy" or "concordance” of a test
method.
3
A measure of the degree to which a test method can be performed reproducibly within and among laboratories
over time. It is assessed by calculating intra- and inter-laboratory reproducibility and intralaboratory
repeatability.
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opinion, that any recommendation for de-novo parallel RPT should be accompanied by astatement, as follows: “The use of rabbits in new parallel tests for the validation of an in-vitro
test should only be conducted after a vigorous search for a scientifically sound, non-animalalternative (i.e., the need for additional animal studies must be justified on a case-by-casebasis).” The inclusion of this statement would reinforce the importance of the 3R’s andwould serve as a reminder of U.S. Federal law.
Review of the ICCVAM Draft Recommendations for Test Method Standardized
Protocols
The Panel agreed that the information provided in the ICCVAM draft BRD supported the
ICCVAM draft recommended protocols for these five in vitro test methods, providing thatthe list of inadequacies4 identified by the Panel with respect to reliability and relevance are
fully addressed.
Review of the ICCVAM Draft Recommendations for Test Method PerformanceStandards
The Panel did not support the statement that the available data and demonstrated
performance in terms of relevance and reliability supported the ICCVAM draft
recommendations for these in vitro test methods in terms of their performance standards. The
Panel noted several inadequacies with regard to the essential test method components foreach in vitro test method and agreed that the demonstrated performance of certain aspects ofseveral of the assays, particularly in terms of relevance, yielded some concern. With regard
to the minimum list of reference substances, the Panel agreed that if the intent of the proposal
was to replace the RPT with one or more of the in vitro test methods under consideration,then the in vitro test methods must be validated for all classes of substances (e.g.,
pharmaceuticals, biologicals, and implants) and medical devices that are tested with the RPT.
The same minority opinion directed towards the issue of parallel testing using the RPT as
detailed above was expressed.
Review of the ICCVAM Draft Recommendations for Future Studies
The Panel agreed that to better determine the relevance of these in vitro test methods, theproposed additional studies should be performed using the ICCVAM proposed protocols,
taking into account the Panel's comments and recommendations. The Panel also agreed that
if the intended use of the in vitro assays were only to detect Gram-negative endotoxin, it
would seem critical to include parallel studies with the BET in any future validation efforts.
However, if the intended use of the in vitro methods is to evaluate substances containing
endotoxin that are unable to be evaluated with the BET, then the parallel testing studies
should include the RPT. The Panel recognized that these test methods could be applicable toa wider range of pyrogens and test materials, provided that they are adequately validated forsuch uses.

4

Based on the list of 20 separate inadequacies outlined in this report, three Panel members felt that thislist would be better described as a list of "many and substantial" inadequacies.
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The same minority opinion directed towards the issue of parallel testing using the RPT as
detailed above was expressed.

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OVERALL PEER REVIEW OUTCOMES
This international independent Peer Review Panel, consisting of 13 expert scientists fromfive different countries, provided comments and recommendations on the usefulness andlimitations of five in vitro pyrogen test methods for the detection and quantification of Gramnegative endotoxin and on the ICCVAM draft test method recommendations on the use of
these in vitro methods as partial replacements for the RPT. These remarks are summarized
below.
• In general, the information presented in the ICCVAM draft BRD was sufficient forthe purpose of determining the usefulness and limitations of these test methods fortheir proposed use and for adequately addressing the applicable validation criteria on
the basis of the currently available evidence.
• The available data and demonstrated performance in terms of their reliability andrelevance do not at this time support the ICCVAM draft proposed use for these test
methods (i.e., as a partial substitute or replacement for the RPT, for the identificationof Gram-negative endotoxin, on a case-by-case basis, subject to product specificvalidation). To better characterize the test methods and more clearly define theirreliability and relevance, the Panel recommended that specific additional studies be
performed using the ICCVAM proposed protocols, taking into account the Panel'scomments and recommendations.
o The lack of parallel testing in the in vitro tests and the RPT, and the resulting
lack of concordance data, was considered to be a major limitation of thevalidation study design. For this reason, the Panel recommended that futurestudies include parallel testing. A minority opinion (Dr. Peter Theran)associated with parallel testing was expressed as follows: “The use of rabbits
in new parallel tests for the validation of an in-vitro test should only be
conducted after a vigorous search for a scientifically sound, non-animalalternative (i.e., the need for additional animal studies must be justified on acase-by-case basis)".
• The available data and demonstrated performance in terms of their reliability andrelevance does not support the ICCVAM draft performance standards for these invitro test methods for regulatory purposes.
• The information provided in the ICCVAM draft BRD supports the ICCVAM draft
recommended protocols for these five in vitro test methods, providing that the list of
inadequacies5 identified by the Panel with respect to reliability and relevance are fully
addressed.
• These test methods could be applicable to a wider range of pyrogens and test
materials, provided that they are adequately validated for such uses.
• It is critical to recognize, despite concerns about the performance of these five in vitro
test methods, that a formal process exists for materials regulated under 21 CFR 610.9

5

Based on the list of 20 separate inadequacies outlined in this report, three Panel members felt that this list
would be better described as a list of "many and substantial" inadequacies.
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to qualify these in vitro methods for the identification of Gram-negative endotoxin ona case-by-case basis, subject to product specific validation.

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A.

REVIEW OF THE VALIDATION STATUS OF IN VITRO PYROGEN TESTMETHODS

1.0

INTRODUCTION AND RATIONALE FOR THE PROPOSED USE OF IN
VITRO PYROGEN TEST METHODS1

1.1

Introduction

1.1.1

Is the historical background provided for the in vitro pyrogen test methods and therationale for their development adequate?
Yes, the Preface, the Executive Summary and Section 1.1.1 of the ICCVAM draft
Background Review Document (BRD) are all informative, clear, and concise with thefollowing exceptions:1.

The action of pyrogens on circulating cells and the mechanism by which thepro-inflammatory cytokines produce pyrexia should be considered in Section
1.1.1 instead of in Section 1.3.2).

2.

The reduction in the use of animals to test medicinal products produced undercurrent Good Manufacturing Practices (GMP) is an obvious goal. However,no information is provided on the current use of the Rabbit Pyrogen Test
(RPT) or the bacterial endotoxin test (BET) (i.e., the approximate number of
rabbits and horseshoe crabs used each year for pyrogen testing), or of
anticipated trends in their use, or of the extent to which the RPT is currently
used in contexts where risk assessments deem endotoxin to be the only
relevant contaminant.

3.

On lines 694-696 of the ICCVAM draft BRD (December 1, 2006), it is statedthat the proposed in vitro tests were selected for their ability to replace the
RPT. In the previous paragraph, it is stated that the RPT is capable ofdetecting both endotoxin and non-endotoxin pyrogens. Elsewhere, it is notedthat these in vitro tests have not been validated for detecting non-endotoxin
pyrogens. If the aim of testing these materials with the RPT is to detect a
range of pyrogens, then these assays cannot, on the basis of information
supplied in the validation dossier, completely replace the RPT.

4.

A more detailed review of the various mechanisms and processes thought tobe involved in the actual induction of fever itself, particularly in the case ofdrugs that are not administered intravenously, would have been useful. A
number of reviews on this subject describe a far more complex picture thanpresented. These additional references include:
Netea et al. (2000) and Saper and Breder (1994).

1

These test methods are referred to in this report as in vitro pyrogen tests in order to maintain consistency withthe designation provided by the test methods' submitter (ECVAM). However, the Panel noted that thisdesignation may be inappropriate because the usefulness and limitations for these test methods have been
defined only for their ability to detect bacterial endotoxin and not other pyrogens.
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1.1.2
Is the previous review of the ECVAM validation studies adequately summarized?
Yes, the previous review of the ECVAM validation studies was adequately summarized. The
questions resulting from the initial review have been answered and included in the ICCVAM
draft BRD. However, it would have been better if the actual ESAC validation statement infull had been appended, as well as any documents used to support the ESAC conclusion. The
ECVAM BRDs (though not the ESAC statement) contain inconsistent text relating to thepossible practical uses of the novel tests that the validation tests were intended to support.
1.2

Regulatory Rationale and Applicability

1.2.1

Are the current regulatory testing requirements and ICCVAM prioritization criteriaadequately discussed and up-to-date?
Yes, the current United States (U.S.) and European Union (EU) regulatory testing
requirements are properly referenced and the relevant documents have been supplied. The
previous product specific acceptance of peripheral blood mononuclear cell (PBMC) data by
the U.S. Food and Drug Administration (FDA) is also mentioned in the Executive Summary.
Inclusion of the following information would have been useful:1.

It should be stated whether the acceptance of the PBMC data by the FDA wasa replacement for the BET or the RPT. The document 21 CFR 610.9 provides
for the use of alternative methods to test for pyrogenic substances as long as
the use of these methods does not compromise the safety, purity or potency ofthe product. The 1987 FDA guideline on the validation of the BET as an endproduct endotoxin test for human and animal parenteral drugs also sets forth
acceptable conditions for the use of the test in lieu of the RPT. However, no
mention is made of the fact that the European Directorate for the Quality of
Medicines (EDQM) also has a working party of experts (apparently
independent of ECVAM and ESAC) reviewing the whole area of in vitro
pyrogens tests and their potential use.

2.

The ICCVAM final BRD should discuss the availability and use of the rFC
that could replace the need for horseshoe crab hemolymph.

3.

The ICCVAM draft BRD gives few insights into how any recommendations,following acceptance by the relevant agencies, would be incorporated into
U.S. Pharmacopeia (USP) and European Pharmacopeia (EP) test
requirements.

Specific comments on the five ICCVAM prioritization criteria outlined in the ICCVAM draft
BRD:
Criterion 1 (Applicability to regulatory testing needs and multiple
agencies/programs): It is clear that the test methods are relevant to the end-producttesting of a variety of healthcare products (for endotoxin) and that the FDA is theprincipal U.S. regulator for such products.
Criterion 2 (Warranted, based on extent of expected use or application andimpact): It is clear from the documents that this criterion is only met with respect
to the detection of endotoxin.
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Criterion 3 (Potential to address any/all of the 3Rs): The tests have the potential toreduce or replace animal use and the associated morbidity and mortality. However,no information is provided in the ECVAM BRDs or in the ICCVAM draft BRD topermit the actual impact on animal use to be accurately assessed.
Criterion 4 (Potential to provide improved prediction): The documents indicatethat the level of protection provided by each of the in vitro test methods isequivalent to that provided by the RPT. However, in the original ECVAM BRDs,
it is recognized that sensitivity may have been underestimated and specificity
overestimated as a consequence of having one of the spiked-sample points set atthe regulatory limit. On lines 777-784 of the ICCVAM draft BRD (December 1,2006), the statement that these methods would better predict the human pyrogenic
response than the RPT because they use human cells is not supported by test
results in the ICCVAM draft BRD. In contrast, it is stated on lines 1299-1303 of
the ICCVAM draft BRD (December 1, 2006) that the pyrogenic response toendotoxin in rabbits and humans is “similar in both species. Based on thesestudies, the rabbit is considered to be predictive of the human response (and may
often overpredict the response).”
Criterion 5 (Other advantages): The new test methods clearly take longer toproduce definitive results. However, no animal facility is required. It was asurprise (in the absence of definitive cost information) that the novel tests wereconsidered to be potentially more expensive than the RPT. Contract researchorganizations should be consulted on potential cost comparisons, as wide
acceptance of these methods may in part be cost-dependent.
1.2.2
Is the description of the intended uses of the in vitro pyrogen tests complete?
These methods are proposed as partial replacements for the RPT. The RPT detects both
endotoxin and non-endotoxin pyrogens, but the in vitro pyrogen tests have not been validated
for non-endotoxin pyrogens. Therefore, they cannot be considered complete replacements for
the RPT.
It is not clear when, or in which situations, the in vitro pyrogen test methods would beappropriate for use. The BET detects endotoxin in most cases and is used instead of the RPT
for this purpose. The application of the in vitro test methods for the detection of endotoxin insample types that cannot be measured in the BET is plausible; however, this proposed use
would represent a very limited application for the in vitro pyrogen tests.
1.2.3 Are the similarities and endpoints measured by the proposed test methods and thereference (RPT) test method adequately described and discussed?
Yes, although the exact causes of the endpoint of the RPT (i.e., fever) are relatively complex
and unclear, it has been known for many years that cytokines, especially those involved in
the inflammatory response (i.e., IL-1, IL-6, and TNF) can induce febrile reactions. The
development of tests based on the production of such cytokines from human white blood
cells or cell lines appears to correlate well with the induction of fever in both the RPT andhumans. However, the RPT detects a whole organ/body fever response; whereas, theproposed test methods detect only cytokine secretion. Evidence to suggest that detection of
IL-1 or IL-6 is necessarily an indication of a febrile reaction is lacking. Additional
information should be included in the ICCVAM final BRD on the relationship between IL-1
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or IL-6 levels produced in cultures of monocytes and the development of fever in humans.
The fact that the cytokine profiles for different endotoxins may vary between rabbits andhumans should also be considered.
1.2.4

Is the description of the use of the proposed test methods in an overall strategy ofhazard or safety assessment adequate?
Yes, the utility of the in vitro pyrogen methods as an addition to the current RPT, especiallywhere non-endotoxin pyrogens are involved, has been clearly discussed. No specific claims
are made for an immediate replacement of the RPT, although future studies may lead to suchan event. The overall demonstration of the applicability of the methods to non-endotoxin
detection is a stated goal. However, this goal does not appear to adequately match themethods employed since non-endotoxin standards were not used. One information gap (in the
ICCVAM draft BRD and ECVAM BRDs) is the extent to which the RPT is currently
performed when risk assessments and regulatory decisions are concerned only with thepresence of endotoxin (that is clearly the intention when only the BET is used). Product-byproduct validation will be required and the full extent of materials for which the new tests are
not suited remains to be defined.
1.3

Scientific Basis for the In Vitro Pyrogen Test Methods

1.3.1

Is the purpose and mechanistic basis of the in vitro test method(s) adequately
described and compared to known and/or suspected mechanisms/modes of action
for fever production in humans?
Yes, the purpose and mechanistic basis of the in vitro test methods appears to be adequate
while acknowledging that, at this point, the reference standard included in the validation
study was Gram-negative endotoxin only. The mechanisms underlying fever induction,
including the production of cytokines involved in the inflammatory cascade, appear to beimportant. The administration of such cytokines can directly induce fevers and their levels
have been shown to dramatically increase during fevers. However, the known and suspectedmechanisms/mode of action of fever may be far more complex than that described (see also
response to Section 1.1.1).
The claim in Section 1.3.1 to 'identify pyrogens' should perhaps be restated to 'detect
pyrogens.'
1.3.2 Are the known similarities and differences of modes of action between the in vitro
pyrogen test methods and the fever response in human and/or rabbits adequately
considered?
Yes, an extensive literature search was performed that covered a wide range of cases
illustrating the similarities and differences between the modes of action between the RPT, thein vitro pyrogen tests, and the induction of fever in humans. The correlation, or lack thereof,
between the tests and human fever induction has been discussed in a scientifically validmanner. It should be noted that the RPT has served as a good predictor of human pyrogen
response. Although there are false positives and false negatives associated with the RPT, it is
not clear that these proposed in vitro assays provide better, similar, or worse results. A major
concern is the lack of validation of these new assays directly compared to the RPT.
The mode of action is oversimplified. See response to Section 1.1.1, especially the reference
to Netea et al. (2000) that provides an excellent review on the multiple-pathway mechanisms
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that link cytokine responses (some of which are monitored by the proposed in vitro assays)
and fever production. Furthermore, no description of the mode of action at either themolecular or cellular level is presented, which may prevent an adequate comparison between
the methods. Specific questions that should be addressed include:1.

Are there any scientific data that compare IL-1 and IL-6 production and fever
response between humans and rabbits?

2.

Is the induction of IL-1 and IL-6 (or even fever) similar or different between
endotoxin and other known TLR-4 ligands?

3.

What is the mechanism of action for pyrogens that do not utilize TLR-4?

4.

TLR-4-mediated IL-1 mRNA induction and the consequent release of mature
IL-1 from cells by stimuli other than pyrogens are regulated by different
molecular mechanisms. Are these mechanisms similar or different in vitro andin vivo, or between humans and rabbits?

1.3.3 Is the range of substances amenable to the in vitro pyrogen test methods, and are thelimits of the test methods adequately characterized?
Yes, given what is known of materials with the potential to interfere with the test system
supplemented by the need for product-by-product validation and the exclusion ofinterference. More work will have to be carried out to understand the types of materials that
could be tested in these assays and how they would be handled (e.g., cell therapies andimplants). However, it must be considered that a manufacturer of a medicinal product would
have to validate the in vitro method they have selected specifically for their particular
product before it would acceptable to any regulatory authority. Thus, comprehensive testingof a wide variety of substances may not be necessary to introduce these tests into general use.
Insufficient information exists at present to be confident that all types of materials that will
demonstrate interference have been identified (e.g., materials that are cytotoxic, contain
immunological adjuvants, or have antipyretic properties) but case-by-case evaluation
provides the necessary safeguards. In addition, although the test methods have been shown tohave the potential to identify non-Gram negative pyrogens, the validation study only
presented detailed data and analysis with respect to the tests’ potential to detect Gramnegative endotoxins (see also Section 1.3.1).
However, with respect to the limits of the test methods, no mention is made of the wide rangeof drugs that are toxic to blood cells or that induce a substantial pro-inflammatory response
and consequently are not amenable to testing by these methods. Many pure, well-establishednon-endotoxin compounds have been shown to activate blood cells, including monocytic
cells, to produce pro-inflammatory cytokines in vitro and in vivo (see suggested additionalreferences [Ishii et al., 2005; Ishii and Akira, 2006] in Section 12.0).
On page 1-5, line 770 of the ICCVAM draft BRD (December 1, 2006) states, “Although thein vitro BET is performed using hemolymph (the equivalent of blood) drawn from Limulus
polyphemus (horseshoe crabs), which are subsequently returned to the wild, there is somemortality associated with the procedure (which requires approximately 20% of the animal’s
total blood volume)". This concern has been largely solved with the commercial introductionof rFC, which was originally cloned from the horseshoe crab. This commercial product iscurrently being compared to the BET for submission for inclusion in the USP. A need for a
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replacement for the RPT for early compound development testing and testing of biologics
that have some propensity to harbor non-endotoxin pyrogens remains to be fulfilled. Thus,
the goals of the overall effort need further refinement. Endotoxin is, of course, the important
standard for validation purposes but non-endotoxin standards need to be characterized tofurther such a test for non-endotoxin testing; this concept is referred to on page 1-7, lines
821-822 of the ICCVAM draft BRD (December 1, 2006).
2.0

IN VITRO PYROGEN TEST METHOD PROTOCOL COMPONENTS

2.1

Overview of How the In Vitro Pyrogen Test Methods are Conducted
Are there gaps or missing information in the overview of how the tests are
conducted?

This section seems adequate and complete. The overview of how the in vitro pyrogen tests
are carried out is brief and to the point. The assays essentially expose human blood cells(either primary or cell line derived) to a test substance that may or may not induce cytokine
release. Any cytokine release is subsequently detected with an immunoassay.
2.2

Description and Rationale for the Test Method Components for ProposedStandardized Protocols
Are the description and rationale for each of the following test method
protocol components for the recommended versions of the in vitro test
methods adequately described and appropriate? Should any protocolcomponents be modified, and, if so, why? Are additional protocolcomponents needed, and, if so, why?

2.2.1
Materials, equipment and supplies
Specific concerns with respect to human blood donors include: diurnal variation, genetic
polymorphisms (i.e., in genes coding for Toll-like receptors [TLRs], cytokine receptors,
response elements, etc.), and number of donors required.
The effect of components in the blood and their effects on the assay systems are not clear(i.e., the effect of variations in the number of monocytes in peripheral blood, which range
from 2 to 10%, as well as the effect of neutrophil or lymphocyte presence on the cytokine
response).
2.2.2
Endpoint(s) measured
The viability of the human blood cells should be monitored before and after incubation withthe test samples. Cytotoxic substances should not be tested with these methods.
2.2.3
Duration of exposure
A fixed exposure time rather than a broad range of exposure times (e.g., 16 to 24 hours)
should be defined.

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2.2.4
Known limits of use
It is suggested that the in vitro pyrogen tests are suitable for the testing of medical devices
and materials by direct contact rather than testing extracts. However, direct contact may notadequately permit the solubilization or leaching of potential pyrogens.
2.2.5
Nature of the response assessed
The nature of the response assessed is accurately summarized. However, a description of theblood cell types known to respond to pyrogens by producing IL-1 and/or IL-6 should be
included.
2.2.6

Appropriate negative, vehicle, and positive controls and the basis for theirselection
The ECVAM BRDs do not discuss why high quality Gram-positive material (Lipoteichoic
acid [LTA]) available from the University of Konstanz was not also used as a 'model'
pyrogen. The inclusion of such non-endotoxin positive controls would be useful in future
validation studies to further characterize the usefulness and limitations of these methods forthe detection of such substances.
2.2.7

Acceptable ranges of negative, vehicle, and positive control responses and thebasis for the acceptable ranges, or procedures for establishing acceptable ranges
The ECVAM BRDs indicate that (refer to Sections 6.1.1), with hindsight, the use of an
endotoxin spike solution at the threshold pyrogen dose (marking the pass/fail level for
regulatory purposes) was not wise. See above (response to Criterion 4, Section 1.2) regarding
possible relevance to determination of sensitivity and specificity of the novel test methods.
2.2.8
Nature of the data to be collected and the methods used for data collection
The description of the nature of the data to be collected and the methods used for datacollection is accurate.
2.2.9
Type of media in which data are stored
The type of data storage media seems to fit the purpose. However, one printed version of thedata should be stored.
2.2.10Measures of variability
The description of the measures of variability reflects the current state of knowledge. Other
relevant physiological variables may exist but the main sources of potential variation seem tohave been addressed.
2.2.11
Statistical or nonstatistical methods used to analyze the resulting data
Generally adequate, but additional clarification is desired. It would seem appropriate to use aconsistent approach across assays. For example, in some places, Dixon’s test was used to
identify outliers, while in others Grubb’s test was used; the reasons and contexts for thesedifferences are not apparent. However, it is accepted that minor problems arise with thecalculation of sensitivity and specificity of the novel test methods from using a spike-point
coincident with the regulatory limit.
The statement that "using an endotoxin curve, the endotoxin content of the product is
calculated" is not true. The in vitro pyrogen test is not specific for Gram-negative endotoxin
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May 2008

and therefore, it is impossible to know whether the response measured is due to endotoxin or
another pro-inflammatory response reactive substance in the sample.
2.2.12

Decision criteria and the basis for the prediction model used to classify a test
substance as positive or negative for the presence of a pyrogenic material
The RPT data used to set the pass/fail criteria were produced in one rabbit strain in one
laboratory and were not obtained concurrently within the validation study.
It is not clear that the criteria used to assign test results as positive or negative are based on
the precise criteria set out in the USP. The significance of any deviations from these criteria
is also not clear.
2.2.13

Information and data that will be included in the study report and availability ofstandard forms for data collection and submission

The descriptions provide a good overview of each test for the purposes of comparing andcontrasting them with one another and with current methods.
2.3

Basis for Selection of Test Method Systems
Is the description of the basis for selection of the test method systems
complete and appropriate?

A brief description of the advantages of each test method have been provided and areappropriate for considering the limitations of the existing tests for pyrogens, namely the RPT
and the BET.
2.4

Proprietary Components
Are proprietary components appropriately identified (if applicable), and are
the procedures adequate for ensuring their integrity from 'lot-to-lot' andover time?

The licensing procedure and availability of the Mono Mac 6 (MM6) cell line is unclear.
Variations in the MM6 cell line (and primary cells) must be properly controlled. A directcomparison of the commercially available enzyme-linked immunosorbent assay (ELISA) kits
should also be included in the ICCVAM final BRD.
2.5

Number of Replicates
Are the numbers of replicate and/or repeat experiments appropriate for eachtest method?

The appropriate number of donors from which to collect blood cells is unclear. Furthermore,some of the test methods permit pooling of blood donors while others do not. The rationale
for these differences is unclear.

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2.6

May 2008

Modifications to the Test Method Protocols Based on ECVAM Validation
Study Results
Are the protocol modifications based on ECVAM validation study results
appropriate for each modified test method?

Yes, only minor modifications were made to two of the five assays, both to improve assay
performance, and therefore the limited explanations are appropriate.
3.0

SUBSTANCES USED FOR THE VALIDATION OF IN VITRO PYROGENTEST METHODS

3.1

Rationale for the Substances or Products Selected for Testing
Is the rationale for the selected test substances appropriate and acceptable?

No, the only rationale given for the choice of test substances is that they represent marketedparenteral pharmaceuticals that were readily available at reasonable cost. According to their
USP monographs, seven of the ten test substances are currently tested in the BET, not in the
RPT. No USP monographs exist for the remaining three because pyrogen testing is not
required. The inclusion of test substances that may interfere with the in vitro responses
should be tested.
Although the test materials spiked with endotoxin are described as having been initiallypyrogen-free and having been approved for clinical use, all that can be said with confidence
is that they did not contain a level of pyrogen above the permissible or tolerable limit. As aresult, in describing the concentration of endotoxin in the spiked sample, it is more correct tostate the minimum level of endotoxin they were known to contain rather than offering an
absolute value.
Non-endotoxin pyrogens should be evaluated because these pyrogens must be tested in the
RPT and they cannot be tested in the BET. The list of test substances should also include
protein- and lipid-containing materials that are used parenterally. No ‘classical’ examples of
biological products or medical devices were included; thus, the validation for either of thesecategories has not been provided.
Although it is stated that endotoxin was chosen as a model pyrogen, insufficient information
exists in the ICCVAM draft BRD or in the supporting ECVAM BRDs to support this claim.
The validation study documents, the ESAC validation statement and the ICCVAM draft
BRD claim only that the test methods are suited for the detection/qualification of Gramnegative endotoxin for regulatory testing.
3.2

Number of Substances
Please comment on the adequacy of the number of substances used in theperformance analyses.

The total number of substances included in the validation study is adequate only forvalidation of a specific class of products. Replacement of the RPT would require a muchlarger number of substances because of the wide range of product classes that would require
testing. Moreover, the test substances should have represented each of the major classes of
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products normally tested in the RPT (e.g., medical devices, biologicals, implants, and those
substances known to interfere with the RPT, the BET, and/or the in vitro pyrogen tests) as
positive controls for interference testing.
3.3

Identification and Description of Substances Tested
Are the test substances adequately identified and described?

The samples included in the validation process are adequately identified and described suchthat they could be readily obtained for future studies. However, more information on theirpurity and batch/lot numbers is needed in order to adequately demonstrate that the same
substances were tested throughout the validation studies. In response to a request for
additional information, ECVAM did provide the lot numbers used in the validation study,
which demonstrated that they were identical. However, some differences in the lots tested in
the catch-up validation study were noted (e.g., two of the ten substances had different lot
numbers due to the lack of availability; one was a different substance with the same active
ingredient).
3.4

Sample Coding Procedure
Were the coding procedures used in the validation studies appropriate?

The coding procedures were adequate for the assessment of relevance during the validation
studies. However, the identity of the substances used in the reproducibility analyses was notblinded (although the spike concentrations were). A reason was not given.
4.0

IN VIVO REFERENCE DATA FOR THE ASSESSMENT OF TESTMETHOD ACCURACY
Are the in vivo reference data used in the validation study appropriate toallow for adequate assessment of test method relevance2(accuracy/concordance, sensitivity, specificity, false positive and false negative
rates) of these in vitro pyrogen test methods as a partial replacement for the
RPT, for materials which may be contaminated with gram-negative
endotoxin, but which cannot be tested by the BET?

No, a summary of the reference data demonstrating whether substances that were shown tobe pyrogenic in humans either passed or failed the RPT, BET or in vitro pyrogen tests would
have been useful.
4.1

Description of the Protocol Used to Generate In Vivo Data
Is the RPT protocol used to generate reference data for the cited studies
appropriate?

2

The extent to which a test method correctly predicts or measures the biological effect of interest in humans oranother species of interest. Relevance incorporates consideration of the "accuracy" or "concordance” of a test
method.
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The RPT protocol and the pass-fail criteria used would not meet the current USP
requirements. The significance of these deviations is not clear. The data are derived from asingle study carried out at the Paul Ehrlich Institute (PEI) where historical controls tested
over five years were accumulated and analyzed. The protocols used at the PEI were based on
the EP monograph for the RPT, although this fact is not explicitly stated in the publications.
Furthermore, the detailed protocol used by this laboratory was not provided.
4.2

Reference Data Used to Assess In Vitro Test Method Accuracy
Is the interpretation of the reference data used to assess in vitro test method
accuracy correct? Is any other data or information needed to determine theaccuracy of the test methods?

The reference data were previously and separately generated by one protocol, in one
laboratory, using one strain of rabbit, and two sources of endotoxin. A second study,
undertaken in Brazil, is cited. The response criteria of the Brazilian study do not match thoseof the PEI study. It is not clear why the Brazilian study was not relied upon for the validation
study.
4.3

Availability of Original Records for the In Vivo Reference Data
Are there any concerns with the availability of the original reference data
records as described?

The data were derived from a single study at the PEI and presented in graphical form. No
additional data were available for analysis. Archived records have not been audited by
ECVAM or ICCVAM.
4.4

In Vivo Data Quality
Are there any concerns with the RPT data quality?

The ECVAM documentation is not sufficiently specific and in the absence of the primarydata, the quality of the RPT data is unknown. The ICCVAM draft BRD does not clearly
indicate the GLP status of the laboratory or of the study. However, the PEI did not haveformal GLP accreditation (refer to Section 5.5, ECVAM response to a request for additional
information).
4.5

Availability and Use of Toxicity Information from the Species of Interest
Is the discussion of the availability of relevant pyrogenicity information forhumans adequate and appropriate? Are there other sources of quality
human data for pyrogenicity that should be considered? Would human data
be compatible with regulatory needs (e.g., exposure duration, individual
sensitivity)?

The available data are limited. However, the availability of information on clinical adverse
events resulting from the administration of medical products producing pyrogenic effects,
and the relevant pre-clinical test data, would be an excellent source of human data. See
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suggested additional reference (McKinney et al. 2006). The data would reflect responses seenusing appropriate human exposure; thus, it should be compatible with regulatory needs.
A discussion of relevant pyrogenicity information for humans is present in the ICCVAM
draft BRD, but additional information is needed. An extensive literature on acute humanpyrogenicity responses exists and this data should be better reviewed. Effects of longer
exposure and individual sensitivity are available in Rylander (2002).
The data in the cited paper by Greisman and Hornick (1969)3 are not accurately described(page 4-6, lines 1299-1301 of the ICCVAM draft BRD [December 1, 2006]).
4.6

Information on the Relevance and Reliability of the In Vivo Test Methods
Is what is known about the relevance and reliability4 of the RPT adequately
discussed and appropriately considered?

The appropriateness of the theoretical assumption model is unclear. It is not clear how thesensitivity and specificity values have been derived using this model. Therefore, reference tothese values as accurate figures (particularly with respect to the 58% sensitivity) is a concern.
The theoretical sensitivity and specificity for the RPT that has been supplied does not seemto reflect its performance in practice or the regulatory decisions and level of patient safetythat RPT data currently supports.
The 'correct' figures for the theoretical specificity of the RPT are confusing. It was stated to
be 83% in Section 4.6 of the ICCVAM draft BRD (December 1, 2006) but given as 88.3% in
the ECVAM response to ICCVAM questions (page 24).
However, this difference has little bearing on the overall interpretation of the results.
5.0

Test Method Data and Results

5.1

Test Method Protocol
Are the in vitro test method protocols used to generate each set of dataconsidered in the ICCVAM draft BRD appropriately described?

The following problems with all five in vitro test method protocols were noted:

3

1.

Quality control (QC) testing of cell viability is not performed. Viability testingof the human cells before and after incubation should be performed.

2.

No microscopic examination for anticipated levels of cell fragments anddebris is described.

3.

Substances should not be tested at cytotoxic concentrations by these methods.

Greisman SE, Hornick RB. 1969. Comparative pyrogenic reactivity of rabbit and man to bacterial endotoxin.
Proc Soc Exp Bio Med 131(4):1154-1158.
4
A measure of the degree to which a test method can be performed reproducibly within and among laboratories
over time. It is assessed by calculating intra- and inter-laboratory reproducibility and intralaboratory
repeatability.
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4.

More detailed source information and the pyrogen status (i.e., pyrogen-free)should be required for all protocol components.

5.

A description of the procedure used for donor recruitment and donor selection
is not provided.

6.

A description of the protocols used for preparation of blood samples for thecytokine assays is not found.

The following problems with specific test method protocols were noted:

5.2

1.

In the Cryo WB IL-1 assay, the incubation of the test sample is performed in
the presence of 10% DMSO (methods for its removal after thawing of the
cryopreserved cell preparation and before its use are not described). DMSO is
known to effect the detection of certain cytokines. In response to a request for
additional information, ECVAM indicated that the DMSO is not removed.

2.

A limit to the passage number should be defined for the MM6 cell cultures.

3.

The use of the terms RPMI-M and RPMI-C (described in the ECVAM
MM6/IL-6 Standard Operating Procedure [SOP]) is confusing in the
ICCVAM protocol.

4.

A typographical mistake appears in the ICCVAM MM6/IL-6 protocol (lines
285 and 286 of the ICCVAM draft BRD [December 1, 2006]) where ‘FBS’ isstated instead of ‘PBS’.

Availability of Copies of Original Data Used to Evaluate Test Method
PerformanceHas the availability of the original data use in the test method performance
evaluation been adequately described?

Yes, the availability of the original source data has been adequately described.
5.3

Description of the Statistical Approaches Used to Evaluate the Resulting Data
Are the statistical and non-statistical approaches used in each cited study toevaluate the in vitro test results appropriate? What other approaches could
have been used?

The statistical approaches appear adequate. However, it is suggested that more emphasisshould have been placed on a quantitative estimate of pyrogen concentrations rather thandichotomizing results based upon a hypothesis test. One would have expected to see a priori
criteria for successful validation in terms of acceptable performance statistics.
The term 'correlation' appears to be used colloquially (e.g., lines 1365 and 1373 of the
ICCVAM draft BRD [December 1, 2006]); a correlation is not a percentage. Therefore,'correlation' should be replaced with 'association' everywhere, except when Pearson’s
correlation is being referenced.
Information on the identification and elimination of aberrant data from Section 4.2 of the
Trial Data report should be included in the ICCVAM final BRD.
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5.4

May 2008

Summary of Results
Is the summary of the results for each test method appropriate and adequate?

No data were presented to confirm that results in the in vitro tests reflect humanphysiological responses or that production of IL-1 or IL-6 in vitro correlates with
pyrogenicity in vivo. A quantitative link between IL-1 and IL-6 concentrations and theirdonor-to-donor variation with physiological effects was not presented. It should be
mentioned that according to Schindler et al. (2006)5, which describes the validation of the
Cryo WB/IL-1 method, testing problems existed with many of the products included in the
study (up to 9 of 10). This is evident by failure of the positive product control (PPC), which
under normal circumstances would invalidate the test. Instead, when the PPC failed, theauthors report that the saline control was used in place of the PPC and the experiment was
still considered acceptable. This practice is unacceptable.
The lack of direct parallel testing in rabbits with the products tested in the validation study
prevents an evaluation of actual physiological effects. It also would have been of assistanceto the Panel if information had been provided to document that the use of human cells could
partially replace the BET and RPT for the detection of substances that are pyrogenic inhumans.
Some of the data (or lack thereof) indicate significant limitations of the in vitro assays.
Specific examples are listed below:

51.

In the ICCVAM draft BRD (December 1, 2006), page 2-7, line 989: The useof a single donor in the WB/IL-1 assay is inadequate.

2.

In the ICCVAM draft BRD (December 1, 2006), page 2-10, line 1050: There
are no data offered to document that the use of human cells will better reflect
human physiological responses or that production of IL-1 or IL-6 in vitro
correlates with pyrogenicity in vivo.

3.

In the ICCVAM draft BRD (December 1, 2006), page 6-2, line 1456: 20 of
150 runs in the Cryo WB/IL-1 assay were not usable. Even then, the false
positive rate of the remaining 120 assays was 18.6%.

4.

In the ICCVAM draft BRD (December 1, 2006), page 6-4, line 1493: 1 of the
3 validation laboratories had a 50% false positive rate for the PBMC/IL-6
assay.

5.

In the ICCVAM draft BRD (December 1, 2006), page 7-7, Table 7-4:
Agreement across three validation laboratories was only 57% for the WB/IL-1
assay.

Schindler S, Spreitzer I, Löschner B, Hoffmann S, Hennes K, Halder M, Brügger P, Frey E, Hartung T,
Montag T. 2006. International validation of pyrogen tests based on cryopreserved human primary blood cells. J
Immunol Methods 316:42-51.
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5.5

May 2008

Use of Coded Chemicals and Compliance with GLP Guidelines
For each set of data for each test method, is whether coded substances were
tested and whether experiments followed GLP Guidelines adequately
documented?

The use of coded substances is adequately documented, but the rationale for not blinding theidentity of the three substances tested in the reliability analyses is not known. The in vitro
pyrogen test studies were conducted 'in the spirit of' GLP requirements. However, gaps andlapses in the information supplied by ECVAM would indicate that none of the testinglaboratories were audited in real-time. In response to a request for additional informationfrom ECVAM, it was stated that:"The initial validation study has been carried out to large extent in laboratories
such as National Control laboratories, which do not operate under GLP. It was,however, agreed to comply with the requirements of GLP, especially with regard
to the creation and management of SOPs. The partner laboratories have received
presentations on the requirements. No auditing was done but various quality
checks and blinding mainly under the responsibility of ECVAM were included. In
the catch-up validation two GLP laboratories and two National Control
laboratories participated.""Raw data: In both studies, the laboratories were asked to transfer readings into
Excel sheets provided by the statistician. This was mostly done by directly
inserting the ASCII files created by the plate reader. However, reader printouts areavailable and can be provided on request."
5.6

Lot-to-Lot Consistency of Test Substances
Is the information on the 'lot-to-lot' consistency of the test substances, thetime frame of the various studies, and the laboratory in which the study or
studies were conducted, adequately described?

Information on specific lots used in the validation studies was not provided in the ICCVAM
draft BRD and therefore, lot-to-lot consistencies cannot be evaluated. Additional information
has been received to demonstrate that the same lots were tested in the validation study, butthere were lot differences in 2 of 10 substances used in the catch-up validation study. In
addition, because one of the substances used in the original validation was no longer
available, a different substance (with the same active ingredient) was used in the catch-up
validation.
Unfortunately, little or no high concentration protein samples (e.g., Factor VIII concentrates
or 5-25% human albumin samples), where lot-to-lot inconsistencies might be expected, were
tested in the validation studies. This exclusion was explained to some extent by ECVAM in
the responses that they provided to the ICCVAM/PWG questions. Interference testing for all
sample types should be tested on multiple lots (also see the specific inadequacy [No. 10, lines
1361-1362] noted in the proposed test method standardized protocols).

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6.0

RELEVANCE OF THE IN VITRO PYROGEN TEST METHODS

6.1

In Vitro Pyrogen Test Method RelevanceMay 2008

Has the relevance (e.g., accuracy/concordance, sensitivity, specificity,
positive, and negative predictivity, false positive and false negative rates) of
the in vitro test methods for detection of pyrogens, as defined by statutes in
the United States Code (see Section 1), or for sterility testing defined by the
U.S. Pharmacopeia or the International Standards Organization, beenadequately evaluated? Are the discussions of the relevance of each in vitro
test method and the reference test method appropriate and accurate?
In general, the evaluation of the relevance of the in vitro pyrogen tests appears to have been
appropriately demonstrated and discussed, but limited by the ability to judge a positive
versus negative response using a cut-off at 0.5 endotoxin units (EU)/mL. Furthermore,because only endotoxin-spiked samples were tested, relevance has been demonstrated only
for the detection of bacterial endotoxin.
This section is entirely focused on comparisons between the in vitro pyrogen test methods
since the RPT was not carried out in parallel, but rather estimates of the RPT performance
were modeled statistically. The validity of this approach remains in question due to thenature of the RPT, where a definitive cut-off point does not exist, but was defined based on
the results generated from the historical database. Therefore, no data exist with which toestablish concordance with the RPT and thus, the discussion on concordance with the RPT is
speculative.
Discrepancies between Table 6-1 and the accompanying text of the ICCVAM draft BRD(December 1, 2006) for the cryopreserved PBMC assay prevented assessment of this method.
6.2

Summary of the Performance Statistics for In Vitro Pyrogen Test Methods
Is the summary of the performance of the test methods adequately
described? Are the strengths and limitations of each in vitro test methodadequately identified?

A more critical description and explanation are needed (i.e., a failure of the prediction modelor a failure of the assay to correctly detect the pyrogen concentration) for the cases where thetest failed to correctly classify the pyrogen concentration.
The discussion of the strengths and limitations of each of the test methods should be
expanded. Specific points include:
1.

Inadequate performance is noted for: a) Cryo WB/IL-1 (false positive rate =18.1%); b) WB/IL-1 (false negative rate = 27.3%); c) WB/IL-1 (false positive
rate = 16.4%). High false positive rates are clearly a concern for
manufacturers since lots may be unnecessarily withheld from release.

2.

The high exclusion rate for individual runs in the case of the Cryo WB/IL-1
test (20% - 30% out of 150 runs) due to excessive variability among the four
replicates, even with a relatively high coefficient of variation (CV) criteria(CV > 45%).
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3.

May 2008

The low sensitivity (only 72%) for the WB/IL-1 assay, resulting in an
extremely high false negative rate (27.3 %). High false negative rates would
obviously be a major concern, as endotoxin-contaminated lots would be
released.

Taken together, these statements could indicate that the WB/IL-1 assays (WB/IL-1 Cryo
WB/IL-1, and WB/IL-1 96-well plate method) do not, in general, perform as well as the otherassays that measure an IL-6 response.
It would have been very interesting to have had the opportunity to compare performance
analysis data for the BET, since only endotoxin spiked samples were used in the validationand endotoxin testing is now the intended use for the in vitro pyrogen tests. Unfortunately,
the BET was not performed in the validation so no direct comparison can be made between it
and the new in vitro assays.
7.0

RELIABILITY OF THE IN VITRO PYROGEN TEST METHODS

7.1

Selection Rationale for the Substances Used to Evaluate the Reliability of In
Vitro Pyrogen Test Methods
Is the selection rationale and the number and types of substances used toevaluate the reliability of the in vitro test methods (intralaboratory
repeatability and intra- and inter-laboratory reproducibility) as well as theextent to which the chosen set of substances represent the range of possible
test outcomes appropriate?

The use of a standard material such as the endotoxin control (WHO-LPS, 94/580) is a valid
choice for conducting the experiments described since it is a well-characterized, welldocumented material. However, the rationale for the selection of the drugs used in the studies
for evaluating reproducibility versus sensitivity/specificity is not clear, except that they were
manufactured under GMP, were licensed products, were reported not to be contaminatedwith unacceptable levels of endotoxin, and were all available at reasonable cost. It would
have been more appropriate to evaluate reliability using a subset of the drugs used in thesensitivity/specificity studies.
7.2

Analysis of Repeatability and Reproducibility
Are the analyses and conclusions regarding the intralaboratory repeatability
and reproducibility and the intra- and inter-laboratory reproducibility ofeach test method appropriate? Should other analyses be considered?

The experiments performed to evaluate intralaboratory repeatability and intra- and interlaboratory reproducibility were overly complicated. However, the analysis based on ‘positiveor negative’ calls suggests that the reliability of these in vitro test methods are generally
acceptable both within and between laboratories, although a more critical description isneeded to explain the lack of agreement among some test results.
It is interesting that the variability of the cell line-based MM6 assay is much reduced
compared to that obtained for the whole blood assays, although this observation did nottranslate into an improved ability to assign a negative or positive status to a sample.
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The following deficiencies were noted:
1.

More discussion is needed about the use of a coefficient of variation (CV)analysis to evaluate the reliability of the in vitro test methods, including how
an 'acceptable' CV was identified (e.g., 45% in the WB/IL-1 assay) and why
the criteria for an acceptable CV was inconsistent among the different in vitro
test methods.

2.

It is not clear which statistical test(s) was used to detect outliers and whether
the test(s) was based on original or log-transformed data. Furthermore, it is notclear how many data points were identified as outliers and how they were
subsequently handled during data analysis. The information provided by
ECVAM addressing these concerns should be integrated into the ICCVAM
final BRD.

3.

A quantitative assessment of the intra- and inter-laboratory variability would
have been more informative than an assessment based on dichotomizing thetest results. The assessment should have included estimates of the amount ofinter- and intra-laboratory variability and the number of replicates needed toestimate the sources of variability. Consistent with general practice,acceptable levels of variability should have been identified a priori, and it
should have been recognized that formal hypothesis testing is essential withthe alternative hypothesis being no different between groups.

4.

Potential problems related to plate-to-plate variation and/or other plate design
issues should be addressed in the ICCVAM final BRD.

5.

The use of the term 'mean value calculated' needs to be clarified.

6.

It is misleading to state that the test substances were spiked at fourconcentrations when two of the spikes are at the same concentration. The
concentrations should be noted explicitly, even in summaries if this is theirfirst reference.

7.

The ICCVAM final BRD should state whether or not the data were logtransformed prior to analysis (as was stated in the ECVAM BRDs).
Furthermore, in the ECVAM BRDs, the decision rationale for performing alog transformation versus a square-root transformation of the data should be
provided. In all ECVAM BRDs, it is not clear whether all analyses used logtransformed data or if transformed data were used only for the t-test in theclassification phase of the analysis (e.g., ECVAM BRD for WB/IL-1, page
25).

8.

The ECVAM BRDs state that all data are log-transformed, but the y-axis on
the graphs is labeled OD 450 (e.g., ECVAM BRD for Cryo WB/IL-1,
Appendix D). The data should be log-transformed if this has not yet been
done. The CV after transformation is of most interest; however, the figuresappear to give data before the transformation indicating that the varianceincreases with the mean. Data after the transformation should also be plottedto show that the relationship of the mean and the variance is well suited to thelog transformation. The analysis with respect to the transformation needs to be
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clarified. The values on the x-axis are unreadable and need to be given in thelegends or in the description that accompanies each figure.
9.

7.3

The notation used in the t-test (e.g., the subscripts on the population andsample means) needs to be defined. In the standard two-sample t-test, thegroups are assumed to be independent. However, it looks like one group is acollection of subgroups and the other group is one of these (i.e., the data fromone group are used in the calculation of both means). This point needs to beclarified.

Historical Positive and Negative Control Data

Is the availability of historical negative and positive control data adequately
considered?
The fact that the in vitro pyrogen test methods are not in routine use except for the two
manufacturers cited (who are unlikely to provide what would be considered proprietary data)
leads to a paucity of historical data.
8.0

TEST METHOD DATA QUALITY

8.1

Adherence to National and International GLP Guidelines
Is the extent of adherence to national and international GLP guidelines forall submitted in vitro and in vivo test data and the use of coded substancesand coded testing adequately presented?

It is clear that SOPs exist and that protocols were developed for all in vitro experiments
performed. However, the precise GLP status of the studies and the test laboratories is notclearly stated and the ICCVAM final BRD should be revised to clarify this information. The
in vivo data are derived from a single published study.
8.2

Data Quality Audits
Are the results of any data quality audits, if conducted, adequately
summarized?

From the information provided, it would seem that no audits were undertaken while thestudies were in progress. However, the ECVAM BRDs state that 'deviations' were recorded
but no further details or information is provided. A summary of the GLP deviations that
occurred would have been useful for determining their overall significance to theexperimental outcome.
8.3

Impact of Deviations from GLP Guidelines
Does the lack of an evaluation of the impact of deviations from GLP
guidelines affect the data analysis?

This question cannot be answered, as no data have been provided on any deviations from
GLP guidelines.
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8.4

May 2008

Availability of Laboratory Notebooks or Other Records
Is the availability of laboratory notebooks or other records for an
independent audit adequately discussed?

Yes, the study authors state that all raw data are available for inspection and have beenarchived appropriately.
9.0

OTHER SCIENTIFIC REPORTS AND REVIEWS

9.1

Have Relevant Data Identified in Other Published or Unpublished Studies
Conducted Using the In Vitro Test Methods Been Adequately Considered?

Although an extensive literature has been cited and discussed, no attempt at a comprehensive
summary of findings or overall conclusions about the relevance of the in vitro pyrogen tests
compared to the BET or the RPT, or the advantages/capabilities or disadvantages/limitationsof the individual in vitro assays, has been presented in the ICCVAM draft BRD.
The following additional references should be included (see Section 12.0 for full citations):

9.2

1.

Marth and Kleinhappl (2002). The studies described here indicate theimportance of monitoring multiple pro-inflammatory cytokine responses. In
the specific case cited, the TNF-α pro-inflammatory cytokine response
appeared to correlate best with fever.

2.

Norata et al. (2005), van Deventer et al. (2000), von Aulock et al. (2003) are
relatively new studies that evaluate the effects of genetic polymorphisms on
TLR-4 responses.

3.

Martis et al. (2005). This paper describes a situation where the PBMC/IL-6
assay was used to help resolve a non-febrile adverse drug reaction issue with alicensed product.

Are the Conclusions Published in Independent Peer-Reviewed Reports or
Other Independent Scientific Reviews of the In Vitro Test Methods
Adequately Discussed and Compared?

Yes, the conclusions are adequate for the published data.
The formal ESAC validation statement and other EU validation expert/panel process
documents should be appended to the ICCVAM final BRD.
9.3

Are There Other Comparative In Vitro Test Method and RPT Data That
Were Not Considered in the ICCVAM draft BRD, But are Available forConsideration?

It is known that manufacturers have parallel test result data for the BET and RPT for specificproducts, which unfortunately are not published or peer reviewed. As a consequence, anumber of companies are now advocating that they should be permitted to use the BET as analternative to the RPT to detect the presence of Gram-negative endotoxin on a case-by-casebasis, such as for testing of established products with documented proof that safe, reliable
and consistent GMP production and QC procedures are in place.
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10.0

ANIMAL WELFARE CONSIDERATIONS (REFINEMENT, REDUCTION,
AND REPLACEMENT)

10.1

How the Five In Vitro Pyrogen Test Methods Will Refine, Reduce, or Replace
Animal Use
Is the extent to which the in vitro test methods will refine (reduce or eliminate
pain or distress), reduce, or replace animal use in the RPT adequately
described?

No numbers are included regarding the current number of rabbits used and/or killed with thistest. These estimates would be helpful when assessing the potential impact of these in vitro
tests. However, given that the proposed use for these test methods is very limited, it is notclear that their application would have a significant impact on animal numbers.
The ICCVAM final BRD should discuss the practice of, and the U.S. Federal restrictions on,
the reuse of rabbits in pyrogenicity testing.
A discussion on the ethical cost of conducting concurrent RPT testing should be added.
10.2

Requirement for the Use of Animals
Is the discussion of the use of cultured human cells and the need forvolunteers for donations of peripheral blood used in the in vitro test methods
appropriate and adequate?

No, the licensing arrangements and the maintenance of the MM6 cell line are unclear.
The discussion that reduction of the use of animals (i.e., rabbits) will be associated with theincreased use of another animal (i.e., humans) is inadequate.
11.0

PRACTICAL CONSIDERATIONS

11.1

Transferability of the In Vitro Pyrogen Test Methods
Are the following aspects of in vitro test method transferability, including an
explanation of how this compares to the transferability of the RPT,
adequately described with regard to the:11.1.1
Facilities and major fixed equipment needs?
Yes, either a sterile tissue culture facility or a laboratory animal facility is needed.
11.1.2
General availability of other necessary equipment and supplies?
Yes, equipment and supplies for both in vitro and in vivo studies are routinely available. In
general, the skills and kits required are available in most diagnostic and testing facilities.
The availability (in ready to use kit form), the convenience, and the lower costs of the BET
will mitigate against widespread use of the in vitro pyrogen tests that are far more work
intensive (e.g., cytokine and endotoxin standard curves must be established, tests must be
performed in quadruplicate, multiple donors are required), less convenient (as yet only one of
the assays is available in kit form), and probably associated with higher costs.
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11.1.3Nature of the drug substance tested?
Yes, the drug substances are adequately described. The overall requirements for the assays
are comparable with most other types of in vitro QC diagnostic assays.
11.2

Personnel Training Considerations
Are the following aspects of the in vitro test method training adequately
considered? Is the explanation of how this compares to the level of training
required to conduct the RPT adequate with respect to:

11.2.1
The required level of training and expertise needed to conduct the test method?
Yes, the individual technical steps and competencies are common to many other laboratory
activities.
11.2.2

Any training requirements needed for personnel to demonstrate proficiency andany laboratory proficiency criteria that should be met?
The training required for adequate conduct of biological assays cannot be overestimated.
Aseptic tissue culture techniques are essential, as is the ability to accurately serially dilute
material. It is necessary to maintain the MM6 cell line and functional and non-activatedmonocytes obtained from whole blood. Activation can be caused by physical disruption orcontaminants. Competency in each of these techniques should be demonstrated prior toallowing personnel to carry out these tests on medicinal products intended for human use orfor certification. It should also be noted that the required expertise needed does not typicallyreside in the laboratories that conduct the test (i.e., RPT) targeted for replacement by the
proposed in vitro tests.
11.3

Cost Considerations
Is the cost involved in conducting a study using the in vitro test method, as
compared to the cost of conducting the RPT, adequately evaluated, and is
this considered to be cost-effective compared to the in vivo method?

No, the direct and indirect costs of operating an animal facility that would be needed to houserabbits are incompletely stated. The in vitro pyrogen tests would seem to be considerably
more cost effective than the RPT. It would be interesting to see pricing costs from contract
research organizations for both classes of tests, mindful that cost considerations will impact
on the level of use.
11.4

Time Considerations
Is the amount of time needed to conduct a study using the in vitro test method
as compared to the time it takes to conduct the RPT adequately evaluated,
and is the in vitro test method considered to be time-effective compared to thein vivo method?

The in vitro pyrogen test methods require two days to complete (twice as long as the BET
and RPT under normal circumstances). Furthermore, the in vitro pyrogen test methods are
dependent on the availability of donors or blood supplies, which might further restrict thefrequency to which these tests can be performed.
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12.0

May 2008

RECOMMENDED ADDITIONAL REFERENCES
Are all relevant publications referenced in the ICCVAM draft BRD? If not,what additional references should be included?

The following references should be included:
Barnett V, Lewis T. 1984. Outliers in Statistical Data, 3rd ed. In: Wiley Series in Probability
and Mathematical Statistics. (V Barnett, T Lewis, eds). New York:John Wiley & Sons.
Brunson KW, Watson DW. 1974. Pyrogenic specificity of Streptococcal exotoxins,
Staphylococcal enterotoxin, and Gram-negative endotoxin. Inf Immun 10(2):347-351.
Burrell R. 1994. Human responses to bacterial endotoxin. Circ Shock 43(3):137-153.
Dinarello CA. 2004. Infection, fever, and exogenous and endogenous pyrogens: someconcepts have changed. J Endotoxin Res 10(4):201-222.
Dixon WJ. 1950. Analysis of extreme values. Ann Math Stat 21(4):488-506.
Gaines Das RE, Brügger P, Patel M, Mistry Y, Poole S. 2004. Monocyte activation test for
pro-inflammatory and pyrogenic contaminants of parenteral drugs, test design and data
analysis. J Immunol Methods 288(1-2):165-177.
Grubbs FE. 1950. Sample criteria for testing outlying observations. Ann Math Stat 21(1):2758.
Hochstein HD, Fitzgerald EA, McMahon FG, Vargas R. 1994. Properties of US standard
endotoxin (EC-5) in human male volunteers. J Endotoxin Res 1(1):52-56.
Ishii KJ, Coban C, Akira S. 2005. Manifold mechanisms of toll-like receptor-ligand
recognition. 2005. J Clin Immunol 25(6):511-521.
Ishii KJ, Akira S. Innate immune recognition of, and regulation by, DNA. 2006. Trends
Immunol 27(11):525-532.
Marth E, Kleinhappl B. 2002. Albumin is a necessary stabilizer of TBE-vaccine to avoid
fever in children after vaccination. Vaccine 20(3-4):532-537.
Martich GD, Boujoukos AJ, Suffredini AF. 1993. Response of man to endotoxin.
Immunobiol 187(3-5):403-416.
Martin MA, Roberts S. 2006. An evaluation of bootstrap methods for outlier detection inleast squares regression. J Appl Stat 33(7):703-720.
Martis L, Patel M, Giertych J, Mongoven J, Tammine M, et al. 2005. Aseptic perontonitis
due to peptidoglycan contamination of pharmacopoeia standard dialysis solution. Lancet
365:588-594.
McKinney BA, Reif DM, Rock MT, Edwards KM, Kingsmore SF, Moore JH, Crowe Jr JE.
2006. Cytokine expression patterns associated with systemic adverse events following
smallpox vaccination. J Inf Dis 194(4):444-453.
Mullington J, Korth C, Hermann DM, Orth A, Galanos G, Holsboer F, Pollmächer T. 2000.
Dose-dependent effects of endotoxin on human sleep. Am J Physiol Regulatory Integrative
Comp Physiol 278(4):947-955.
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Netea MG, Kallberg BJ, van der Meer JWM. 2000. Circulating cytokines as mediators offever. Clin Inf Dis 31(Suppl 5):S178-S184.
Norata GD, Garlaschelli K, Ongari M, Raselli S, Gigore L, Benenuto F, Maggi FM,
Catapano AL. 2005. Effect of Toll-like receptor 4 (TLR-4) variants on intima-media
thickness and monocyte-derived macrophage response to LPS. J Int Med 258(1):21-27.
Rylander R. 2002. Endotoxin in the environment. J Endo Res 8(4):241-252.
Saper CB, Breder CD. 1994. The neurological basis of fever. New England J Med
330(26):1880-1886.
van Deventer SJH, Buller HR, ten Cate JW, Aarden LA, Hack EC, Sturk A. 1990.
Experimental endotoxemia in humans: Analysis of cytokine release and coagulation,
fibrinolytic, and complement pathways. Blood 76(12):2520-2526.
van Deventer SJH. 2000. Cytokine and cytokine receptor polymorphisms in infectious
disease. Intensive Care Med 26(Suppl 1):S98-S102.
von Aulock S, Schroder NWJ, Gueninzius K, Traub S, Hofffmann S, Graf K, Dimmeler S,
Hartung T, Schumann RR, Hermann C. 2003. Heterozygous Toll-like receptor 4
polymorphism does not influence lipopolysaccharide-induced cytokine release in humanwhole blood. J Inf Dis 188(6):938-943.

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13.0

May 2008

Summary of Validation Status of the In Vitro Pyrogen Test Methods

Does the Panel agree that the applicable validation criteria have been adequately
addressed in order to determine the usefulness and limitations of these in vitro test
methods, to serve as a substitute for the RPT, for the identification of Gram-negative
endotoxin on a case-by-case basis, subject to product specific validation?
Yes, the information is adequate with which to make an informed decision.
Does the Panel agree that the performance of these test methods in terms of theirreliability and relevance support the proposed use of these test methods (i.e., thedetection of Gram-negative endotoxin in materials that are currently tested in the RPT,
subject to product specific validation to demonstrate equivalency to the RPT)?
No, refer to the reasons indicated in the responses to Sections 1.0 to 12.0.
Minority Opinion # 1 (Drs. Karen Brown, Albert Li, and Jon Richmond): The qualificationin the above statement 'subject to product specific validation' should allow for a vote of yes.
Minority Opinion #2 (Dr. Peter Theran): It is not clear that the qualification included in theabove statement would preclude the use of the in vitro test methods as replacements for the
RPT in those circumstances where the BET is currently serving to replace the RPT.

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B

REVIEW OF ICCVAM DRAFT TEST METHOD RECOMMENDATIONS

1.0

Proposed Test Method Usefulness and Limitations

Does the Panel agree that the available data and demonstrated performance in terms of
relevance (i.e., accuracy/concordance, sensitivity, specificity, positive, and negative
predictivity, false positive and false negative rates) and reliability (i.e., intralaboratory
repeatability and intra- and inter-laboratory reproducibility) support the ICCVAM
draft recommendations for these in vitro test methods in terms of the proposed test
method usefulness and limitations6?
The Panel does not agree with this statement for the following reasons:
The usefulness of these in vitro test methods for detecting Gram-negative endotoxin has notbeen properly assessed for concordance with the RPT or for relevance in comparison to the
BET. Therefore, it is not possible to truly assess their usefulness and limitations. It isregrettable that their ability to detect non-endotoxins could not be demonstrated and validated
due to the limitations of the validation and performance evaluation studies conducted.
Test materials in pure form may directly promote the formation and release of cytokines andthus, they may not be suited to evaluation by the in vitro methods.
As much effort as possible should be placed on truly demonstrating that these assays can bereliably used to detect non-endotoxin pyrogens in actual manufacturing settings for a wide
variety of products. Otherwise, these assays have little advantage over the already establishedand widely used BET.
Mechanisms exist for test method developers to qualify their method on a case-by-case basis(i.e., 21 CFR 610.9). Therefore, the use of any recommended method should be subject toproduct specific validation to demonstrate equivalence as regulated by 21 CFR 610.9.
Minority Opinion (Dr. Peter Theran): This Panel has considered the failure to undertake
additional RPTs a significant flaw in this validation study and therefore proposed that, in the
future, similar validation studies should use the RPT to provide concordance data. I have no
objection to the performance of in vitro tests in parallel with rabbit tests, which are alreadyscheduled to be performed, in order to achieve concordance data. But, it is my opinion, thatany recommendation for de-novo parallel RPT should be accompanied by a statement, as
follows: “The use of rabbits in new parallel tests for the validation of an in-vitro test should
only be conducted after a vigorous search for a scientifically sound, non-animal alternative(i.e., the need for additional animal studies must be justified on a case-by-case basis).” The
inclusion of this statement would reinforce the importance of the 3R’s and would serve as areminder of U.S. Federal law.
2.0

Proposed Test Method Standardized Protocols

Does the Panel agree that the available data and demonstrated performance in terms of
relevance (i.e., accuracy/concordance, sensitivity, specificity, positive, and negative

6

The ICCVAM draft recommendations state that there is sufficient information, based on validation studies witha limited number of pharmaceuticals, to substantiate the use of these test methods for the detection ofpyrogenicity mediated by Gram-negative endotoxin in materials that are currently tested in the RPT, subject toproduct specific validation to demonstrate equivalency.
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predictivity, false positive and false negative rates) and reliability (i.e., intralaboratory
repeatability and intra- and inter-laboratory reproducibility) support the ICCVAM
draft recommendations for these in vitro test methods in terms of the proposed test
method standardized protocols?
The Panel agrees with this statement, provided that the following list of inadequacies7 within
the proposed standardized protocols are fully addressed:

7

1.

Donor-to-donor inflammatory response variation is problematic and therefore
multiple donors should be used and the number used appropriately justified.

2.

Restricting the protocols to a ‘limits’ test design, based on the intravenous
fever threshold, for all test materials independent of administration route could
be considered inappropriate. The threshold concentration for intrathecallyadministered materials would be lower because of the reduced permissible
endotoxin limit associated with these types of products. The use of a‘benchmark reference lot comparison’ test design would alleviate thenecessity to use such strict permissible ‘limits’. Interestingly, in the two
known examples where in vitro pyrogen test data have been considered by the
FDA for release testing (cited in the ICCVAM draft BRD), ‘benchmark
reference lot comparison’ test design protocols have been used.

3.

The protocols do not include sufficient descriptions of donor selection criteria(e.g., volunteer or paid, recruitment process, etc.) and conditions forvenipuncture (e.g., qualified phlebotomists, number and frequency of
venipunctures, etc.). In practice, the requirement for blood donors to havetaken no medication and the recommended CO2 concentrations are more
stringent than the provisions suggested in the draft recommendations.

4.

The protocols are inconsistent in their acceptance criteria with respect to thenumber of blood donors. The IL-6 primary cell assays require four donors to
be used for each test with acceptance criteria applied to each donor. The IL-1
assays show equal variability between donors, but do not require these
acceptance criteria.

5.

The suggested dilution scheme for the initial endotoxin standard and for thesubsequent dilutions should not be recommended. The initial dilution of theendotoxin standard in two of the assays uses 20 µL into 1980 µL. The marginof error with a micropipette is such that even the smallest error at this initial
dilution could affect the whole assay and is often the cause of a substantial
proportion of assay variability. To reduce this potential problem, it isrecommended that alternative dilution schemes be developed based on theaccuracy of the micropipetters.

6.

The use of in-house ELISA assays should not be recommended due to poortransferability and the potential for poor interlaboratory reproducibility
associated with these assays.

Based on the list of 20 separate inadequacies outlined in this report, three Panel members felt that this list
would be better described as a list of "many and substantial" inadequacies.
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7.

The protocols should clearly specify the need for resonication and/or
vortexing of any reference endotoxin solution prior to each use.

8.

To adequately test for interference, spiked test samples containing endotoxinmust be pre-incubated for a specified time prior to addition to the blood cells.

9.

The following should be included in the revised protocols: a consistent
number of donors to be used for all test methods; the acceptable range ofcytokine response for each test method; the rules and the rationale for
exclusion of low and/or high responders.

10. Three separate lots should be included in the pre-qualification of any test
material, similar to the protocol used for the BET.
11. The protocol for the MM6 cell line describes procedures that would be used
for collecting blood from donors. This point obviously is not required for thisparticular protocol.
12. The ECVAM protocols are very complete as to sources for all solutions,equipment, etc. required for testing. The ICCVAM protocols are less specific.
More specific details on all test method protocol components should be
included.
13. Intellectual property issues, as identified in the ICCVAM draft BRD, should
be addressed in the ICCVAM protocols.
14. To prevent inactivation of LPS binding protein, it should be specified that
FBS is heat inactivated at 55°C.
15. The symbols for correlation coefficient (r and r2) are interchanged
inappropriately.
16. On pages 14 and 21, lines 298 and 450 respectively in the PBMC/IL-6
protocol, the basis for the definition of low responders must be justified.
17. On page 15, line 325 in the PBMC/IL-6 protocol, the performance ofmonocyte counts using a hemocytometer is inaccurate compared to modern
flow cytometric methods.
18. If a hemocytometer is used, specifications for the number of replicate
determinations (e.g., at least duplicate), the minimum number of cells counted,
and the magnification used must be stated.
19. On page 14, line 295 in the WB/IL-1 protocol, the statement "not taken any
drug" is not sufficiently inclusive. This statement must also specify no overthe-counter medications or recreational drugs.
20. On page 20, line 411 in the WB/IL-6 protocol, the statement “If necessary, . . .
endotoxin concentration can be modified” is insufficient. The modification ofendotoxin concentration must be defined.

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3.0

May 2008

Proposed Test Method Performance Standards

Does the Panel agree that the available data and demonstrated performance in terms of
relevance (i.e., accuracy/concordance, sensitivity, specificity, positive, and negative
predictivity, false positive and false negative rates) and reliability (i.e., intralaboratory
repeatability and intra- and inter-laboratory reproducibility) support the ICCVAM
draft recommendations for these in vitro test methods in terms of the proposed test
method performance standards?
The Panel does not agree with this statement, based on the inadequacies within the proposed
performance standards outlined below.
Essential Test Method Components
1.

A uniform CV criterion should be defined, which is adequately stringent. The
reported range of 20% - 45% is inappropriate.

2.

The number of individual blood donors used and/or the number of donors to
be included in a pool of multiple donors should be defined, if deemed
appropriate.

3.

The stringency by which the endotoxin curves are validated should be defined(either by using a four-parameter logistic model or by checking that the OD
concentration values ascend in a sigmoidal manner).

4.

The use of CVs or any other measure of variability should be appropriately
justified. If the data have been log-transformed, then CVs are not informative.

5.

The following issues may overestimate the performance of the test methods:a) The nature of the prediction model used for dichotomizing the results; b)Experimental design and data analysis that might lead to overestimation of thesensitivity of the tests; c) The nature and interpretation of the in vivo data usedin the study; d) The nature and cause of incorrect results and the lack ofagreement within and between laboratories; e) Whether the tests accurately
estimate the actual concentration of the pyrogen and whether results met somepre-defined criteria of success.

6.

In Section 2.3.3.1, a ‘significant increase’ is not defined. In Section 2.3.6,
consideration should be given to adding Quality Assurance data and known
biological properties under the ‘test substances and control substances’heading.

Accuracy and Reliability Values
The demonstrated performance of certain aspects of several of the assays, particularly interms of accuracy or relevance, yields some concern. Two of the assays have false positive
rates in excess of 16%, which essentially means that approximately 1 in every 6 production
lots could be unnecessarily prevented from being released, a rate unlikely to be accepted bymanufacturers. A number of these performance characteristic issues can probably be
explained by the fact that some of the spike concentrations used were very close to the ‘limit’concentration criterion set.

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If the intended use of the in vitro assays were only to detect Gram-negative endotoxin, it
would seem very important to compare their performance in parallel validation studies thatshould include the BET. If the intended use of the in vitro methods is to evaluate substances
containing endotoxin that are unable to be evaluated with the BET, then the parallel testingstudies should include the RPT. This type of comparison has neither been made from the
RPT (2-way parallel testing was also not performed on the endotoxin-spiked sample sets
included in the validation studies cited in the ICCVAM draft BRD) nor the BET standpoint.
The last thing one wants to recommend is an inferior performing assay to the one that isalready established; similar or superior is fine.
Minimum List of Reference Substances
If the intent of the proposal was to replace the RPT with one or more of the in vitro test
methods under consideration, then the in vitro test methods must be validated for all classesof substances (e.g., pharmaceuticals, biologicals, and implants) and medical devices that canbe tested with the RPT. Validation of the in vitro test methods with pyrogens (e.g., LTA,
components of viruses and fungi) other than endotoxin also needs to be conducted.
Minority Opinion (Dr. Peter Theran): This Panel has considered the failure to undertake
additional RPTs a significant flaw in this validation study and therefore proposed that, in the
future, similar validation studies should use the RPT to provide concordance data. I have no
objection to the performance of in vitro tests in parallel with rabbit tests, which are alreadyscheduled to be performed, in order to achieve concordance data. But, it is my opinion, thatany recommendation for de-novo parallel RPT should be accompanied by a statement, as
follows: “The use of rabbits in new parallel tests for the validation of an in-vitro test should
only be conducted after a vigorous search for a scientifically sound, non-animal alternative(i.e., the need for additional animal studies must be justified on a case-by-case basis).” The
inclusion of this statement would reinforce the importance of the 3R’s and would serve as areminder of U.S. Federal law.
4.0

Proposed Additional Studies

Does the Panel agree that the available data and demonstrated performance in terms of
relevance (i.e., accuracy/concordance, sensitivity, specificity, positive, and negative
predictivity, false positive and false negative rates) and reliability (i.e., intralaboratory
repeatability and intra- and inter-laboratory reproducibility) support the ICCVAM
draft recommendations for these in vitro test methods in terms of the proposed
additional studies?
The Panel agrees that to better determine the potential of these test methods, the proposed
additional studies should be performed using the test methods described in the ICCVAM
draft BRD, taking into account the comments and recommendations detailed previously. The
Panel recognizes that these test methods could be applicable to a wider range of pyrogens
and test materials, provided that they are adequately validated for such uses. Wherever
possible, either historical data from parallel studies conducted concurrently should be
retrospectively evaluated or parallel testing should be conducted concurrently with RPT data
generated for regulatory purposes.

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The following additional recommendations are given:
1.

A repository of test materials that have been identified clinically as pyrogenic
would be invaluable for use in future validation studies and may allow suchstudies to be conducted without the further use of animals.

2.

A ‘limit’ test design protocol and a ‘benchmark reference lot comparison’ test
design protocol for each assay should be included.

3.

Both endotoxin-spiked and non-endotoxin spiked samples should be included.

4.

The non-endotoxin standards should be characterized as completely as
possible prior to their use in any study and should satisfy the requirements set
forth by ICCVAM for reference standards that are stated in the ICCVAM draft
BRD.

5.

The endotoxin-spike concentrations used for the performance assessment
studies should not be so close to the positive test concentration limit,
especially considering the relatively large enhancement and inhibition range
permitted in the sample specific qualification investigations.

6.

All aspects of the studies should be completely GLP compliant andimportantly, the laboratories and results should be independently audited. This
would include pre- and post-study audits of the laboratories.

7.

The substances tested in the studies should also include products that have
intrinsic pro-inflammatory properties.

8.

A prospective study that includes 3-way parallel testing, with all of the in vitro
assays (using both of the above mentioned protocol designs) being comparedto the RPT and the BET, should be included to allow for complete
concordance analysis and comparative performance assessment. These studies
may be conducted with historical RPT data provided that the same substances(i.e., same lot) are tested in each method. Based on ethical and scientificrationale, the design of any side-by-side studies should be limited only tothose that can gain more data than already available in the literature (i.e., datafrom parallel testing), most likely on the ability of the RPT and the in vitro
pyrogen tests to detect non-endotoxin pyrogens.

9.

Test substances that better represent all categories of sample types intended
for testing by the methods (e.g., pharmaceuticals, biologicals, and medical
devices) should be included. If relevant, extraction procedure protocols for thedetection of pyrogens in medical device materials should be included.

10. The effects of direct administration of IL-1 and IL-6 to rabbits and thecomparison of the resulting pyrogenic response with endotoxin-mediatedpyrogenicity should be evaluated.
11. The correlation of IL-1and IL-6 levels in the in vitro tests with levels
produced in rabbits using similar doses of endotoxin should be evaluated.

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The following statistical recommendations are noted:
1.

For reliability assessments, formal hypothesis testing is essential with thealternative hypothesis being no different between groups.

2.

For any additional studies, formal sample size calculations for equivalence
testing should be made to determine that the required number of replicatesneeded to reject the null hypothesis (i.e., that there is a difference inreliability) at a given level of significance and power. If the study is notprospectively powered, the posterior power should be provided along with theobserved significance level.

3.

The proposed strategy for the Cryo WB/IL-1 test method is to retest if a test
fails because of too much variability. The statistical properties of thismultistage procedure should be characterized.

Minority Opinion (Dr. Peter Theran): This Panel has considered the failure to undertake
additional RPTs a significant flaw in this validation study and therefore proposed that, in the
future, similar validation studies should use the RPT to provide concordance data. I have no
objection to the performance of in vitro tests in parallel with rabbit tests, which are alreadyscheduled to be performed, in order to achieve concordance data. But, it is my opinion, thatany recommendation for de-novo parallel RPT should be accompanied by a statement, as
follows: “The use of rabbits in new parallel tests for the validation of an in-vitro test should
only be conducted after a vigorous search for a scientifically sound, non-animal alternative(i.e., the need for additional animal studies must be justified on a case-by-case basis).” The
inclusion of this statement would reinforce the importance of the 3R’s and would serve as areminder of U.S. Federal law.

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C.

May 2008

OVERALL PEER REVIEW OUTCOMES

This international independent Peer Review Panel, consisting of 13 expert scientists fromfive different countries, provided comments and recommendations on the usefulness andlimitations of five in vitro pyrogen test methods for the detection and quantification of Gramnegative endotoxin and on the ICCVAM draft test method recommendations on the use of
these in vitro methods as partial replacements for the RPT. These remarks are summarized
below.
• In general, the information presented in the ICCVAM draft BRD was sufficient forthe purpose of determining the usefulness and limitations of these test methods fortheir proposed use and for adequately addressing the applicable validation criteria on
the basis of the currently available evidence.
• The available data and demonstrated performance in terms of their reliability andrelevance do not at this time support the ICCVAM draft proposed use for these test
methods (i.e., as a partial substitute or replacement for the RPT, for the identificationof Gram-negative endotoxin, on a case-by-case basis, subject to product specificvalidation). To better characterize the test methods and more clearly define theirreliability and relevance, the Panel recommended that specific additional studies be
performed using the ICCVAM proposed protocols, taking into account the Panel'scomments and recommendations.
o The lack of parallel testing in the in vitro tests and the RPT, and the resulting
lack of concordance data, was considered to be a major limitation of thevalidation study design. For this reason, the Panel recommended that futurestudies include parallel testing. A minority opinion (Dr. Peter Theran)associated with parallel testing was expressed as follows: “The use of rabbits
in new parallel tests for the validation of an in-vitro test should only be
conducted after a vigorous search for a scientifically sound, non-animalalternative (i.e., the need for additional animal studies must be justified on acase-by-case basis)".
• The available data and demonstrated performance in terms of their reliability andrelevance does not support the ICCVAM draft performance standards for these invitro test methods for regulatory purposes.
• The information provided in the ICCVAM draft BRD supports the ICCVAM draft
recommended protocols for these five in vitro test methods, providing that the list of
inadequacies8 identified by the Panel with respect to reliability and relevance are fully
addressed.
• These test methods could be applicable to a wider range of pyrogens and test
materials, provided that they are adequately validated for such uses.
• It is critical to recognize, despite concerns about the performance of these five in vitro
test methods, that a formal process exists for materials regulated under 21 CFR 610.9
to qualify these in vitro methods for the identification of Gram-negative endotoxin ona case-by-case basis, subject to product specific validation.
8

Based on the list of 20 separate inadequacies outlined in this report, three Panel members felt that this list
would be better described as a list of "many and substantial" inadequacies.
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ICCVAM Test Method Evaluation Report: Appendix A2Appendix A2
Summary Minutes from Peer Review Panel Meeting on
February 6, 2007

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Meeting Summary
Independent Peer Review Panel Meeting
Five In Vitro Test Methods Proposed for Assessing Potential Pyrogenicity of
Pharmaceuticals and Other Products
National Institutes of Health (NIH), Natcher Conference Center
Bethesda, MD
February 6, 2007
8:30 a.m. – 5:00 p.m.

Panel Members:
Karen Brown
Brian Crowe
Nancy Flournoy
Ihsan Gursel
Ken Ishii
Jack Levin
Albert Li
David Lovell
Melvyn Lynn
Anthony Mire-Sluis
Jon RichmondPeter Theran
Kevin Williams

DRL Pharma and Pair O'Doc's Enterprises
Baxter Vaccine AG
University of Missouri-Columbia
Bilkent University
ERATO, Japan Science & Technology Agency,
Osaka University
University of California-San Francisco
In Vitro ADMET Laboratories
University of Surrey
Eisai Medical Research
AMGEN, Inc.
UK Home Office
MSPCA
Eli Lilly

ICCVAM and ICCVAM Pyrogenicity Working Group (PWG) Members:
Mustafa Akkoyunlu
FDA/CBER
Peter Amin
FDA/CBER
Kimberly Benton
FDA/CBER
Joseph George
FDA/CBERDavid Hussong
FDA/OPS
Abigail Jacobs
FDA/CDER
Jodie Kulpa-Eddy (ICCVAM
USDA/APHIS
Vice Chair)
Robert Mello
FDA/CDER
Richard McFarland (PWG Chair) FDA/CBER
Penelope Rice
FDA/CFSAN
William Stokes
NIEHS
Raymond Tice
NIEHS
Daniela Verthelyi
FDA/CDER
Marilyn Wind (ICCVAM Chair)
CPSC
Jiaqin Yao
FDA/CDER
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Public Attendees:
Allen Dearry
Basil Golding
Thomas Hartung
Coty Huang
Sue Leary
Thomas Montag
Michael Myers
Steven Myers
Seishiro Naito
Michael Scott
Kristie Stoick
Michael Timm
Rachel Waltman

NIEHS
FDA/CBER
ECVAM
FDA/CBER
ARDF
ECVAM
FDA/CVM
Becton, Dickinson & Company
NIID
FDA/CVM
PCRM
University of Copenhagen
USDA/APHISNICEATM Staff:
David Allen
Elizabeth Lipscomb
Linda Litchfield
Debbie McCarley
James Truax
Douglas Winters

ILS, Inc.
ILS, Inc.
ILS, Inc.
NIEHS
ILS, Inc.
ILS, Inc.

May 2008

Abbreviations: APHIS = Animal and Plant Health Inspection Service; ARDF = Alternatives Research andDevelopment Foundation; CBER = Center for Biologics Evaluation and Research; CDER = Center for Drug
Evaluation and Research; CFSAN = Center for Food Safety and Applied Nutrition; CPSC = Consumer Product
Safety Commission; CVM = Center for Veterinary Medicine; ECVAM = European Centre for the Validation of
Alternative Methods; ERATO = Exploratory Research for Advanced Technology; FDA = U.S. Food and Drug
Administration; ILS = Integrated Laboratory Services; MSPCA = Massachusetts Society for the Prevention of
Cruelty to Animals; NIEHS = National Institute of Environmental Health Sciences; NIID = National Institutefor Infectious Diseases; OPS = Office of Pharmaceutical Science; PCRM = Physicians Committee for
Responsible Medicine; USDA = U.S. Department of Agriculture

Call to Order
Dr. Karen Brown (Panel Chair) called the meeting to order at 8:30 a.m. and introduced
herself. She then asked all Peer Panel members, National Toxicology Program Interagency
Center for the Evaluation of Alternative Toxicological Methods (NICEATM) staff, membersof the Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM) and the ICCVAM Pyrogenicity Working Group (PWG) in attendance, theEuropean Centre for the Validation of Alternative Methods (ECVAM) liaison to the PWG,
and members of the public to state their name and affiliation for the record. Dr. Brown asked
all individuals to identify themselves when they spoke and to use the provided microphones.
Dr. Brown stated that three public comment sessions were scheduled during the meeting andshe reminded individuals who wished to speak to register at the registration table. Dr. Brownemphasized that there was no need to repeat the same comments at each comment session.
Welcome from the ICCVAM Chair
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Dr. Marilyn Wind, Consumer Product Safety Commission and Chair of ICCVAM, welcomed
everyone to the Peer Review Panel meeting and thanked the Panel members for their
participation. Dr. Wind stressed the importance of an independent scientific peer review to
the ICCVAM test method evaluation process.
Welcome from the Director, NICEATM, and Conflict of Interest Statements
Dr. William Stokes, Director of NICEATM, welcomed everyone and reiterated Dr. Wind's
appreciation to the participants for agreeing to serve on the Panel. Dr. Stokes stated that hewould be serving as the Designated Federal Official for the public meeting. He stated thismeeting was being held in accordance with the Federal Advisory Committee Act regulations
and that the Panel was constituted under the NIH Special Emphasis Panel charter. Dr. Stokes
read the conflict of interest statement and asked the Panel members to declare if they had any
direct or indirect conflicts, and to recuse themselves from discussion and voting on any
aspect of the meeting where there might be a conflict. None of the Panel members declared aconflict of interest.
Overview of the ICCVAM Test Method Evaluation Process
Dr. Stokes provided an overview of the ICCVAM test method evaluation process. He statedthat the international Panel was made up of 13 scientists from five different countries(Austria, Japan, Turkey, United Kingdom, and United States). Dr. Stokes described that the
purpose of the Panel was to assist ICCVAM by carrying out an independent scientific peer
review of the information provided in the ICCVAM Background Review Document (BRD)
on the validation studies of five in vitro test methods proposed for assessing the potential
pyrogenicity of pharmaceuticals and other products. He stated that Panel members wereexperts selected and appointed by the National Institute of Environmental Health Sciences(NIEHS) to ensure sufficient scientific expertise to carry out a comprehensive review ofthese test methods.
Dr. Stokes listed the 15 ICCVAM member agencies and provided a brief review of
ICCVAM's history. He summarized the ICCVAM Authorization Act of 2000 (available at:
http://iccvam.niehs.nih.gov/docs/about_docs/PL106545.pdf) and detailed the purpose andduties of ICCVAM as mandated in the Act. He noted that one of ICCVAM's duties is toreview and evaluate new, revised, and alternative test methods applicable to regulatory
testing. He stated that all of the reports produced by NICEATM are available from the
ICCVAM/NICEATM website or directly from NICEATM. Dr. Stokes pointed out thatICCVAM does not carry out research, development, or validation studies, but instead,facilitates these processes by convening scientific symposia, workshops, and expert Panelreviews such as this one.
Dr. Stokes then described the ICCVAM test method evaluation process, which begins with atest method nomination or submission. NICEATM conducts a prescreen evaluation tosummarize the extent to which the proposed submission or nomination addresses the
ICCVAM prioritization criteria. A report of this evaluation is then provided to ICCVAM,
which in turn develops recommendations regarding the priority for evaluation. ICCVAM
then seeks input on their recommendations from the Scientific Advisory Committee on
Alternative Toxicological Methods (SACATM) and the public. Given sufficient regulatory
applicability, sufficient data, resources, and priority, a method will move forward to a formal
evaluation. A draft BRD is prepared by NICEATM in conjunction with an ICCVAM
working group for the relevant toxicity testing area (e.g., pyrogenicity), which provides a
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comprehensive review of all available data and information. ICCVAM then considers all of
the available information and prepares draft recommendations for 1) proposed usefulness andlimitations of the test methods, 2) test method protocol, 3) performance standards, and 4)
future studies. The draft BRD is then made publicly available for review and comment. An
independent peer review Panel is then convened to provide comments and recommendations
on the draft BRD, public comments, and ICCVAM draft test method recommendations. A
Peer Review Panel Report is published and considered by ICCVAM, along with public and
SACATM comments, when their final recommendations are forwarded to the appropriate
ICCVAM agencies.
Dr. Stokes reviewed the criteria for adequate validation. He stated that validation is definedby ICCVAM as the process by which the reliability and relevance of a procedure areestablished for a specific purpose, and that adequate validation is a prerequisite forconsideration of a test method by U.S. Federal regulatory agencies. Dr. Stokes listed the
ICCVAM acceptance criteria for test method validation and acceptance.
ICCVAM Charge to the Panel
Dr. Stokes reviewed the charge to the Panel, which was to: 1) review the draft BRD forcompleteness and identify any errors or omissions; 2) determine the extent to which each of
the applicable criteria for validation and regulatory acceptance had been addressed for the
proposed use; and 3) consider and provide comment on the extent to which the ICCVAM
draft test method recommendations including the proposed use, recommended protocols,
performance standards, and recommended additional studies are supported by the
information provided in the BRD.
Dr. Stokes thanked the PWG, ICCVAM, and NICEATM for their work on this project, andhe acknowledged the NICEATM staff for organizing the Panel meeting and preparing thematerials being reviewed.
Overview of Pyrogenicity Testing Requirements and Current Pyrogenicity Testing
Procedures
Dr. Richard McFarland, Food and Drug Administration (FDA), Center for Biologics
Evaluation and Research (CBER) and Chair of the PWG, thanked the PWG members fortheir efforts in producing the draft BRD, and thanked the Panel members for their
participation in the peer review process. Dr. McFarland discussed the scientific need for
pyrogenicity testing and its relationship to the regulatory mandate for protection of publichealth. He discussed the need for risk management, especially the detection of endotoxin andnon-endotoxin pyrogen-contaminated products, and he noted the need for classification andlabeling of products as pyrogen-free (i.e., the product does not exceed established endotoxin
limits). Dr. McFarland then summarized the U.S. and European legislation and statutory
protocol requirements for pyrogen testing.
Overview of the Five In Vitro Pyrogen Test Method Protocols
Dr. Thomas Hartung, Head of ECVAM and invited test method expert, remarked that he has
been closely involved in the ECVAM validation studies and as such recognized his
considerable conflict of interest. Dr. Hartung summarized the disadvantages of the rabbit
pyrogen test (RPT) and the bacterial endotoxins test (BET), and related these limitations to
the development of the in vitro pyrogen test methods.
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Dr. Hartung indicated that a typical in vitro pyrogen test method consists of two parts: 1)
incubation of the test sample in a cellular cytokine release system (i.e., whole blood [WB],
Mono Mac 6 [MM6] cells, Peripheral blood mononuclear cells [PBMC]); and 2) cytokine
detection using a specific enzyme-linked immunosorbent assay (ELISA) (e.g., Interleukin[IL]-1β or IL-6). He stated that the European Commission granted $2.5 million for thevalidation of these novel test methods, but that this sum was only sufficient to cover "thebasics". Dr. Hartung then made the following comments regarding the design of the ECVAM
validation study:•

For the validation study, the endotoxin threshold was set at 0.5 Endotoxin
Units (EU)/mL, based on the positive response of 50% of the most sensitive
rabbit strain to 50 pg of endotoxin. A substance was considered pyrogen-free
if the endotoxin level in an in vitro test method corresponded to less than 0.5
EU/mL. A positive product control (PPC) was used in a pretest to insure that
there is no interference. Specific criteria were used to minimize assay
variability (e.g., blood donors, coefficient of variation).

•

In 1988, Dr. Stephen Poole described an IL-6 cytokine assay using isolated
leukocytes. The PBMC test method evolved from this study and has
subsequently been used by Novartis for U.S. Food and Drug Administration(FDA) release of one product (i.e., after product-specific validation and in
conjunction with the rabbit pyrogen test).

•

Two of the assays included in the validation exercise, WB/IL-1β and WB/IL6, utilize human WB. Many research studies have described using these test
systems for routine pyrogen testing of up to 80 pharmaceutical products
against a variety of pyrogens. A commercial kit has been developed using
theWB/IL-1β test system.

•

A catch-up validation study was performed using the Cryo-WB/IL-1β test
method, which was not available during the original validation study. This
assay utilizes cryopreserved WB pooled from several donors. Although thecells remain in diluted dimethyl sulfoxide, an effect on cell morphology or
viability is not observed.

Overview of the Draft In Vitro Pyrogen Test Method Background Review Document(BRD)
Dr. David Allen, Integrated Laboratory Systems, Inc. (the NICEATM support contractor),presented an overview of the ICCVAM draft BRD. Dr. Allen indicated that five BRDs were
submitted by ECVAM in June 2005. A Federal Register notice was used to request data fromover 100 interested stakeholders, but no additional data were submitted. Following thisrequest, a comprehensive ICCVAM draft BRD, which describes the current validation statusof the five in vitro test methods based on U.S. Federal regulatory standards, was compiled
and made available to the public on December 1, 2006.
Dr. Allen briefly summarized the performance characteristics of the in vitro test methods,
which are detailed in the ICCVAM draft BRD (available at:
http://iccvam.niehs.nih.gov/methods/pyrogen/pyrodocs/pyroBRDUdocs/PyroBRD01Dec06.p
df).
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Dr. Allen noted that Dr. Marlies Halder, ECVAM liaison to the PWG, provided additional
information requested by the Panel, including data audits, evidence of Good Laboratory
Practice (GLP) compliance of testing laboratories, information on the protocol used for thehistorical RPT studies, and lot numbers of the test substances. He also stated that a request
was made for the ECVAM Science Advisory Committee (ESAC) peer-review documents,
but that these documents are not available to the public.
Peer Review Panel Evaluation:
Dr. Brown introduced the relevant Panel Group Leaders for each BRD Section: (Dr. Melvyn
Lynn - Sections 1, 2, and 11; Dr. Jack Levin - Sections 3, 5, and 6; Dr. Anthony Mire-Sluis Sections 7 and 8; Dr. Jon Richmond - Sections 4, 9, and 10). The Group Leaders presented
the draft responses to the Evaluation Guidance Questions for consideration by the entire
Panel. The Panel discussion and their recommended revisions to each section of the
ICCVAM BRD are reflected in the Independent Peer Review Panel Report: Five In Vitro
Test Methods Proposed for Assessing Potential Pyrogenicity of Pharmaceuticals and Other
Products, published in April 2007 (hereafter, the Panel report, available at:
http://iccvam.niehs.nih.gov/docs/pyrogen/PrRevPanFinRpt.pdf).
Public Comments (Session 1)
Ms. Mary Lou Chapek - President and Chief Executive Officer of MBP Laboratories,
Inc.
Dr. David Allen read the written comments submitted by Ms. Chapek to
ICCVAM/NICEATM. Her comments are summarized as follows:•

Ms. Chapek expressed disappointment in the number of test methods
reviewed by ICCVAM and accepted by federal agencies over the past 15
years. She commented that the pyrogenicity BRD and recommendations
currently under discussion indicate a lack of focus. Ms. Chapek noted that
substantial work remains to be done for validation of these test methods andshe suggested the phased strategy outlined below.

•

Phase I should concentrate on replacement of the BET, not the RPT. A large
array of test substances compatible with the BET could be spiked withendotoxin to determine their accuracy and sensitivity and to determine thelevel of interference, if any, with each of these test systems.

•

Phase II should consist of validation of one or two in vitro test methods for
replacement of the RPT. Cell-based assays that do not depend on blood, which
has an impractical limited time domain, would be preferable and could becompared directly to RPT data. The task would still be complex, but with asmaller focus. Phase II would also require evaluation and validation of allmaterials currently tested in the RPT, as well as the pyrogens detected in the
RPT. Some of these standards would have to be developed. Although thesestudies may take years for completion, replacement of the RPT by one or twoof the in vitro pyrogen tests in Phase II would constitute an achieved goal by
ICCVAM.

Dr. Thomas Montag - Paul Ehrlich Institute (PEI) - Germany
Dr. Montag provided the following comments:
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He stated that the PEI is responsible for the quality and safety of biologicaldrugs in general and that his laboratory has been involved in alternative
pyrogen testing with Dr. Hartung for over 12 years. While the data isproprietary, he confirmed that he has used the WB/IL-1β assay for
approximately two years.

•

Dr. Montag commented that donors are now pooled (up to 10 at a time) tominimize variability, especially for detection of non-endotoxin pyrogens. For
the Cryo WB/IL-1β pyrogen test, the blood is typically pretested for reactivity
after pooling. In response to the PPC issue mentioned previously, he remarked
that this was a design flaw that had been corrected in the ECVAM Standard
Operating Procedure. He also stated that an expert Panel from the EDQM is
now in the process of creating a draft of this alternative test method for
publication.

Final Review of the BRD for Errors and Omissions
Dr. Brown asked the Panel to review the recommended revisions for each BRD section,
taking into account the public comments, and to decide if additional changes are necessary.
No changes were made to the draft recommendations based on the public comments.
Validation Status of the In Vitro Test Methods
Dr. Brown asked the Panel if they agreed that the applicable validation criteria had beenadequately addressed in the ICCVAM BRD in order to determine usefulness and limitationsof these in vitro test methods, to serve as a substitute for the RPT, for the identification ofGram-negative endotoxin on a case-by-case basis subject to product-specific validation.
The Panel agreed that the information was adequate with which to make an informed
decision.
Dr. Brown asked the Panel if they agreed that the performance of these test methods in termsof their relevance and reliability support the proposed use for the detection of Gram-negative
endotoxin in materials that are currently tested in the RPT, subject to product-specificvalidation to demonstrate equivalency to the RPT.
The Panel did not agree with this statement based on the reasons indicated in the responses tothe questions related to Sections 1.0 to 12.0 of the ICCVAM BRD. Two minority opinions
were expressed. Responses to these questions, and the associated minority opinions aredetailed in the Panel Report.
Public Comments (Session 2)
Dr. David Hussong - FDA, Center for Drug Evaluation and Research (CDER)
Dr. Hussong commented that the Code of Federal Regulations (CFR), Section 211.167, statesthat if a drug is to be labeled as pyrogen-free, an appropriate test is required. The U.S.
Pharmacopeia (USP) provides guidelines for the RPT and the BET. While the BET is notconsidered equivalent to the RPT, data from the BET is accepted. The USP states that use ofalternative tests is permitted and that they may be used in lieu of the BET, provided that thealternative test uses a reference standard for comparison. It should be noted that the FDA
CDER approves drugs, not test methods, but welcomes the use of alternative test methods.
Dr. Thomas Hartung - Head, ECVAM - Italy
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Dr. Hartung stated that the in vitro pyrogen tests were designed to determine the thresholdlevel of endotoxin in the most sensitive rabbit strain. This design was ambitious andconsequently, resulted in the low sensitivity (58%) and specificity (83%) observed. It should
be noted that some assays had values of 80% or 90% at this critical concentration andperformed better than the RPT.
ICCVAM Draft Recommendations for In Vitro Pyrogen Test Methods
Presentation of Draft ICCVAM Recommendations
Dr. Brown asked the Panel to evaluate the extent to which the ICCVAM draft
recommendations are supported by the information and data provided in the ICCVAM draft
BRD. Dr. Brown reminded the Panel that the purpose is not to approve or disapprove of the
ICCVAM draft recommendations, but rather to comment on the extent to which they aresupported by the information contained in the ICCVAM BRD. The Panel discussion andassociated conclusions relevant to each of the ICCVAM recommendations are reflected in
the Panel Report.
Public Comments (Session 3)
Ms. Kristie Stoick - Physicians Committee for Responsible Medicine
Ms. Stoick reviewed written comments that she previously submitted to
ICCVAM/NICEATM. She stated that the pace of acceptance of alternative methods, such asthese in vitro pyrogen tests, in the opinion of the animal protection community, is
unacceptably slow. She continued to state that too much time is spent debating every
scientific detail and that the ultimate goal is lost. She closed by asking ICCVAM to take into
account her comments when considering the Panel's recommendations for the validation ofthese assays.
Final Review of the ICCVAM Draft Recommendations
Dr. Brown asked the Panel to review the ICCVAM draft recommendations, taking into
account the public comments, and to decide if additional changes are necessary. No changes
were made to the draft recommendations based on the public comments.
Concluding Remarks
Dr. Brown thanked the Panel and ICCVAM/NICEATM for their help. She expressed hopethat this peer review process helped to establish a focus for ICCVAM and that the reduction
in animal use would be the ultimate outcome. Dr. Stokes thanked the Panel for their hard
work, thoughtful and objective deliberations, and advice. Dr. Stokes stated that the ICCVAM
PWG and ICCVAM would consider these recommendations as they move forward with thisprocess and the results of this meeting would culminate in a Peer Review Panel Report thatwould be released to the public toward the end of March for additional comment.
Adjournment
The meeting was adjourned at 5:47 p.m.

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William S. Stokes, D.V.M.
NIEHS
P.O. Box 12233
MD-EC17
Research Triangle Park, NC 27709

Dear Dr. Stokes:
The Meeting Summary, Peer Review Panel Public Meeting, Five In Vitro Test Methods
Proposed for Assessing Potential Pyrogenicity of Pharmaceuticals and Other Products, dated
February 6, 2007, accurately summarizes the Peer Review Panel meeting of February 6,
2007, in Bethesda, MD.
Sincerely,--------------------------------------------------------------------------

Signature

Printed Name

Date

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ICCVAM Test Method Evaluation Report: Appendix A3Appendix A3
Pyrogenicity Peer Review Panel Biographies

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Karen Brown, Ph.D. (Panel Chair)
Dr. Brown received her Ph.D. in Microbiology and Biochemistry at Oklahoma State
University in Stillwater. She is President, Pair O’ Docs Enterprises, consulting withcompanies and with the U.S. Department of Agriculture Animal and Plant Health Inspection
Service (APHIS) Center of Veterinary Biology on development of in vitro assays to replace
animal tests for release of veterinary vaccines and is a Consultant, sharing the CEO position
for MVP Laboratories. Dr. Brown’s resume indicates her broad expertise in in vitro and invivo pyrogen testing and thorough knowledge of regulatory requirements for drug andproduct development. Early in her career, Dr. Brown developed bench and supervisory
experience working in Quality Control conducting animal testing, including the rabbit
pyrogen test and in vitro Limu endotoxin testing. She initiated an in vitro development groupat Bayer as Head of Biological Research and Development that specialized in developing andvalidating ELISAs for Limulus Amebocyte Lysate (LAL) testing and antigen quantitation for
release of biological products. Dr. Brown remains involved in endotoxin testing byconsulting with various companies to determine correlations in endotoxin levels in various
veterinary products to reactions produced by some of these products when used to vaccinate
animals. She has expertise in microbiology, veterinary medicine, vaccine and biologicals
development and safety testing, in vitro methods development, and technical government
relations (European Union [EU] and United States [U.S.]). Dr. Brown was Chairman of the
In Vitro Working Group of the Veterinary Biologics Section of the Animal Health Institute(AHI) and APHIS liaison (regulatory) for registration of new vaccine and diagnostic
products. Dr. Brown has conducted or managed research and development to register 44 new
drug products, pharmaceuticals, vaccines, or diagnostic products or technologies and she has
44 publications and presentations and 23 U.S. patents. She is a member of the AHI,
Veterinary Biologics Section, the Association of Veterinary Biological Companies, theAmerican Society for Microbiology, and the U.S. Animal Health Association.
Brian Crowe, Ph.D.
Dr. Crowe received his Ph.D. in Microbiology from Trinity College in Dublin, Ireland. He is
the Director of Immunology (Vaccines) at Baxter Vaccine AG in Austria and has
responsibility for two research departments (Molecular Immunology and Humoral
Immunology) and a quality control department (Biological Control) comprised of three
quality control laboratories (Bacteriology, in vitro and in vivo testing). Dr. Crowe’s resume
demonstrates a significant and broad level of expertise in pyrogen test methodology andknowledge of laboratory, manufacturing, and validation procedures. Dr. Crowe has
responsibility for general safety and toxicity testing and heads the Rabbit Pyrogen Testingand Endotoxin (LAL) Testing Units for Baxter Bioscience in Austria with testing rates of
3,000 to 26,000 samples per year. Dr. Crowe has extensive experience with high throughput
screening, cytokine response assays, cytotoxicity testing, inflammatory response assays,
complement testing, and other molecular, cellular, and humoral immunological response
testing. He is also well versed in Good Manufacturing Practice and Good Laboratory Practice(GLP) standards and in issues of validation and audit requirements and procedures. Dr.
Crowe has authored or coauthored 25 publications and 4 patents. His research interests arefocused on bacterial and viral vaccines.

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Nancy Flournoy, M.S., Ph.D.
Dr. Flournoy received a M.S. degree in Biostatistics from the University of California at Los
Angeles, and a Ph.D. in Biomathematics from the University of Washington. She is Professor
and Chair of the Department of Statistics at the University of Missouri-Columbia. Her
research interests include adaptive designs, bioinformatics, chemometrics, clinical trials, andenvironmetrics. She has an extensive list of edited volumes and papers on statistical theory,
statistical genetics and immunology, epidemiology in immune suppressed subjects, clinical
trials for prevention and treatment of viral infection, transplantation biology and its effects ondigestion, lungs, eyes, mouth, and central nervous system, optimization of statisticalprocessing, and additional papers, interviews, and technical reports. She has editorial
responsibilities for numerous statistical journals, serves on numerous advisory boards, andnominating committees. She is a member and past Chair of the Council of Sections of theAmerican Statistical Association, and served in various other statistical, medical andtoxicological societies or programs as Chair or as a member of the Board of Directors. She isa former member of the Scientific Advisory Committee on Alternative Toxicological
Methods. She also served on the Expert Panels for the National Toxicology Program
Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)
and the Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM) that evaluated the Revised Up-and-Down Procedure; the Current Validation
Status of In Vitro Test Methods for Identifying Ocular Corrosives and Severe Irritants; andFive In Vitro Pyrogen Test Methods.
Ihsan Gursel, MSc, Ph.D.
Dr. Gursel received his MSc. and Ph.D. degrees from the Middle East Technical University,
Department of Biological Sciences in Ankara, Turkey. He is an Assistant Professor in the
Department of Molecular Biology and Genetics at Bilkent University in Ankara. Dr. Gursels’
resume indicates that he has significant experience studying the receptor family believed tomediate pyrogenic responses (i.e., Toll-like receptors [TLR]). Dr. Gursel’s research interests
include studies on the roll of TLR and TLR-ligand interactions in an innate immune
response, gene expression and transcriptional profiling of immune cells via high throughput
screening methods, design of controlled release systems for oligodeoxynucleotide targetingand delivery, and application of biodegradable natural polymers for biomaterials, tissue
engineering, and drug delivery. Dr. Gursel has received numerous awards and grants to
support his work and has authored or coauthored more than 45 publications, 7 patents, andhas given 28 formal presentations related to his research. He has also refereed papers for the
Journal of Leukocyte Biology, Immunopharmacology and Immunotoxicology, and Vaccine.
Ken Ishii, M.D., Ph.D.
Dr. Ishii received his M.D. and Ph.D. degrees from the School of Medicine at Yokohama
City University in Kanagawa, Japan. He is a Group Leader for the Akira Innate Immunity
Project at the Exploratory Research for Advanced Technology, Japan Science andTechnology Agency, Osaka University. Dr. Ishii’s resume indicates that he has extensive
regulatory experience that includes pyrogen testing requirements for pharmaceuticals. Dr.
Ishii was formerly a Staff Scientist at the Food and Drug Administration (FDA) Center for
Biologics and Evaluation Research (CBER). His work experience includes regulation of
Investigational New Drug applications related to DNA-based vaccines and immunotherapy
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using DNA vaccine and immunostimulatory DNA (CpG DNA). Dr. Ishii also has regulatory
experience related to vaccines and immunotherapies against infectious diseases and allergy.
He has authored or coauthored 58 publications in peer-reviewed journals and holds 17
patents.
Jack Levin, M.D.
Dr. Levin received an M.D. from the Yale University School of Medicine in New Haven,
CT. He is an Independent Investigator at the Marine Biological Laboratory at Woods Hole,MA. Dr. Levin is also a Professor of Laboratory Medicine and Professor of Medicine at the
University of California School of Medicine in San Francisco. He previously held various
academic positions (e.g., Professor of Medicine) at Johns Hopkins Hospital in Baltimore andholds additional positions (e.g., Associate Member of the Cancer Research Institute at
University of California at Santa Cruz, attending physician and Director of the
Anticoagulation Clinic at the Veterans’ Administration Medical Center in San Francisco).
Dr. Levin is board-certified in Internal Medicine by the American Board of Internal
Medicine. Dr. Levin’s resume indicates that he has extensive experience studying the
pyrogenic response and pyrogen testing (e.g., research in hemoglobin-lipopolysaccharide
interactions and pioneered gel-clot LAL technology). Dr. Levin is a former editor-in-chief of
the Journal of Endotoxin Research, a member of the American Society of Hematology(serving on various committees), a member of the Corporation, Marine Biological
Laboratory, a Fellow of the American College of Physicians, a member of the American
Society for Experimental Pathology, American Society for Clinical Investigation, theCalifornia Academy of Medicine, the International Endotoxin Society, and numerous othersocieties. Dr. Levin has co-organized nine international conferences and has 246 publications
in peer-reviewed journals, book chapters, or edited series.
Albert Li, Ph.D., MBA
Dr. Li received his Ph.D. in Biomedical Sciences from the University of Tennessee, Oak
Ridge and an Executive MBA from the University of Maryland University College inCollege Park. Dr. Li co-founded three companies to advance drug development. He is
Chairman and CSO of ADMET Group, LLC; Founding Chairman, President, and CEO of In
Vitro ADMET Laboratories in Rockville, MD; and Founding Chairman, President, and CEO
of Advanced Pharmaceutical Sciences in Baltimore, MD. Dr. Li’s resume indicates that hehas a broad level of experience in validation of in vitro and alternative methods. Dr. Li has
secured multiple research grants to advance a drug candidates from the preclinical laboratory
through clinical trials, developed proprietary technology of interest to the pharmaceuticalindustry, and established a GLP laboratory for in vitro efficacy, metabolism, and toxicity
testing. Dr. Li has published over 130 scientific papers, numerous books/special journal
issues, and is frequently invited to speak in national and international conferences.
David Lovell, Ph.D., FIBiol, CBiol, F.S.S., CStat
Dr. Lovell received a Ph.D. from the Department of Human Genetics and Biometry,
University College, London. He is currently Reader in Medical Statistics at the Postgraduate
Medical School at the University of Surrey. Previously, he was Associate Director and Headof Biostatistics support to Clinical Pharmacogenomics at Pfizer Global Research andDevelopment in Sandwich, Kent providing data management and statistical support topharmacogenetics and genomics. He joined Pfizer in 1999 as the Biometrics Head of Clinical
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Pharmacogenetics. Before joining Pfizer, Dr. Lovell was the Head of the Science Division at
BIBRA International, Carshalton, which included Molecular Biology, Genetic Toxicology,
Biostatistics and Computer Services. At BIBRA, Dr. Lovell managed the statistical andcomputing group providing specialized statistical support to BIBRA’s Clinical Unit andcontract research work. He conducted and managed research programs on genetics, statistics
and quantitative risk assessment for the EU and United Kingdom (U.K.) Government
Departments. His research interests at BIBRA were in the use of mathematical and statisticalmethods together with genetic models in the understanding of toxicological mechanisms andrisk assessment problems. Dr. Lovell had previously been a Senior Research Officer with the
U.K. Medical Research Council (MRC) Experimental Embryology and Teratology Unit, avisiting Postdoctoral Fellow at the National Institute of Environmental Health Sciences inNorth Carolina, U.S., a Geneticist at the MRC Laboratories, Carshalton and a Research
Assistant in Cytogenetics at Birmingham University. He has acted as a consultant to a numberof organizations, has considerable experience of working with Regulatory Authorities, has many
publications related to his work and has wide experience of making presentations to a wide
range of audiences. He is a member of the U.K. Government’s advisory Committee on
Mutagenicity of Chemicals in Food, Consumer Products and the Environment and theIndependent Scientific Advisory Committee for Medicines and Healthcare Products
Regulatory Agency database research. He served on the NICEATM-ICCVAM Expert Panels
that evaluated the Frog Embryo Teratogenesis Assay - Xenopus, In Vitro Test Methods for
Identifying Ocular Corrosives and Severe Irritants, and Five In Vitro Pyrogen Test Methods.
Melvyn Lynn, M.S., Ph.D.
Dr. Lynn received M.S. and Ph.D. degrees in Microbiology from Rutgers University in New
Brunswick, New Jersey. He is currently Senior Director and Global Head, Sepsis and AntiInfectives Therapeutic Area at Eisai Inc. Dr. Lynn’s expertise in the area of pyrogenicity isevidenced from his involvement in the clinical development of TLR4 antagonists andantimicrobials. Dr. Lynn directs global clinical development of a TLR-4 antagonist andantimicrobials and is head of a multifunctional, international project team, for which heregularly interacts with FDA and international regulatory agencies. Dr. Lynn has participatedin global Standard Operating Procedure process development teams and served on the Eisai
Global Clinical Development Global Development Board to address globalization of clinicaldevelopment of drugs and clinical processes. Dr. Lynn has authored or coauthored 24 peerreviewed publications, a review, two book chapters, a research letter, and 28 abstracts. Dr.
Lynn has additional drug development experience during his tenure at the Bristol-Myers
Squibb Company.
Anthony Mire-Sluis, Ph.D.
Dr. Mire-Sluis received his Ph.D. in Cell Biology and Biochemistry from the Department of
Haematology at the Royal Free Hospital Medical School. He is Senior Director – ProductQuality and External Affairs at AMGEN, Inc. with former positions as Head of the Cytokine
Group at the National Institute for Biological Standards and Control, Director of
Bioanalytical Sciences at Genentech, Inc., Head of Analytical Science and Standards in theCenter for Biologics Evaluation and Research (CBER) at the FDA and Principal Advisor for
Regulatory Science and Review in the Office of Biotechnology Products and Office ofPharmaceutical Sciences in the Center for Drug Evaluation and Research at the FDA. Dr.
Mire-Sluis’s resume demonstrates his expertise in regulatory science associated with pyrogen
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testing with experience in product quality and development of biologicals, in immunology,
and prior experience with the FDA. Dr. Mire-Sluis has managerial and product development
experience including management of analytical and product quality departments of up to 75
staff (postdoctoral and technical levels). He is involved in strategic planning of developmentof biotechnology-derived products, including toxicology, assay development, and quality
control. Dr. Mire-Sluis has expertise in the detection, measurement, and characterization of
biological materials using immunological, molecular biological, and cell biological
technology (cytokines, growth factors, enzymes, monoclonal antibodies). He is involved inhigh throughput screening technology, bioassay and immunoassay designs, risk assessment
and process validation. He is a member of the World Health Organization consultative
committee for therapeutic drug standardization, Chairman of the International Union of
Immunological Societies Standardization Committee and of the human therapeutics
committee of the International Association for Biologicals, a board member for the Journal of
Immunological Methods, a member of the U.S. Pharmacopeia Biological Assay Statistical
Analysis Expert Working Group and the Biological Assay Validation Expert Working
Group. Dr. Mire-Sluis has authored almost 100 peer-reviewed publications.
Jonathan Richmond, BSc (Hons) Med.Sci., MB ChB, FRCSEd, FRMS
Dr. Richmond received a Bachelor of Science in Medical Science with Honors (B.Sc. [Hons]Med.Sci.) and Bachelor of Medicine and Bachelor of Surgery (MB ChB) degrees with
Distinction in Medicine and Therapeutics from Edinburgh University. Presently, he is headof the Animals Scientific Procedures Division at the Home Office. He is a Fellow of theRoyal College of Surgeons of Edinburgh (FRCSEd) and a Fellow of the Royal Society of
Medicine (FRMS). Other appointments include convener of the U.K. interdepartmentalgroup on the 3Rs, board member U.K. National Centre for the 3Rs, convener of theInternational Standards Organization Technical Corrigendum 194/Working Group 3(Biocompatibility of Medical Device Materials), and member of related expert working
groups. He is a former member of the EU Committee on Scientific and Technical Progress
and past Chairman of the European Commission Technical Expert Working Group on ethicalreview. He served as chair of the peer review panel for the reduced murine local lymph node
assay (LLNA) test protocol and prediction model for ESAC in 2007 and has been designatedas an ESAC peer reviewer for ECVAM's performance standards for the standard LLNA. He
served on the NICEATM-ICCVAM Expert Panel that evaluated Five In Vitro Pyrogen Test
Methods. He has a variety of publications in peer-reviewed journals and national andinternational meetings, on the principles and practice of surgery, regulation of biomedical
research, principles of humane research, bioethics, and public policy.
Peter Theran, V.M.D.
Dr. Theran holds a Doctor of Veterinary Medicine degree from the University ofPennsylvania. He has had many years of experience both as a veterinary internal medicine
specialist at the Massachusetts Society for Prevention of Cruelty to Animals’ Angell
Memorial Animal Hospital in Boston, and as the director of Boston University Medical
Center's Laboratory Animal Science Center. He presently serves on a number of government
committees as an animal welfare member, and is a member of the Board of Directors of theInstitute for In Vitro Sciences in Gaithersburg, MD and Chimp Haven in Shreveport,
Louisiana. He served on the NICEATM-ICCVAM Expert Panels that evaluated the In Vitro
Test Methods for Identifying Ocular Corrosives and Severe Irritants, and Five In Vitro
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Pyrogen Test Methods. He is a former member of the Advisory Committee on Alternative
Toxicological Methods and SACATM. He is presently working as a consultant.
Kevin Williams
Mr. Williams received a B.S. degree in Microbiology from Texas A&M University. He is a
Microbiologist in the Quality Control Laboratory at Eli Lilly & Company. Mr. Williams’
resume indicates that he is a well-noted expert in pyrogen testing (Bacterial Endotoxin Test
[BET] and LAL) and validation and he has authored several books on endotoxins. His
responsibilities include bacterial endotoxin testing and validation, automation of BET,
depyrogenation validation, automated microbial identification system validation, validationof sterility tests, preservative effectiveness testing, microbial purity testing and validation,and bioburden testing and validation. Mr. Williams is a member of the LAL User Steering
Committee, the Parenteral Drug Association, and the American Society for Microbiology. He
has developed a method to calculate tolerance limits for excipients based on unit formula
content of finished drug and developed novel methods of recovering endotoxin fromparenteral drug packaging components. Mr. Williams served as editor of the textbook,“Microbial Contamination Control in Parenteral Manufacturing,” and contributed a chapter
on “Historical and Emerging Themes in Parenteral Manufacturing Control.” He also edited
the textbook “Endotoxins,” and contributed chapters on endotoxin and contamination control.

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Appendix B
Relevant Federal Pyrogenicity Regulations and Testing Guidelines
B1

B2
B3

B4
B5

U.S. Code of Federal Regulations (CFR) ............................................................B-3
B1-1 21 CFR 211.167 - Current Good Manufacturing Practice for
Finished Pharmaceuticals: Special Testing Requirements(April 1, 2007)...........................................................................................B-5
B1-2 21 CFR 314.50(d)(1)(ii)(a) - Applications: Content and Formatof an Application (April 1, 2007) .............................................................B-6
B1-3 21 CFR 610.9 - General Provisions: Equivalent Methods andProcesses (April 1, 2007) ........................................................................B-17
B1-4 21 CFR 610.13 - General Biological Products Standards:
Purity (April 1, 2007) .............................................................................B-19
International Organization for Standardization - ISO 10993-11 Biological Evaluation of Medical Devices - Part 11: Tests for
Systemic Toxicity (First Edition 1993-12-15)....................................................B-21
U.S. Pharmacopeia (USP) 30-NF25...................................................................B-23
B3-1 (85) - Bacterial Endotoxins Test ............................................................B-23
B3-2 (151) - Pyrogen Test ...............................................................................B-23
B3-3 (1041) - Biologics ....................................................................................B-23
European Pharmacopeia 5.0 .............................................................................B-25
B4-1 2.6.8 - Pyrogens ......................................................................................B-25
B4-2 2.6.14 - Bacterial Endotoxins .................................................................B-25
Guideline on Validation of the Limulus Amebocyte Lysate Test
as an End-Product Endotoxin Test for Human and Animal Parenteral
Drugs, Biological Products, and Medical Devices (December 1987)................B-27

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Appendix B1
U.S. Code of Federal Regulations (CFR)
B1-1
B1-2
B1-3
B1-4

21 CFR 211.167 - Current Good Manufacturing Practice for FinishedPharmaceuticals: Special Testing Requirements (April 1, 2007)
21 CFR 314.50 (d)(1)(ii)(a) - Applications: Content and Format of an
Application (April 1, 2007)
21 CFR 610.9 - General Provisions: Equivalent Methods and Processes(April 1, 2007)
21 CFR 610.13 - General Biological Products Standards: Purity (April 1, 2007)
These documents are available at:
http://www.access.gpo.gov/cgi-bin/cfrassemble.cgi?title=200442

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Food and Drug Administration, HHS§ 211.167§ 211.170

Special testing requirements.

(a) For each batch of drug productpurporting to be sterile and/or pyrogenfree, there shall be appropriate laboratory testing to determine conformanceto such requirements. The test procedures shall be in writing and shall befollowed.
(b) For each batch of ophthalmic
ointment, there shall be appropriate
testing to determine conformance tospecifications regarding the presence of
foreign particles and harsh or abrasive
substances. The test procedures shallbe in writing and shall be followed.
(c) For each batch of controlled-release dosage form, there shall be appropriate laboratory testing to determine
conformance to the specifications forthe rate of release of each active ingredient. The test procedures shall be inwriting and shall be followed.

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21 CFR Ch. I (4–1–07 Edition)from any source. FDA will maintainguidance documents on the format andcontent of applications to assist applicants in their preparation.
(a) Application form. The applicant
shall submit a completed and signed
application form that contains the following:(1) The name and address of the applicant; the date of the application; theapplication number if previously issued(for example, if the application is a resubmission, an amendment, or a supplement); the name of the drug product, including its established, proprietary, code, and chemical names; thedosage form and strength; the route ofadministration;
theidentification
numbers of all investigational new
drug applications that are referenced
in the application; the identification
numbers of all drug master files andother applications under this part thatare referenced in the application; andthe drug product’s proposed indications
for use.
(2) A statement whether the submission is an original submission, a505(b)(2) application, a resubmission, ora supplement to an application under§ 314.70.
(3) A statement whether the applicant proposes to market the drug product as a prescription or an over-thecounter product.
(4) A check-list identifying what enclosures required under this section theapplicant is submitting.
(5) The applicant, or the applicant’s
attorney, agent, or other authorized official shall sign the application. If theperson signing the application does notreside or have a place of business within the United States, the application isrequired to contain the name and address of, and be countersigned by, an
attorney, agent, or other authorized official who resides or maintains a placeof business within the United States.
(b) Index. The archival copy of theapplication is required to contain acomprehensive index by volume number and page number to the summary
under paragraph (c) of this section, thetechnical sections under paragraph (d)of this section, and the supporting information under paragraph (f) of thissection.

Subpart B—Applications§ 314.50 Content and format of an application.
Applications and supplements to approved applications are required to besubmitted in the form and contain theinformation, as appropriate for the particular submission, required under thissection. Three copies of the application
are required: An archival copy, a review copy, and a field copy. An application for a new chemical entity will generally contain an application form, an
index, a summary, five or six technicalsections, case report tabulations of patient data, case report forms, drug
samples, and labeling, including, if applicable, any Medication Guide required under part 208 of this chapter.
Other applications will generally contain only some of those items, and information will be limited to that needed to support the particular submission. These include an application of
the type described in section 505(b)(2)of the act, an amendment, and a supplement. The application is required tocontain reports of all investigations of
the drug product sponsored by the applicant, and all other information
about the drug pertinent to an evaluation of the application that is received
or otherwise obtained by the applicant

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Food and Drug Administration, HHS§ 314.50(c) Summary. (1) An application is required to contain a summary of the application in enough detail that thereader may gain a good general understanding of the data and information in
the application, including an understanding of the quantitative aspects ofthe data. The summary is not required
for supplements under § 314.70. Resubmissions of an application should
contain an updated summary, as appropriate. The summary should discuss all
aspects of the application, and synthesize the information into a wellstructured and unified document. The
summary should be written at approximately the level of detail required for
publication in, and meet the editorial
standards generally applied by, refereed scientific and medical journals.
In addition to the agency personnel reviewing the summary in the context of
their review of the application, FDA
may furnish the summary to FDA advisory committee members and agency
officials whose duties require an understanding of the application. To the extent possible, data in the summary
should be presented in tabular andgraphic forms. FDA has prepared a
guideline under § 10.90(b) that provides
information about how to prepare asummary. The summary required
under this paragraph may be used by
FDA or the applicant to prepare theSummary Basis of Approval documentforpublicdisclosure(under§ 314.430(e)(2)(ii)) when the application
is approved.
(2) The summary is required to contain the following information:(i) The proposed text of the labeling,including, if applicable, any Medication Guide required under part 208 of
this chapter, for the drug, with annotations to the information in the summary and technical sections of the application that support the inclusion ofeach statement in the labeling, and, if
the application is for a prescription
drug, statements describing the reasons for omitting a section or subsection of the labeling format in § 201.57
of this chapter.
(ii) A statement identifying the pharmacologic class of the drug and a discussion of the scientific rationale forthe drug, its intended use, and the po-tential clinical benefits of the drug
product.
(iii) A brief description of the marketing history, if any, of the drug outside the United States, including a listof the countries in which the drug has
been marketed, a list of any countries
in which the drug has been withdrawn
from marketing for any reason related
to safety or effectiveness, and a list of
countries in which applications formarketing are pending. The description is required to describe both marketing by the applicant and, if known,
the marketing history of other persons.
(iv) A summary of the chemistry,
manufacturing, and controls section of
the application.
(v) A summary of the nonclinicalpharmacology and toxicology sectionof the application.
(vi) A summary of the human pharmacokinetics and bioavailability section of the application.
(vii) A summary of the microbiology
section of the application (for anti-infective drugs only).
(viii) A summary of the clinical data
section of the application, including
the results of statistical analyses of
the clinical trials.
(ix) A concluding discussion that presents the benefit and risk considerations related to the drug, including a
discussion of any proposed additional
studies or surveillance the applicant
intends to conduct postmarketing.
(d) Technical sections. The applicationis required to contain the technicalsections described below. Each technical section is required to containdata and information in sufficient detail to permit the agency to make aknowledgeable judgment about whether to approve the application or whether grounds exist under section 505(d) of
the act to refuse to approve the application. The required technical sections
are as follows:(1) Chemistry, manufacturing, and controls section. A section describing thecomposition, manufacture, and specification of the drug substance and thedrug product, including the following:(i) Drug substance. A full descriptionof the drug substance including its
physical and chemical characteristics
and stability; the name and address of

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21 CFR Ch. I (4–1–07 Edition)

its manufacturer; the method of synthesis (or isolation) and purification of
the drug substance; the process controls used during manufacture andpackaging; and the specifications necessary to ensure the identity, strength,
quality, and purity of the drug substance and the bioavailability of the
drug products made from the substance, including, for example, tests,
analytical procedures, and acceptancecriteria relating to stability, sterility,
particle size, and crystalline form. The
application may provide additionallyfor the use of alternatives to meet anyof these requirements, including alternative sources, process controls, andanalytical procedures. Reference to thecurrent edition of the U.S. Pharmacopeia and the National Formulary
may satisfy relevant requirements inthis paragraph.
(ii)(a) Drug product. A list of all components used in the manufacture of the
drug product (regardless of whether
they appear in the drug product) and astatement of the composition of the
drug product; the specifications foreach component; the name and addressof each manufacturer of the drug product; a description of the manufacturing
and packaging procedures and in-process controls for the drug product; thespecifications necessary to ensure theidentity, strength, quality, purity, potency, and bioavailability of the drug
product, including, for example, tests,
analytical procedures, and acceptancecriteria relating to sterility, dissolution rate, container closure systems;
and stability data with proposed expiration dating. The application may
provide additionally for the use of alternatives to meet any of these requirements, including alternative components, manufacturing and packaging
procedures, in-process controls, and analytical procedures. Reference to thecurrent edition of the U.S. Pharmacopeia and the National Formulary
may satisfy relevant requirements inthis paragraph.
(b) Unless provided by paragraph(d)(1)(ii)(a) of this section, for eachbatch of the drug product used to conduct a bioavailability or bioequivalence study described in § 320.38 or§ 320.63 of this chapter or used to conduct a primary stability study: The

batch production record; the specification for each component and for the
drug product; the names and addressesof the sources of the active andnoncompendial inactive components
and of the container and closure system for the drug product; the name andaddress of each contract facility involved in the manufacture, processing,packaging, or testing of the drug product and identification of the operation
performed by each contract facility;
and the results of any test performedon the components used in the manufacture of the drug product as requiredby § 211.84(d) of this chapter and on the
drug product as required by § 211.165 ofthis chapter.
(c) The proposed or actual masterproduction record, including a description of the equipment, to be used forthe manufacture of a commercial lot of
the drug product or a comparably detailed description of the production
process for a representative batch of
the drug product.
(iii) Environmental impact. The application is required to contain either aclaim for categorical exclusion under§ 25.30 or 25.31 of this chapter or an environmental assessment under § 25.40 of
this chapter.
(iv) The applicant may, at its option,
submit a complete chemistry, manufacturing, and controls section 90 to 120
days before the anticipated submissionof the remainder of the application.
FDA will review such early submissions as resources permit.
(v) The applicant shall include astatement certifying that the fieldcopy of the application has been provided to the applicant’s home FDA district office.
(2) Nonclinical pharmacology and toxicology section. A section describing,with the aid of graphs and tables, animal and in vitro studies with drug, including the following:(i) Studies of the pharmacological actions of the drug in relation to its proposed therapeutic indication and studies that otherwise define the pharmacologic properties of the drug or are
pertinent to possible adverse effects.
(ii) Studies of the toxicological effects of the drug as they relate to the
drug’s intended clinical uses, including, as appropriate, studies assessing

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Food and Drug Administration, HHS§ 314.50
and the bioavailability or bioequivalence, or both, of the drug product.
(4) Microbiology section. If the drug isan anti-infective drug, a section describing the microbiology data, including the following:(i) A description of the biochemicalbasis of the drug’s action on microbial
physiology.
(ii) A description of the antimicrobial spectra of the drug, including results of in vitro preclinical studies to demonstrate concentrations of
the drug required for effective use.
(iii) A description of any known
mechanisms of resistance to the drug,
including results of any known epidemiologic studies to demonstrateprevalence of resistance factors.
(iv) A description of clinical microbiology laboratory procedures (for example, in vitro sensitivity discs) neededfor effective use of the drug.
(5) Clinical data section. A section describing the clinical investigations of
the drug, including the following:(i) A description and analysis of eachclinical pharmacology study of thedrug, including a brief comparison of
the results of the human studies withthe animal pharmacology and toxicology data.
(ii) A description and analysis of eachcontrolled clinical study pertinent to a
proposed use of the drug, including theprotocol and a description of the statistical analyses used to evaluate thestudy. If the study report is an interim
analysis, this is to be noted and a projected completion date provided. Controlled clinical studies that have notbeen analyzed in detail for any reason(e.g., because they have been discontinued or are incomplete) are to be included in this section, including a copyof the protocol and a brief descriptionof the results and status of the study.
(iii) A description of each uncontrolled clinical study, a summary of
the results, and a brief statement explaining why the study is classified as
uncontrolled.
(iv) A description and analysis of any
other data or information relevant toan evaluation of the safety and effectiveness of the drug product obtainedor otherwise received by the applicant
from any source, foreign or domestic,
including information derived fromthe drug’s acute, subacute, and chronic
toxicity; carcinogenicity; and studies
of toxicities related to the drug’s particular mode of administration or conditions of use.
(iii) Studies, as appropriate, of the effects of the drug on reproduction andon the developing fetus.
(iv) Any studies of the absorption,
distribution, metabolism, and excretion of the drug in animals.
(v) For each nonclinical laboratory
study subject to the good laboratory
practice regulations under part 58 astatement that it was conducted incompliance with the good laboratory
practice regulations in part 58, or, if
the study was not conducted in compliance with those regulations, a brief
statement of the reason for the noncompliance.
(3) Human pharmacokinetics and bioavailability section. A section describing
the human pharmacokinetic data andhuman bioavailability data, or information supporting a waiver of the submission of in vivo bioavailability data
under subpart B of part 320, including
the following:(i) A description of each of the bioavailability and pharmacokinetic studies of the drug in humans performed byor on behalf of the applicant that includes a description of the analyticalprocedures and statistical methods
used in each study and a statementwith respect to each study that it either was conducted in compliance with
the institutional review board regulations in part 56, or was not subject tothe regulations under § 56.104 or § 56.105,
and that it was conducted in compliance with the informed consent regulations in part 50.
(ii) If the application describes in thechemistry, manufacturing, and controls section tests, analytical procedures, and acceptance criteria needed
to assure the bioavailability of the
drug product or drug substance, orboth, a statement in this section of therationale for establishing the tests, analytical procedures, and acceptancecriteria, including data and information supporting the rationale.
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21 CFR Ch. I (4–1–07 Edition)clinical investigations, including controlled and uncontrolled studies of usesof the drug other than those proposedin the application, commercial marketing experience, reports in the scientific literature, and unpublished scientific papers.
(v) An integrated summary of thedata demonstrating substantial evidence of effectiveness for the claimed
indications. Evidence is also required
to support the dosage and administration section of the labeling, including
support for the dosage and dose interval recommended. The effectivenessdata shall be presented by gender, age,and racial subgroups and shall identify
any modifications of dose or dose interval needed for specific subgroups. Effectiveness data from other subgroupsof the population of patients treated,when appropriate, such as patientswith renal failure or patients with different levels of severity of the disease,also shall be presented.
(vi) A summary and updates of safety
information, as follows:(a) The applicant shall submit an integrated summary of all available information about the safety of the drug
product, including pertinent animal
data, demonstrated or potential adverse effects of the drug, clinically significant drug/drug interactions, andother safety considerations, such asdata from epidemiological studies ofrelated drugs. The safety data shall bepresented by gender, age, and racial
subgroups. When appropriate, safety
data from other subgroups of the population of patients treated also shall bepresented, such as for patients withrenal failure or patients with different
levels of severity of the disease. A description of any statistical analyses
performed in analyzing safety data
should also be included, unless alreadyincluded under paragraph (d)(5)(ii) of
this section.
(b) The applicant shall, under section
505(i) of the act, update periodically its
pending application with new safety information learned about the drug that
may reasonably affect the statement ofcontraindications,warnings,
precautions, and adverse reactions in thedraft labeling and, if applicable, any
Medication Guide required under part
208 of this chapter. These ‘‘safety up-

date reports’’ are required to include
the same kinds of information (fromclinical studies, animal studies, andother sources) and are required to besubmitted in the same format as theintegrated summary in paragraph(d)(5)(vi)(a) of this section. In addition,
the reports are required to include thecase report forms for each patient who
died during a clinical study or who did
not complete the study because of an
adverse event (unless this requirement
is waived). The applicant shall submit
these reports (1) 4 months after the initial submission; (2) following receipt ofan approvable letter; and (3) at othertimes as requested by FDA. Prior to
the submission of the first such report,
applicants are encouraged to consult
with FDA regarding further details on
its form and content.
(vii) If the drug has a potential forabuse, a description and analysis ofstudies or information related to abuseof the drug, including a proposal forscheduling under the Controlled Substances Act. A description of any studies related to overdosage is also required, including information on dialysis, antidotes, or other treatments, if
known.
(viii) An integrated summary of thebenefits and risks of the drug, including a discussion of why the benefits exceed the risks under the conditions
stated in the labeling.
(ix) A statement with respect to eachclinical study involving human subjects that it either was conducted incompliance with the institutional review board regulations in part 56, or
was not subject to the regulations
under § 56.104 or § 56.105, and that it was
conducted in compliance with the informed consent regulations in part 50.
(x) If a sponsor has transferred any
obligations for the conduct of any clinical study to a contract research organization, a statement containing thename and address of the contract research organization, identification of
the clinical study, and a listing of theobligations transferred. If all obligations governing the conduct of thestudy have been transferred, a generalstatement of this transfer—in lieu of alisting of the specific obligations transferred—may be submitted.

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May 2008

Food and Drug Administration, HHS§ 314.50(xi) If original subject records wereaudited or reviewed by the sponsor in
the course of monitoring any clinicalstudy to verify the accuracy of the casereports submitted to the sponsor, a list
identifying each clinical study so audited or reviewed.
(6) Statistical section. A section describing the statistical evaluation ofclinical data, including the following:(i) A copy of the information submitted under paragraph (d)(5)(ii) of thissection concerning the description andanalysis of each controlled clinicalstudy, and the documentation and supporting statistical analyses used in
evaluating the controlled clinical studies.
(ii) A copy of the information submitted under paragraph (d)(5)(vi)(a) of
this section concerning a summary of
information about the safety of the
drug product, and the documentationand supporting statistical analyses
used in evaluating the safety information.
(7) Pediatric use section. A section describing the investigation of the drug
for use in pediatric populations, including an integrated summary of the information (the clinical pharmacology
studies, controlled clinical studies, or
uncontrolled clinical studies, or otherdata or information) that is relevant tothe safety and effectiveness and benefits and risks of the drug in pediatricpopulations for the claimed indications, a reference to the full descriptions of such studies provided underparagraphs (d)(3) and (d)(5) of this section, and information required to besubmitted under § 314.55.
(e) Samples and labeling. (1) Upon request from FDA, the applicant shallsubmit the samples described below to
the places identified in the agency’s request. FDA will generally ask applicants to submit samples directly to
two or more agency laboratories thatwill perform all necessary tests on thesamples and validate the applicant’s
analytical procedures.
(i) Four representative samples of thefollowing, each sample in sufficientquantity to permit FDA to perform
three times each test described in theapplication to determine whether the
drug substance and the drug productmeet the specifications given in the application:(a) The drug product proposed formarketing;(b) The drug substance used in the
drug product from which the samplesof the drug product were taken; and(c) Reference standards and blanks(except that reference standards recognized in an official compendium neednot be submitted).
(ii) Samples of the finished market
package, if requested by FDA.
(2) The applicant shall submit thefollowing in the archival copy of theapplication:(i) Three copies of the analytical procedures and related descriptive information contained in the chemistry,
manufacturing, and controls section
under paragraph (d)(1) of this section
for the drug substance and the drug
product that are necessary for FDA’s
laboratories to perform all necessary
tests on the samples and to validate
the applicant’s analytical procedures.
The related descriptive information includes a description of each sample;
the proposed regulatory specifications
for the drug; a detailed description of
the methods of analysis; supporting
data for accuracy, specificity, precisionand ruggedness; and complete resultsof the applicant’s tests on each sample.
(ii) Copies of the label and all labeling for the drug product (including, if
applicable, any Medication Guide required under part 208 of this chapter)for the drug product (4 copies of draft
labeling or 12 copies of final printed labeling).
(f) Case report forms and tabulations.
The archival copy of the application isrequired to contain the following casereport tabulations and case reportforms:(1) Case report tabulations. The application is required to contain tabulations of the data from each adequate
andwell-controlled
study
under
§ 314.126 (Phase 2 and Phase 3 studies as
described in §§ 312.21 (b) and (c) of thischapter), tabulations of the data fromthe earliest clinical pharmacology
studies (Phase 1 studies as described in§ 312.21(a) of this chapter), and tabulations of the safety data from otherclinical studies. Routine submission ofother patient data from uncontrolled

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21 CFR Ch. I (4–1–07 Edition)

studies is not required. The tabulations
are required to include the data oneach patient in each study, except thatthe applicant may delete those tabulations which the agency agrees, in advance, are not pertinent to a review ofthe drug’s safety or effectiveness. Upon
request, FDA will discuss with the applicant in a ‘‘pre-NDA’’ conference
those tabulations that may be appropriate for such deletion. Barring unforeseen circumstances, tabulations
agreed to be deleted at such a conference will not be requested during
the conduct of FDA’s review of the application. If such unforeseen circumstances do occur, any request for
deleted tabulations will be made by the
director of the FDA division responsible for reviewing the application, in
accordance with paragraph (f)(3) of thissection.
(2) Case report forms. The applicationis required to contain copies of individual case report forms for each patient who died during a clinical study
or who did not complete the study because of an adverse event, whether believed to be drug related or not, including patients receiving reference drugs
or placebo. This requirement may be
waived by FDA for specific studies if
the case report forms are unnecessary
for a proper review of the study.
(3) Additional data. The applicant
shall submit to FDA additional case report forms and tabulations needed to
conduct a proper review of the application, as requested by the director of
the FDA division responsible for reviewing the application. The applicant’s failure to submit information requested by FDA within 30 days after receipt of the request may result in theagency viewing any eventual submission as a major amendment under§ 314.60 and extending the review period
as necessary. If desired by the applicant, the FDA division director willverify in writing any request for additional data that was made orally.
(4) Applicants are invited to meetwith FDA before submitting an application to discuss the presentation andformat of supporting information. If
the applicant and FDA agree, the applicant may submit tabulations of patient
data and case report forms in a form

other than hard copy, for example, on
microfiche or computer tapes.
(g) Other. The following general requirements apply to the submission of
information within the summary underparagraph (c) of this section and within
the technical sections under paragraph(d) of this section.
(1) The applicant ordinarily is not required to resubmit information previously submitted, but may incorporate the information by reference. A
reference to information submitted
previously is required to identify thefile by name, reference number, volume, and page number in the agency’s
records where the information can befound. A reference to information submitted to the agency by a person otherthan the applicant is required to contain a written statement that authorizes the reference and that is signed by
the person who submitted the information.
(2) The applicant shall submit an accurate and complete English translation of each part of the applicationthat is not in English. The applicant
shall submit a copy of each original literature publication for which an
English translation is submitted.
(3) If an applicant who submits a new
drug application under section 505(b) of
the act obtains a ‘‘right of reference oruse,’’ as defined under § 314.3(b), to an
investigation described in clause (A) ofsection 505(b)(1) of the act, the applicant shall include in its application awritten statement signed by the ownerof the data from each such investigation that the applicant may rely on in
support of the approval of its application, and provide FDA access to, theunderlying raw data that provide thebasis for the report of the investigation
submitted in its application.
(h) Patent information. The application is required to contain the patent
information described under § 314.53.
(i) Patent certification—(1) Contents. A
505(b)(2) application is required to contain the following:(i) Patents claiming drug, drug product,or method of use. (A) Except as provided
in paragraph (i)(2) of this section, acertification with respect to each patent issued by the United States Patent
and Trademark Office that, in the opinion of the applicant and to the best of

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Food and Drug Administration, HHS§ 314.50

its knowledge, claims a drug (the drug
product or drug substance that is acomponent of the drug product) on
which investigations that are relied
upon by the applicant for approval of
its application were conducted or thatclaims an approved use for such drug
and for which information is required
to be filed under section 505(b) and (c)of the act and § 314.53. For each suchpatent, the applicant shall provide thepatent number and certify, in its opinion and to the best of its knowledge,
one of the following circumstances:(1) That the patent information has
not been submitted to FDA. The applicant shall entitle such a certification‘‘Paragraph I Certification’’;(2) That the patent has expired. The
applicant shall entitle such a certification ‘‘Paragraph II Certification’’;(3) The date on which the patent will
expire. The applicant shall entitle sucha certification ‘‘Paragraph III Certification’’; or(4) That the patent is invalid, unenforceable, or will not be infringed by
the manufacture, use, or sale of the
drug product for which the application
is submitted. The applicant shall entitle such a certification ‘‘Paragraph IV
Certification’’. This certification shallbe submitted in the following form:

proved patented drug or that claims anapproved use for such a drug.
(ii) No relevant patents. If, in the opinion of the applicant and to the best of
its knowledge, there are no patents described in paragraph (i)(1)(i) of this section, a certification in the followingform:
In the opinion and to the best knowledge of(name of applicant), there are no patents thatclaim the drug or drugs on which investigations that are relied upon in this application
were conducted or that claim a use of such
drug or drugs.

(iii) Method of use patent. (A) If information that is submitted under section
505(b) or (c) of the act and § 314.53 is fora method of use patent, and the labeling for the drug product for which theapplicant is seeking approval does notinclude any indications that are covered by the use patent, a statement explaining that the method of use patentdoes not claim any of the proposed indications.
(B) If the labeling of the drug productfor which the applicant is seeking approval includes an indication that, according to the patent information submitted under section 505(b) or (c) of theact and § 314.53 or in the opinion of theapplicant, is claimed by a use patent,
the applicant shall submit an applicable certification under paragraph(i)(1)(i) of this section.
(2) Method of manufacturing patent.
An applicant is not required to make acertification with respect to any patent that claims only a method of manufacturing the drug product for which
the applicant is seeking approval.
(3) Licensing agreements. If a 505(b)(2)application is for a drug or method ofusing a drug claimed by a patent andthe applicant has a licensing agreement with the patent owner, the applicant shall submit a certification underparagraph (i)(1)(i)(A)(4) of this section(‘‘Paragraph IV Certification’’) as tothat patent and a statement that it has
been granted a patent license. If thepatent owner consents to an immediate
effective date upon approval of the505(b)(2) application, the application
shall contain a written statement fromthe patent owner that it has a licensing agreement with the applicant andthat it consents to an immediate effective date.

I, (name of applicant), certify that Patent No.
llllll (is invalid, unenforceable, or will
not be infringed by the manufacture, use, or
sale of) (name of proposed drug product) for
which this application is submitted.

The certification shall be accompanied
by a statement that the applicant willcomply with the requirements under§ 314.52(a) with respect to providing a
notice to each owner of the patent or
their representatives and to the holderof the approved application for the
drug product which is claimed by thepatent or a use of which is claimed by
the patent and with the requirements
under § 314.52(c) with respect to thecontent of the notice.
(B) If the drug on which investigations that are relied upon by the applicant were conducted is itself a licensed
generic drug of a patented drug first
approved under section 505(b) of theact, the appropriate patent certification under this section with respect
to each patent that claims the first-ap-

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211069ICCVAM Test Method Evaluation Report: Appendix B1

May 2008§ 314.50

21 CFR Ch. I (4–1–07 Edition)(4) Late submission of patent information. If a patent described in paragraph(i)(1)(i)(A) of this section is issued andthe holder of the approved application
for the patented drug does not submit
the required information on the patent
within 30 days of issuance of the patent, an applicant who submitted a505(b)(2) application that, before thesubmission of the patent information,
contained an appropriate patent certification is not required to submit anamended certification. An applicant
whose 505(b)(2) application is filed aftera late submission of patent information or whose 505(b)(2) application waspreviously filed but did not contain an
appropriate patent certification at thetime of the patent submission shallsubmit a certification under paragraph(i)(1)(i) or (i)(1)(ii) of this section or astatement under paragraph (i)(1)(iii) of
this section as to that patent.
(5) Disputed patent information. If an
applicant disputes the accuracy or relevance of patent information submitted to FDA, the applicant may seeka confirmation of the correctness ofthe patent information in accordancewith the procedures under § 314.53(f).
Unless the patent information is withdrawn or changed, the applicant must
submit an appropriate certification foreach relevant patent.
(6) Amended certifications. A certification submitted under paragraphs(i)(1)(i) through (i)(1)(iii) of this section
may be amended at any time before theeffective date of the approval of the application. An applicant shall submit anamended certification as an amendment to a pending application or byletter to an approved application. If an
applicant with a pending application
voluntarily makes a patent certification for an untimely filed patent, theapplicant may withdraw the patentcertification for the untimely filed patent. Once an amendment or letter forthe change in certification has been
submitted, the application will nolonger be considered to be one containing the prior certification.
(i) After finding of infringement. An applicant who has submitted a certification under paragraph (i)(1)(i)(A)(4) of
this section and is sued for patent infringement within 45 days of the receipt of notice sent under § 314.52 shallamend the certification if a final judgment in the action is entered finding
the patent to be infringed unless the
final judgment also finds the patent to
be invalid. In the amended certification, the applicant shall certify
under paragraph (i)(1)(i)(A)(3) of thissection that the patent will expire on a
specific date.
(ii) After removal of a patent from thelist. If a patent is removed from thelist, any applicant with a pending application (including a tentatively approved application with a delayed effective date) who has made a certification with respect to such patentshall amend its certification. The applicant shall certify under paragraph(i)(1)(ii) of this section that no patents
described in paragraph (i)(1)(i) of thissection claim the drug or, if other relevant patents claim the drug, shallamend the certification to refer only tothose relevant patents. In the amendment, the applicant shall state the reason for the change in certification(that the patent is or has been removed
from the list). A patent that is the subject of a lawsuit under § 314.107(c) shallnot be removed from the list until FDA
determines either that no delay in effective dates of approval is required
under that section as a result of thelawsuit, that the patent has expired, orthat any such period of delay in effective dates of approval is ended. An applicant shall submit an amended certification as an amendment to a pending application. Once an amendment
for the change has been submitted, theapplication will no longer be considered to be one containing a certification under paragraph (i)(1)(i)(A)(4) of
this section.
(iii) Other amendments. (A) Except asprovided in paragraphs (i)(4) and(i)(6)(iii)(B) of this section, an applicant shall amend a submitted certification if, at any time before the effective date of the approval of the application, the applicant learns that the submitted certification is no longer accurate.
(B) An applicant is not required toamend a submitted certification when
information on an otherwise applicablepatent is submitted after the effective
date of approval for the 505(b)(2) application.

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211069ICCVAM Test Method Evaluation Report: Appendix B1

May 2008

Food and Drug Administration, HHS§ 314.50(j) Claimed exclusivity. A new drug
product, upon approval, may be entitled to a period of marketing exclusivity under the provisions of § 314.108.
If an applicant believes its drug product is entitled to a period of exclusivity, it shall submit with the new
drug application prior to approval thefollowing information:(1) A statement that the applicant isclaiming exclusivity.
(2) A reference to the appropriate
paragraph under § 314.108 that supports
its claim.
(3) If the applicant claims exclusivity
under § 314.108(b)(2), information toshow that, to the best of its knowledge
or belief, a drug has not previously
been approved under section 505(b) of
the act containing any active moiety
in the drug for which the applicant isseeking approval.
(4) If the applicant claims exclusivity
under § 314.108(b)(4) or (b)(5), the following information to show that theapplication contains ‘‘new clinical investigations’’ that are ‘‘essential to approval of the application or supplement’’ and were ‘‘conducted or sponsored by the applicant:’’(i) ‘‘New clinical investigations.’’ A certification that to the best of the applicant’s knowledge each of the clinicalinvestigations included in the application meets the definition of ‘‘new clinicalinvestigation’’
set
forth
in
§ 314.108(a).
(ii) ‘‘Essential to approval.’’ A list of
all published studies or publicly available reports of clinical investigations
known to the applicant through a literature search that are relevant to theconditions for which the applicant isseeking approval, a certification thatthe applicant has thoroughly searched
the scientific literature and, to thebest of the applicant’s knowledge, thelist is complete and accurate and, in
the applicant’s opinion, such publishedstudies or publicly available reports do
not provide a sufficient basis for theapproval of the conditions for which
the applicant is seeking approval without reference to the new clinical investigation(s) in the application, and an
explanation as to why the studies orreports are insufficient.
(iii) ‘‘Conducted or sponsored by.’’ If
the applicant was the sponsor named in

the Form FDA–1571 for an investigational new drug application (IND)
under which the new clinical investigation(s) that is essential to the approvalof its application was conducted, identification of the IND by number. If theapplicant was not the sponsor of the
IND under which the clinical investigation(s) was conducted, a certificationthat the applicant or its predecessor ininterest provided substantial support
for the clinical investigation(s) that isessential to the approval of its application, and information supporting thecertification. To demonstrate ‘‘substantial support,’’ an applicant must
either provide a certified statementfrom a certified public accountant thatthe applicant provided 50 percent ormore of the cost of conducting thestudy or provide an explanation of why
FDA should consider the applicant tohave conducted or sponsored the study
if the applicant’s financial contribution to the study is less than 50 percent
or the applicant did not sponsor the investigational new drug. A predecessor
in interest is an entity, e.g., a corporation, that the applicant has taken over,merged with, or purchased, or fromwhich the applicant has purchased allrights to the drug. Purchase of nonexclusive rights to a clinical investigation after it is completed is not sufficient to satisfy this definition.
(k) Financial certification or disclosurestatement. The application shall containa financial certification or disclosurestatement or both as required by part
54 of this chapter.
(l) Format of an original application—(1) Archival copy. The applicant must
submit a complete archival copy of theapplication that contains the information required under paragraphs (a)through (f) of this section. FDA will
maintain the archival copy during thereview of the application to permit individual reviewers to refer to information that is not contained in their particular technical sections of the application, to give other agency personnel
access to the application for official
business, and to maintain in one placea complete copy of the application. Except as required by paragraph (l)(1)(i)of this section, applicants may submit
the archival copy on paper or in electronic format provided that electronic

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May 2008§ 314.52

21 CFR Ch. I (4–1–07 Edition)

submissions are made in accordancewith part 11 of this chapter.
(i) Labeling. The content of labelingrequired under § 201.100(d)(3) of thischapter (commonly referred to as the
package insert or professional labeling), including all text, tables, and figures, must be submitted to the agency
in electronic format as described inparagraph (l)(5) of this section. This requirement is in addition to the requirements of paragraph (e)(2)(ii) of this section that copies of the formatted label
and all labeling be submitted. Submissions under this paragraph must be
made in accordance with part 11 of thischapter, except for the requirements of§ 11.10(a), (c) through (h), and (k), andthe corresponding requirements of§ 11.30.
(ii) [Reserved](2) Review copy. The applicant must
submit a review copy of the application. Each of the technical sections, described in paragraphs (d)(1) through(d)(6) of this section, in the review copy
is required to be separately bound witha copy of the application form required
under paragraph (a) of this section anda copy of the summary required underparagraph (c) of this section.
(3) Field copy. The applicant must
submit a field copy of the applicationthat contains the technical section described in paragraph (d)(1) of this section, a copy of the application form required under paragraph (a) of this section, a copy of the summary required
under paragraph (c) of this section, anda certification that the field copy is atrue copy of the technical section described in paragraph (d)(1) of this section contained in the archival and review copies of the application.
(4) Binding folders. The applicant may
obtain from FDA sufficient folders tobind the archival, the review, and thefield copies of the application.
(5) Electronic format submissions. Electronic format submissions must be in aform that FDA can process, review, andarchive. FDA will periodically issue
guidance on how to provide the electronic submission (e.g., method oftransmission, media, file formats, preparation and organization of files).

Sections Affected, which appears in the
Finding Aids section of the printed volume
and on GPO Access.

[50 FR 7493, Feb. 22, 1985]
EDITORIAL NOTE: For FEDERAL REGISTER citations affecting § 314.50, see the List of CFR

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May 2008§ 610.1

21 CFR Ch. I (4–1–07 Edition)

Subpart B—General Provisions§ 610.9 Equivalent methods and processes.
Modification of any particular test
method or manufacturing process or
the conditions under which it is conducted as required in this part or in the

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Food and Drug Administration, HHS§ 610.11

additional standards for specific biological products in parts 620 through
680 of this chapter shall be permitted
only under the following conditions:(a) The applicant presents evidence,in the form of a license application, ora supplement to the application submitted in accordance with § 601.12(b) or(c), demonstrating that the modification will provide assurances of the safety, purity, potency, and effectivenessof the biological product equal to or
greater than the assurances provided
by the method or process specified in
the general standards or additional
standards for the biological product;and(b) Approval of the modification isreceived in writing from the Director,
Center for Biologics Evaluation andResearch or the Director, Center for
Drug Evaluation and Research.
[62 FR 39903, July 24, 1997, as amended at 70
FR 14984, Mar. 24, 2005]

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Food and Drug Administration, HHS§ 610.13§ 610.13

Purity.

Products shall be free of extraneousmaterial except that which is unavoidable in the manufacturing process described in the approved biologics license application. In addition, products
shall be tested as provided in paragraphs (a) and (b) of this section.
(a)(1) Test for residual moisture. Eachlot of dried product shall be tested forresidual moisture and shall meet andnot exceed established limits as specified by an approved method on file inthe biologics license application. The
test for residual moisture may be exempted by the Director, Center for Biologics Evaluation and Research or the
Director, Center for Drug Evaluation
and Research, when deemed not necessary for the continued safety, purity,and potency of the product.
(2) Records. Appropriate records forresidual moisture under paragraph(a)(1) of this section shall be preparedand maintained as required by the applicable provisions of §§ 211.188 and211.194 of this chapter.
(b) Test for pyrogenic substances. Eachlot of final containers of any productintended for use by injection shall be

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May 2008§ 610.14

21 CFR Ch. I (4–1–07 Edition)
imum temperature rises does not exceed 3.7 °C.

tested for pyrogenic substances by intravenous injection into rabbits as provided in paragraphs (b) (1) and (2) ofthis section: Provided, That notwithstanding any other provision of Subchapter F of this chapter, the test for
pyrogenic substances is not required
for the following products: Products
containing formed blood elements;
Cryoprecipitate; Plasma; Source Plasma; Normal Horse Serum; bacterial,
viral, and rickettsial vaccines andantigens; toxoids; toxins; allergenic extracts; venoms; diagnostic substances
and trivalent organic arsenicals.
(1) Test dose. The test dose for eachrabbit shall be at least 3 milliliters per
kilogram of body weight of the rabbit
and also shall be at least equivalent
proportionately, on a body weightbasis, to the maximum single humandose recommended, but need not exceed 10 milliliters per kilogram of bodyweight of the rabbit, except that: (i)Regardless of the human dose recommended, the test dose per kilogramof body weight of each rabbit shall be
at least 1 milliliter for immune
globulins derived from human blood;(ii) for Streptokinase, the test doseshall be at least equivalent proportionately, on a body weight basis, to the
maximum single human dose recommended.
(2) Test procedure, results, and interpretation; standards to be met. The test for
pyrogenic substances shall be performed according to the requirements
specified in United States Pharmacopeia XX.
(3) Retest. If the lot fails to meet thetest requirements prescribed in paragraph (b)(2) of this section, the test
may be repeated once using five otherrabbits. The temperature rises recorded
for all eight rabbits used in testing
shall be included in determining
whether the requirements are met. The
lot meets the requirements for absenceof pyrogens if not more than three ofthe eight rabbits show individual rises
in temperature of 0.6 °C or more, and if
the sum of the eight individual max-

[38 FR 32056, Nov. 20, 1973, as amended at 40
FR 29710, July 15, 1975; 41 FR 10429, Mar. 11,
1976; 41 FR 41424, Sept. 22, 1976; 44 FR 40289,
July 10, 1979; 46 FR 62845, Dec. 29, 1981; 49 FR
15187, Apr. 18, 1984; 50 FR 4134, Jan. 29, 1985;
55 FR 28381, July 11, 1990; 64 FR 56453, Oct. 20,
1999; 67 FR 9587, Mar. 4, 2002; 70 FR 14985,
Mar. 24, 2005]

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211071ICCVAM Test Method Evaluation Report: Appendix B2

May 2008

Appendix B2International Organization for Standardization
ISO 10993-11 - Biological Evaluation of Medical Devices - Part 11: Tests for Systemic
Toxicity (First Edition 1993-12-15)
This document is available for purchase at:
http://www.iso.org/iso/store.htm

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May 2008

ICCVAM Test Method Evaluation Report: Appendix B3

May 2008

Appendix B3
U.S. Pharmacopeia (USP) 30-NF25
B3-1
B3-2
B3-3

(85) - Bacterial Endotoxins Test
(151) - Pyrogen Test
(1041) - Biologics
These documents provide a description of the respective biological test and areavailable for purchase at:
http://www.usp.org/products

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B-24

May 2008

ICCVAM Test Method Evaluation Report: Appendix B4

May 2008

Appendix B4European Pharmacopeia 5.0
B4-1
B4-2

2.6.8 - Pyrogens
2.6.14 - Bacterial Endotoxins
These documents provide a description of the respective biological test and areavailable for purchase at:
http://www.edqm.eu/site/Online_Publications-581.html

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May 2008

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

Appendix B5
Guideline on Validation of the Limulus Amebocyte Lysate Test as an End-Product
Endotoxin Test for Human and Animal Parenteral Drugs, Biological Products, andMedical Devices (December 1987)
This document is available at:
http://www.fda.gov/cder/guidance/old005fn.pdf

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May 2008

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

GUIDELINE ON
ON
GUIDELINE
VALIDATION OF
OF THE
THE LIMULUS
LIMULUS AMEBOCYTEAMEBOCYTE LYSATELYSATE TESTTESTVALIDATION
AS AN
AN END-PRODUCTEND-PRODUCT ENDOTOXINENDOTOXIN TESTTEST FOR
FOR HUMAN
HUMAN
AS
AND ANIMALANIMAL PARENTERALPARENTERAL DRUGS.
DRUGS, BIOLOGICALBIOLOGICAL PRODUCTS.
PRODUCTS, AND
AND
AND
MEDICALDEVICESMEDICAL DEVICES
December 1987
1987
DecemberPreparedCenter for
Drug Evaluation
Evaluation
andResearch
Prepared by:
by: Center
for Drugand ResearchCenterfor
Biologic
Evaluation
and ResearchCenter for Biologic Evaluation andResearchCenter forfor Devices
Devices and
HealthCenterand RadiologicalRadiological HealthCenterfor
Veterinary
Medicine
Center for Veterinary Medicine

Maintained
by: Divisionof Manufacturing
and ProductQuality(HFN-320)
Maintained by: Division of Manufacturing and Product Quality (HFN-320)
OfficeofCompliance
Office of ComplianceCenter for Drug Evaluation
and ResearchCenterandforDrug
Drug
Evaluation and Research
Food
Administration
Food and Drug Lane
Administration
5600 Fishers
5600
Fishers
Lane
Rockville,
MD 20857
Rockville, MD 20857

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ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

CONTENTSCONTENTSINTRODUCTIONINTRODUCTION
I.I.

Background
Background ......................................................
••......•••.....•................................•.... 1

II.II.

Legal
Legal Effect
Effect ....................................................
....•.......................•......••....••••....... 33

III.
III.

Regulatory...4 4
Regulatory Requirements
Requirements ......................................
••....•••..••............................

IV.
IV.

Human
...6 6
Human andand Animal
Animal Drugs
Drugs andand Biological
Biological Products
Products ...............
.....•...•.••.•.••A.
A. Validation
Validation ofof thethe LAL
LAL Test
Test ...................................
.•.......•......•.............•.... 66
B.
B. Testing
Testing ofof Drugs
Drugs byby thethe LAL
LAL Test
Test .............................
•.....•...................... 88

V.
V.

Medical................................................
11
Medical Devices
Devices .•..••....................................•.....
11
A.
..................................
11
A. Validation
Validation ofof thethe LAL
LAL Test
Test ...•..............................
11
B.
;est..........................14
B. Testing
Testing ofof Devices
Devices byby thethe LAL
LAL ~est...............••..•.•.... 14

VI,
VI.

Appendices.....................................................
16
Appendices .............................•.•.•.............•.....
16
A. Initial
InitialQualityA.
Quality Control.....................................1
Control •.••.•••.•••.•..•................... . 177
B. USPXIV Bacterial
Bacterial
Endotoxins
Test....................1B.
USP XXI/NF
XXI/NF XIVEndotoxins
Test..................•. 188
C. Determinationof thethe Relationship
Relationship
Betweenthe Control
ControlC.
Determination of
Between theStandard
Endotoxin
and the ReferenceStandard
Endotoxin.....loStandard
Endotoxin andthe Reference Standard
Endotoxin
D. Maximum
Maximum Valid
Valid
Dilution
Calculation..........................2
D.
Dilution
Calculation

19222

E. Maximum
Maximum Dose
Dose andand Endotoxin
Endotoxin
Limit
Table......................2
E.
Limit
Table
..................•.•. 255

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ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

INTRODUCTIONINTRODUCTION

This
This

guideline
guideline

sets
sets

forth
forth

Limulus
Limulus Amebocyte
Amebocyte Lysate
Lysateacceptable
acceptableconditions
conditions

for
for

ItIt also
also describes
describes

test.
test.

use
use

of
of thetheprocedures
procedures

for
for

using
using this
this methodology
methodology asas anan end-productend-product endotoxin
endotoxin test
test forfor human
human
injectableinjectable drugs
drugs (including(including biologicalbiological products),
products),
drugs,
drugs, andand medical
medical devices.
devices.

animalanimal injectableinj ectableThe
in lieuof
The procedures
procedures may
may bebe usedused inlieu ofthethe rabbit
rabbit pyrogen
pyrogen test.
test.

For
of thisguideline,
For thethe purpose
purpose ofthis guideline,
reagent”
reagent"

refer
refer

CenterCenter

for
for

test”
test"

means
means

only
only

to
to

Limulus
Limulus

the
the

terms
terms

“lysate”"lysate"Amebocyte
LysateAmebocyte
LysateBiologic
Biologic Evaluation
Evaluation andand Research.
Research.
that
that

aa

test
test

is
is

referenced
referenced

licensedlicensed

The
The
in
in

or
or

aa

term
term

“lysate"lysate
by
by

the
the

“official"official

UnitedUnitedStatesStatesPharmacopeia
drug monograph,monograph,
Drug Application,
Application,
New Animal
AnimalDrug
Pharmacopeia
drug
aa New
New DrugNew
Drug
Applicationor aa Biological
Biological
License.
Application or
License.

B-31

I’

ICCVAM Test Method Evaluation Report: Appendix B5I.
I.

May 2008

BACKGROUNDBACKGROUNDIn
In aa notice
notice ofof January
January 12,
12, 1973
1973 (38(38 FR
FR 1404),1404)) FDA
FDA announced
announced thatthat Limulus
Limulus
Amebocyte
Lysate(LAL),
derivedfromcirculating
blood
cells
Amebocyte
Lysatederivedcells
fromcirculating
blood
(La),
(amebocytes)ofis(amebocytes)of thethe horseshoe
horseshoe crab,
crab, (Limulus(Limulus polyphemus),polyphemus),is aa biologicalbiologicalproduct.
Aslicensingrequirements
asinproduct.
As such,such, itit isis subject
subject totolicensingrequirements
as provided
providedin
section
351 ofHealth
Act
262).
Since
sectionof thethe Public
PublicHealth Service
Service
Act (42(42 U.S.C.
U.S.C.
262).
Since
1973,
indicatorof
1973, LAL has proved
proved to be
be aa sensitive
sensitive
indicatorof thethe presence
presenceofbacterial
endotoxins(pyrogens).
Becauseof
thisdemonstrated
bacterial
endo toxins
Becauseof
thisdemonstrated(eyrowns).
sensitivity,
LAL
the
or
sensitivity,
I&L can be ofof value
value inin preventing
preventing
the administration
administrationor useuseofwhichshock,
toof products
products
which may
may produce
produce fever,
fever,
shock, andand deathdeath if
if administeredadministeredtoorendotoxinsareor used inin humans
humans oror animals
animals when bacterial
bacterial
endotoxins
are present.
present.

ot

When
12,
was
available
data
When thethe January
January
12, 1973 notice
noticewas pUblished,published,available
data and
andexperiencewith
its
adoptionasexperience
with LAL were
were notnot adequate
adequate toto support
supportits
adoptionas thethefinal
pyrogen
test
which
final
pyrogentest inin place
place ofof thethe rabb:'
rabbtt t pyrogen
pyrogen test,
test,
which had been
been
accepted
and recognizedforIn
aa data
acceptedrecognized
for many
many years.
years.
In orderorder toto establish
establish
data
base
with
permitted
thethat notice
noticepermitted
thebase and gain
gain experience
experience
with thethe use ofof LAL,
LAL, that
introductionofwithout
aa license.
This
introductionof LALLAL into
into thethe marketp"lacemarketpiace
without
license.
This was
was
upon
that
to thetestingofupon thethe condition
conditionthat its
its use be limited
limitedthe in-processin-process
testingofdrugs
products,
thatthedecision
to
use
it
be
reacheddrugs and otheritreachedotherproducts,
thatthedecisiontovoluntarily
by affected
firms,and theon LAL statethatthevoluntarily
byaffected
firms,
the labelinglabeling
state
that
the
test was
was not
not suitable
asfor
pyrogentest
suitable
as aa replacement
replacement
for thethe rabbit
rabbitpyrogen test.
test.
Since
time,
production
te~hniques
have
improved
andSince that
that
time,
production
techniques
have been
been greatly
greatly
improved
andstandardized
so thatthey
consistently yield
LAL withan endotoxin
standardized
consistently
yield
with
endotoxinthat
theysensitivity
over 100 times
thanobtained.
Moreover,sensitivity
overtimes greater
greaterthan originallY
originallyobtained.
Moreover,itis widelywidely recognizedrecognized that
more economical,
and
it is
that thethe LAL test
test isis faster,
faster,economical,
requires aa smaller
volume of
than
test.
requiressmaller volume
of productproduct
than does thethe rabbi
rabbit t pyrogen
pyrogentest.
In
addition, the
is
than
test,
In addition,
the procedure
procedureis lessless labor
labor intensive
intensive
than thethe rabbit
rabbittest,
making it
possible toto perform
tests inin aa single
single day.
makingit possible
perform many tests
day.
In
notice publishedinFederal Register
Register ofNovember 4,
In a notice
publishedin thethe Federalof November
4, 1977 (42(42 FRFR
57749), FDA described
described conditions
conditions forthe use ofof LAL as an end-productend-product57749))
for thetest forfor endotoxins
endotoxins inbiological products
products and medical
devices.
test
in human biological
medicaldevices.
The noticestated further
further thatthat theapplication ofof LAL testing
testing toto human
The
noticestatedthe application
drug products
products would
would be thethe subject
subject of
aa ~uture
Federal Register
RegisterdrugoffutureFederalpublication.
publication.
The thenthen Bureau
Bureau ofof Medical
Devices, now FDA‘s
FDA' s Center
Center forfor Devices
Devices andThe
Medical
Devices,
Radiologic Health
Health (CDRH),(CDRH), issued
issued recommended proceduresfor thethe use ofRadiologic
procedures
forof
LAL testing
testing as anan end-productend-product endotoxin
endotoxin test
test on March
26, 1979.
1979. These
These
LAL
March 26,
procedures were
were revised
revised as a result
result ofof thethe comments received
received fromproceduresfrominterested parties
parties .
interested
As a direct
direct result
result ofof CDRH’s
CDRH's experience
experience inin approving
approving petitions
petitions fortheASfor theuse ofof thethe LAL
LAL test
test inin place
place ofof thethe rabbit
rabbit pyrogentest, several
severalusepyrogentest,
procedures forfor usingusing thethe LAL
LAL test
test have
have evolved
evolved and have
have been
been adopted
adopted forprocedures
fordevices.
devices.
In thethe FEDERALFEDERAL REGISTERREGISTER ofof January
January 18,
18, 1980
1980 (45(45 FR
FR 3668))3668), FDA
FDA announced
announcedIntheavailability
of
a
draft
guideline
that
set
forth
proceduresfor usethe availabilityofdraft
guidelinethat
set forthproceduresforof thethe LAL
LAL test’test" as anan end-productend-product testing
testing method
method forfor endotoxins
endotoxins inin humanofand animal
animal injectableinjectable drug
drug products.
products. This
This draft
draft guideline
guideline was mademadeand
- I -l-

B-32

I

(

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

availableinterested
partiesmanufacturers,
especiallythoseavailable toto interested
parties toto permit
permit manufacturers,
especially
those
who
test
in
with
the rabbit
rabbitpyrogentest,
towho hadhad used
used thethe LAL
LAL test
in parallel
parallel withthepyrogentest, tosubmitcouldbeconsideredin thethepreparationof anyany final
final
submit data
data thatthat could
be consideredinpreparationofguideline.
guideline.
Incomments
receivedon thethe January
January
18 draftdraft
guideline,
In response
response toto comments
received on
guideline. FDA
madesignificant
changes(i.e.
Endotoxin
limitschangedandmade several
several significant
changes(i.e.
Endotoxin limitschangedanddeletionon Absenceof Non-endotoxin
Pyrogenic
Substances),deletion ofof section
section on
Absence ofNon-endotoxin Pyrogenic
Substances),andchanges.
The agency
also
determinedthatand many
many minor
minor editorial
edi torial changes.
The
agency also
determinedthataasingledocument
shouldberegulatedsingledocument should
be mademade available
available .covering
covering all
all FDA
FDA regulated
products
be subjsubjectect toPrimarily
becauseoftheproducts thatthat may
may beto LALLAL testing.
testing.
PrimarilY
becauseof theadditionof biologicaland medical
medicaldevicestheaddition of
biological products
products anddevices toto thethe guideline,
guideline, thethemade, inin theFEDERAL REGISTERREGISTER of
29, 1983
1983 (43(43 FR
13096),
the agency
agency made,the FEDERAL
of March
March 29,
FR 13096),
another
draft
of
the
guideline
availableforpubliccomment.
another draft of the guideline available for pUblic comment.
Basedcomments
receivedon theMarch 29
29 draftdraft
guideline,
FDA
has
the comments
received on
the Marchguideline,
FDA hasBased onon theThe
comments
used
in
made
changes
in
this
final
guideline.
made several
several changes
in thisfinal guideline.
The comments
used inof
thesechangesmaybeviewedat
FDA’s
Dockets
Management
supportsupport of these changes may be viewed at FDA's Dockets Management
Branch,4-62,
5600
MD betweenpmBranch, Room
Room 4-62,
5600 Fishers
Fishers Lane,
Lane, Rockville,
Rockville, MD
between 99 am
am andand 44 pm
Briefly,changesmade are:MondayFriday.
Monday throughthrough Friday.
Briefly, thethe significant
significant changes
made
are:
A.
A.

validation
criteria
forthechromogenic,
InclusionofInclusionofvalidation
criteria
forthechromogenic,
endpoint-turbidimetricandkinetic-turbidimetric
LAL
techniques.
endpoint-turbidimetric and kinetic-turbidimetric LAL techniques.

B.
B.

Any
chromogenic
or turbidimetricj
can beAny technique
technique (gel-clot,(gel-clot, chromogenicorturbidimetric) can
be
used
in
testinga
product
forendotoxin.
However,ifagel-clot
used in testing a product for endotoxin. However, if a gel-clot
lysateis usedused
in aa different
different
techniquetheresultsmust
lysate isintechniquethe resultsmust bebeinterpreted
using
the
criteria
forthetechnique
being
used.
interpreted using the criteria for the technique being used.

C.
C.

Elimination
Elimination ofof thethe requirement
requirement
rabbitpyrogentestingcolony.
rabbit pyrogen testing colony.

D.
D.

The
Centerfor
Devicesand RadiologicalRadiological
Healthhas
The Centerfor
DevicesandHealth (CDRH)(CDRH) has
adoptedthe!JSP
Endotoxin
Reference
Standardandrevisedtheadopted the USP Endotoxin Reference Standard and revised thelimit
expressions
from
ng/mL
to EU/mL.
The
new
limit
for
limit expressions from ng!mL to EU!mL.
The new limit for
medical.
devicesis 0.5 EU/mL except
for devices
in contact
withmedical devices is 0.5 EU!mL except for devices in contact withcerebrospinal
fluid
for whichis 0.06
These
cerebrospinal fluidforwhich thethe limit
limi t is
0.06 EU/rnL.
EU!mL.
These
limitsfor devices
are equivalent
to thosefor drugs
for a 70 Kg
limitsfordevicesareequivalent
to
those
for
drugs
for
a
70
Kg
man when consideration
is givento the following:

to testof aa
totest thethe sensitivity
sensitivity of

man when consideration is given to the following:
1.
1.

In the worst
case situation,allendotoxinpresent
inIn
the worst rinsingscase situation,
all endotoxinpresent in
the combinedof 10 devices
could have come fromthe combined rinsings of 10 devices could have come fromjust
one device.
A widevariationinbioburdenisjust one device.
A wide variation in bioburden iscommon to some devices,

common to some devices.

2.
2.

E.

E.

Published

FDA

studies

indicate

that

less

than

half

of

Published
FDA studies
indicate fromthat less·half ofa
added
endotoxinisrecovereddevices than using
added
endotoxinisrecoveredfromdevicesusinganon-pyrogenic
water
rinse.
non-pyrogenic water rinse.

The Center
forDrug Evaluation
and Research(CDER) has added a
The
Centerfor
Evaluation
and Research (COER)
addedthea
the Drughour has andlistingofmaximum
doses
per
Kg
per
listingofthemaximum
doses
per
Kg
per
hour
and
the
endotoxin
limits
for
most
of
the
aqueouscorresponding
corresponding
endotoxin
limits
for
most
of
the
aqueousThis
injectabledrugs
and biologics
currently
on the market.
injectabledrugs and biologicsuniformity
currently amongon thecompanies
market. making
Thislisting
was added to promotelisting
was
added
to
promoteuniformity
amongcompanies
makingthe same product.

the same product.

-2-

2

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ICCVAM Test Method Evaluation Report: Appendix B5

II.
II.

May 2008

LEGAL EFFECTEFFECT OF
OF THETHE GUIDELINEGUIDELINE
LEGAL

This
guidelineisissuedunder
section10.90(b)(21 CFR
10.90(b))
ofThis
guideline isissuedunder
section 10.90(b)(21
CFR 10.90(b»of
FDA’s's administrative
administrative
regulations,which
provides
for
use
FDA
regulations,
IoIhichprovides foruse ofof guidelines
guidelines
to outline
proceduresor standardsof generalgeneralapplicability
that
are
to
outline proceduresor
standards ofapplicabili
ty thatareacceptable
to
FDA
for
a
subject
matter
withinits
statutory
authority.
acceptable to FDA for a subject matter loIithin its statutory authority.
Althoughguidelinesarenot legal
legal
requirements,
person,
who follolols
follows
an
Although guidelines
are not
requirements,
aa person
IoIhoan
agency guideline
may beassuredthatthe procequresproceduresor standards
standards
willbeagencyguideline may
be assured
that
the
or
loIill be
acceptableto FDA.
FDA.
The
following
guideline
hasdeveloped
toinformacceptableto
The follololing
guideline
has been
been developed
to inform
manufacturers
of
human
drugs
(including
biologicals),animaldrugs,
manufacturers of human drugs (including biologicals), animal drugs, andandmedicaldevicesof procedures
FDA considers
considersnecessaryto validate
the useuse
medicaldevicesofprocedures FDA
necessary tovalidate theofas an
an end-productend-productendotoxin
test.
A manufacturer
manufacturer
who
adherestoof LALLAL asendotoxin
test. A
IoIho adheres
totheguideline
wouldbeconsideredincompliancewiththe gUideline loIould be considered in compliance
loIith relevant
relevant provisions
provisionsofapplicable
FDA
current
goodof thethe applicable
FDA current
good manufacturing
manufacturing practicepractice regulations
regulations(CGMP)
applicablerequirements.
As(CGMP) forfor drugs
drugs andand devices
devices andand otherother applicablerequirements.
Asin 21
10.90(b),persons
who
and techniques
notprovidedprovided in
21 CFRCFR 10.
90(b), persons
who useuse methods
methods andtechniques notprovided
the guideline
shouldbethroughprovided inin theguideline shouldbe ableable toto adequately
adequately assure,assure, throughvalidation,thatthemethodortechnique
they
use
is
adequatetodetectvalidation, that the method or technique they use is adequate to detecttheendotoxin
limit
for
the
product.
the endotoxin limit for the product.
.

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ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

REGULATORY PROVISIONSTHAT PERM!TPERMIT INITIATIONINITIATIONREGULATORYPROVISIONS THATOF
END-PRODUCTTESTINGWITHLAL
OF END-PRODUCT TESTING WITH LAL

III.
III.

The regulatory
regulatory
provisions
firm mustmeet before
LAL test
test
The
provisions thatthat aa firmmust meet
before using
using thethe LALasan
end-product
test
are
not
the
same
for
all
categories
ofproducts
as an end-product test are not the same for all categories of products
different
provisions
becauseoftheapplicablestatutory
andbecauseofthedifferent
applicablestatutory
provisions
and
These
provisionsareasfollows:
regulations.
regulations. These provisions are as follows:
A.
A.

ADDliCatiOnS(NDAs)
or
Human
Drun
Human Drugs
Drugs subject
subject toto New
New Drug
ApPlications(NoAa)or(ANDAs).
Antibiotic
Drug
Abbreviated
New Drum ADDlications
Abbreviated New Drug Applications (ANnAs), Antibiotic Drug
ADDliCatiOnS.
and animal
animaldrugs
subject
to New
New Animal
AnimalDrug
Applications,
and
drugs
subject
to
Drug(NADAs).
andAnimalDrug
ADDliCatiOnS
AbbreviatedNew
Applications(NADAs),andAbbreviatedNew
AnimalDrug
Auolication.

Application.
For

these

classesofdrugs,

manufacturers

Droductsforhuman use.

are

to

submitathemanufacturing

For these classes of drugs, manufacturers are to submit
However, asupplemental
application
to provide
for LAL testing.
supplemental application to provide forfor human
LAL testing.
However,
use and 21 CFR
under 21 CFR 314.70(c)
for
drugs
under
21 CFRfor 314.70(c)drugshumanchanges
use and
21 madeCFR
514.8(d)(3)drugs for foranimaluse forvarious
may be514.8(d)(3)
for
drugs
for
animaluse
variouschangesmay
bemadebefore FDA approval.
Under these sections
changes in testingof
beforeUnderin testingofa humanFDA
or approval.
animal drugthat thesigive sections
increased changes
assurance
that
the
adrug
human
or
animal
drug
that
give
increasedassurance
that
the
will
have the characteristics
of purity
it purports
or is
drug
will havecharacteristics
purityintoit purports
isrepresented
to thepossessshouldbe ofplaced
effectator therepresented
to possessshouldeffect theat LALtheearliestpossibletime.
Therefore, be ifplaceda firminto
validates
earliest
time. Therefore,
firm validates
the drug
LAL
test
for possible
a particulardrug
productif acoveredby a newtest
foraparticulardrug
product
covered
by
a
new
drug
in thisguideline
using
a LAL
applicationby the procedures
applicationby theby procedures
this Biologic
guidelineEvaluation
using a LAL
reagent
licensed
the Centerin forandreagent licensed
Center being
for Biologic
Research(OBER) for bythe thetechniqueused, the Evaluation
change may andbeResearch(OBER) for the
being used,of thethechange
may bemadeconcurrently
with techniquethesubmission
supplement
madethe submissionof theThewithsupplement
should containinitial supplement
qualityprovidingconcurrently
for it.
providing
for
it.
The
supplement
shouldcontaininitialqualitycontroldata,inhibition/enhancement
data
and theendotoxin
controlfor data,
inhibition/enhancement
data and the endotoxin
limit
the drugproduct.
limit for the drug product.
B.

Biolopical

B.

Biological products for human use.
Under

21 CFR 601.12

significant

changesinUnder
601.12 significant
the bemanufacturingmethods 21 of CFR
biologicalproducts
arechanges
required in toreportedtomethodsof andbiologicalare required
be reported
tothe agency
may not products
become effective
until to approvedby thethe agency OBER.
and mayTherefore,
not become a effectiveby theDirector,
manufactureruntil ofapproveda biologicalproduct
an approved
amendment ofto a its biologicalproduct
Director, shall
OBER. obtain
Therefore,a manufacturer
end-productlicensechanging anto approved
the use amendmentof LAL in toan its
product before
shall obtainproduct
test,
irrespectiveof the validation
used.an end-productlicensebefore changing
to the use procedureof LAL in
test, irrespective of the validation procedure used.
C.

Drugs not subject

C.

Drugs not subject to premarket approval.
A manufacturer

to oremarket
ofaunroval.

an injectable

drug

for

human oranimaluse

thatmanufacturer
is not subject
premarket drug
approval
would beto useAof an toinjectablefor humanor ableanimalthetest subject
as an toend-producttest
for would
endotoxinsthat LAL
is notpremarket approval
be able without
to useany asinformation
to the test
agency.
CGMPs require withoutthesubmitting
the LAL test
an end-productfor endotoxins
manufacturer
to
have
data
on
file
to
validate
the
use
of
the
LAL
submitting any information to the agency.
CGMPs require thetestforeach toproductfor whichbeing used.
manufacturer
have dataon fileit tois validate
the use of the LAL
test for each product for which it is being used.
D.

D.

Medical

Devices.

Medical Devices.

in
basis
of extensive
experiencesinceNovember
1977,
CDRH
believes
basis of extensive experience in- 4 CDRH
devices since November 1977,
believes

On thedevicesOn the-I~reviewof LAL data
on
that
the
LAL
test,
review of LAL data onthat the LAL test,

-

B-35

I

I

I

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

whenvalidatedaccording
to
this
least
guideline,
is
at
when
validatedaccordingtothisguideline,
is
at
least
equivalent
to thethe rabbit
rabbitpyrogentest
as an
an end-productend-producttestforequivalenttopyrogen
test
as
test
for
medicaldevices.
A
manufacturer
labeling
deviceasmedicaldevices.
A
manufacturer
labeling
a deviceasnon-pyrogenic
must validate
validatethe LAL
LAL test
test
for thatthatdevicein thethenon-pyrogenic
must
the
for
deviceintestlaboratory
to beused forfor end-productend-producttesting
beforeusing
testlaboratory to
be usedtesting
before using
the
LAL
test
as
an
end-productendotoxin
test
for
any
device.
the LAL test as an end-product endotoxin test for any device.
The data
discussed
under
SectionV ofof thisthisguideline
beThedata discussed
under
SectionV
guideline may
may beexpressedgraphicallyor inin tabulartabularform andand should
shouldbe on
on filefile
expressed graphically
or
form
be
at themanufacturing
site;
nopreclearanceprior
to useof theatthe manufacturing
sitej
no preclearan~eprior touse ofthe
LAL test
as an
an end-productend-producttest
isrequired
ifif it
it
is
used
LAL
test astest
isrequiredisusedaccordingto this
this
FDA
guideline.
Voluntary
submissionofLAL
accordingto
FDA guideline.
Voluntary
submissionof LAL
validation
andinhibitiondata
obtainedfollowingissuanceofvalidation and inhibition dataobtained following
issuanceof
thisguideline
willbeacceptedfor
CDRH
review
andcomment.
this guideline will be accepted for CDRH review and comment.
Whenmanufacturerplans
LAL test
When aa manufacturer
plans toto useuse LAL
test procedures
procedures that
that
deviatefromsignificantly
guideline,
deviate significantly
from thethe LAL
LAL gUideline,
aa premarket
premarket
notification
under section
section510(k)of theFederal
Food,
Drug,
and
notification under
SlO{k)
ofthe Federal
Food, Drug,and
Cosmetic
Act
(the
Act)
or
a
Premarket
Approval
Application(PMA)
Cosmetic Act (the Act) or a Premarket Approval Application (PMA)
supplement
under
section
515 ofActshouldsubmitted.
supplement
under section
515
of thethe Actshould be
be submitted.
Significant
deviations
would-include-but
notnecessarilybeSignificant
deviations would-include-but
notnecessarily belimitedto-higher
endotoxin
concentration
releasecriteria,
limi ted to-- higher endotoxin concentration release cri teria,
sampling
fewer
than
lots
for
sampling fromfrom fewer
than three
three lots
for inhfbition/en.hancement
inhi.bition/enhancement
testing,sensitivity
tothetesting, lesser
lesser sensitivity
to endotoxin,
endotoxin, rabbit
rabbit retest
retest when
when theLAL
methodshows
endotoxin
above
therecommended
allowable
LAL method shows endotoxin above the recommended allowableendo toxin
dose,
devicerinsinginendotoxin
dose, andand aa device
rinsing protocol
protocol resulting
resulting in
greaterdilutionofendotoxin
thanthatrecommendedin
thisgreater dilution of endotoxin than that recommended in thisguideline.
guideline.
CDRH will
CDRH will also
also consider
consider submissions
submissions inin thethe form
form ofof aa premarket
premarket
notification
or
PMA
supplementfor
another
deviation
notification or PMA supplement for another deviation fromfrom this
this
draft
of endotoxin
draft guideline;
guideline; processprocess control
control ofendotoxin contamination
contamination withwithreducedend-producttesting,i.e.,reduced end-product testing) Le.) aa decrease
decrease inin thethe number
number ofofdeviceslotundergoing
end-producttesting.
The
perdevicesperlot
undergoing
end-producttesting.
Themanufacturer
must demonstrateadequate
controlof the production
manufacturer must demonstrate adequate control of the production
processby the use of routine
checksfor endotoxin
stagesprocess by the use of routine checks for endotoxin at
at keykey stagesof production
exceptwhere
it has been shown that no possibilityof production except
where it has been shown that no possibilityof contamination
exists.

of contamination exists.

Tofacilitate
subsequent
PMA
review,
providersofTo
facilitate devices
subsequent
PMA
review,
providersofinvestigational
subject
to 21 CFR part812 or 813 areinvestigational devices subject to 21 CFR part 812 or 813 areencouraged
to use thisguideline
when a non-pyrogenic
deviceisencouraged to use this guideline when a non-pyrogenic device is
to be manufactured.

to be manufactured.

- 5 -

5

B-36

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

IV.
IV. HUMAN AND
AND ANIMALANIMAL DRUGS AND BIOLOGICALBIOLOGICAL PRODUCTS
PRODUCTS

GENERAL
GENERAL REQUIREMENTREQUIREMENT
Manufacturers
Manufacturers shall
shall use
use anan LALLAL reagent
reagent licensed
licensedvalidation,
in-process,andend-productvalidation, in-process, and end-product LAL
LAL tests.
tests.
A.
A.

by
by

CBER
CBER

in
in

all
all

VALIDATIONVALIDATION OF THE
THE LAL
LAL TESTTESTValidation
Validation ofof thethe LAL
LAL test
test asas anan endotoxin
endotoxin test
test forfor thethe release
release ofofdrugs
includesfollowing:
human
and
animalthe(1)human and animal drugs includes the following:(1) initial
initialqualification
qualification ofof thethe laboratory,laboratory, andand (2)(2) inhibition
inhibition andand enhancement
enhancement
tests.
tests.
1.
1.

INITIALINITIAL OUALIFICATIONQUALIFICATION OF
OF THE
THE LABORATORYLABORATORY
Variousforthe detectionofVarious methodologies
methodologies have
have been
been described
described forthedetection of
using
limulusamebocyte
lysate.
Currently,
endotoxin,
endotoxin,using
1imulusamebocyte
lysate.
Currently,
commerciallyclot,commercially available
available licensed
Hcensed lysates
lysates use
use thethe gelgel clot,
endpoint-turbidimetricchromogenic,
chromogenic,
endpoint-turbidimetric oror kinetic-turbidimetrickinetic-turbidimetric
Other
methods
techniques.
techniques.
Other
methods which
which have
have been
been reported
reported show
show
potentialforincreasing
LAL
potential forincreasing further
further thethe sensitivity
sensitivity ofof thethe LALmethod.
method.
shouldassessManufacturers
Manufacturers should
assess thethe variability
variability ofof thethe testing
testinglaboratory
any officalperformed.
Each analyst
analystlaboratory before
before any
offical tests
tests areare performed.
Each
using
of LAL
singlelotofusing aa single
single lot
lot of
LAL andand aa single
lotof endotoxin
endotoxin should
shouldfor
confirmation
performthetestoflabeledLAL
reagent
perform the test for confirmation of labeled LAL
reagent
sensitivity
Appendixsensitivity oror ofof performance
performance criteria.
criteria.
Appendix AA gives
gives thetheprocedures
and test
licensedtechniques.
procedures andtest criteria
criteria forfor thethe current
current licensed
techniques.

2.
2.

INHIBITION
AND ENHANCEMENTINHIBITION AND
ENHANCEMENT TESTINGTESTINGThe
degreeofinhibition
or enhancement
enhancementofthe LAL
The degreeof productproduct inhibition
orof theLAL
procedure
determinedfor eacheach drug
drug
formulation
beforeprocedure should
should be
be determinedforformulation
beforethe LAL test
used
to assessthe endotoxin
any
the LAL test is
is usedto assess theendotoxin content
content ofof anydrug.
Allvalidation
tests
shouldbe performedon undiluteddrug.
All validation tests should be performed on undiluted
dilution.
drug productor on an appropriate
Dilutionsshoulddrug
product oron an appropriate dilution.
Dilutionsshouldnotexceedthe Maximum Valid
Dilution(MVD) (seeAppendixD).
not exceed the Maximum Valid Dilution (MVD) (see Appendix D).
At
least
three
production
batchesof
each
finishedproduct
At least three production batches of each finished productshouldbe tested
for inhibitionand enhancement.

should be tested for inhibition and enhancement.
a) GEL-CLOT

TECHNIOUEa) GEL-CLOT TECHNIQUE
Inhibition/enhancement
testingshouldbe conductedaccordingInhibition/enhancement testing should be conducted accordingtothe directionsin the preparatory
sectionof the USP
to the directions
inTestthe (seepreparatory
sect
ion Briefly,of the USP
Bacterial
Endotoxins
AppendixB).
theBacterial
Endotoxins
Test
(see
Appendix
B).
Briefly,
concent
rat ioncontainingthe
methodinvolves
takinga drugmethodinvolves takingof aa standard
drug concentration
varying
concentrations
endotoxinthat containing
bracketvarying
concentrationsofastandard
endotoxinthatbracketthesensitivityof thelysateand comparingitto a series
the thesensitivityof the concentrations
lysate and comparing
to a series
Theofsame endotoxin
in water it alon:.
of
the
same
endotoxin
concentrat
ions
in
water
alon2.
withendotoxin
andthendiluted The
is“spiked”
drug productdrug
product isdrug"spiked"
and concentration
then diluted
the drugwithadditional
product with(so endotoxin
that
with
additional
drug
product
(so
that
the
drug
concentration
constant)
to
the
same
endotosin
concentrations
inremainsremains constant) to - the
6 - same endotoxin concentrations in
- 6 -

B-37

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

water.
Results ofof endotoxin
endotoxfn
determinationin waterwater and thethewater.
Results
determination
inshouldFall
drug
product
withinplus/minus
twofold
drug
productshouldfall
withinplus/minus
aa twofold
sensitivity.
dilutionof thethe labeledlabeled sensitivity.
If thethe undiluted
undiluted
drug
dilutionofIf
drug
product
shows inhibition,
inhibition,
the drug
drug productproduct
diluted,productshows
thecan be diluted,
will
be
not toto exceed
exceed thethe MVO,
MVD, with
with thethe samesame diluent
diluent
that
not
that
will
be
in
the
releasetesting
procedure
the
above
used
in
the
releasetesting
and theabove
procedure
repeated.
Negative controls
controls(diluent
plus lysate)lysate)should be
repeated. Negative(diluentplus
shouldincludedin allall inhibition/enhancement
inhibition/enhancement
testing.
includedintesting.

b)b)

CHROMOGENICAND
AND ENDPOINT-TURBIDIMETRIC
ENDPOINT-TURBIDXMETRIC TECHNIQUESTECHNIOUESCHROMOGENIC
In inhibition/enhancement
inhibition/enhancement
testingby thesethesetechniques,In
testingbytechniques,aadrug concentration
concentration
containing
lambda concentration
concentrationof thethedrugcontaining
44 lambda
of
RSE oror
CSE (lambda(lambda
is
equal
to
the
lowestendotoxin
RSE
CSE
is
equal
to
the
lowestendotoxin
concentration
used toto generate
generate
the standard
standard
curve)is tested
testedconcentrationused
thecurve)
isinduplicateaccordingtothelysatemanufacturer’sinduplicateaccordingtothelysatemanufacturer's
methodology.
The standard
standard curve
curve forfor these
these techniques
techniques
shallmethodology.
The
shall
consist
of
at
least
four
RSE
or
CSE
concentrationsinwater
consist of at least four RSE or CSE concentrations in waterthat extend
extend over
over thethe desired
desired range.
If thedesired
range
is
that
range.
If
the desired
range isgreater
one log,log,
additional
standards
concentrations
greater than
than one
additional
standards
concentrations
shouldbe included.
The standard
standard
curve
must
meet thetheshouldbeincluded.
The
curve
must meetcriteria
forlinearityasoutlinedinAppendixA(2).
The
cd teria for lineari ty as outlined in Appendix A( 2) .
The
detected
amount ofendotoxia
inspikeddrug must
must bebedetected
amountof endotoxL'lin thethe spiked
drug
withinor minus
minus 25%
25% ofof thethe 44 lambda
concentrationforwi thin plus
plus orlambda concentrationforthe drug
drug concentration
concentration
to be
be considered
considered
to neither
neitherenhancethetotoenhancenorinhibittheassay.
Iftheundiluted
drug
productshows
nor inhibit the assay.
If the undiluted drug product showsthedrug
product
canbediluted,nottoexceedinhibition,
inhibition, the drug product can be diluted, not to exceed
theMVD, andt:.
test repeated.
the MVD,
and tJ:.
test
repeated.
An
as describedabove
An alternate
alternate procedure
procedure may
may bebe performed
performed as
described above
exceptthe
RSE/CSEstandard
curve
ispreparedinLAL
except the RSE/CSE standard curve is prepared in LAL
negative
drug
product,i.e.
nodetectableendotoxin,
insteadnegative drug product, i.e. no detectable endotoxin, insteadof LALnegativewater.
The
curve
must
the
of
LAL negative
water.
The standard
standard curve
must meet
meet thetest
for linearity,
i.e.
r
equal
to
or
greater
than
0.980,
test for linearity, Le. r equal to or greater than 0.980,
and in addition
the difference
between the O.D. readingsforand in addition the difference between the 0.0. readings forthelowestand highest
endotoxin
concentrations
must
bethe lowest and highest endotoxin concentrations must be
greaterthan 0.4 and less than 1.5 O.D.
units.
If
thegreater than 0.4 and less than 1.5 D.D. units.
If thestandard
curve
does notmeet thesecriteria,
standard curve does not meet these criteria, thethe drugdrug
product
cannot be tested by the alternate
procedure.
product cannot be tested by the alternate procedure.

c) KINETIC-TURBIDIMETRICTECHNIOUE
c) KINETIC-TURBIDIMETRIC TECHNIQUE
In inhibition/enhancement
testingby thistechnique,a drugIn inhibition/enhancement
this technique,a drugconcentration
containing
4testing
lambda byconcentrationof theRSE
concentration
containing
4
lambdaconcentration
of
the
RSE
or
CSE (lambda
is
equal
to
the
lowestendo toxinorCSE(lambda
is
equal
to
the
lowestendotoxin
concentration
used to generate
the standard
curve)is tested
concentration used to generate the standard curve) is tested
induplicateaccordingtothelysatemanufacturer’sinduplicateaccordingtothelysatemanufacturer' s
methodology.
The standard
curve shallconsistof at least
methodology.
The standard curve shall consist of at least
four RSE or CSE concentrations.
If
the desiredrange
isfour RSE thanor CSE
Ifstandard
the desired
range isgreater
one concentrations.
log,
additional
concentrations
greater bethan
one log, The
additional
standard
concentrations
shouldincluded.
standard
curve
must meettheshouldbeincluded.
The
standard
curve
must
meet meanthecriteria
outlinedin Appendix
A(3).
The calculated
criteria of outlinedA(3). drug
The calculatedmeanamountendotoxin in inAppendix
thespikedproduct,whenamount of toendotoxinin thecurve,spiked
product,plus whenmust drug
be withinorreferenced
thestandardreferenced
standard tocurve,
be withininhibit orminus
25% toto betheconsideredneithermustenhance
nor plus
minus
25% to be If considered
to neitherdrugenhancetheassay.
theshows
undiluted
productnor inhibit
theassay.
Iftheundiluted
drug
productshows-7- 7 -B-38

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

inhibitionorenhancement,
the drug
drug productdiluted,inhibition
or enhancement,
theproduct can
can bebe diluted,
notto
exceedthetestrepeated,
not to exceed the MVD,
MVD, andand thethe test
repeated.
An
wherebythe RSE/CSE
An alternate
alternate procedure
procedure may
may bebe performed
performed whereby
theRSE/cSE
curve
isstandard
preparedin
drug
product
orproduct
standard curve is prepared in drug product or
product
dilution
insteaddrug
product
cannothave
dilution
instead ofof water.
water. The
The drugproduct
cannothave aabackgroundtoxin
concentration
of
more
than
10%
background endo
endotoxin
concentration of
more
than
lOr.
(estimatedline)of thethe(estimated byby extrapolation
extrapolation ofof thethe regression
regression line)oflambdaconcentration(lambdaequalsthelowestconcentration
lambda concentration (lambda equals the lowest concentrationusedcurve).
The
curve
used toto generate
generate thethe standard
standard curve).
The standard
standard curve
must meet the testforlinearit’y,i.e.
r equalto or lessmust meet the test for linearit'y, Le. r equal to or lessin additionof the regression
thanthan -0.980,-0.980, andand inaddition thethe slopeslope ofthe regression
must
be lessthan-0.1
and greater
than
-1.0.
If
the
must be less than -0.1 and greater than -1.0.
Ifthestandard
curve
does
not
meet
these
criteria,thedrugstandard cannot
curve be does
notby meetthese criteria,
the drugproducttestedthe alternate
procedure.

product cannot be tested by the alternate procedure.

when
In
those
instances
the
drug
is
manufacturedinvariousIn those instances
the drugin various
concentrationsof active wheningredient
while is themanufactured
othercomponentsof theco~centrations of active ingredient while the other components of theformulation
remainconstant,
only
the highest
and lowest
concentration
formulation remain Ifconstant,
the highest difference,and lowest concentration
there
isonlya significant
i.e.
greaterneed be tested.
needbetested.
If
there
is
a
significant
difference,
betweentheinhibition
endpoints
or
if i.e.the greater
drug
than
twofold,
than twofold, per
betweenthe inhibition
or if thesolutionsendpoints
is different,
then drug
eachconcentration,
mL, in the test
concentration,
per roL, inshould
the test
solutionseach
remaining
concentrations
be tested.
If is
the different,
drug
productthen shows
remaining
concentrations
shouldbetested.
Ifthedrug
product
showsinhibition
or enhancement
at the MVD, when testedby the proceduresininhibition
or enhancement
the MVD,
theabove sections,and is at
amenableto whenrabbit tested
testing, by thethenprocedures
the rabbit in
the above
testing,thenwill sections,stillbeandtheis amenableappropriate to rabbit
test
for
that
drug. the Ifrabbit
the
test
test
will
still
be
the
appropriate
test
for
that
drug.
If theinhibitingorenhancing
substances
canbeneutralized
without
inhibiting theor sensitivity
enhancing ofsubstances
iswithoutmoreaffectingthe test canor ifbe theneutralized
LAL test
affecting than
the thesensitivityof the testtesttheor MLif test
the LALtest
sensitive
rabbitpyrogencan beused.is moreForsensitive
rabbit pyrogen
the LAL
test canused. Forthosedrugs than
not theamenable
to rabbit test
pyrogentesting,
the bemanufacturer
those drugs
not amenable
to rabbit
pyrogenmanufacturer
shoulddeterminethesmallestquantityof testing,endotoxin the thatcanbeshould determine
smallestquantity toof the endotoxin
detected.
This datathe should
be submitted
appropriate thatFDA can
Office be
detected.
This data should be submitted to the appropriate FDA Office
for review.

for review.
The inhibition/enhancement
tests
must be repeatedon one unitof the
The inhibition/enhancement
tests must be is repeatedofproductifthelysatemanufacturerchanged. on one
If unitthelysatetheproduct ifis thelysatethe manufacturer
is enhancementchanged.
Iflysatetechniquechanged,inhibition
andtests the mustbetechnique using
is changed,
the inhibition
and manufacturing
enhancement tests
bethreebatches.
repeated
When theprocess, must therepeated formulation,using threethebatches.
the manufacturing
process,
theproduct
sou,rce of Whena particularingredientof the drug
product
sou,rcea particularingredient
drug
formulation, formulation,or lysate the lot
is ofchanged,the positive
productof thecontrolcanbe used to orreverifyof thetest
forproductthe product.
formulation,lysate the
lot validity
is changed,
the LALpositive
controlFirms
are reverify
obtaining the anvalidity
ingredient of the
fromLALa test
new manufacturer
arecan be thatused tofor the product.
encourged
includeas part an ofingredient
theirvendorthe rabbitare
Firms thatto are
obtainingfrom qualificationa new manufacturer
todeterminethat their
the vendor
ingredientqualification
does
notthe containpyrogenencourged testto include
as part of
rabbitnon-endotoxin
pyrogens.
pyrogen test to determine that the ingredient does not containnon-endotoxin pyrogens.
B.

RoutineB.

Routine Testing of Drugs by the LAL Test.

Testinvof Drugs

by the LAL Test.

based
on
data
from
the
End-producttestingistobeSamples,
inhibition/enhancement
testingasoutlinedinSectionA(2).
End-producttesting
is
to
be
based
on
data
from
the
standards,
positive
product
controls
and negative
controls
beinhibition/enhancement
testingas outlined
in Section
A(2). should
Samples,testedat least
in duplicate.
standards,
positive
product controls and negative controls should betested at least in duplicate.

- 8 8

B-39

‘-i

i

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

Forthe gel-clot
gel-clottechnique,an endotoxin
endotoxin
standard
seriesdoes not
not
For
the
technique,anstandard
series
does
have
to
be
run
with
each
set
of
tests
if
consistencyofstandard
have to be run with each set of tests if consistency of standard
endpoints
has been
been demonstrated
demonstrated
in thethe test
test
laboratory.
It should
shouldendpoints
has
inlaboratory.
It
be runrun atat least
least
once aa day
day withwiththe first
first
set ofof tests
tests
and repeated
repeated
be
once
the
set
and
if
there
is
changeinlysatelot,
endotoxin
lot
or
test
any
if
there
is
any
changeinlysatelot,
endotoxin
lot
or
test
conditions
duringtheday.
Anendotoxin
standard
seriesshouldbeconditions during the day.
An endotoxin standard series should be
runwhen
confirming
end-productcontamination.
Positive
productrunwhen
confirming
end-productcont~~ination.
Positive
product
controls(( twotwo lamda
lamda concentration
concentrationof standard
standard
endotoxin
in product)
product)
controlsofendotoxin
in
must
be
positive.
If
your
test
protocols
state
that
you
are
using
must be positive.
If your test protocols state that you are using
the
USP
Bacterial
Endotoxin
Test,
rememberthatitrequiresthe USP Bacterial Endotoxin Test, remember that it requires aa
standard
seriesto be
be runrun witheach test.
test.
The
above deviation
deviation
must
standard series
towith each
The abovemust
be notedin your test
protocol.
be noted in your test protocol.
Forthechromogenic
andendpoint-turbidimetrictechniques,an
For the chromogenic andendpoint-turbidimetrictechniques,anendotoxin
standard
seriesdoes not have to be run witheach set of
endotoxin standard series does not have to be run with each set oftests
if consistencyof standard
curveshas been demonstrated
in thetests if consistency of standard curves has been demonstrated in thetestlaboratory.
Itshouldbe runat least
once a day withthetest laboratory.
It should be run at least once a day with the
first
set
of tests
and repeatedif
there
is any change
in lysate
first endotoxin.
set of tests
if thereany day.
change in lysate
lot,
lotorand testrepeatedconditions
during is the
However,atlot,
endotoxin.lot
or
test
conditions
duringtheday.
However, andat
least
duplicatesof a 4 lambda
standard
concentrationin waterleastof
a 4 lambda
in water withandin eachduplicatesproduct(positive
productstandard
control) concentrationmust
be includedin
each
product(positive
product
control)
mustbeincluded
wi
th
each
run
of
samples.
The meanendotoxin
concentrationoftheeach run mustof samples.
Themean endotoxinof thestandard
be withinplus/minus
25% of the concentration
actualconcentration
standard
must be productwithin plus/minusof theconcentrationand the positive
control
must 25%meet
the actual
same criteria
after
and
the
positive
product
control
must
meet
the
same
cri
after
subtractionofany endogenous
endotoxin.
An endotoxin teria
standard
subtraction
endogenous
seriesshould ofbe any
run whenconfirmingendotOXin.
end-product An endotoxin
contamination. standard
If
seriesshouldberunwhen
confirming
end-productcontamination.
the alternate
procedureis used,a standard
in productseriesmustIfthe conducted
alternateeach
procedure
used, ais standard
in product series mustbetime the isproducttested.
be conducted each time the product is tested.
For the kinetic-turbidimetrictest,
it
is notnecessary
to runaFor
it is end
not productnecessary
to run a
standardthe kinetic-turbidimetriccurve
each day or when test,
confirming
contamination
curve ofeachstandard
day or curves
when confirming
product contamination
ifstandard
consistency
has been end
demonstrated
in the test
if consistency However,of standard
curves duplicateshas been demonstrated
test
laboratory..
at leastof a 4 lambdain thestandardlaboratory. .
However,
4 lambda standard
concentrationin water atandleastin duplicateseach
productof a (positive
product
concentrationeach runproduct(positive Theproductcontrol)
must inbe waterincluded and within eachofsamples.
meancontrol) must
be included
with standard
each runsamples.
The meanendotoxin
concentrationof thewhenof calculatedusing
anendotoxin standard
concentrationof thestandard
calculatedusing anarchived
curve(See
AppendixC), whenmust bewithinplus/minusarchived(See Appendix
25% of thestandard
actual curve
concentration
and C),
the must
positive be within
product plus/minus
control
25% ofmeetthe' theactualthe positive
controlmustsame concentration
criteria
after and subtractionof productany
endogenous
must
same
criteria after
endogenous
endotoxin.meet theIfthealternateproceduresubtractionisused, of aanystandard
inproduct
series If must
conducted
each time thetested.
endotOXin.
the bealternate
procedureis productused, a is standard
inproduct series must be conducted each time the product is tested.
Beforea new lot of lysate
is used,the labeledsensitivityof thelysatebe sensitivity
confirmed
byof thethe lotperformance
criteria
shouldlabeled
Before aor newof lysate isused, thethelaboratory,
using performancethe procedures criteria
in Appendix
lysate or theshouldA. be confirmed by thelaboratory, using the procedures in Appendix A.
The samplingtechniqueselected
and the number of units
to be tested
shouldbe basedtechniqueon the selected
manufacturingand theprocedures
the tobatch
size.
The sampling
number ofandunits
be tested
Ashould
minimumof threeunits,manufacturing
representing procedures
the beginning,andbe basedon theand the middle,
batch size.
end,minimum
testedunits,fromrepresenting
a lot.
canberunshould of bethree
These beginning,
units
A
the
middle,andindividuallyorpooled.
If
the
units
are
pooledandanyendotoxin
end, should be tested from a lot.
These units can be run
The and
LAL any
test endotoxin
may beisindidetected,
testing I f canvidually repeator pooled.
the beuniperformed.
ts are pooled
repeated
no morethan testing
twice.
Thebe first
repeatconsists
twicemay theis detected,
repeatcanperformed.
The LAL oftest
beinitialreplicatessamplein consists
question
examinerepeated number
no moreof than
twice. ofThe thefirst
repeatofto twicethethepossibility
that replicates
extrinsic
contaminationin theto initial
initialnumber ofofthe sample occurredin question
examinethe possibility that extrinsic contamination occurred in the initial-9- 9 -B-40

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

assay procedure.
procedure.
On pooledpooled samples,
samples,
assay
On
if
is
in
if anyany endotoxin
endotoxin
is detected
detected
in
the
first
repeat,proceedto secondsecondrepeat.
thefirstrepeat,proceedtorepeat.
The
repeatThe second
secondrepeatconsistsof anan additional
additional
10 unitD
units
testedindividually,
consistsof
10
testedindividually.
None
None ofof thethe
10 units
units
testedin thethe second
second repeat
repeatmay containcontainendotoxin
in excessexcess10testedinmayendotoxin
inof thethe limit
limit
concentration
for thethe drug
drug product.
product.
ofconcentrationfor
The following
following
shouldbe considered
consideredTheshouldbelimit t forfor allalltheendotoxin
theendotoxin
limiparenteraldrugs to meet iiff thethe LAL test
test
is to
to be used
used asas ananparenteraldrugs
isend-productendotoxin
test:
end-productendotoxin
test:
1.
1.

K/M:
KIM:

parenteralFor
drug
exceptthoseadministeredFor
any parenteraldrug
exceptthoseadministered
limit
for
endotoxinisintrathecally,
the endotoxin
endotoxin
intrathecally,
the
limit
for
endotoxiniswhich equals
equals thethe amountamount ofdefinedas KIM,
K/M, whichof endotoxin
endotoxin
definedK is
is equalequal to 5.0
5.0
allowed per
per ng or mL
mL ofof product.
product.
(EU) allowed
K
EU/Kg. (SEE
(SEE appendix
appendix D forfor definition
definitionof M).
M).
EU/Kg.
of

drugs
that
have
an
For
parenteral
For parenteral
drugs
that
have
an
K
is
equal
to
0.2
EU/Kg.
administration,
administration, K is equal to 0.2 EU/Kg.

intrathecal
intrathecal

route
route

of
of

limitsare:Drugs exempted
exempted fromfrom thethe above
above endotoxin
endotoxin
limits
are:
Drugs
1.
1.

Compendia1 drugs
drugs
Compendia1
established.
established.

for
for

which other
other
which

endotoxin
endotoxin

limitshave been
been
limits
have

2.
2.

drugs
coveredbynew
Non-compendia1
drug
applications,Non-compendia1
drugs
coveredbynew
drug
applications,newanimaldrug
applications,andantibiotic
drug
applications,antibiotic drug applications, new animal drug applications, andbiologicalproduct
licenseswheredifferent
limitshave
beenbiological product licenses where different limi ts have been
approved
the agency.
agency.
approved byby the3.
3.

Investigational
drugs orfor whichan IND
IND oror INADINAD
Investigationaldrugs
or biologicals
biologicals forwhich anexemption
has
been
filed
and
approved.
exemption has been filed and approved.

4.
4.

Drugs oror biologicals
biologicals
which cannot
be tested
tested by
the LAL
LAL method.
Drugs
which
cannot beby themethod.

Atest should
should not
not beretested
A batchbatch which
which fails
fails aa validated
validated LAL
LAL release
release test
be retested
itpasses.
Due tothe highhighbyby thethe rabbitra;bit test
test andand released
released ifif it
passes.
Dueto thevariability
and
lack
of
reproducibilityoftherabbittest
asvariability and lack of reproducibility of the rabbit test as ananendotoxinappropriate
retestendotoxin assay
assay procedure,
procedure, we
we do
do notnot consider
consider itit anan appropriate
retestprocedure
procedure forfor LAL
LAL failures.
failures.

-

10 -

10 -

B-41

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

V.
MEDICAL DEVICES
DEVICESV. MEDICAL

GeneralReauirements
GeneralRequirementsThe
the results
resultsof theStudy
for
The CDRH has
has reviewed
revie ....ed theofthe “HIMA"HIMA Collaborative
Collaborative Studyforthe
Evaluationof a Reference
USP Rabbit
Rabbitthe Pyrogenicity
Pyrogenicity EvaluationofReference Endotoxin
Endotoxin by
by thethe US?
Test.”
This
study,
recommends
0.1 ng/mL
ng/mL(10 mL/kg)
Test."
This
study' recommends
0.1(10
mL/kg) ofof E.
Bo. colicoli 055:,B5055:.B5
Difco
Laboratories
level .of.of endotoxin
endotoxin
which
endotoxin
endotoxin fromfrom Difco
Laboratories as
as thethe level
which should
shouldbethe LAL
when used
testingof
be detectabledetectable inin the
LAL test
test whenused forfor end-productend-product testingofThissensitivity
(0.1
ng/mL
given
10 mL/kg)
ismedicalmedical devices.
devices.
This sensitivity(0.1 ng/mL
given
10
mL/kg) issufficienttesting
andforretestofinrabbits.
sufficient forfor LAL
LAL testing
and forretestof devices
devices inrabbits.
Accordingstudiesin thethe rabbit
rabbitpyrogenAccording toto recentrecent collaborative
collaborative studiesinpyrogen andand LAL
LAL
tests,
nanogramofcoli
055:B5endotoxin
similarintests, one
one nanogramof E.
E. coli
055:85endotoxin isis similarin potency
potency toto
55 EU‘LISP Endotoxin
endotoxin
limit
for
EU of
of thetheUSP
Endotoxin Reference
Reference Standard.
Standard. The
The endotoxin
limit formedicalbeen converted
and is
0.5 EU/mL
themedical devices
devices has
has been
converted to
to EU
EU andis now
now 0.5
EU/mL using
using therinsevolumerecommendedinSection2
below.
Liquiddevicesshouldberinse volume recommended in Section 2 below. Liquid devices should be
moreappropriately
validatedandtestedaccordingtotherequirementsformore appropriately validated and tested according to the requirements fordrugs
maximum humandose perweightperdrugs byby takingtaking thethe maximum
human dose
per kilogram
kilogram ofof body
body weightperhour
hour into
into considerationconsideration (See(See Section
Section IV,B).
IV,B).
Manufacturers
failureswiththe LALtest
or aa USP
Manufacturers may
may retest
retest LAL
LAL test
test failures....ith the
LAL testor
USP
rabbiteluatehas
rabbit pyrogen
pyrogen test.
test. IfIf thethe endotoxin
endotoxin level
level inin aa device
device eluate
has been
been
quantitated
at 0.50.5 EU/rnL
endotoxinorgreater,thenretestinquantitated byby LALLAL at
EU/mL endotoxin
or greater,then retestinrabbitsisnotappropriate.
Medicaldevicesthat
contact
cerebrospinal
rabbits is not appropriate.
Medical devices that contact cerebrospinalof endotoxin.
These
values
fluid
0.06
EU/mL
fluid should
should have
have lessless than
than 0.06
EU/mL ofendotoxin.
These
valuescorrespond
to
those
set
by
the
CDER
for
intrathecal
drugs.
correspond to those set by the CDER for intrathecal drugs.
Manufacturers
shall
use
an
LAL
reagent
licensed
Manufacturers shalluse anLAL
reagent
licensedvalidation,
in-process,andend-product
LAL
tests.
validation, in-process, and end-product LAL tests.
A.
A.

by
by

OBRR
OBRR

in
in

all
all

Validationof theLAL Test
Test
Validation of
the LAL1.
1.

Sensitivity:
Data
demonstrating
Sensitivity:
Data
demonstrating
reproducibility
of
the
LAL
reproducibility of the LAL test.
test.

2.

Inhibition/Enhancement
Testing:
Each productline
of devices
Inhibltion/Enhancement Testing:
Each product lineof devices
utilizing
differentmaterials
or methodsof manufacture
utilizing different materials or metl10ds of manufacture should
shouldbe checked
for inhibition
or enhancementof the LAL test.

2.

the
the

sensitivity
sensitivity

and
and

be checked for inhibition or enhancement of the LAL test.

Furtherof the above points
as follows:
Further explanation
explanation of
the above points is
is given
given asfollows:
1.

1.

SENSITIVITYSENSITIVITY

A manufacturer
must be able to demonstrate
a sensitivityof atA manufacturer must be able to demonstrateatendo toxin a sensitivity
selected
as of theleast
0.5
EU/mL.
Thelevel
ofleast 0.5 EU/mL.
The level of endotoxin selected as thepass/failpoint
forevaluating
pyrogenicity
of products
usingpass/fail point for evaluating pyrogeniclty of products level.
using
the
LAL
test
must
be equivalent
to
or
below
this
the
LAL
test
must
be
equivalenttoorbelo....
thislevel.
ifa reproducible
Manufacturers
useanother
endo toxin
may
Manufacturers
may useanother endotoxin if
a reproducible
correlation
betweenit and the USP Reference
Endotoxin
Standardc~rrelation between it and the US? Reference Endotoxin Standard
has been demonstrated
in theirlaboratory(see appendix
C).

has been demonstrated in their laboratory (see appendix C).

-

11 -

- 11 -B-42

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

The sensitivity
sensitivityof thethe LAL
LAL technique
techniqueused should
shouldbe determined
determined
The
of
used
be
the
procedures
andcriteria
inAppendixA.
Routinebytheprocedures
andcriteria
inAppendixA.
Routinebyperformanceof thethe LAL
LAL test
test
shouldincludestandards(runin
performanceofshouldincludestandards(runin
duplicate)
and aa negative
negative
control.
endotoxin
standard
seriesduplicate)andcontrol.
An endotoxin
standard
seriesis usefulusefulfor checking
checking
lysatesensitivity
and thethe competencecompetence
of
is
for
lysatesensitivity
and
of
the
technician,andforidentifying
otherproblems
such
as
the
the technician, and for identifying other problems such as thecontaminationof glassware.
glassware.
contaminationof
The stability
stabilityof thethe endotoxin
endotoxin
standards
and appropriate
appropriate
storageTheofstandards
andstorageconditions
shouldalso
beconsidered;diluteendotoxin
solutionsconditions should also be considered; dilute endotoxin solutions
are not
not asas stable
stableas moremore concentrated
concentrated
solutionsundercertainareassolutionsundercertainconditions.
conditions.
2.
2.

INHIBITION
AND ENHANCEMENTENHANCEMENT TESTINGTESTINGINHIBITIONAND
Lack ofof productproduct
inhibition
or enhancement
enhancementof thethe LAL
LAL test
test
shouldLackinhibition
orofshouldbe shown
shownfor eacheach type
type ofof devicedevicebeforeuse ofof thethe LAL
LAL test.
test.
be
for
beforeuse
Possible
inhibitionofdifferent
chemicalcomponentsofsimilarPossible inhibition of different chemical components of similar
devicesshouldbe considered.
considered.
A manufacturer
manufacturer
may _logically.logicallydevicesshouldbeAmay
divideits
deviceproducts
into groups
groupsof products
products
accordingtodivideits
deviceproducts
intoofaccordingtocommon chemicalchemicalformulation;
andthenqualifyonlymaycommonformulation;
and
may
then
qualify
only
aa
from
representative
producteachsuchgroup.
Ideally,therepresentative product from each such group.
Ideally, theproductchosen fromfrom each
each group
group would
would bebe thethe oneone withwiththe largest
largestproductchosenthesurfacearea
contacting
bodyor fluid
fluidforadministration
to aa
surfacearea
contacting
body orfor
administrationtopatient.
patient.
At
least
three
lots
of eacheach producttypeshouldbe
At least
three production
production lotsofproduct typeshouldbeinhibition.
fn
general,tested
for
use
of
the
sampling
tested forinhibition.
Ingeneral,useofthesampling
techniqueselectedshouldresultin aa random
samplingoftechnique selectedshouldresultinrandom samplingof aa
finishedproduction
lot.
CDRH
recommends
testing2
devicesforfinished production lot. CDRH recommends testing 2 devices for
lot
sizes
under
30,
3
devices
for
lot
sizes
30-100,andlot siz.es under 30, 3 devices for ] ot sizes 30-100, and 33
percent
above size
size
100, upUP to
to aa maximum
maximum of
10 devices
devices
percent ofof lotslots above
100,
of 10
per lot.

per lot.

The
processofeluate/extractforpyrogen
The processof preparing
preparing anan eluate/extractforpyrogen oror
inhibition/enhancement
testing
may
vary
for
each
device.
Someinhibition/enhancement testing may vary for each device.
Some
medical devices
can be flushed,
have toto be
be immersed
medical devices canbe flushed, Somesome may
may have
immersed ininthe non-pyrogenic
rinse solution,
while others may be tested
the non-pyrogenic rinse solution, while others may be tested bybydisassemblingdisassembling oror by
by cutting
cutting thethe device
device into
into pieces
pieces priorprior toto
In general,for devices being flushed,
extractionbyimmersion.
extraction by immersion. In general, for devices being flushed,
the non-pyrogenic
rinse
solutionshould be heldin the fluid
the non-pyrogenic rinse solution
should be held in the fluid
room temperaturefor
one hourat(above18”
C);pathway
pathway for
one behourcombined.
at room If temperature(above18°
C);shoulda deviceistoundergoeffluents
effluents shouldbecombined.
Ifadeviceistoundergoa minimum extraction
time should be 15 minutes
atextraction,
extraction, a minimum extraction time (above
should 18”
be 15 minutes atC) or other37” c, one hour at room temperature
3]0 C, one hourat room
temperature (above 18° C) or otherdemonstrated
equivalentconditions.

demonstrated equivalent conditions.

Guidelines

Guidelines forfor rinse
rinse volumes
volumes include
include thethe following:
following:a.

a.

Each of the 10 test units should be rinsed with 40 mL of
Each of the 10water.
test un! ts should be rinsed wi th 40 mL of
non-pyrogenic

non-pyrogenic water.

- 12 -- 12 -B-43

ICCVAM Test Method Evaluation Report: Appendix B5b.
b.

May 2008

For unusually
unusuallysmall
or large
large
devices,the surface
surface
area
For
small
or
devices,theareaof thethe device
devicewhichcomes inin contact
contactwiththe patientpatient
of
which
comes
with
the
may
be usedused asas anan adjustment
adjustment
factorin selecting
selecting
the
may
be
factorintherinsingor extracting
extracting
volume.
rinsingorvolume.
The
limit
can
The endotoxin
endotoxin
limit
canbe adjusted
adjustedaccordingly.
accordingly.
be

The rinsing
rinsing
scheme should
shouldnot result
resultin aa greater
greaterdilutionofThe
schemenotindilutionofendotoxin
than used
used inin USPLJSP rabbit
rabbitpyrogentestingof transfusiontransfusion
endotoxin
thanpyrogentestingofand infusion
infusionassemblies.
For
inhibition/enhancement
testing,andassemblies.
For
inhibition/enhancement
testing,boththerinsing/extraction
anddevicesolution
the
both
the
rinsing/extraction
solutionandthedeviceeluate/extract
shouldtestedas prescribedprescribed
below
under
the
eluate/extract
shouldbe tested
as
below
under
the
specific
techniquebeing used.
used.
specific
techniquebeing
GEL-CLOT TECHNIQUETECHNIOUEa) GEL-CLOT
In inhibition/enhancement
inhibition/enhancement
testing,deviceeluateiextract
In
testing,aa deviceeluate/extractcontaining
varying
concentrationsof aa standard
standard
endotoxin
containing
varying
concentrationsofendotoxinthatbracketthesensitivityofthelysateiscomparedwiththat bracket the sensitivity of the lysate is compared withseriesofthe samesame endotoxin
endotoxin
concentrations
in waterwater
aa seriesoftheconcentrations
inalone.
The device
deviceeluate/extractis "spiked"“spiked”
withendotoxin
alone.
The
eluate/extractiswithendotoxin
and thenthen diluted
dilutedwithadditional
eluate/extract
to thethe same
same
and
with
additional
eluate/extract
toendotoxin
concentrations
as
in
the
water
series.
Results
ofendotoxin concentrations as in the water series. Results ofendotoxin
determinationin waterwater
and thethedeviceproduct
endotoxin
determinationinanddeviceproduct
eluate/extract
shouldfall
withinplus/minus
twofold
eluate/extract
shouldfall
withinplus/minus
aa twofold
thelabeledsensitivity.
devicedilutionofIfthedilution of the labeled
sensi
ti vi ty.
If
thedeviceeluate/extract
shows
inhibition,thegel-clottechniqueeluate/extract shows inhibition, the gel-clot techniquecannotbe used
used
to
test
the
device.
Negative
controls
cannotbeto testthedevice.
Negative
controls(diluentplus
lysate)shouldbeincludedinall(diluentplus
lysate)shouldbeincludedinallinhibition/enhancement
testing.
inhibition/enhancement
testing.
b)b)

CHROMOGENIC AND
AND ENDPOINT-TURBIDIMETRIC
ENDPOINT-TURBIDIMETRICTECHNIOUESCHROMOGENICTECHNIQUESIn
inhibition/enhancement
testingbytechniques,a’In
inhibition/enhancement testingby these
these techniques,adeviceeluateiextractcontaining
4
lambdaconcentrationofdevice eluate/extract containing 4 lambda concentration of
theor CSECSE (lambda(lambdais equal
equal
to thethelowestendotoxin
the RSERSE oristolowestendotoxin
concentration
usedtogeneratethestandard
curve)
istestedconcentration used to generate the standard curve) is tested
induplicateaccordingtothelysatemanufacturer’sinduplicateaccording tothelysate manufacturer's
methodology.
The standard
for
these
shall
methodology.
Thestandard curve
curve forthese techniques
techniques shallconsistof at leastfour
RSE or CSE concentrations
in waterconsist of at least four RSE or CSE concentrations in waterthatextendover the desired
If the desiredisthat extend
over
the desired range.
range. If
the desired range
range isgreater
than
one
log,
additional
standard
concentrations
greater than one log, additional standard concentrations
shouldbe
The standard
standard
curve
shouldbe included.
included.
The
curve must
must meet
meet thethecriteria
forlinearity
as outlinedin Appendix
A(2).
The
criteria for linearity as outlined in Appendix A(2). The
detected
amountof endotoxinin the spiked
eluate/extract
detected amount of endotoxin in the spiked eluate/extract
4 lambda
must
bewithin
plus
or
minus
25% ofthemust
be withinplUS or minus
25% of the to 4 neither
lambdaconcentrationforthe device
to be considered
concentration
forthedevicetobeconsideredtoneitherenhance
nor inhibit
the assay.
If the device
eluateiextract
enhance inhibition,
nor inhibit thethe assay.
If the device
eluate/extractshows
devicecannotbe testedby
thisshows inhibition, the device cannot be tested by thistechnique.

technique.

An alternate
procedure
may be performed
as describedabove
An alternate
procedurestandard
may be performed
describedin above
curve
is asprepared
LAL
exceptthe
RSE/CSE
except the device
RSE/CSE eluate/extract,
standard curve i.e.
is preparedin LALdetectablenonegative
negativedeviceeluate/extract,i.e.
no The detectable
standard
insteadof L.AL negative
water.
endotoxin,
endotoxin,
insteadof
LAL
negativewater.
curve
must meet the testforlinearity,
i.e.
r The
equalstandard
to or
curve mustmeet the andtest in foraddition
lineari ty,
e. r equalbetween
to or
the 1.
difference
greaterthan 0.980,
greater than 0.980, andin
additionthedifference
between- 13 - 13 -B-44

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

thethe O.D.
O.D. readings
readings forfor thethe lowest
lowest andand highest
highest endotoxin
endotoxin
concentrations
concentrations must
must bebe greater
greater than
than 0.4
0.4 andand less
less than
than 1.5
1.5
O.D.
O.D. units.
units. If If thethe standard
standard curve
curve does
does notnot meetmeet these
thesecriteria
criteria thethe device
device cannot
cannot bebe tested
tested byby thethe alternate
alternateprocedure.
procedure.

B-45

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

DEPARTMENT
OF HEALTHHEALTH &.
& HUMANHUMANSERVICES
DEPARTMENTOfSERVICES

15, 1991

PublicHealth
HealthServic.e
ServicePublicFood andand Drug
Drug Adminisltation
AdmInistration
Food

eMlYa

Interim
Guidancefor Human
Human andand Veterinary
Veterinary
Drug
Product6 andand &&$!~i!&$)857
Interim
Guidancefor
Drug
Products
i~~l1rP857
KINETIC LAL
LAL TECHNIQUESTECHNIQUESKINETIC
Until we
we update
update thethe guideline
guideline
the fo11owi.nq
following
guidance andand thethe lyeate
lyoate manufacturers
manufacturers
Until
the
guidance
approved
procedure8
ueed.
kinetic
technique0
should be
be donedone
approved procedures
can be used.
The kinetic
LAL technique.
shouldaccording toto thethe lysate
lyeate manufacturers
UIanUfaCtUrer8
recormaended procedures,procedures,i.e.,eample/Lyeate
accordin9
recommendedLe.,sample/lyBate
temperature
ratio,
incubationand times,
times,
measurement
wavelength,etc.
rati~,
incubation
temperatureandmeasurement
wavelength,etc.
Instrumentation
other than
than thethe oneone recommended
recommended byby ly••telymate manufacturer
manufacturer
cantnstrWllSnt&tion
other
can
.&a.
used.
The
performance
characterietic#(elope,andcorrelation
y-intercept
ueed.
The
performance
characteristics(slope,y-interceptAnd
correlation
coefficient),forthe lyaate
lylrate
lot,
eent
by
the manufacturer
manufacturer
will
not bebe valid.
valid.
coefficient),
for
the
lot,
sent
by
the
will
not
New porformance
performance
characterietfc
have
to be
be establishedeetabliehed
for eacheach lot
lot byby performing
performingNewcharacteristic
have
to
for
the
in Appendix
Appendix A.
A.
the procedures
procedures outlined
outlined in
JNNIBITION/ENHRNCEHNTTESTXNGXNHIBtTION/E~CEKENT TESTINC
Zn inhibition/enhancement
FnhFbition/enhancementtestingtesting ofof aa productproduct byby kinetic
kinetic
techniques,temt aa drug
drugIntechnique8,
teat
concentration
containing
quantityof the
RSE oror
CSE
between0.1
and
0. S EU/mL
EU/&
concentration containing
aa quantity
of
the RSE
CSE
between0.1
and0.5
or 1.0
1.0 and5.0 EU/mL depending on its Paee/
Fail
Cutoff(PFC) in duplicateorand 5.0 EU/mL depending on its Pa••' Fail Cutoff (PVC) in dupli.cate
accordingthe lysate
ly6atamdnufacturer'e
methodology.
The 44 lambda
lambda spike
epike
according toto themanufacturer'e
methodoloqy.
Thein thethe current
current
guideline,
procedure,ia still
Still
valid
and cancan bebe usedused inin thetheprocedure,inguideline,
is
valid
and
kinetic
procedure should
should be
cautionif lambda
ie les8leeskinetic techniguae.
techniques. This
Thill procedure
be usedused with
with caution
if
lambda isthan
0.01 EU/mL.
EU/mL.
than 0.01
The
Paee/Fsil
Catoffequal8 theendotoxin
limitof thethe productproduct
solution(KU/a)
The
Pass/Fiil
Cutoff equale
the endotoxin
lLmit ofsolution(EU/mL)timee
thepotencyofthep'oduct
dividedbytheproduct
dilutionueedfortimes the potency of the p~~duct divided by the product dilution used for the
the
teet.
For
PFCe lees
1.0 EU/mL
endotoxin
epike'ehould
be
test.
For
PFCs
les8 than
than oror equalequal toto 1.0EU/mL thethe endotoxin
spike'should be
between0.1
and0.5
EU/mL,
otherwise
theendotoxin
epikeshouldbe
between1.0
between 0.1 and 0.5 EU/mL, otherwi.e the endotoxin spike should be between 1.0
and
5.0 EU/mI..
EU/mL.
and 5.0The
curve shall
shall COnSiat
Of
at least
leaat three
USE or CSE concentrations.
The etandard
standard
curve
of at
three
~SE or CSE concentrations.
Additional
etdndards
shouldconsist
be included
tobracketeach
logincreaee inin thethe range
Additional
standards
shouldbeincludedtobracketeach
log increase
range
of
the
etandard
curve
60
that
there
is
at
leant
one
etdndardperlogincrementofthestandard
curve
80 that there is at least one standard per log increment
The standard curve
muet
meet
the criteria
outlinedin Appendix A.
oftherange.
of therange. The
standard
curve
mU8t meet
thereferenced
criteria outlinedin Appendix
The
calculatedmeanamountofendotoxinwhentothe standard
etandard
curve,A.
The
calculatedmeanamountofendotoxin
whenreferenced
tothecurve,
minue any meaeurable endogenoue endotoxinin the epiked drug product,muat
beminus any
the spiked drug
product,
must bewithinpluemeasurable
or minue endogenous
50% of the endotoxin
known
epikein concentration
to beconeideredtowithinor minus
50\ of thethe away.
known spike concentration to be considered toneither plus
enhanceor inhibitnomeaeurable endogenous
endogenoue
If there
there isis noneither enhance
inhibitIf
endotoxinin the or
product
the thevalueassay.
will usually
be equaL
tomeAsurable
or Leee than plue or
endotoxin
valuevalue.
will usuallyequal to or
les8productthan plusorminue
25% inof thethe productstandard thecurve
If thebe undiluted
drug
shows
minus
standard thecurvedrug
value.
the beundiluted
drug
inhibition25\ ofor theenhancement,
productIf candiluted,not productexceeding showstheinhibition
enhancement, the drug prOduct can be diluted, not exceeding theMM,and testor repeated.
MVD, and test repeated.
An alternate
procedure may be used,
in which the RSE/CSE standard ia preparedin
An alternate
be used,inetead
in which~SE/CSE standard is prepared in
drug
product procedureor product maydilutionof thewater.
The drug product(at thedrug product or
product
dilutionwater. cannot~he drug product (at theconcentration
used
to prepare
the instead
etandardof curve),
have an endotoxin
concentration
used to than
preparethe standard
curve) usedfcannothave antheendotoxin
concentration
greaterthe lowest
concentration
to generate
product
concentration
than the lowest
used togeneratewhen referenced
againet concentrationa etandard curve
preparedin thewater.productstandard curve, greaterThestandard standard
curve, whenagainstcurve preparedinwater.to The
product
curvereterenced
must meet
the testa standard
for linearity,i.e.,
r equalor
product thanst&ndard
curve must
meet
testandforelope
linearity,i.e., r equal
greatertheabeolute
Valueof the0.980,of the regreseionline tomuetorgreatervaluethanof 0.980,
andof the regression-0.1 absoluteand greater-1.0.
If slope
the etandard
curve doee line
not must
meetbeleee than
than thebelessthan ~O.l-1.0.be If
the standard
curve does
not meettheeecriteria,
theanddrug greater
productthan
cannotteetedbythe alternate
procedure.
these criteria, the drug product cannot be tested by the alternate procedure.
ROUTINE TESTING

ROUTINE TESTIt!C

The standard curveehall
consiet of at leant three RSE or CSE concentrationsin
The standardAdditional
curve shallconsist shouldof at least
three ~S£Or CSE concentrations
duplicate.
standards
be included
to bracket
each
log increasein
duplicate.
shouldincludedbracketcurve
so that be thereisat to leaet
one eachstandard log increase
per login the range Additionalof rhe standardstandards
in the range
scandard
ac least
one standard
The curve
standard so thatcurve there
must ismeet
thecriteria
outlined per log
znincrementof ot
the~herange.
increment A.of For
the therange.
standard curveche criteria
inkineticThe techniquea,
it muscie notmeet
nsc;cooary
LO run outlined
a ecandard
Appendix
Appendix A. For the kinetic techniquAB, i t is not neccao4.y La run a standard

B-46_,..---------~--------:--I

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

each dayday ififconsiotencyofcurves
is
shown
each
consistencyof standard
standard curves
isshown inyour test
in your
test
Determineconsistency
by regression
regreeeion
analysisthe data
data point0
laboratory.
laboratory.
Determineconsistency byanalysis ofof thepoint.s fromfront
the standard
standard curves
over three
three consecutive
test days
daye (minimum(minimum ofof threethecurves generated
generated overconsecutive teetthree
If thecoefficientofcorrelation,
r,r, meet6
curve61. If
curve.).
the coefficientof correlation,
meets thethe criteria
criteria in
in Appendix
Appendix
A thenthenconsistency
iscurve
becomee"archived
curve:
A
consietency
is provenproven andand thethe curve
becomes thethe "archived
curve.If
If rr
does notnot meetcriteria
then consistency
coneistency
in yourlaboratory
has not
been shOwn
shown
meet thethe eriteria
theninyour laboratory
not been
and you
you cannot
cannot use an archived
archived
curve
in routine
testing.
Thearchived
curve iscurve inroutine testing.
The archived
curve
is
only
lysate/endotoxin
lot combination.
combination.
If
you use an
an archived
archived
only valid
valid forfor aa lysate/endotoxinlot
I f you
at Least
duplicatesof
standard
curve , atstandard
endotoxin
concentration,
standard
curve
least duplicate.
of aa standard
endotoxin
concentration,
equal
mid-Pointonbasis, between
the endotoxin
concentrationof theequAL toto thet.he miCS-pointon aa log
log baBis,
between theendotoxin concentrationofthehighest andand Loweetin thethe standard
curve,
in
water
must
be included
includedhigh••tlowe.t standards
standards instandard curve,
in watermust beThe
thisetandard
with each
each runrunof sample..
sampleev.
withof
The meanmean endotoxin
endot.o~in concentration
concent.ration ofof this8tandard
control
must bewithinplus/minus
25ti ofof thethe standard
standard curve
curve concentration
concentration
when
control must
be withinplus/minus 25\
whencalculated
standard curve.
curve.
of using
Independentcalculated using
using thethe archived
archive4 Beandard
IndependentofuBing an endotoxin
endotoxin
at least
least
duplkatea
ofendotoxin
in eacheach productproduct orstandard
curve,
standard
curve, atduplicate.
of aa standard
BtandarCS endotoxin
inor
product
dFLutFon (positive(paeLtive
product
control),equal toeither
0.1 -- 0.5
0.5 or
1.0 -product dilution
product control),equal
to either
0.1
or 1.0
5.0
depending on
on itaits
PFC
or 44 lambda,
lambda,must
each run
5.0 EU/mL
EU/mL depending
PFC ormust bebe included
included with
with eachrun ofof
The meanendotoxin
concentrationofpositive
product control
control
when
eamplee.
sampLe..
Themean endotoxin
concentrationof thethe positive
product
whenreferenced
tostandard curvemust
be within
withinplus/
minue
50% ofthe known
referenced
to thethe standard
curve mustbeplus'
minus 50\of theknown
concentration
after
eubtractionofany endogenous
endogenoua
endotoxin.
An endotoxin
endotox inconcentration after
8ubtra.ctionof anyendotoxin.
Anetandard
eerie6shouldberun
when
reteetingtodetermineif
end-productstandard series should be run when reteBting to deter'll'line if end-productalternateIfyou
endotoxin
contami.natFon
exceed8 productendotoxin
contamination
exceeds
product 1iai.t.
limit.
If
you useuse thethe alternate
standard
curve
prepared
Fn productmust
be conductedconducted
withprocedure,
procedure, aa standard
curve preparedin
product mustbewith each
each
product
product tea;.
teu.;.

curve
curve

APPENDIXAPPENDIX A
A
Usingor CSECSE ofknown
pOtenCy,in endotoxin
unite,
aeeay
at
least
3
Using
aa RSERSE orof known
potency, inendotoxin unJ.ts,
assay
at
least
3
that
extentiover thedeairedendotoxin
range.
concentrations
in triplicate
concentrations
intriplicate that
extenrl overthe desired
endotoxin range.
Additional
standards
ehouldbe included
to bracket
log increase
increase ~nin therange
Additional
standards
should be
included tobracket each
each log
the rangeof thestandard curve
curve eo
that
their
ie at
at lea8t
leaet
one standardper
Log incrementofthe standard
so
that
their
is
one
standardperlogincrement
Do regression
Correlationoftheanalyeieon theReactionTimeofthe range.
range.
Do regression -- correlation
analysisonthe Loglog ReactionTimeversus
the
Log
of
the
endotoxin
concentrationfor
each
replicate.
DO
NOT
AVEWKX
the log of the endotoxin concentra.tion for each replieate. DO NOT AVERAGE
verSUBTHE REACTION TIXBS OF REPLICATES OF EACH STANDARD BEFORE PERFORMING RECRESSIONTHE
REACTION ANALYSIS.
TIMES OF REPLICATES OF EACH STANDARD BEFORE PERFORMING REGRESSIONCORRELATIONCORRELATION ANALYSIS.
The coefficientof correlation,r, shallshall bebe greaterthan oror equalequal toto theabsoluteThecoefficientr,greaterthanthe absoluter is Leoe than
the absolute
valueof 0.980
the causeof thevalue
of 0.980.
0.980. ofIfIfcorrelation,
value
of
r
is
le.s
than
the
absolute
value
of
0.980
the
cause
of thenon-Linearity
shouldbe determined
and test
non-linearity shouldbedetermined andtest repeated.
repeated.

B-47

ICCVAM Test Method Evaluation Report: Appendix B5

VI.
VI.

May 2008

APPENDICESAPPENDICES- 16 - 16

B-48

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

APPENDIXAPPENDIX BBBACTERIALBACTERIAL ENDOTOXINSENDOTOXINS TESTTESTUnitedUnited States
States Pharmacopeia
Pharmacopeia XXI/National
XXI/National Formulary
Formulary XVI
XVI
and
and
First
First Supplement
Supplement toto USPUSP XXJ/NF
XX~/NF XVI
XVI- 18 -- 18 -B-49

I

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008(85) BACTERIALBACTERIAL(85)
ENDOTOXINSENDOTOXINS TEST
TEST
Tht,
3 lestesttmattng
thewnccntrJtwn
01
Thl~ chapter
dapt~r provtdes
provide~ atc,t forfor cstlmatln~
the cunCCntrall<lnI)fbactcrtal
cndotoxmspresent
tn
or 011on thethe \arnple
bactcrlalcndolO~ins thatthat may
may bebe present
In';r,ample ofvI' thethcarticle(,)
ied
ustng
Ltmulus
articleb) IO
to whichwhich thethe test
tcst I$is appl
applieduSing limulu,..\mebwvtc\mebocytc
Lysa~e
(LAL)
which
ha>
obtainedfromwhichha.' been
been obtained
from ~qucous
J<jUl;OUS extractsc.~tra<·IS 01
oflysate: (lAl)the circulating
amcbocytcsof the horscshoc
cr.rb. L/ntultr.r
P~/I.the cIrculating ::lmdxxytes vf the horseshoe crab. Llmu/IIJ ('0/'"phcmus.
phemus. and;lnd which
which has
has been
been preparedprepared andand charactcrtrcd
eharactertled forfor useusc
3s 3 LAL reagent
for gel-clot
formatton.
as a lAL reagent forgel-clot formation.
Where
the ICSI IS conductedlimtt ICSI. the >pectmen
dcWherethe lest IS conducted as
as aa limll
lest. the ,peclmen I>
I, ,1(tcrmmcd
IO bc posittve
or negative
IO the WI Judged
aga~nb~ theterminedtobepositiveornegativeto«het~SI Judged against thc
cndotoxin
concentration
specifiedin the tndivtdunl
endotoxin
concentration
specilicd
in the indiVidual monograph.
monugraph.
Where
the lest is conducted
as an assay of the conccntratton
of
the
test
is
conducted
asanassay ofthe conc:entratlonvf
Where
endotoxin.
with calculationof confidence
limttsof the result
abendotOXin.
with
calculationof
confidence
limitsvf
theresultobtaincd.
the specimen
is judged
to comply
with the requirements
iftained. the specimen is judged
to comply with thelimttrequirements
if
the result does not exceed(a) the concentration
specifiedintheresultdocs
notexceed(a)theconcentration
limit
specifiedin
the individual
monograph.
and (b) the specified
confidence
limttsthe theindividual
and the(b) determination
the specilied conlidence
limitsforassay. monograph.
In eithercaseof the reaction
for the assay.
eitherdilutions
case thefrom
determination
the reaction
end-point
is made In withthe material of underWI
in
is made with
with parallel
dilutionsfrom thetest inend-poim
directcomparison
dilutionsof amaterialreference undercndotoxin.
directcomparison
withparalleldilutionsofareference
endotoxin.
and quantities
of cndotoxin
are expressedin deftned
Endotoxin
andUnits. quantities of endotoxin are expressed in defined Endotoxin
Units.
Since
LAL
reagents
have also been formulated
to be used forSince LAL (including
reagents havebeen formulated
used forturbidimetrickinetic alsoassays)
or colorimctric to bereadings.
turbidimetric (including kinetic assays) or colorimetric readings.
such tests may be used if shownto comply
with the rquiremcnts~uchtests may methods.
be used if shownto comply
requirements
for alternative
Thcsc tests
require withthe theestablishmentofforalternative
methods.
These
tests
requirethecontent
establishment
a standard
regression
curve and the rndotoxinof the testof
amaterialstandard
regression
curve
and
the
endotoxin
content
lJf
Ihe
lest
is determinedby interpolation
frum the curve.
The promaterialinclisudedetermined
The procedures
incubation by inlerpolation
for a pre-selectedfrom thetime curve.
of reacting
cncedures
includeincubalionforapre-selected
limeofreacting
endotoxin
and control
solutionswith LAL
Rsngcnt
and retdingof
thedotoxinandcontrolsolutionswithLA
L
Reagent
andreadingofthespcctrophotometriclight absorbance
31 suitablewavclcngths.
In
spectropholometriclight absorbanceat thesuit:lble
thecase of the turbidimetric
procedure
reading wavelengths.
is made imme- In
the caseatofthetheendturbidimetricmade assays.
immediatelyof the incubationprocedure
period. the orreadingin the iskinetic
diatelyat the endof the incubation
or in theperi
kinetic
assays.
the absorbanceis measured
throughout period.
the reaction
od
and
rate
the absorbanceis measured
the reaction
and rate
valuesare determinedfrom throughQut
those readings.
In theperiodcolorimc:ric
the colorimc:ricvalues are thedetermined
procedurereaction
isfromarrestedthoseat readings.
the end of theIn prc-selectedtimeby
the additionof an appropriateacid solutton.time
the reactionis arrested atamountthe end ofof acetic
lhe pre·selectedpnx"'dureprior
the readings.
A possible
advantage
the mathematicalby theto additionof an appropriate
amount of inacetic
acid solutIon.
treatment
results.
if thetest bc otherwisevalidated
and the assay
A possible
advantage in the mathematicalprior to theof readings.
suitably
couldif thebe testthe beapplication
tests of assay
validtty
otherwiseofvalidated
and theassay
treatmentdesigned.
of results.
and the calculation
ot’the beconlthe
idenceapplicationinterval of andof potency
suitably
designed. could
tests limitsof assayvalidity
from thethecalculationinternal
evio(
dence
of each assay
andand
the confidence
intervalitselfand (seelimitsDesignof potency
Ana/.vsis.-Ways of each
( I I I )assay).
from the o/Biologico/internal evidence
itself (see Dtsign andAna(vsis of Biologiral Assays ( III »).

Reference

Standard

and Control

Standard

Reference Standard
and Control Standard
Endotoxins
Endotoxins
The reference
standard
ertdotoxin(RSE)
is the USP Endotoxin
Reference
Standard standard
which endotoxin
has a definedof 10.000
USP
The reference(RSE)potency
is the USP
Endotoxin
Endotoxin
Units (EU) which
per vial.
the entireof 10.000
contents USP
ofReferen~e Sta.ndard
has aConstitutedefined potency
I vial of the Umts
RSE (EU)
with 5permL vial.
of LALConstitute
Reagent theWater.’
vortexforof
EndotolllO
entire ncontents
notlessof than
20 minutes.,t ofand lAlUSC Reagent
this concentrate
making for
I vialthe RSE
with 5 mL
Water. I for·vortellappropriate
dilutions.
Preserve
the concentrate
in a refrignot less .thanserial
2~ mi~ut~s'.1
and
use thisconcentrate formakingcrater.
for making
subscqucnt
dilutions,for concentrate
not more thanin a14refrig·
days.
Preserve
theappropriate
senal dIlUtions.
.Allow
it
to
reach
room
temperature.
ifaplicable,andvortexiterator. for making subsequent dilutions. for not more than) 4 days.
vigorously
not less than S minutes
Farc USC. Vortex
each·.Allow it tofor reachroom temperature. ifbcapplicable.
and vortex
itdilutionfor not less than I minute
before proceeding
IO make thevIgorously
for not less than 5 minutes
before use. Vortex
eachnexl dilution.
,t
Do not use stored
dilutions.
A control
standard
dilution for(CSE)
not less thanJ minute before proceeding to make thecndotoxin
endotoxin
preparation
than the RSE
next dilution'.1 isDoannot
useagainst
storeddilutions. otherA control
standard
that has been standardized:hc RSE.
If a CSE is a prcpais
anendotoxin
preparationotherthan theshoulRSE
endotoxin(CSE)
rationnot alreadyadequately
characterized.
its evaluationdtha.t hascharacterizing
been standardized
against both
the RSE.
If a CSEquality
is a prepainclude
parameters
for cndotoxin
andratiOnnotalreadyadequately
characterized.
its
evaluation
shouldperformance(such as reactionin the rabbit),and for suitabilityoflI1c1udecharacterizing
parameters
bothforendotoxin
qualityandthe material
IO serve as a reference(such as uniformity
and stabilperforma~ce(suchasreactionintherabbit).
and
for
suita
bililyofity).
.Detaiied
proccdura
for its weighing
and/orconstitution!o serve
as a reference(such as uniformity
and stabilthe matenaland
USC to assure
consistency
in performance
shouldalso
be inits weighing
constitution
Ity). ·Detalled
proceduresof forcluded.,,Standardizationa CSEagainsttheand/or
RSE usinga LAL
consistency
andReagentuse tofor assure
the gel-clot
procedure in performance
may bc effectedshouldby alsoassayingbe in-a
cluded.• , ofStandardization
againslthe RSE
using awhereLAL
minimum
4 vials of thcCSE of aorCSE
4 corresponding
aliquots.
R7a~ent forof the.
procedure
effectedby assaying
appltcablc.
the gel-clotbulk CSEandI vial mayof thebe RSE.
as directedunder amlOlmumof 4 VIals
the CSEor 4 corresponding
Tesr Proccdurc.
but ofusing
4 replicate
reactionlubes aliquolS.
a~ each wherelevel

applicable. of the bulk CSE and I vial of the RSE. as directed under
Tut Procedurt. but using 4 replicate reaction tubes at each level’ LAL
Reagent
Water-Stcrilc
Water
for Injection
or otherwaterthat shows no reaction
with the specific
LAL
Rcagcnt
with
Injectionor olher
which, LAL
it is Reagent
IO be used.Water-Sterile
31 the limit of Water
sensitivity for ofsuch reagent.

watcr that shows no reaction with the specilic LAL Reagent with
whIch It IS be used, at the limit of sensitivity of such reagent.

,aulvf theRSE 3rdrcactwnrubcs
thc dllu~lon
dllullon scrws,crlcs for(Qr thelhe RSEand .t4 rcpllccltc
replicate reaCllon
tubes~tmtlarlyof thedtluttons)imllarly forfor wcheach veal
VIal orvr aliquot
aliquot ofof thethe CSE.
CSE IfIf 411
all ofthe dilutions
for thethe 4
4 vealsor aliquots
aliquotsofthe CSECSE cannot
cannotbc(orVIals orof thebe .rscommcdatcd
accommodated wth
withthedilutionsforthe II vtalof thesame rack
the dilutions
for theVial ofthe RSERSE onon rhcthe samerack (orfor tncuIncubation.
bc usedused forfor accommodatingof
bation. additional
additional racks
racks may
may beaccommodating comesome oftheof thethe racks
the replicate
replicate dilutions
dilutions forfor thethe CSE.
eSE. but
but allall of
racks contatntng
containing
theof the
RSE andthe CSEarc mcubntcd
the dilutions
dilutions of
the RSEand theCSE arelI1cubaled as
as aa block.
blod.
However.
mcases, thedilutton
scrtc) fromfrom thevtal
However.
In suchsueh cases.
the replicate
replicate dilution
seriesthe II Vialof thetogetheronrack andofthe RSERSE arc
are accommodated
accommodated together
on aa single
SIngle rackand thetherc liwtc dilution
dilution
scrtesfrom any
one ofof thevials or
replicateseries fromanyonethe 44 vials
or aliquots
aliquots ofof thethe
C!iE are not divided
betweenracks.
lThe
antilogor.hcdifference
CSE are not diVided between racks. .The antilog of .he differencebctwccnthemeanlogtoend-point
of
the
RSE
and
the
mean
logto
between themean 10~lo end-point of thepotency
RSE andthe mean loglo
end-pointof the CSE is the standardizedof the CSE which
end-polOtof the CSE IS the standardized potencyof the CSE whichthen 1s to be.1 convcrtcd
IO and expressedin Units
then IS !O be. 1 converted to and expressed in Units per
per ngng underunderstateddryingconditions
for the CSE. or in Units
per container.
dryi.ng conditions
forthe CSE. or in Units percontainer.
statedwhicheveris appropriate.
Standardize
each new lot of CSE priorwhichever IS appropriate.
Standardize
each new lot of CSE prior
IO USC in the test.
Calibrationof a CSE in terms of the RSE mustto
usc in Ihe test. Calibrationof a CSEandin terms
ofprocedure
the RSE mustbc with the specific
lot of LAL
Reagent
withbe withit the
specific lot ofSubsequent
LAL Reagenl
and thethe MI
test
procedure
with
which
is to bc used.
lots of LAL
Reagent
from the
which
it
is
to
be
used.
SUbsequent
lots
of
LAL
Reagent
fromIhesame source and with similar
characteristics
need only chcokingofsame
and with.Thesimilar
characteristics
need only checking
of
thc$tencysource ratto.
inclusionof one or more dilution
seriesthepotency
ratJo.
·The
inclusion
of
one
or
more
dilutionseriesma c from the RSE when the CSE is used for testing
will enable
made fr~m the
RSE whenis used potency
for testing
will enable
observationof whether
or thenot CSEthe relativeshownby theobservatIOn
whether
not the relative
thelatterremains ofwithin
the ordctcrmincd
cortlidcncc potency
limits.,,shown Abylarge
confidence
limits.•,A
large
latterremains
withinthedetennined
lot of a CSE may, however,be characterized
by a collaborativeassay
Jot aofa
CSE may.
by relative
a collaborativeofsuitable
design however.
to provide be characterizeda rcprcscntative
potency assay
andofsuitable design to provide
a representative relative
the a within-laboratory
and between-laboratory
variance. potency
l .t andtheA within-laboratory
between-laboratory
suitable
CSE has aandpotencyof not loss than variance.
2 Endotoxin • ••Units
of nOt lessEndotoxin
per Angsuitable
and not CSE
more hasthana potency
50 Endotoxin
Units thanper 2ng.
where in Unitsbulk
per ngunderand not
more thanUnits pere.g..ng.towherein bulk
form,
adopteduniform SO Endotoxin
dryingconditions,a particularform.moistureunder adopted
conditions.
particularlowcontent uniform
and otherdrying
spccificd
conditionse.g.• toof a USC,
and a
low
and other range
specifiedpotencymoisture
within content
a corresponding
whereconditions
filledin ofvials use.
of anda ho- apotency within
mogeneouslot. a corresponding range where filled in vials of a ho-mogeneous 101.

Preparatory

Testing

Preparatory Testing

Use a LAL
reagentof confirmed
label or determinedsensiltvity.
reagent
label orin determined
In Useaddition,a LAl
where
there of ~nfinned
is IO tx a change
lot of CSE. sensitivity.
LAL RcIn addition.
there isconduct
to be a change
agent
or anotherwherereagent.
1~1s of ina 101
prior ofCSE.
satisfactoryLAL Refat
of
CSE,or another
LAL
and/orotherconduct
reagent testsin ofparallelchangeover. lot
agent
reagent.
a prior onsatisfactory
Treat
any LAL
containersand/oror Other
utensils reagent
employedin parallel
so as toon
destroy
extraof CSE.
changeover.
neous
cndotoxins or utensils
that mayemployed
be present.so as
suchto destroy
as by heatrng
any containers
extraTreat surface
ineousn an ovenat 250’or above thatfor may
sufficientlimc.z such as by heating
surfaceendotoxins
be present.
2
validityat 2,50
of 0 test
results forfor sufficient
bac:erial
endotoxins
requiresanin Thean ovenor abovetime.
adequatedemonstrationthat spbcimenrof theendotoxins
article.
or ofrequires
solulions. an
The validityof test results
for bacterial
washings,
orextracts
thcruof
IO
which
the
test
is
to
bc
appl
i
e
d
do
no1
adequate demonstration that specimens of the article. or of solutions.
ofwashings.
themselves or extracts
inhibitorenhance to which
the reaction
otherwiseinterfere
theroofthe test oris tobe applied
do notwiththemselves
the test. inhibit
Validationor enhanceis accomplishedby otherwise
testing
untreatedofthe reaction orinterfere
specimens
or appropriate
dilutions
thereof.
concomitantlywith andwith the test.
Validationis accomplishedby testing untreated
withoutknown
and demonstrable
amounts
of RSE orwitha CSE.
specimens
or appropriate
dilutionsaddcd
theroof.
concomitantly
andandcomparing
the results
obtained. added
Appropriate
without
known anddemonstrable
amounts ofnegative
RSE orcontrols
a CSE.
aremust be repeated
if the negative
LAL
Reagent
and included.
comparing Validation
the results obtained.
Appropriate
controls
sourceor the methodof manufacture
or formulationof theReagent
articlc
are included.
Validationmust be repeated
if the LAL
is changed.or the method of manufacture or formulation of the article
sourceTest jar conjirmariono/ labeled
LAL Rcogcn:
sensiriuiryis changed.
Vonfirm.,
the labeledsensitivityof LALthe particularreagcnf
Ttst for confirmationof labtltd
Rtagtnt LAL
stnsitivitywith
the RSE the(or labeledCSE) usisensitivity
nn
not lessof than
4 replicate
vials. reagent
under·Confirm.lthe particularLAlconditions
shown(or CSE)
to achieve-anof the under
test.
with the RSE
using notacceptableless than 4vatiability
replicate vials.
viz.. the antilogshownof thegcotnctrio anmean
logtu lysatc
gel-clotsensitivity
to achieve
acceptablevariability
the test.
conditions
is within
0.5X to of2.0X
where X is mean
the lab&d
sensitivity
in Endotoxin
viz..
the antilog
the geomelric
IogIO lysate gel-<:Iot sensitivity
Thewhere
RSE A(orGE)labeledconcentrations
inUnits
per 0.5>'
mL:,t to 2.0A,
is withinis thesensitivity in sclcctcd
Endotoxin

orUnits per mL.-.
tency concentrations
shouldbracket selected
the slated inconfirming
rbc LAL 1 The
reagent RSElabel(or CSE)
onftrmthe labeledsensitivityofsensitivity
LAL reagent
reagent. labelc?potency
confirmingofthetheLAL
should bracket the statedeach new lot of LAL
to use in the lest.
sensitivity of the LA Lreagent
reagent.prior Confirm
the labeled sensitivity oflnhibirion
or Enhanccmenr
Test-Conduct
assays with standardeach
new lotof LAL reagentprior
to use inthere
the
test.
endotoxin.
of untreatedspecimens
in which
is no endogencous
Inhibition
orEnhanrtmt'nt
Ttst-Conduct
assayswith standard
cndotoxin
dctcctablc.
and of the same specimens
to which cndotoxin
endotoxin.
oruntreated
specimens
in
which
there
is
nobutendogeneous
has been added.
as directedunder
Test k’roccdure,using notendoloxin
detectable.reaction
and of thesame specimens to which endotoxin
less than 4 replicate
tubes at each level of the dilution
serieshas
beenadded.
asdirected
under
Ttst
Proctdurt.
butusing notfor each untreatedspecimenand for each specimen
IO whichenlessthan
4
replicate
reaction
tubes
at
each
level
of
the
dilutionseriesdotoxtn
has been added.
Recordthe end-points(E. in Unils
per
ror
each
untreated
specimen
and
for
each
specimentowhichenmL) observedin the replicates.
Take the logarithms(c) of the
dotoxinhas
been
added.
Recordtheend-points
(E.
in
Units
cnd.points.
and compute
Ihc geometricmeans of the log end-pomts per
mLltheobserved
the replicates.
Take thelogarithms
for
RSE (or inCSE).
for the untrcatcd
specimensand (t)for ofspcci-theof thex:e//.
log end-points
end·mcns points,
containingand computecndotoxin thebygeomctric
the formulameans
antilog
in whtchforthe untreated
and forCr theIS theRSE
sum (orof CSEl.
the lee for
end-pointsof the specimens
dilution
scrtcsused speciandmens containing endotoxin by the rormula antilog kt If. in whichtc~t forvaliditylog end-points
of proccdurc offortheinactivationof endotoxins.
~<' r ISForthea sumof thedilution senes
used andsee “Dry-heat
Sterilization’ under S/~ri/irorionandS/crili~,~Asfor va.lidity.ofr,rocedureof endotoxins.
surancc or aoflest
Compendi
a1
Arricies(I 21forI ).inactivation
USC a LAL
Reagent
having Dry-heata sensitivitySterilizationof not less underthan Stuilizotion
0.15 Endotoxin andUnit
per mL.
sec
Stui/itv
Assuranrt of CO'!'f'tndial Artidts (1211). Use a LA L Reagent
ha vms a sensItIvIty of not less than 0.15 Endotoxin Unit per In L.

2.:

B-50

ICCVAM Test Method Evaluation Report: Appendix B5

f is thethe number
numberofofreplicate
replicateend-points
end· points inin each
each case.
case. Compute
Compute thethe/isamount ofofcndo1oxm
endotoxin inin 1hc
the specimen
specimen toto which
whichcndotoninendotoxin has
has beenbeen
amountadded. The
Thelesttest isisvalid
valid forfor thethearticle
article ififthis
this result
result isis within
within twofold
twofold
added.
the known
knownadded
added amountamount ofofcndotoxin.
endotoxin. Allcrnativcly,
Alternatively,ififthethe test
test
ofofthehas
has been
been appropriately
appropriately se1
set up,up. thethe test
test isis valid
valid forfor thethe articlc
article ifif thethegeometric mean
mean end-pointend· point dilution
dilution forfor thethe specimen
specimen CO
to whichwhich cnenncomcIric
dotoxin has
has been
been addid
added isis within
within one
one Z-fold.dilution
2·fold dilution ofofthe
the corrccarre·
dotoxinsponding ncomctricgeometric mean
mean end-point
end-point dilution
dilution ofof thethe standard
standard cnensoonding&toxinovtoxin. the result
result obtained
obtained forfor thethe specimens
specimens IOto which
which cndotoxin
endotoxin has
has
IfIfthebeen
been added
added isis oulsidc
outside thethe specified
specified limit.
limit. thethe article
article isis unsuitableunsuitable
for
oxi&
Tesr.
for 1hethe Bacterial
Dacluia! &ndol
Endoroxins
Tesr. or,or. inin thethe casecase ofof Injections
Injections oror
solutionssolutions forfor parcntcral
parenteral adminis1ration.
administration. iIit may
may bcbe rendered
rendered suitable
suitablebyby diluting
diluting specimens
specimens approprialcly.
appropriately.
RcncaI
Repeat thethe test
test forfor inhibition
inhibition oror cnhanccmcm
enhancement using
using spccimcns
specimens
diluteddiluted byby aa factor
factor not
not cxazding
exceeding thatthat given
given byby thelhe for&la’x/Xformula X/A (sue
(see
Use
the
least
dilution
Maximum
id
Dilurion.
Maximum Val
Valid
Dilution, below).
below). Use the least dilution suflicicnt
sufficientCO
to ovcrcomc
Ovc;rcome thethe inhibition
inhibition oror cnhanccmcnt
enhancement ofof thethe known
known added
added
cndotoxin,
endotoxin. forfor subsequent
subsequent assays
assays ofof cndotoxin
endotoxin inin test
test specimens.
specimens.
IfIf cndogcncous
endogeneous cndotoxin
endotoxin isis detectabledetectable inin thethe untreateduntreated specimens
specimens
under
under thethe conditions
conditions ofof thethe test,
test. thethe article
article isis unsuitableunsuitable for{or thethefnhibirion
Inhibition oror Enhancemenr
Enhancement Tesr,
Test. or,or, itit may
may bebe rendered
rendered suitable
suitablebyby removing
removing thethe cndotoxin
endotoxin present
present by
by uhra-filtration,
ultra-filtration, oror byby ap
appropriate
propriate dilution.
dilution. Dilute
Dilute thethe untreateduntreated specimen
specimen (as(as constituted.
constituted.
where
where applicable,
applicable, forfor administration
administration oror us-c).
use). toto aa level
level notnot cxaedingexceedingthethe maximum
maximum valid
valid dilution,
dilution. atat which
which nono endotoxin
endotoxin isis detectable.
detectable.
. ml Repeat the test for lnhibirion
Repeat the test for Inhibition oror Enhancemenf
Enhancement using
using thethespecimens
specimens atat those
those dilutions.
dilutions.

·.1

Test
Test Procedure
ProcedureinIn preparing
preparing forfor andand applying
applying thethe test.
test. obscrvc
observe precautions
prC1:autions inin
handhng
handling thethe specimens
specimens inin order
order IO
to avoid
avoid gross
gross microbial
microbial contamicontami·nalion.
Washings
nation.
Washings oror rinsings
rinsings ofof dcviccs
devices must
must bebe withwith LALLAL Reagent
Reagent
Water
involumes
appropriate
IO
their
use
and,
where
applicable,
Water in volumes appropriate to their use and. where applicable,ofthesurface
area
which
comes
into
contactwithbodytissuesof the surface area which comes into contact with body tissues oror
fluids.
fluid hasfluids. Use
Use suchsuch washings
washings oror rinsings
rinsings ifif thethe extrading
extracting fluid
has been
been
inin contact
oontact with
with thethe relevant
relevant pathway
pathway oror surface
surface forfor not
not less
less than
than II
hour(IS” IO
m30°m1).
Suchhour atat controlledcontrolled room
room temperaturetemperature (15°to -30°.d.
Such cxextracts
combined,
whcrc
appropriaIc.
The uhimate
tracts may
may bebe combined.
where appropriate.
The
ultimate rinse
rinseorwash volume
as IO
result 10
In possibleof anyor washvolume isis such
such asto resultpossible dilution
dilution of
any conoontainedlevel notless than
than thatthat suitable
suitable forin thetained endotoxin
endotoxin IO
to aa level
not less
for useuse inthe
Pyrogcn
Tess
(
IS
I
)
under
Transjusion
andInfusion
Assemblies
Pyrogen Tesr (151) under Transfusfon and Infusion Assemblies(161).
(161 ).
For validating
the ICSI for an article. for endoIoxin
For
validating thetest for an article. for endotoxin limit
limit testsor
tests orassays,or forfor special
special purposes
purposes where
where soso specified.
spccilicd. testing
Iuting ofspeciassays.
orof specimens is conducted
quantitatively
to determine
response end-points
mens is oonducted quantitatively to determine response end-points
forreadings.
Usually gradedgraded strengths
strengths of
specimen andandfor gel-clot
ge1-<:lot readings.
Usuallyof thethe specimen
standard
cndotoxin
are made by multifold
dilutions.
?Sclcct
standard
endotoxin
are
made
by
multifolddilutions.
·Select
dilutions.1
so Iha they correspondto a geometricsorics in which
dilutions_,
sothat they correspond toa geometric series
in whicheach step is greater than the next lower by a cons(ant ratio...
DO
each
step
is
greater
than
the
next
lower
by
a
constant
ratio. 1 Do
not store diluted cndotoxin.
because of lass of activity by adsorption.
not
store
dilutedendotoxin.
becauseofloss of activity by adsorption.
In the abscncc of suowrtina.
data to the contrary,negativeandIn the absence of supporting data
to the contrary,and. negative
positive con1rols arc fn&po;atcd
in 1hc test.
positive
comrols are incorporated in
the test.
USC not less than 2 replicate reaction tubes at each lcvcl of theUsenotless than 2 replicate reaction tubes at each level
the oftestth.eisdilutionseries for each specimen under rest. Whether
dilutionseriesfor each specimen under test. Whether the test IS
employed
as a limit test or as a quantitative
assay, a standard
enemployed
as a limit
test or as a quantitative
assay, a standard endotoxin
dilutionseries involving
not less than 2 replicate reaction
dotoxindilutionseriesinvolvingi notless than 2 replicate reaction
tubes is conducted in parallel.
set of standard enbotoxin
dilutiontubes isisconductedin parallel.
of standard
dilutionseriesincludedforeach blockA setof tubes.
which endotoxin
may consistof aisincluded
for
each
block
of
tubes.
whichmayconsistofaseriesnumberof racks for incubation
together.
..I provided
the envinumber
of
racks
for
incubation
together.
providedtheenvi·ronmcntal
conditions
within blocks arc uniform.
ronmental
conditions withinblocksandare uniform.
Preparafion-Since
the form
amountper conlainer
of

-.1

-.1

Preparation-Since the form and amount per container of.

May 2008·.1

standard
standard endotoxln
endotoxin andand of<.If LAL
LAL rcagcnt
reagent may
may vary.
vary. consritution
constitution
and/orshould be
be as
as dirtial
directed rnIn thethe labeling.*,,labeling.
and/or dilution
dilution ofofcuntcnts
contents should
The
The pH
pH ofof thethe test
test mlxturc
mIxture ofof thethe specimen
specimen andand thethe LALLAL RcagcnI
Reagent
isis 1nin Ihethe range
range 6.0
6.0 toto 7.5
7.5 unlessunless spccilicallyspecifically dircctcd
directed othcrwisc
otherwiseinin Ihc
theindividual
individual monograph.
monograph. The
The plipH may
may bcbe adjusted
adjusted byby 1hc
the addition
addition
ofof stcrilc,
sterile, cndotoxin-freeendotoJlin-free sodium
sodium hydroxidehydroxide oror hydrochloric
hydrochloric acidacid oror
suitablesuitable buffers
buffers toto thethe specimen
specimen priorprior IOto testing.
testing.
Maximum
Maximum Vu/id
Valid Dilution
Dilution lMYD)-The(MVD)- The Maximum
Muimum Valid
Valid
Dilution
Dilution isis appropriaIc
appropriate toto Injections
Injections or.Io
or to solutions
solutions forfor parcnteralparenteraladministration
arlministration In10 thethe form
form cnnstitutcd
constituted orQ( diluted
diluted forfor administration.
administration,oror whcrc
where applicable,
applicable. toto thethe amountamount ofofdrug
drug byby weightweight ifif thethe volume
volumeofof 1hC
the dosage
dosage form
form forfor administration
administration could
could bebe varied.
varied. .Whcrc·Where thethe
cndoIoxin
endotoxin limit
limit concentra1ion
concentration isis specified
specified inin thethe individual
individual mono
monographgraph inin terms
terms ofof volume
volume (in(in EU
EU perper mL).
mL), divide
divide thethe limit
limit byby A.A.
which
which isis thethe labeledlabeled sensitivity
sensitivity (in(in EU
EU perper mL)mL) ofof thethe lysatc
lysate cmemWhere
ploycdployed inin thethe assay,
assay, toto obtainobtain thethe MVD
MVD factor.
factor. Where thethe endoendotoxin
toxin limit
limit conccntracion
concentration isis specified
specified inin thethe individual
individual monographmonographinin 1crms
terms ofof weightweight oror ofof Units
Units ofof active
active drug
drug (in(in EU
EU perper mgmg oror inin EUEU
perper UniI),
Unit), multiply
multiply thethe limit
limit byby thethe concentration
concentration (in(in mgmg perper mLmL
oror inin Units
Units perper mL)mL) ofof thethe drug
drug inin thethe solution
solution tested
tested oror ofof thethe drugdrug
constituted
constituted according
according toto thethe labellabel instructions.
instructions, whichever
whichever isis appliapplicable.
X toto obtainthe multiplication
multiplication byby,\,obtaincable. andand divide
divide thethe productproduct ofof thethethe MVD
MVD factor.
factor. The
The MVD
MVD factor
factor so
so obtained.1
obtained. I isis thethe limi1
limit
dilution
dilution factor
factor forfor thethe preparation
preparation forfor thethe test
test toto be
be valid.
valid.
Procedure-To~01s ofof theProcedure- To IO10· XX 75.mm
75-mm tut.tubcs
test.tubes add
add alialiquots
the apapt led volumes
propriately
propriately constituted
constitu~ed LAL
LAL reagent,
reagent, and~nd thespecI
the specified
volum~~ ofofspecimens.
and aa positive
specimens, cndotoxin
endotOXin standard,
standard. negativeneg~ttve controls,
controls. flnd
posl.tlve
product
product control
control consisting
consisting ofof thethe article.
article. oror ofof solutions,solutions, washings
washings
oror cxlracts
extracts thereof
thereof toto which
which thethe RSERSE (or(or aa standardized
standardized CSE)
CSE) has
has
been
added
at
a
concentration
ofcnddtoxin
of
2X
for
tha1
been added at a concentration of endotoxin of 2A for that LAL
LAL rcreaxcnt
(see
under
Test
for
confirmation
of
l
o
bel
e
d
LAL
Reagent
agent (see under. Test for conflrma~i()" of labele,d LA.L Reag~nt
s&.&ity).
Swirlc&h
gcntjy
to
mix,
and
place
in
an
incubating
sensitivity). SWirl each gently to mix. and place In an Incubat!ng
deviceheating block.
recordingas aa water
water bath
bath oror heating
block. accurately
accuratelyeachrecordingdevice such
such asthearc sothe timetime at
at whichwhich thethe tubes
tubes areso placed.
placed. Incubate
Incubate each tube.
tube.
undisturbed,
I ‘, andremovefor 6060 f:::I: 22 minuIcs
minutes atat j737 f:::I: 1°,
and carefully
carefully remove
undisturbed, foritit foris characterized
the forfor observation.
observation. AA positive
positive reaction
reaction ischaracterized byby theformation
remainswhen
invcrtcd
through180”.
mation ofof aa firm
firm gelgel thatthat sitivc
remains
when
invertedthrough180°.
(+).
A negative result is characRecord
Record such
such aa result
result asas positive(+). A negative r~uJt is c~arac­a gel or by the formationof a viscousterizedbv
theabscncc oofp”suchterizedbytheabsence
such
a
gel
or
by
the
formation of
a VISCOUS
gel that does no1 maintainits integrity.
Record such a result as
gel
that
does
not
maintainits
integrity.
Recordsu~h a re.sult as
ncgativc(-).
Handlethe tubes with care, and avoid subjecting
negative (-). Handle
the tubes
with care. and aVOid subjecting
them IO unwanted
vibrations,or false negative observations
them to The
unwanted
vibrations. or
false negative observations may
may
rcsul1.
test is invalid if the positive product control or Ihe enresult. The
test isdoes
invalid
if the positive productcontrol or theto endotoxin
standard
no1 show the end-poinI
concentrationbc
dotoxinstandard does
not showthe end-point concentration to bewithin f l twofold
dilutionsfrom the label claim scnsitiviIY of thewithin± I twofoldfrom thelabel claim
the
LAL
Reagent
or ifdilutions
any negative
controlshows sensitivity
a gel-clot ofendLAL Reagent or if any negative control shows a gel-clot endpoint.
point.
.

Calculation andand Interpretation
Interpretation
Calculation
Calcularion-Calculate
the concen1raIionof endotoxin(in Unitsof endotoxin(in byUnits
per Calculatioll-Calculate
mL or in Units per g or themg) concentrationin or on the article
under testtheper mL orin Units
per g .S
or ismg)or on ofthethearticle
under mean
test bylog,,,
theformulaoS/U.
in which
the inanIiloggeometric
formulapSI U. in which
S is theantilog of thegeometricmeanof
1hc end-points.
expressed
in Endotoxin
Units(EU) permL loglo
forofend-points.
expressedin theEndotoxin
Units
per emLforthe theS1andard
Endotoxin.
U isantilog of.Ze/f,(EU)whereis thetheStandard
Endotoxin,
Uistheantilogofi:.f/f.
where
e
is
the
10810 of the end-point
dilutionfactors. expressedin decimal fracloglo
dilution
expressedintionsj’isof thetheend-point
numberof replicate factors.
reaction
tubes
readdecimalaI the fraecndlions./isthenumberofreplicate
reactiontubes
readat thefactor
end.
poinl level for the specimen under 1cs1. and p is the correction
point
level
for
the
specimen
under
test.
and
p
is
the
oorrection
factorfor those cases where a specimenof the article cannot bc taken diforcannot soluIion.
be taken directlythoseintocases(es1where
but aisspecimen
processed of the
as article
an extract.
or
rectlywashing. into test but is processed as an extract, solution. or
washing.
Where the tes1 is conducted
as an assay wi1h sufficient
replication
test is conductedasassay with resulrs.
sufficient
replication
to Whereprovide thea suirable
numberof an
independentcalculatefortoof independent
results, calculateforeachprovide
rcplicalca suitableassay number
1hc concentrationof cndotoxin
in or on theeachreplicateassay
theconcentrationofendotoxin
in
or
on
the
arriclc under 1es1 from the antilog of the geometricmean log cndarticle
under testfrom thelog from
endpoin1 raIios.
Calculalc
the antilog
mean ofandthethegeometricconfidence mean
limi1spoint
ratios.
Calculate
the
mean
and
the
confidence
Ihc rcphcalc logarithmicvalues of all the obtained assaylimits
resultsfrom
bythereplicatesta1is1ical
logarithmicvalues(seeof all
assay results bya
a suitablemcrhodCulrulthe
arion obtainedo/Porency/rom
aSinglesuitablestatisticalAssoy( I I I ) ).method (sec Calculation of Potency from aA.r.w.l' ( II ) ». arliclc mec1s the requircmcnts
SingleInrcr~rcrurion-Theof 1hc ICSI
InlerflrctationThearticle meetsor the testinof 1hcconcewarion
ofcndoroxin
dots theno1 requirements
exceed 1ha1 specified
ifthetheindividual
concentrationof endoto,jndocs sonotspecified
exceed thaI
monograph.
and u hcrc
in thespecified
lndivldual intheindiVidualor monograph.
"here
so specified
monograph
III thls chapcr, and thewnlidcncc
limits In ofthetheIndiVidual
assay domonopraphur In (hiS
chapler. (he Lonfidence limits of the assay do
no1 crcced
those
qwi~f~cd.
not e~eecd those speCified.

B-51

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

APPENDIXCAPPENDIX CDETERMINATION
THE RELATIONSHIPTHE
DETERMINATION OF
OF THERELATIONSHIP BETWEENBETWEEN THE
CONTROL STANDARD
STANDARD ENDOTOXINENDOTOXIN (CSE)
THE REFERENCE
REFERENCECONTROL
CCS~} ANDAND THESTANDARD ENDOTOXINSTANDARD
ENDOTOXIN (RSEZ
CRSE}
If aa manufacturer
to useuse anendotoxin
preparation(CSE)
If
manufacturer chooses
chooses toan endotoxin
preparation
(CSE)

other
than
other
than
the
Standard
Endotoxin(RSE),
the CSECSE
the United
United States
States Pharmacopeia
Pharmacopeia Reference
Reference Standard
Endotoxin (RSE),
the
RSE.
If
the
CSE isis notnot
will
will have
have toto be
be standardized
standardized against
against the
the RSE.
If
the CSEaa
commercial
preparation
which
has
been
adequately
characterized,
itshouldbecommercial preparation which has been adequately characterized, it should bestudiedandfully
characterized
as totostabilityoftheuniformity,
studied andfully characterizedasuniformity,
stability
of
the
The
relationship
of
the
CSE
to
the
RSE
shouldbepreparation,etc.
preparation, etc.
The relationship of the CSE to the RSE should bedetermined
prior
to
use
of
a
new
lot,
sensitivity,ormanufacturerofthedetermined prior to use of a new lot, sensitivity, or manufacturer of theLALor aa newsource oror manufacturer
the CSE.
LAL ornew lot
lot source
manufacturer ofof theCSE.

A.
TECHNIQUE
A. GEL-CLOT
GEL-CLOT TECHNIQUE
The
following
is an exampleofThe
followingof theis CSEan exampleofrelationshipto the RSE:
relationship of the CSE to the RSE:aaprocedure
proceduretotodetermine
determinethethe

At least
4 samples (vials)
fo;the lotof CSE should
be assayed.
Atsamples
assayed.
Stateleastin 4ng/mL
the (vials)
endpoint fa:for thethe lot
CSEofandCSEin should
EU/mL beof the
RSE.
State
in
ng/mL
the
endpoint
for
the
eSE
and
in
EU/mL
of
the
RSE.
The values
obtainedshouldbe the geometricmean of the endpoints
The
obtainedusing values
a minimumof 4 should
replicates.be the geometric mean of th.:! endpoints
using a minimum of 4 replicates.
Example:LAL end:iints
for the RSE and CSE are as follows:
Example: LAL end: iints for the RSE and CSE are as follows:
RSE = 0.3 EU/mL
RSE ==: 0.3
CSE
0.018 EU/mL
ng/mL
CSE =: 0.018 ng/mL
The EUs per ng of CSE are calculated
as follows:
The EUs per ng of eSE are calculated as follows:
RSE _ 0.3 EU/mL= 16.7 EU/ng
Q.3 EU/mLiCSE
l l =: 0.018= 16.7 EU/ng
ng/mL
CSE
0.018 ng/mL
This
indicatesthat 0.018 ng of the CSE is equal to 0.3 EU ofThis
indicates
0.018
ng of 16.7
the CSEthe RSE.
Thus, that
the CSE
contains
EU/ng.is equal to 0.3 EU of
the RSE. Thus, the CSE contains 16.7 EU/ng.
B. CHROMOGENIC AND ENDPOINT-TURBIDIMETRICTECHNIQUES
B. CHROMOGE~Ie AND ENDPOINT-TURBIDIMETRIC TECHNIQUESAt least
4 samples(vials)for the lotof CSE should
be assayed.
In least
addition 4 samples
to a water
blank, for assay
dilutions
RSE which
fall
in
At
(Vials)the lotof CSEof should
be assayed.
the addition
linearrangea waterand dilutionsof dilutions
theCSE. of Linear
regression
In
toblank, assay
RSE whichfall inanalysisis performed
the absorbancethe RSE regression
standards
the linear
range and ondilutionsof thevalues
CSE. of Linear
endotoxinof the concentrations
analysis
is versus
performedtheiron the respective
absorbance values
RSE standards(Y-=is)(x-axis).
Calculatethe EU/ngrespectiveof the CSEendotoxin
by inserting concentrations
the average(y-axis)versustheir
CSE
O.D. readings
concentration
whichinserting
falls
in
RSE
(x-axis).
Calculatefor theeach
EU/ngof the CSE by
the theaveragestandard
into forthe each
RSE concentration
straight
line
equation. fallsThein resulting
CSE
o. D. range
readings....hichthe RSECSE values range(in intoEU) theareRSE thendividedline by
theircorresponding
standard
straight
equation.
The
resulting
Thesedivided
values by are their
then corresponding
averagedtoconcentrations
CSE
values (in (in EU) ng/mL).
are thenobtainthepotency
of
the
CSE
lot.
concentrations (in ng/mL).
These values are then averaged toobtain the potency of the CSE lot.

- 19 - 19 -B-52

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

EXAMPLE
EXAMPLE::RSE Standard
Standard Curve
Curve
Concentration
Concentration

O.D.
0.0.

0.1
0.1

0.11
0.11
0.26
0.26
0.49
0.49
1.06
1.06

RSE (EU/mL)(ElJ/mL)

0.25
0.25
0.5
0.5
1.0
1.0
y-intercepty-intercept =:::: -0.008-0.008slope
1.056
slope::::= 1.056

rr =:::: 0.999
0.999

Straight
Line Equation
Equation(Y) ::::= -0.008-0.008++ (1.056
Straight Line(Y)
(1.056 ** X)
X)
CSE
CSE Standard
Standard Curve
Curve
CSE
CSE
Cont.
Cone. (ng/mL)(ng/mL)AVERAGE O.D.
AVERAGE
O. D.

0.01
0.01
0.025
0.025
0.05
0.05
0.1
0.1

Corresponding
Corresponding
RSE (EU/mL)
RSE(EU/mL)

0.12
0.12
0.31
0.31
0.60
0.60
1.23
1.23

0.119
0.119

0.301
0.301
0.626
0.626
1.291
1.291

EU/ng
EU/ng
(RSE/CSE)(RSE/CSE)
11.9
11.9
12.0
12.0
12.5
12.5
12.9
12.9

Mean
EU/ng ::::
= 12.3
12.3
Mean EU/ng
TECHNIOUEC.
C. KINETIC-TURBIDIMETRICKINETIC-TURBIDIMETRIC TECHNIQUEIn
EUs toto aa eSE,
CSE, theshouldbe
In orderorder toto assign
assign EUs
the following
following shouldbe performed
performed
on
4
vials
from
the
same
CSE
lot.
on 4 vials from the same CSE lot.
Twofold
be madein therange
of
1.0
Twofold dilutions
dilutions ofof thethe RSERSE should
should be
made inthe rangeof 1.0
EU/mL
to
0.03
EU/mL.
Determine
the
Time
of
Reaction(T)
forat
EU/mL to 0.03 EU/mL. Determine the Time of Reaction (T) for at
least
duplicatesof
eachstandard
concentration.
Construct
least duplicates of each standard concentration.
Construct aa
standard
curve(Log10T
versusLog10
endotoxin
concentration
standard curve (LoglO T versus LoglO endotoxin concentration
Calculatethe mean
1.0
andEU/mL.
These
T’s
(El).
(E».
Calculate themean TT forfor 1.0
and 0.03
0.03 EU/mL.
These T's
define
RSE standard
range.
define thethe RSEstandard range.
For each of the fourvialsof CSE make twofold
dilutionssuch that
For each of the four vialsofCSE make twofold dilutions
such
that
the T values
for at least
3 concentrationsof the CSE are within
the T values for at least 3 concentrations of the CSE are within
therange.
DeterminetheT values
for
at
least
the RSERSE standard
standard range.
DeterminetheTvalues forat
least
duplicatesof each endotoxin
concentration.
Calculatethe EU/ng ofduplicates of each endotoxin concentration. Calculate the EU/ng of
CSE by insertingthelog mean CSE T valuesforeach
endotoxin
CSE by inserting the log mean CSE T values for each endotoxin
concentration
which
falls
in the RSE standard
range
into
the RSE
concentration which fallsThein resulting
the RSE standard
the then
RSE
CSE values range(in intoEU) arestraight
line
equation.
straight
line
equation.
The
resultingCSE
values
(in
EU)
are
then
These
dividedby theircorresponding
concentrations(inng/mL).
divided by their corresponding concentrations (in ng/mL).
Thesevaluesare averaged
to obtain
the potencyof the CSE lot.

values are averaged to obtain the potency of the eSE lot.

- 20 - 20 -B-53

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

EXAMPLE:
EXAMPLE:RSE Standard
Standard
Curve
aSE
Curve
Straight
Line Equation
Equation(Y) == 3.03
3.03 +t (-0.181(-0.181X)
Straight
Line
(Y)
** X)
RSE Standard
Standard
Range =
= 10371037 -- 2235
2235 seconds
seconds (17.3-37.3(17.3-37.3
minutes)
RSE
Range
minutes)
CSE Stand~rdStandard
Curve
CSE
Curve
Vial
Vial

0.1
0.1

11
2
33
44

1018.81018.8
990.7
990.7
998.2
998.2
1003.41003.4

Note:
Note:

Endotoxin
Concentration(ng/mL)
Endotoxin
Concentration(ng/mL)
0.05
0.025
0.0125
0.006
0.05
0.025
0.0125
0.006

0.003
0.003

1114
1114
1090.61090.61116.81116.81086.11086.11740.71740.71780.01780.01800.91800.91781.01781.01218.6
1218.61249.81249.81227.81227.81198.51198.51402.71402.71406.41406.41411.0 01411.
1415.61415.6

1548.71548.71586.01586.01554.11554.1
1593.91593.9Each TT inin thethe above
above table
table
is expressed
expressed
in seconds
seconds
and
Each
is
in
and
represents
the meanmean ofof at
at least
least duplicate
duplicatedeterminations.
represents
thedeterminations.

Mean T
T (sec.)(sec.)
Mean
Log meanmean T
Log

1002.8*1002.8*
3.001
3.001

1223.;
1223.73.088
3.088

1101.91101.93.042
3.042

1408.91408.93.149
3.149

1570.71570.7
3.196
3.196

1775.71775.73.249
3.249

Calculations:
Calculations:
Solving
for EU/mL
EU/mL equivalentequivalent
by substituting
substituting
onset times
times generated
generatedwithSolving
for
by
onset
with
CSE
(ng/mL)
into
the
above
RSE
standard
line
equation,X=(
CSE (ng/mL) into the above RSE standard line equation, X = ( yY -3.03)/-0.181= log
onset time
time andX == log
log EU/ml
ElJ/ml equivalent.
equivalent.
3.03)/-0.181 where
where YY=
log meanmean onset
and X
CSE
CSE Endo.
Endo. Cont.
Cone.
(nn/mL)(ng/mL)
0.1*
0.1*
0.05
0.05
0.025
0.025
0.01250.0125
0.006
0.006
0.003
0.003

Log
Log Mean
Mean
T
3.001
3.001
3.042
3.042
3.088
3.088
3.149
3.149
3.196
3.196
3.249
3.249

EU/mL
Equivalent
EU/mL Equivalent
(RSE
Std.
(RSE Std. Line)
Line)
Log
Antilog
Log
Antilog
0.16
0.16-0.066-0.066-0.32-0.32-0.657-0.657-0.917-0.917-1.210-1.210

1.45
1.45
0.859
0.859
0.479
0.479
0.22
0.22
0.121
0.121
0.062
0.062

EU/ng
EU/ng
14.5
14.5
17.2
17.2
19.2
19.2
17.6
17 .6
20.2
20.2
20.6
20.6

Mean
Mean EU/ng
EU/ng == 19.0
19.0 (SD(SO == 1.52)
1.52)*

the RSE standard
range* Outside
Outside the RSE standard range -- notnot used
used inin calculation
calculation ofof mean.
mean.

The values
for the y-intercept
and slope of the four CSE curves used forthe y-intercept and slope of the four CSE curves used for
The values
theEU/ng fordetermination
may be stored
for
use in routine
testing
the EU/ng determination may be stored for use in routine testing(archived
standard
curve)
insteadof runninga seriesof standards
each(archived standard curve) instead of running a series of standards each
day.
Using
the EU/ng conversion
factor,CSE
standards
withintherange
day.the Using
the EU/ng
within theoutsiderangeof
RSE curve
can beconversion
made up infactor.
endotoxinCSE standards
units.
Standards
of
the
RSE
curve
can
be
made
up
in
endotoxin
units.
Standards
outsidethisrange require
the use of RSE and a new RSE standard
curve.
If CSE
this range require the use of RSE and a new RSE standard curve. If CSE
standards
outsidethe
RSE standard
range
are
requiredtheEU/ng
standards outside
the be
RSE determined
standard forrangethe areEU/ng
conversion
factormust
new requiredrange
as thedescribed
conversion
factormust
bedetermined
for
the
new
range
as
described
above..
above..

- 21 - 21 -B-54

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

APPENDIXAPPENDIX DD

MAXIMUM
MAXIMUM VALID
VALID DILUTION
DILUTIONTo
To determine
determine how
how muchmuch thethe productproduct can
can bebe diluted
diluted andand still
still bebe ableable totodetectdetect thethe limit
limit endotoxin
endotoxin concentration,
concentration, thethe following
following two
two methods
methods will
will
determine
determine thethe Maximum
Maximum Valid
Valid Dilution:
Dilution:
METHODMETHOD II
ThisThis method
method isis used
used when
when there
there isis an
an official
official USP
USP limit
limit oror whenwhen thethelimitslimits listed
listed inin Appendix
Appendix EE are
are used.
used.
MVD
MVD == Endotoxin
Endotoxin Limit
Limit XX Potencv
Potency ofof ProductProduct
A;...
For
For drugs
drugs administeredadministered on
on aa weight-per-kilogram
weight-per-kilogram
potencyisexpressed
as
ts/mL
and
potency is expressed as mg
mg oror uniunits/mLandadministeredadministered on
on aa volume-per-kilogram
volume-per-kilogram basis,
basis, the
the
equal
equal toto 1.01.0 m.L/mL.
mL/mL.

basis,
basis, thetheforfor drugs
drugs
potencypotency isis

METHODMETHOD IIIIThis
is usedno official
theThis method
method isused when
when there
there isis no
Official USP
USP limit
limit andand thelimitslistedinlimits listed
in Appendix
Appendix EE are
are not
not used.
used.
Step
Step 1.
1.

Minimum Valid
Minimum
Valid Concentration
Concentration (MVC)
(MVC)
MVC=
MVC
= ,&M
A M
K

K

Where
Where::A=~=GEL CLOT: Labeledsensitivity-EU/mI,.
GEL
CLOT: Labeled sensitivity-EU/mL.
CHROMOGENIC, TURBIDIMETRIC
and KINETIC-TURBIDIKETRIC:CHROMOGENIC, TURBIDIMETRIC and KINETIC-TURBIDIMETRIC:
The lowest
point
used in the standard
curve.
The lowest point used in the standard curve.

M = Rabbit
Dose or Maximum Human Dose/Kgof body weightthatM= would
Rabbit beDoseor Maximum
Human Dose/Kgofbody weightthat
administeredin a single
one hour period,
whicheverwould be administered in a single one hour period, whicheveris larger.
Forradiopharmaceuticals,
M equals
the rabbit
isForhumanradiopharmaceuticals,
M equals
the rabbidose larger.
or maximum
dose/Kg
at the productexpiration
date tdoseormaximum
humandose/Kgattheproduct
expirationdateor time.
Use 70 Kg as the weight of the average human whenortime. Usethe70 maximum
Kg as theweight of the average
human
when
Also,
if
the
calculating
human dose per Kg.
calculatingthemaximum
human
dose
per
Kg.
Also,
i f thepediatric
dose/Kgis higher
than the adultdose then it
pediatric dose/Kg is higher than the adult dose then it
shall be the dose used in the formula.
shall be the dose used in the formula.
K = 5.0

EU/Kg

for

parenteraldrugs

execptthose

K = 5.0
EU/Kg for 0.2
parenteralexecpt drugs
those
W/Kg
for drugs
intrathecal
intrathecally;
intrathecally; 0.2 EU/Kg for intrathecal drugs- 22 22 -B-55

administeredadministeredICCVAM Test Method Evaluation Report: Appendix B5

May 2008

APPENDIX D
D (cont.)(cont.)
APPENDIX
Step 2.
2.
Step

Maximum Valid
Valid Dilution
Dilution(MVD)
Maximum(MVD)
MVD == Potency
Potency ofof ProductProduct
MVD
MVC
MVC

For drugs
drugs
administeredon aa weight-per-kilogram
weight-per-kilogrambasis,
theForadministeredonbasis,
thepotencyisexpressed
as mg
mg or
or
drugs
units/ml
andforpotencyisexpressed
asuni
ts/mL andfor
drugs
administeredon aa volume-per-kilogram,volume-per-kilogram,the potency
potency
is equalequal
toadministered
on
the
is
to
1.0 mL/mL.
m.L/mL.
1.0
METHOD II EXAMPLES
EXAMPLESMETHOD
Endotoxin
Limit
Expressed
bv Weight:
Weight:Endotoxin
Limit
Expressed
byProduct:
Cyclophosphamide
Injection
Product:
Cyclophosphamide
Injection
Potency:
20 mg/mL
mg/mL
Potency:
20'
Lysate Sensitivity
Sensitivity(A):
0.065 EU/mL
EU/mL
Lysate(;l):
0.065
Endotoxin
Limit(AppendixE): 0.17
0.17 EU/mg
EU/mg
Endotoxin
Limit(Appendix E):
MVD =
MVD;

0.17 EU/mP
EU/mg X
mg/ml
0.17
X 20 mr/ml
0.065
EU/mL
0.065 EU/mL

=
3.4
=
0.065
0.065

= 1:52.3
1:52.3
or
1:52=
or 1:52

Endotoxin
Limit
Expressedbv
Volume:'Endotoxin
Limit Expressedby Volume:
Product:
Product: 5%
5% Dextrose
Dextrose Injection
Injection
LysateSensitivity
EU/mL
Lysate Sensitivity (A):(it): 0.065
0.065 EU/mL
Endotoxin
Limit(Appendix
E):
0.5
Endotoxin Limit (Appendix E): 0.5 EU/mL
EU/mL
MVD
0.5 EU/mL
MVD == .0.5
EU/!!tf.t XX 11 mL/mL
mL/mL
0.065
0.065 EU/mL
EU/mL= _,l:WO.~2_
0.5
== 1:
1: 7.77.7= 0.065
0.065

METHODMETHOD IIII EXAMPLES
EXAMPLES
PARENTERAL DRUGS EXCEPT INTRATHECALPARENTERAL DRUGS EXCEPT INTRATHECAL
Drug Administeredon a Weight-Der-Kilogram
Basis
Drug Administered on a Weight-per-Kilogram Basis
Product:
Cyclophosphamide
Injection
Product: Cyclophosphamide Injection
Potency:
20 mg/mL
Potency: 20 mg/mL
Maximum Dose/Kg ( M ): 30 mg/Kg
Maximum Dose/Kg ( M ): 30 mg/Kg
Lysate Sensitivity(A):
0.065 EU/mL
Lysate Sensitivity ()w): 0.065 EU/mL
WC = h M= 0.065 EU/mL X 30 mn/Kg= 0.065 EU/mL X JO mg/K&
MVC = Ll M
K
5.0 EU/Kg
5.0 EU/Kg
K
MVD = Potencv
of Product=20 mn/mL
MVD = Potency of Product = 20 mg/mL
Mvc
0.390
mg/mL
MVC
0.390 mg/mL- 23 23

B-56= 0.390 mg/~
= 0.390 mg/mL= 1:51.2
or 1:51= 1:51.2 or 1:51

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

APPENDIXAPPENDIX DD (cont.)(cont.)
Drup
Drug Administered
Administered on
on a Volume-per-Kilogram
Volume-oer-Kilogram Basis
Basis
Product:
Product: 5%
5% Dextrose
Dextrose inin Water
Water
Maximum
Maximum Dose/Kg
Dose/Kg ( MM ):): 10.0
10.0 mL/Kg
mL/Kg
LysateLysate Sensitivity
Sensitivity (A):(rl): 0.065
0.065 EU/mL
EU/mL
MVC=
MVC=

AM
?..M
K
K

==

0.065
0.065 EU/mL
EU/mL X
X 10.010.0 mL/Kg
mL/Kg == o,13
0.13 ~~~
mL/mL
5.0
EU/Kg
5.0 EU/Kg

MVD
MVD == Potency
Potency ofof ProductProduct
MVC
MVG

1.0
mL/mL
== __
1 .....O,-",mL=/,-,mL..-.._
0.13
0.13 mL/mL
mL/mL

=

=

1:7.7
1:7.7

INTRATHECALINTRATHECAL DRUGS
Drug
Administeredon
Drug Administeredon aa WeiPht-per-Kilopram
Weight-oer-Kilogram Basis
Basis
Product:
Gentamicin
SulfateProduct: Gentamicin
SulfatePotency:Potency: 2.0
2.0 mg/mL
mg/mL
Maximum
0.11 mg/Kg
Maximum Dose/Kg
Dose/Kg (( MM ):): 0.11
mg/Kg
LysateSensitivity(A):
Lysate Sensitivity (;L): 0.1
0.1 EU/mL
EU/mL
MVC
MVC =&
= AM
K
K

= 0.1

EU/mL

X 0.11

mP/Kg

X 0.11 mg/Kg= 0.1 EU/mL 0.2
0.2 EU/Kg
EU/Kg

MVD
of ProductProduct ==Potency of
MVD == Potency
MVC
MVC

2.0 mg/mL
mn/mL
2.0
0.055
0.055 mg/mL
mg/mL= 0,055 mg/~
0.055 mg/mL-= 1:36.4
1:36.4:;:;:Drug
Drug Administered
Administered on
on aa Volume-per-Kilogram
Volume-oer-Kilogram Basis
Basis
Product:
Injection
Product: Lidocaine
Lidocaine Hydrochloride
Hydrochloride Injection
Maximum
Dose/Kg
(
M
):
0.057
mL/Kg
Maximum Dose/Kg ( M ): 0.057 mL/Kg
Lysate Sensitivity
Sensitivity(2):0.1 EU/mL
Lysate(~): 0.1 EU/mL
MIJC = -&.IMVe=
AMK
K

=

O-1 EU/mL
0.1
EU/mL

Potency of Product
MVD = Potencyof Product
MVD
MVC
MVC

=

X
d/Kg
= 0.0285
0.0285 mL/mL
d/a
X 0.057
0.057 mL/Kg=
0.2 EU/Kg
0.2 EU/Kg
1.0 mL/mL= 1.0= 1:35.0
mL/mL= 1:35.0= 0.0285
mL/mL
0.0285 mL/mL

-

B-57

24

-

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

APPENDIXAPPENDIX EEMAXIMUM
MAXIMUM DOSEDOSE ANDAND ENDOTOXINENDOTQXIN LIMIT
LIMIT TABLE
TABLE
Drug
Drug Name
Name

Dose
Dose 0(M)Endotoxin
Endotoxin Limit
Limit(EU/mg,ml,units(EU/mg,ml,unitsofof product)
product)-A-AAcetic
Acetic Acid
Acid Irrigation
Irrigation
Acetazolamide
Acetazolamide Sodium
SodiumAcetylcysteine
Injection
Acetylcysteine Injection
Acyclovir
Acyclovir Sodium
SodiumAdenosine
Adenosine Phosphate
Phosphate
Albumin,Normal
Human Serum
Serum (25%)(25%)
Albumin,Normal HumanAlbumin,Normal
Human
Serum
(20%)
Albumin,Normal Human Serum (20%)
Albumin,Normal
Human Serum (5%)
Albumin,Normal Human Serum (5%)
Albuterol
SulfateAlbuterol Sulfate
Alcohol and Dextrose Injection
Alcohol and Dextrose Injection
Alfentanil Hydrochloride
Alfentanil Hydrochloride
Alkaloids of Belladonna
Alkaloids of Belladonna
Alpha,-Proteinase
Inhibitor
Alphat-Proteinase Inhibitor
Alphaprodine HCI Injection
Alphaprodine
HCl Injection
Alprostadil(Postaglandin)
Aiprostadil (Postaglandin)
Alteplase
Alteplase
AmdinociIlin
Amdinocillin
Amikacin Sulfate Injection
Amikacin
Injection
Amino AcidSulfate
Injection
Amino
Amino Acid
Acids Injection
and Electrolytes
Amino
Acids
andAcids
Electrolytes
Essential Amino
and Dextrose
Essential
Amino
Acids
and
Aminocaproic Acid Injection Dextrose
Aminocaproic
Injection
Aminohippurate Acid
SodiumInjection
Aminohippurate
SodiumInjection
Aminophylline Injection
Aminophylline
Injection
Amitriptyline HCIInjection
Amitriptyline
HCI
Injection
Ammonia N 13 Injection
13 Injection
Ammonia
Ammonium N Chloride
Injection
Ammonium
Chloride
Injection
Amobarbital Sodium
Amobarbital
Sodiumand Suspension
Amoxicillin, Sterile
Amoxicillin, Sterile and Suspension

B-58

10.00
mL
10.00 mL10.00
mg10.00 mg150.00mg
150.00 mg
30.00 mg
30.00 mg0.71 mg0.71 mg
3.00 mL
3.00 mL
3.75 mL
3.75 mL
10.00 mL
10.00 mL
0.008 mg0.008
mg1.79 mL
mL
1.79
250.00 mcg
250.000.007mcg
mg
0.007mg
mg
60.00
60.00
0.60 mgmg
0.60
6.00 mg
mcg
6.00
mcg
1.25 mg1.25 mg
10.00
mg
10.00
mg
15.00 mg
15.00 mg
25.00
mg
25.00
mg
25.00 mg25.00 mg25.00 mg
100.00mg
100.00125.00 mg
mg
125.00mg
5.00 mg
5.00
0.42 mg
0.42
7.00 mgmL
7.00
2.90 mL
mEq CI
2.90 mEq
14.30
mg CI
14.30
mg
20.00 mg
20.00 mg35
350.50 +
0.50 +
0.50 l0.50 +
0.03
0.03
0.17
0.17
7.04
7.04
1.67
1.67
1.33
1.33
0.50
0.50
625.00625.00
2.70
2.70
0.02
0.02
714.29714.29
0.08
0.08
8.33
8.33
0.83
0.83
4.00
4.00
0.50 +
0.50
0.33 ++
0.33
0.20 +
0.20
0.20
0.20
0.05
0.05
0.04 +
0.04
1.00 +
1.00
12.00
12.00
25.00 f
1.72 +
25.00
0.40 f1.72
0.25 ++
0.40
0.25 +

April
April ,1992,1992

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

1.00
Amphotericin
Amphotericin B
B forfor Injection
Injectionmg
1.00 mg0.01
mg“Amphotericin
Bfor
Injection
If< Amphotericin B for Injection
0.01 mg
Ampicillin
33.30
Ampicillin Sodium
Sodium33.30 mgmg
Ampicillin
andSulbactam
28.60
mg
Ampicillin and Sulbactam
28.60 mg
Amrinone
10.00
10.00 mgmg
Amrinone Lactate
LactateAnileridine
0.70
mg0.70 mgAnileridine
Anticoagulant
22.00·mL.OO’mL
Anticoagulant Heparin
Heparin Solution
Solution
Anticoagulant,-.-~.-- mL
mL
Anticoagulant, Citrate
Citrate Dextrose
Dextrose Sol.
Sol.
-.-mL
Anticoagulant,
CitratePhosphate
Dextrose
Anticoagulant, Citrate Phosphate Dextrose-.-~ mL
Anticoagulant,
Anticoagulant, Citrate
Citrate Phosphate
Phosphate Dextrose
Dextrose
AdenineSolution-.-Adenine Solution-.-- mL
mL
Antihemophilic
10.00
10.00 units
units
Antihemophilic Factor
FactorAntihemophilic
3.00
3.00 mL
hr. atat 56-57oC)
56~57oC)
mL
Antihemophilic Plasma(1Plasma(1 hr.
3.00
Antirabies
3.00 mL
mL
Antirabies Serum
Serum
3.00 mL
Antitoxin
Antitoxin (Gas
(Gas Gangrene)
Gangrene)
3.00 mL
Antivenom
3.00
mL
Antivenom
3.00 mL
0.09 mg
Apommorphine
HCl Tablets for Injection
Apommorphine HCI Tablets for Injection
0.09 mgArginine HCl Injection
500.00 mg
Arginine HCI Injection
500.00 mg
4.20 mg
Ascorbic Acid
4.20 mg
Ascorbic Acid
Asparaginase for Injection
1000.00 Iu
1000.00
Asparaginase
for
Injection
Atracurium Besylate
0.50 IVmg
0.50 mg
mg
Atracurium
Besylate
Atropine
Sulfate
0.09
0.09
mg
Atropine
Sulfate0.70 mg
Aurothioglucose Suspension
0.70
mg
Aurothioglucose
Suspension
5.00 mg
Azathioprine Sodium for Injection
5.00
mg
Azathioprine
Sodiumfor
Injection
Azlocillin
75.00 mg75.00 mg
mg
Azlocillin for Injection
Aztreonam
28.60
28.60 mg
Aztreonam for Injection

5.00
5.00
20.00
20.00
0.15
0.15 ++
0.17
0.17
0.50
0.50
7.20
7.20 ++
2.50
2.50
5.56
5.56
5.56
5.56
5.56
5.56
0.50
0.50
1.67
1.67
1.67
1.67
1.67
1.67
1.67
1.67
55.56
55.56
0.01
0.01
1.20 +
1.20 #+
0.01
0.01
10.00 #
10.00 +
55.60
55.60
7.14 +
7.14
1.00 +
1.00 ++
0.07
0.07
0.17 ++
0.17 +-B--B-

Bacitracin
Bacitracin
Bacitracin Zinc
Bacitracin
Zinc HCI
Benzquinamide
Hel Injection
Benzquinamide
Benztropine Mesylate
Benztropine
Mesylate
Injection
Benzylpenicilloyl
Polylysine
Benzylpenicilloyl
Polylysine
Betametbasone Acetate andBetamethasone
Betamethasone Acetate
Sodium andPhosphate
Betamethasone Sodium
Sodium Phosphate
Phosphate
Betamethasone
Betamethasone
Sodium Phosphate
Betazole HCl Injection
Betazole
HCIChloride
Injection
Bethanechol
Bethanechol
Chloride
Biperiden Lactate
Injection
Biperiden Lactate Injection

500.00 units
500.00 units
500.00units
500.00units
1.00 mg1.00
mg0.09 mg
0.09 mg0.004
mL
0.004 mL
0.17 mg
0.17 mg
0.17
2.86 mg
2.86
0.20 mg
0.20
0.06 mg
0.06 mg
36
36

B-59

0.01 +
0.01
0.01 +
0.01
5.00
5.00
55.60 +
55.60 +
1250.00
1250.00
29.20 +
29.20
29.20 ++
1.75 +
29.20
1.75
25.00
83.30 +
25.00
83.30 +ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

Bleomycin
Bleomycin Sulfate
SulfateBretylium
Bretylium Tosylate
Tosylate Injection
Injection
Bretylium
Bretylium Tosylate
Tosylate in Dextrose
Dextrose
Brompheniramine
Brompheniramine Maleate
Maleate Injection
Injection
Bumetanide
Bumetanide
Bupivacaine
Bupivacaine Hydrochloride
Hydrochloride Injection
Injection
Bupivacaine
Hydrochloride
andBupivacaine Hydrochloride andEpinephrine
Epinephrine Injection
Injection
Bupivacaine
HCI
Bupivacaine HCI andand Dextrose
Dextrose
Buprenorphine
Buprenorphine HCI
HCI
Butorphanol
Butorphanol Tartrate
Tartrate

0.50
0.50 unit
unit
25.00
25.00 mgmg
25.00
25.00 mgmg
0.14
0.14 mgmg
0.01 mgmg
2.50
2.50 mgmg

10.00
10.00 #+
#+
0.20
0.20
0.20
0.20
35.71
500.00500.00 ++2.50
2.50 ++

3.20
3.20 mgmg
0.11
mg
0.11 mg
0.004
0.004 mg
mg
0.057
mg
0.057 mg

1.60
1.60 ++
1.80.+
1.80.+
1250.00
1250.00
88.00
88.00 ++

20.00 mg
20.00
mg
7.14
7.14 mgmg
0.007
0.007 mgmg
4.00
4.00 IuIV
0.05
0.05 mcg
mcg
14.30
14.30 mgmg
25.00
25.00 mgmg
35.00
35.00 mgmg
15.70 mg
15.70
mg
28.60
28.60 mg
mg

0.25
0.25
0.70
0.70 ++
714.30714.301.25
1.25
100.00100.00
0.35
0.35
0.20
+
0.20 +
0.143
0.143
0.32
0.32 ++
0.17
0.17 ++

1.43 mgmg
1.43
0.14 mL
mL
0.14
14.30
14.30 mgmg
0.14
mg
0.14 mg
100.00 mg
mg
100.009.26 mg9.26
mg0.007 meg
mcg
0.007
5.14 mg
mg
5.14
33.30
mg
33.30 mg33.30 mg
mg
33.30
-.-mg
-.-- mg14.30 mg
14.30
mg
28.57
mg
28.57 mg20.00
mg
20.00 mg28.50 mg28.50 mg28.60 mg28.60 mg

3.50
3.50
35.70
+
35.70 +
0.35
-t0.35 +
34.96
34.96
0.05 ++
0.05
0.54 ##
0.54
714.30+714.30 +1.00 #
#
1.00
0.15
+
0.15 +
0.15 +
+
0.15
0.20
+
0.20 +
0.35 +
+
0.35
0.20
+
0.20 +
0.25 +
0.25 +
0.20 +
0.20 +
0.17
0.17-C-CCaffeine
Caffeine Citrated
Citrated
Caffeine
Sodium Benzoate
Caffeine andand Sodium
Benzoate
Calcitonin
Human
Calcitonin - Human
Calcitonin
SalmonCalcitonin -- Salmon
Calcitriol
Calcitriol
Calcium
Calcium Ascorbate
Ascorbate
Calcium
Chloride
Calcium Chloride
Calcium
EdetateCalcium Disodium
Disodium Edetate
Calcium Gluceptate
Gluceptate Injection
Calcium
Injection
Calcium
Gluconate
Calcium Gluconate
Calcium
Glycerophosphate
Calcium GlycerophosphateandCalcium lactate
and Calcium
lactateCalcium
Levulinate
Calcium Levulinate
Capreomycin
Capreomycin Sulfate
SulfateCarbazochrome
Carbazochrome Salicylate
Salicylate
Carbenicillin Disodium
Disodium
Carbenicillin
Carboplatin
Carboplatin
Carboprost Tromethamine
Tromethamine
Carboprost
Carmustinefor Injection
Injection
Carmustine for
Cefamandole
Nafateand Sodium
SodiumCefamandole Nafate andCefazolin Sodium
SodiumCefazolin
Cefmetazole
SodiumCefmetazole Sodium
Cefonicid Sodium
SodiumCefonicid
Cefoperazone
SodiumCefoperazone Sodium
Ceforanide
Ceforanide
Cefotaxime Sodium
SodiumCefotaxime
Cefotetan Disodium
Cefotctan Disodium

37
37

B-60

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

Cefoxitin Sodium
SodiumCefoxitin
Ceftazidime
Ceftazidime
Ceftizoxime Sodium
SodiumCeftizoxime
Ceftriaxone Sodium
SodiumCeftriaxone
Cefuroxime Sodium
SodiumCefuroxime
Cephacetrile Sodium
Sodium forfor Injection
Injection
Cephacetrile
Cephaloridine
Cephaloridine
Cephalothin Sodium
Sodium Injection
Injection
Cephalothin
Cephapirin
SodiumCephapirin Sodium
Cephradine forfor Injection
Injection
Cephradine
Cerulitide
diethylamine
Cerulitide diethylamine
Chloramphenicol Sodium
Sodium Succinate
Succinate
Chloramphenicol
Chlordiazepoxide
HCI
Chlordiazepoxide HCIChloroprocaine
HCI
Chloroprocaine HCI
Cholecystokinin
Cholecystokinin
Chorionic
Chorionic Gonadotropin
Gonadotropin
Chlormerodrin
Injection
Chlormerodrin Hg197
Hg197 Injection
Chlormerodrin
Hg203
Injection
Chlormerodrin Hg203 Injection
Chlormerodrine
Chlormerodrine
Chloroquine HCIHCI Injection
Injection
Chloroquine
Chloroprocaine HCl
Chloroprocaine
HCI
Chloroprocaine HCIHCI -- Epinephrine
Epinephrine
Chloroprocaine
Chlorothiazide Sodium
Chlorothiazide
SodiumChlorpheniramine
Chlorpheniramine Maleate
MaleateChlorpromazine
HCI
Chlorpromazine HCIChlorprothixene
Chlorprothixene Injection
Injection
Chlortetracycline
Chlortetracycline HCl
HCI
Chromate
Sodium Cr51
Chromate Sodium
Cr51 Injection
Injection
Chromic
Chloride
Injection
Chromic Chloride Injection
Chromic
Chromic Phosphate
Phosphate P32
P32 Suspension
Suspension
Chymopapain
Chymopapain
Chymotrypsin
Chymotrypsin
SterileSodiumSterile Cilastatin
Cilastatin Sodium
Cimetidine
Cimetidine HCIHCI Injection
Injection
Cisplatin
Cisplatin forfor Injection
Injection
CitricCitric Acid,
Acid, Magnesium
Magnesium Oxide,
Oxide,&
SodiumCarbonate
Irrigation& Sodium Carbonate Irrigation
Citrate,Phosphate,Dextrose,Adenine
Citrate,Phosphate,Dextrose,Adenine
Clindamycin
Clindamycin Phosphate
Phosphate Injection
Injection
Cloxacillin
Cloxacillin
Codeine Phosphate Injection
Codeine Phosphate Injection

40.00 mg50.00 mg50.00 mg28.60 mgmg
50.00 mg80.00 mg14.30 mg60.00’mg
60.00mg28.60 mg25.00 mgmg
25.00
0.30 mcg
25.00 mg
mg
25.00
1.40
mg
1.40 mg11.43 mg
mg
11.43
1.00
IDU
1.00IDU
142.90 units
142.90
units
7.00
mL
7.00 mL
7.00 mL
7.00
mL
1.40
mg
1.40 mg
7.50
7.50 mgmg
20.00
mg
20.00 mg
20.00 mgmg
20.00
15.00 mg
15.00
mg
0.57
mg
0.57 mg0.72
mg0.72 mg
0.72
0.72 mgmg
5.00
mg
5.00 mg
7.00
7.00 mL
mL
0.30
ug
0.30 ug
7.00
7.00 mL
mL
42.90
pKat
42.90 pKat
4.30
4.30 units
units
-.-mg
-.-- mg
10.00 mg
10.00
mg
2.57
mg
2.57 mg+
0.13 +
0.10 ++
0.10 ++
0.20 ++
0.10 +
0.06
0.35
0.35
0.08 ++
0.17+
0.17 +
0.20 ++
0.20
16.67
0.20 +
+
0.20
3.57
+
3.57 +
0.45
0.45
5.00
5.00
0.03 +
+
0.03
25.00
+
25.00 +
25.00 +
+
25.00
3.57
3.57
0.70 +
+
0.70
0.25
0.25
0.25
0.25
0.30 +
+
0.30
8.80
+
8.80 +
6.90 ++
6.90
6.90
6.90 ++
1.00+
1.00 +
25.00 +
25.00
+
16.70
16.70
25.00 ++
25.00
0.12
0.12
1.16
1.16
0.23 +
+
0.23
0.50
0.50
1.90 ##
1.90

mL-.-- mL
0.90
0.90 mL
mL
8.60 mg
8.60 mg12.50
12.50 mgmg
0.86 mg
0.86 mg

0.50 +
0.50 +
5.56
5.56
0.58 +
0.58 +
0.40 +
0.40 +
5.80 +
5.80 +

-.--

38
38

B-61

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

0.03
166.700.03 mgmg
166.70 ++2.50
2.00
2.50 mgmg
2.00 ++
mg
0.36
13.89
0.36 mg
13.89
1.60
3.10
1.60 units
units
3.10 ++
5.00
mg
1.00
5.00 mg
1.00
3.57
1.40
3.57 mcg
mcg
1.40
1.86
1.86 CSR
CSR units
units 2.69
2.69
0.02
250.000.02 ing
ing
250.00 ++0.02
250.000.02 mgmg
250.00 f+
14.30
mcg
0.40
14.30 mcg
0.40 ++
1.00
5.00
1.00 mg
mg
5.00
30.00
mg
0.20
30.00 mg
0.20 #+
#+
0.12
42.00
0.12 mL
mL
42.00 +
+
7.14
mg
0.70
+
7.14 mg0.70 +
3.00
0.07
3.00 mgmg
0.07 -t+
1.93
mg
0.10
#
1.93 mg
0.10 #Colchicine Injection
Injection
Colchicine
Colistimethate Sodium
SodiumColistimethate
Conjugated Estrogens
Estrogens
ConjugatedCorticotropin, Gel,
Gel, Zinc
Zinc && Re.
Re.
Corticotropin,
Cortisone Acetate
AcetateCortisone
Cosyntropin
Cosyntropin
Cryptenamine
Cryptenamine Acetate
AcetateCupricChloride
Cupric Chloride Injection
Injection
CupricCupric Sulfate
Sulfate Injection
Injection
Cyanocobalamine
Cyanocobalamine andand Repository
Repository
Cyclizine
Cyclizine Lactate
LactateCyclophosphamide
Cyclophosphamide
Cyclosporine
Cyclosporine Injection
Injection andand Cont.
Cone.
Cysteine
HCI
Cysteine HCI
Cytarabine
Cytarabine“Cytarabine*Cytarabine-D-DDacarbazine
Dacarbazine forfor Injection
Injection
Dactinomycin
forDactinomycin for Injection
Injection
Dantrolene
Dantrolene Sodium
SodiumDaunorubicin
HCI
Daunorubicin HCI
Decamethonium
BromideDecamethonium Bromide
Deferoxamine
Mesylate
Deferoxamine Mesylate
Dehydrocholate
Sodium Injection
Injection
Dehydrocholate Sodium
Deslanoside
Deslanoside
Desmopressin
AcetateDesmopressin Acetate
Desoxycorticosterone
Desoxycorticosterone Acetate
AcetateDesoxycorticosterone Pivalate
Pivalate Sus.
Sus.
Desoxycorticosterone
Dexamethasone Acetate
Acetate Suspension
Suspension
Dexamethasone
Dexamethasone
Sodium
Phosphate
Dexamethasone Sodium Phosphate
Dexpanthenol
Dexpanthenol
Dextran 40
40
DextranDextran 40
40 inin Sodium
Sodium Chloride
Chloride
DextranDextran70
Dextran 70
Dextrose << 5%
5%Dextrose
Dextrose5%-70%
Dextrose- 5%-70%
Dextrose andand Sodium
Sodium Chloride
Chloride
Dextrose
Dezocine
Dezocine

9.60
9.60 mgmg
0.05
mg
0.05 mg
10.00 mg
10.00
mg
1.16
mg
1.16 mg
0.043
0.043 mg
mg
15.00
mg
15.00 mg150.00 mg
150.00mg
0.03
mg
0.03 mg
0.30 meg
mcg
0.30
0.07 mg
mg
0.07
1.80
mg
1.80 mg
0.23 mg
mg
0.23
0.16
mg
0.16 mg7.10 mg
mg
7.10
5.00
mL
5.00 mL
5.00 mL
mL
5.00
10.00
mL
10.00 mL
10.00 mL
mL
10.00
0.50
gm
0.50 gm
0.50 gm
gm
0.50
0.29
mg
0.29 mg
39

39

B-62

0.52
0.52 ##
100.00100.00 #+
#+
0.50
0.50
4.30
4.30 ##
116.30116.30
0.33
0.33
0.04
0.04
167.00167.0016.70
16.70
71.40 +
+
71.40
2.78
+
2.78 +
21.74 +
+
21.74
31.30
+
31.30 +
0.70
0.70
1.00
1.00
1.00
1.00
0.50
0.50
0.50
0.50
10.00
10.00
10.00
10.00
17.24
17.24

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

Diatrizoate Meglumine
Meglumine Injection
Injection 60%
60%
Diatrizoate
30%
30%
Diatrizoate Meglumine
Meglumine
Diatrizoate
and Diatrizoate
Diatrizoate Sodium
Sodium 66%
66% -- 10%10%and
60% -- 30%
30%
60%
52%
8%
52% - 8%50%
25%
50% - 25%
34.3% -- 35%
35%
34.3%
28.5%
29.1%
28.5% - 29.1%
Diatrizoate Meglumine
Meglumine
Diatrizoate
and Iodipamide
Iodipamide Meglumine
MeglumineandDiatrizoate Sodium
Sodium50%
Diatrizoate
50%
25%
25%
20%
20%
Diazepam
Injection
Diazepam Injection
D&oxide Injection
Injection
Diazoxide*Dibucaine*Dibucaine
Dibucaine Hel
HCI andand Dextrose
Dextrose
Dibucaine
Dicloxacillin Sodium
SodiumDicloxacillin
Dicyclomine
HCI
Injection
Dicyclomine HCI Injection
Diethylstifbestrol Injection
Injection
Diethylstilbestrol
Diethylstilbestrol
Diphosphate
Diethylstilbestrol Diphosphate
Digitoxin Injection
Injection
Digitoxin
Digoxin
Injection
Digoxin Injection
DigoxinDigoxin Immune
Immune FabFabDihydroergotamine
Dihydroergotamine Mesylate
Mesylate
Dihydroergotamine
Mesylate,
HeparinDihydroergotamine Mesylate, Heparin
Sodium
Sodium && Lidocaine
Lidocaine HCIHCIDihydrostreptomycin
Dihydrostreptomycin Sulfate
SulfateDihydrotachysterol
Dihydrotachysterol
Diluent
Diluent forfor Meningococcal
Meningococcal Vaccine
VaccineDimenhydrinate
Injection
Dimenhydrinate Injection
DimethylSulfoxide Irrigation
Dimethyl Sulfoxide Irrigation
Dimercaprol
Dimercaprol
Dinoprost Tromethamine
Dinoprost Tromethamine
Diphenhydramine HCI Injection
Diphenhydramine HCI Injection
Diphenidol
Diphenidol
Diphtheria Antitoxin,Pur.Conc.(equine)
Diphtheria Antitoxin,Pur.Conc. (equine)
Dipyridamole
Dipyridamole
Dobutamine HCI
Dobutamine HCI

1.00 mL
mL
1.00
4.40 mL
mL
4.40

5.00
5.00
1.10
1.10

2.30 mL
mL
1.40 mL
mL
1.40
2.80 mL
mL
mL
2.80 mL
2.80 mL
mL
2.80
2.80 mL
mL
2.80

2.17
3.57
1.80
1.80
1.80
1.80
1.80
1.80
1.80
1.80

0.11 mL
mL
0.11
1.00 mL
mL
1.00
4.00
mL
4.00 mL
0.90 mL
mL
0.90
0.43
mg
0.43 mg
10.00 mg
mg
10.00
0.14
mg
0.14 mg0.07
mg0.07 mg
0.29
0.29 mgmg
0.29
0.29 mgmg
7.14
mg
7.14 mg
7.14
7.14 mgmg
0.045
0.045 mg
mg
0.025
mg
0.025 mg
5.00 mg
5.00
mg
0.014
0.014 mg
mg

45.45
5.00
1.25
1.25
5.56
5.56
11.60
+
11.60 +
0.50 +
+
0.50
35.70
+
35.70 +
71.43
71.43
16.70 +
+
16.70
17.20
+
17.20 +
0.70 ++
0.70
0.70 ++
0.70
111.00+111.00 +200.00+200.00 +1.00
1.00
357.00357.001667.00
1667.00 units
units(Heparin)(Heparin)
10.00 mg
10.00
mg
0.03
mg
0.03 mg
5.00
5.00 mL
mL
1.25
mg
1.25 mg
10.00
10.00 mL
mL
5.00
mg
5.00 mg
0.57
0.57 mgmg1.50
mg1.50 mg
0.30
0.30 mgmg
3.00
mL
3.00 mL
0.14 mg
0.14 mg0.90
mg0.90 mg

0.003 ++
0.003

40

40

B-63

0.50
0.50 ++
166.67166.67
1.00
1.00
4.00
4.00
0.50
0.50 ++
1.00
1.00
8.77
8.77
3.40
3.40 ++
16.67
16.67
1.67
1.67
37.70
37.70
5.56
5.56

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

Dopamine HCIHCIDopamine
Dopamine
HCI
Dextrose
Dopamine HCl inin Dextrose
Doxapram HCIHCI Injection
Injection
Doxapram
Doxorubicin HCIHCI forfor Injection
Injection
Doxorubicin
Doxycycline Hyclate
Hyclate forfor Injection
Injection
Doxycycline
Dromostanolone Propionate
Propionate
Dromostanolone
Droperidol
Droperidol
Dyphylline Injection
Injection
Dyphylline

0.30 mgmg
0.30
0.30 mgmg
0.30
1.50 mg
mg
1.50
1.93
1.93 mgmg
4.40
4.40 mgmg1.40
mg1.40 mg
0.14
0.14 mgmg
7.10’
mg
7.10 mg

16.67
16.67 ++
16.67 ++
16.67
3.30
3.30 ++
2.20#+
2.20 #+
1.14
1.14 ++
3.57
3.57
35.70
35.70
0.70
0.70 ++

50.00
50.00 mg
mg
25.00
mg
25.00 mg
0.60
0.60 mgmg
10.00
mL
10.00 mL--.---.-- mL
mL

0.10
0.10 ++
0.20
0.20
8.33
8.33 ++
0.50
0.50
0.50
0.50

--.---.-- mL
mL

0.50
0.50

--.---.-- mL
mL

0.50
0.50
5.40 ++
5.40
280.00280.001.70 ++
1.70
357.00+357.00 +200.00200.000.035
0.035
1250.00
1250.000.80
0.80
357.00 ++357.001.00+
1.00 +
10.00
10.00
250.00-I250.00 +192.31
192.31-E-EEdetate
Edetate Calcium
Calcium Disodium
Disodium
EdetateEdetate Disodium
Disodium
Edrophonium
Edrophonium Chloride
Chloride Injection
Injection
Electrolyte
SolutionsLVP
Electrolyte Solutions- LVP .
MultipIe
Multiple Electrolytes
Electrolytes TypeType 1 &&2
Multiple
Electrolytes
andInvertMultiple Electrolytes and Invert Sugar
Sugar
Type
and 3
Type 1,2,
1,2, andMultipleand Dextrose
Dextrose
Multiple Electrolytes
Electrolytes andType
1,2,3,and
4
Type 1,2,3,and 4
EmetineHCl
Emetine HCIEnalaprilat
Enalaprilat
Ephedrine
Injection
Ephedrine Sulfate
Sulfate Injection
Epinephrine
Injection
Epinephrine Injection
Epinephrine Suspension
Suspension
Epinephrine
Ergocalciferol (D2)
(D2)
Ergocalciferol
ErgoloidMesylates
Ergoloid Mesylates
Ergonovine Maleate
MaleateErgonovine
Ergotamine Tartrate
Tartrate
Ergotamine
Erythromycin
GluceptatelLactobionate
Erythromycin Gluceptate/Lactobionate
Esmolol
Esmolol
Estradiol (aqueous)(aqueous)
Estradiol
Estrogens(Combined) Aqueous
Aqueous
Estrogens (Combined)

0.93
0.93 mgmg
0.018
0.018 mgmg
3.00 mgmg
3.00
0.014 mgmg
0.014
0.025 mgmg
0.025
142.80units
142.80 units
0.004 mg
mg
0.004
6.00
mcg
6.00 mcg
0.014 mg
mg
0.014
5.00
mg
5.00 mg
0.50 mgmg
0.50
0.02 mg
mg
0.02
0.026
mg
0.026 mg
EstroneEstrone0.36 mgmg
0.36
0.057 mg
mg
0.057
0.057
mg
0.057 mg
1.00 mg
mg
1.00
1.40
mg
1.40 mg0.029 mgmg
0.029

Estrogens ConjugatedConjugatedEstrogens
Estrogenic
Substances oror Estrogens
Estrogens
Estrogenic Substances
Estrone Aqueous
Aqueous Suspension
Suspension
EstroneEthacrynate
SodiumEthacrynate Sodium
Ethamivan Injection
Injection
Ethamivan
Ethylnorepinephrine
HCI Injection
Injection
Ethylnorepinephrine Hel

41

41

B-64

14.00
14.00
88.00
88.00
88.00 ++
88.00
5.00 +
5.00 +
3.60
3.60
172.40 +172.40 +ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

EtidocaineHCI
HC)
Etidocaine
Etidocaine
HCI
and Epinephrine
Epinephrine
Etidocaine HCI and
Etidronate Disodium
Disodium
Etidronate
Etomidate
Injection
Etomidate Injection
Etoposide Injection
Injection
Etoposide
Evans Blue
Blue Injection
Injection
Evans

5.50mgmg
5.50
5.50 mgmg
5.50
7.50 mg
mg
7.50
0.60
0.60 mgmg
2.57
2.57 mgmg0.36
mg0.36 mg

0.90
0.90
0.08
0.08
0.67
0.67
8.35
8.35
1.95
1.95 ##
14.00
14.00 ++

~F~

Factor IX
IX
FactorFamotidine
Famotidine
Fat Emulsion
Emulsion
Fat
Fentanyl Citrate
CitrateFentanyl
Fentanyl
Citrate andand Droperidol
Droperidol
Fentanyl Citrate
Ferrous Citrate
Citrate Fe59 Injection
Injection
FerrousFibrinogen
Fibrinogen
Fibrinogen, Dried
Dried
Fibrinogen,and Desoxyribonuclease
Desoxyribonuclease
Fibrinolysin andFibrinolysin
18
Injection
Flurodopa
F
Flurodopa
Injection
Floxuridine
Floxuridine
Fluorodeoxyglucose F18
Fluorodeoxyglucose
Fluorescein Sodium
Fluorescein
Sodium Injection
Injection
Fluorouracil
Fluorouracil Injection
Injection
Fluphenazine
Fluphenazine HCIHCI
Flupenthixol Decanoate
Decanoate
Flupenthixol
FolateFolate Sodium
SodiumFolic Acid
Acid Injection
Injection
Folic
Fructose
Fructose
Fructose andand Sodium
Sodium Chloride
Chloride
Fructose
Furosemide Injection
Injection
Furosemide

50.00
50.00 units
units
0.30
0.30 mgmg(10%)(10%) 3.20
3.20 mL
mL(20%)(20%) 1.60
1.60 mL
mL
0.10
0.10 mgmg
0.004
mg0.004 mg
Fentanyl
Fentanyl
7.00
7.00 mL
mL
30.00
mg30.00 mg
30.00
30.00 mg
mg
1.00
units
1.00 units
7.00
7.00 mL
mL
0.60
mg0.60 mg
7.00 mL
7.00
mL
10.70 mgmg
10.70
15.00 mg
mg
15.00
0.03
mg
0.03 mg
0.57 mgmg
0.57
0.01 mg
mg
0.01
0.014
mg
0.014 mg
10.00 mL
mL
10.00
10.00
mL
10.00 mL1.40
mg1.40 mg

0.10
0.10
16.67
16.67
1.56
1.56
3.13
3.13
50.00
50.00
1250.00
1250.00
25.00
25.00 ++
0.17
0.17
0.17
0.17
5.00
5.00
25.00
25.00 ++
8.33#
8.33 #
25.00
25.00
0.47
0.47
0.33 ##
0.33
166.67166.678.77
8.77
500.00500.00357.10 +357.10 +0.50 +
0.50 +
0.50 +
0.50 +
3.60 +
3.60 +-G-~G-

1.00 mgmg
1.00
mL
7.00 mL
7.00
3.00
mg
3.00 mg0.11
mg0.11 mgmL
1.00 mL
1.00
0.04
units
0.04 units

Gallamine Triethiodide
Triethiodide
Gallamine
Gallium
CitrateGa67 Injection
Injection
Gallium Citrate Ga67
Gentamicin Sulfate
SulfateGentamicin*Gentamicin
Sulfate*Gentamicin Sulfate
Globulins (Humans)(Humans)
Globulins
Glucagonfor
Injection
Glucagon for Injection

42

42

B-65

5.00 +
5.00 +
25.00 +
25.00 +
1.70 +
1.70 +
45.46
45.46
5.00
5.00
125.00125.00ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

GlycineGlycine Irrigation
Irrigation
Glycopyrrolate
GJycopyrrolate
GoldGold Au198
Au198 Injection
Injection
Gold
SodiumGold Sodium Thiomalate
Thiomalate Injection
Injection
Gonadorelin
Gonadorelin HCl
HCI--.---.-- mL
mL

0.009
0.009 mgmg
7.00
7.00 mL
mL
1.00
mg1.00 mg1.40
1.40 mcg
mcg

0.50
0.50 ++
555.50555.50 ++25.00
25.00 -t+
5.00
5.00
3.60
3.60

0.07
0.07 mgmg
4.00
mg
4.00 mg
143.OO units
143.00units
143.00units
143.00 units
10.00
10.00 mL
mL
10.00
mL
10.00 mL--.-mg--.-- mg
20.00
20.00 mL
mL
0.60
mg0.60 mg0.04
mg0.04 mg0.071
mg0.071 mg71.40
71.40 -I+
1.25
1.25
0.03
0.03
0.03
0.03
0.50
0.50
0.50
0.50
0.30
0.30 ++
0.25
0.25
8.35
8.35
125.00125.00 ++
70.42
70.42

2.14
2.14 units
units
3.50
mg
3.50 mg
4.00
4.00 mgmg
1.07
mg
1.07 mg4.00
mg4.00 mg4.00
mg4.00 mg
0.057 mg
0.057
mg
14.30
mcg
14.30 mcg
14.30
mg
14.30 mg4.50
mg4.50 mg1.40 mg
mg
1.40
0.007
mg
0.007 mg0.007
mg0.007 mg2.30+
2.30 +
1.45+
1.45 +
1.25+
1.25 +
4.67
4.67
1.25
+
1.25 +
1.25+
1.25 +
88.00+
88.00 +
0.40 +
+
0.40
0.35
0.35
1.10 +
+
1.10
3.60
+
3.60 +
714.30 ++714.30714.29714.29

-H-HHaloperidol,
Haloperidol, Decanoate
Decanoate andand Lactate
LactateHemin
for
Injection
Hemin for Injection
Heparin
Heparin Sodium
Sodium andand Calcium
Calcium
HeparinSodiumInjection
Heparin Sodium Injection
Heparin
Heparin Lock
Lock Flush
Flush Solution
Solution
Heparin
Heparin andand Sodium
Sodium Chloride
Chloride
Hetacillin
Potassium
Hetacillin Potassium
Hetastarch
Hetastarch
Hexafluorenium
Hexafluorenium Bromide
Bromide Injection
Injection
Histamine
Phosphate
Histamine Phosphate
Hyaluronate
Hyaluronate Sodium
SodiumHyaluronidase
Hyaluronidase Injection
Injectionandand forfor Injection
Injection
Hydralazine
Hydralazine HCIHCI Injection
Injection
Hydrocortisone
Suspension
Hydrocortisone Suspension
Hydrocortisone
Hydrocortisone Acetate
AcetateHydrocortisone
SodiumHydrocortisone Sodium Phosphate
Phosphate
Hydrocortisone
Hydrocortisone Sodium
Sodium succinate
succinate
Hydromorphone HCL
Hydromorphone
HCL
HydroxocobalaminHydroxocobalaminHydroxyprogesterone Caproate
Hydroxyprogesterone
Caproate
Hydroxystilbamidine
Isethionate
Hydroxystilbamidine Isethionate
Hydroxyzine
HCI Injection
Hydroxyzine HCIInjection
Hyocyamine
SulfateHyocyamine Sulfate
Hyocyamine Sulfate
Sulfate andand ScopoIamine
Hyocyamine
Scopolamine-I-10.31 mg
mg
0.31
30.86 mg30.86 mg-.-- mg-.-- mg7.14 mg7.14 mg
1.00 mg
1.00 mg

Idarubicin HCIHCI Injection
Injection
Idarubicin
Ifosfamide
Ifosfamide
Imipenem
Imipenem
Imipenem andand Cilastatin
Ciiastatin
Imipenem
Imipramine
HCI
Injection
Imipramine HCI Injection

43
43

B-66

16.13 #
16.13 #
0.16 #+
0.16 #+
0.23 +
0.23 +
0.70
0.70
5.00 -I5.00 +ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

Immune Serum
Serum Globulin
Globulin
Immune5.50 mL
mL
5.50
Indigotindisulfonate
Sodium Injection
Injection
Indigotindisulfonate Sodium
1.00 mL
mL
1.00
Indium In111
In111 Oxyquinoline
Oxyquinoline
Indium7.00 mL
mL
7.00*Indium Pentetate
Pentetate Inlll,Injection
In111' Inj~tion*Iridium0.50 mL
mL
0.50
Indium Chlorides
Chlorides Inll3m
In113m Injection
Injection
Indium2.00 mL
mL
2.00
Indocyanine Green
Green
Indocyanine
0.70 mgmg
0.70
Indomethacin Sodium
SodiumIndomethacin
0.20
0.20 mgmg
InsulinInsulin2.00 Units
units
2.00
Insulin Human
Human
w-m
InsulinInterferon Alfa-nl
Alfa-nl
77142.00 units
unitsInterferon
77142.00
Interferon
Alfa-n33571.00
units
Interferon Alfa-n33571.00 units
Interferon Alfa
Alfa -- 2a428571.00 units
unitsInterferon
428571.00
Interferon Alfa
Alfa -- 2b514285.00
unitsInterferon
514285.00 units
Inulin50.00 mg
mg
Inulin50.00
Invert Sugar
Sugar
2.38
mL
Invert2.38 mL
Iodamide meglumine
meglumine - 24%
24%
mL
Iodamide4.30 mL
Iodide Sodium
Sodium 1123 Solution
Solution
7.00 mL
mL
Iodide7.00
Iodinated
1125
Albumin
Injection
7.00
mL
Iodinated
Albumin Injection
7.00 nil2
Iodide Sodium
Sodium 1125 Solution
Solution
7.00 mL
mL
Iodide7.00
Iodinated
1131
Albumin
Injection
7.00
mL
Iodinated
Albumin Injection
7.00 mL
Iodinated 1131 Albumin
Albumin AggregatedAggregated Injection
Injection 7.00 mL
mL
Iodinated
Iodohippurate
Sodium1131
Injection
7.00
mL
Iodohippurate Sodium 1131 Injection
mL
Bengal Sodium
Injection
7.00 mL
Rose Bengal
Sodium 1131 Injection
7.00
mL
IodideSodium1131
Solution
7.00
mL
Iodide Sodium 1131 Solution
mL
Iodipamide Meglumine
Meglumine Injection
Iodipamide
Injection - 52%
0.60 mL
mL- 10.5%1.40 mL
mL
Iodipamide Meglumine
Iodipamide
Meglumine Diatrizoate meglumine
0.14 mL
Diatrizoate
megluminemL*Iohexol*Io hex01
43.70 mg I
*Iopamidol
40.00 mg I
*Iopamidol*IophendylatemL
0.22 mL*Iophendylate Injection
Injection
Iothalamate
Meglumine Injection
80%Iothalamate Meglumine
Injection
80%- 1.40
1.40 mL
mL
mL
60%- 2.00 mL
43%- 5.70 mL
mL
30%- 4.30 mL
mL
17.2% -5.70 mL
mL
17.2%
Iothalamate
Iothalamate Meglumine
Meglumine Iothalamate
1.50
1.50 mL
mL
Iothalamate Sodium
Sodium 52%-26%
52%-26%Iothalamate
Sodium1.50
1.50 mL
mL
66.8% -Iothalamate SodiummL
54.3%
0.90 mL
54.3% loxaglate
Meglumine
3.00 mL
mL
3.00
Ioxaglate Meglumine
44
44

B-67

0.91
0.91
5.00
5.00 ++
25.00
25.00 ++
28.00
28.00 -t+
87.50
87.50 ++
7.10
7.10 ++
25.00
25.00
2.50
2.50
0.80
0.80 ++
0.65/10,000
0.65/10,000 ##0.14/1000.14/100 ##0.10/10,000
0.10/10,000 #0.10/10,000
0.10/10,000 #0.10
0.10 ++
2.10
2.10
1.20
1.20
25.00
25.00 ++
25.00
25.00 ++
25.00
25.00 f+25.00
25.00 l-+25.00
25.00 -i+
25.00 +
25.00
+
25.00
25.00 ++
25.00 i-+
25.00
8.33
8.33
3.60 +
+
35.71
0.11
0.6 +
+
0.90 +
3.57
2.50
0.90 +
1.16
0.90
0.90

f
3.35 ++
3.35 +
5.56
1.67
1.67

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

IoxaglateSodium
SodiumIoxaglate
Iron
Dextran
Injection
Iron Dextran Injection
Iron Sorbitex
Sorbitex
Iron
Isobucaine
Isobucaine HCIHCI andand Epinephrine
Epinephrine
Isoniazid
Isoniazid
Isoproterenol
Isoproterenol HCIHCI Injection
Injection
Isosulfan Sulfate
SulfateIsosuifan
Isoxsupine
Isoxsupine HCIHCI Injection
Injection

3.00
mL
3.00mL
0.90
0.90 mgmg
0.50
mL
0.50 mL
0.14
0.14 mL
mL
20.00
mg
20.00 mg
0.014
0.014 mgmg0.71
mg0.71 mg
0.14
0.14 mg
mg

1.67
1.67
5.60
5.60
10.00
10.00 ++
35.70
35.70
0.30
0.30 ++
350.00350.00
7.00
7.00
35.70
35.70 ++

-K-KKanamycin
Kanamycin Sulfate
Sulfate Injection
Injection
Ketamine
HCI
Ketamine HCl

7.50
7.50 mgmg
13.00
mg
13.00 mg

0.67
0.67 ++
0.40
0.40 ++-L-LLabetalol
Labetalol HCI
HCILeucovorin
Leucovorin Calcium
Calcium Injection
Injection
Leuprolide
AcetateLeuprolide Acetate
Levallorphan
Levallorphan Tartrate
Tartrate Injection
Injection
Levarterenol
Levarterenol
Levorphanol
Tartrate Injection
Levorphanol Tartrate
Injection
Levothyroxine
SodiumLevothyroxine Sodium forfor Injection
Injection
Lidocaine HCIHCI Injection
Injection (with(with D5W)
D5W)
Lidocaine
Lidocaine
HCI
withEpinephrine
Lidocaine HCI with Epinephrine
Lincomycin HCI
HClLincomycin
Liver Derivative
Derivative Complex
ComplexLiver
Lorazepam
Lorazepam
Loxapine
Loxapine

4.30
mg
4.30 mg
2.57
2.57 mgmg
0.03
mg
0.03 mg
0.04
0.04 mgmg
0.06
0.06 mgmg
0.04
mg
0.04 mg0.007 mgmg
0.007
4.50 mg
mg
4.50
7.00
mg
7.00 mg
10.00 mg
mg
10.00
0.03
mg
0.03 mg
0.05 mg
mg
0.05
0.71
mg
0.71 mg

1.20
1.20 ++
1.95
1.95
16.67
16.67
125.00125.0083.33
83.33
125.00 +125.00 +714.00714.001.10 +
1.10 +
0.70 +
0.70 +
0.50 +
0.50 +
166.67166.67100.00 +100.00 +7.00
7.00-M-M57.10 mg
57.10 mg
11.00 ug
11.00 ug
11.00 ug
11.00 ug
120.00 mg
120.00 mg
2.00 g2.00 g120.00 mg
120.00 mg
0.40 mg
0.40 mg
14.30
mg
14.30
mg
0.20 rng
0.20 mg

Magnesium Sulfate
SulfateMagnesium
Manganese Chloride
Chloride Injection
Injection
Manganese
Manganese
SulfateManganese Sulfate
Mannitol << =
= 10%
10%
MannitolMannitol>
10%
Mannitol > 10%
Mannitol andand Sodium
Sodium Chloride
Chioride
Mannitol
Mechlorethamine
HCI
for Injection
Injection
Mechlorethamine HCI for
Medroxyprogesterone Acetate
AcetateMedroxyprogesterone
Menadiol
SodiumDiphosphate(K-4)
Menadiol Sodium Diphosphate (K-4)

45

45

B-68

0.09 +
0.09
0.45 ++
0.45
0.45 ++
0.45
0.04 +
0.04
2.50
2.50
0.04 +
0.04
12.50 +#-k12.50
0.35 #+
0.35 +
25.00
25.00 +ICCVAM Test Method Evaluation Report: Appendix B5

Menadione
Menadione
N.
N. Meningococcal
Meningococcal Polysaccharide
Polysaccharide
Pm.
Pur. Bulk,
Bulk, Group
Group AA
N.Meningococcal
N.Meningococcal Polysaccharide
Polysaccharide
Pur.
Pur. Bulk,
Bulk, Group
Group CC
Meningococcal
Polysaccharide
Meningococcal Polysaccharide Vaccine
VaccineGroup
Group AA
Meningococcal
Meningococcal Polysaccharide
Polysaccharide Vaccine
VaccineGroup
CGroup C
Meningococcal
Meningococcal Polysaccharide
Polysaccharide Vaccine
Vaccine
Group
A
and
C
Group A and C
Menotropin
Menotropin
Meperidine
Meperidine HCl
HCI Injection
Injection
Mephentermine
Mephentermine Sulfate
SulfateMepivacaine
Mepivacaine HCl
HCI
Mepivacaine
Mepivacaine HCl
HCI andand Levonordefrin
Levonordefrin
Meprobamate
Injection
Meprobamate Injection
Meprylcaine
Meprylcaine HCIHCI andand Epinephrine
Epinephrine
Mercaptomerin
SodiumMercaptomerin Sodium
Mersalyl
Mersalyl with
with theophylline
theophylline
Merethoxylline
MerethoxyUine Procaine
Procaine
Mesoridazine
Mesoridazine Besylate
Besylate Injection
Injection
Metaraminol
Bitartrate
Metaraminol Bitartrate
Methadone
Methadone HCIHCIMethandroil
Methandroil
Methapyrilene HCI
HClMethapyrilene
Methicillin Sodium
SodiumMethicillin
Methiodal
SodiumInjection
Methiodal Sodium Injection
Methocarbamol Injection
Injection
Methocarbamol
Methohexital
SodiumMethohexital Sodium
Methotrexate Sodium
Sodium Injection
Injection
Methotrexate
Methotrimerprazine
Methotrimerprazine
Methoxamine HCI
HCl
Methoxamine
Methyldopate
HCl
Methyldopate HCl
Methylene Blue
Blue Injection
Injection
Methylene
Methylergonovine
MaleateMethylergonovine Maleate
Methylprednisolone Acetate
Acetate Sus
Sus
Methylprednisolone
Methylprednisolone
Sodium
Succinate
Methylprednisolone Sodium Succinate
for Injection
Injection
for
Metoclopramide
Metoclopramide
Metocurine Iodide
Iodide
Metocurine

May 2008

0.09
mg0.09 mg58.30
58.30 ++

0.25
0.25 ug
ug

20.00
20.00

0.25
0.25 ug
ug

20.00
20.00

0.025
0.025 ug
ug

200.00200.000.025
0.025 ug
ug

200.00200.000.05
0.05 ug
ug
2.00
units
2.00 units
2.14
2.14 mgmg
mg
0.64
0.64 mg
6.60
6.60 mgmg
6.60
mg6.60 mg
1.00
1.00 mgmg
mg
6.60
6.60 mg
3.57
3.57 mgmg2.90
mg2.90 mg
2.90
2.90 mgmg0.71
mg0.71 mg1.43
1.43 mgmg
0.57
mg
0.57 mg1.43 mg
1.43
mg
0.60
mg
0.60 mg50.00 mgmg
50.00
1300.00 mg
mg
1300.00
28.60
mg
28.60 mg10.00
mg10.00 mg-.-- mg
-.-mg
0.28
mg
0.28 mgmg
0.25 mg
0.25
10.00
mg
10.00 mg2.00 mL
mL
2.00
2.90
mcg
2.90 mcg
0.80 mgmg
0.80

100.00100.002.50
2.50 ++
2.40
2.40 f+7.80
7.80 ++
0.80
0.80 ++
0.80
0.80 ++
5.00
5.00
0.80
0.80 ++
1.40
1.40
1.72
1.72
1.72
1.72
7.00 ++
7.00
3.50
3.50 f+8.80 ++
8.80
3.50
3.50
8.33
8.33
0.10
+
0.10 +
0.004
0.004
0.20 +
+
0.20
0.50
+
0.50 +
2.00 +
+
2.00
17.90
+
17.90 +
20.00 +
+
20.00
0.50
+
0.50 +
2.50 +-t2.501.70 +
1.70 +
6.25
6.25

30.00 mgmg
30.00
2.00 mg2.00 mg
0.40 mg
0.40 mg

0.17 -I0.17 +2.50 +
2.50 +
12.50 +
12.50 +

46

46

B-69

ICCVAM Test Method Evaluation Report: Appendix B5MetoprololTartrate
Tartrate
Metoprolol*'Metrizamide“Metrizamide
Metrizamide
Metrizamide
Metrizoic
Metrizoic
MetronidazoleHCI
HCI
Metronidazole
MetyraponeTartrate
TartrateInjection
Injection
Metyrapone
Mezlocillin
SodiumMezlocillin Sodium
MiconazoleInjection
Injection
Miconazole*'Miconazole
Injection*Miconazole Injection
Midazolam
HCI
Midazolam HCI
Minocycline I-ICI
HCI
Minocycline
Mithramycin forfor Injection
Injection
Mithramycin
Mitomycin forfor Injection
Injection
Mitomycin
Mitoxantrone HCl
HCI
Mitoxantrone
Molybdenum
Molybdenum
Morphine Sulfate
SulfateMorphine*'Morphine Sulfate
Sulfate*Morphine
Morrhuate Sodium
SodiumMorrhuate
Moxalactam
Moxalactam
Muromonab-CD3
Muromonab-CD3

May 2008

0.20
0.20mgmg
4.29
4.29mg
mgI I
634.00634.00mg
mgI I
73%73%- - 2.90
2.90mLmL
46.18%
46.18%- - 1.00
1.00mLmL
15.00
15.00mgmg
15.00
15.00 mgmg
87.50
87.50 mg
mg
40.00
mg
40.00 mg
0.29
0.29 mgmg
0.35
mg0.35 mg
4.00
4.00 mgmg
0.05
mg
0.05 mg
6.51
0.51 mgmg0.36
mg0.36 mg2.30
2.30 ug
ug
0.29
mg
0.29 mg
0.014
0.014 mgmg
3.60
mg3.60 mg
100.00100.00 mgmg
0.10
0.10 mgmg

25.00
25.00++
1.17
1.17
0.008
0.008
1.72
1.72
5.00
5.00
0.35
0.35 ++
0.35
0.35
0.06
0.06 ++
0.10
0.10 ++
0.69
0.69
14.30
14.30
1.25
1.25 ++
100.00100.009.80
9.80 ## ++13.90
13.90 ##
2.17
2.17
17.00
17.00
14.29
14.29
1.40
+
1.40 +
0.05
0.05 +
+
50.00
50.00

-N-NNafcillin
Nafcillin
Nalbuphine
Nalbuphine HCI
HClNalorphine Hel
HClNalorphine
Naloxone Hel
HCl Injection
Injection
Naloxone
Neomycin Sulfate
SulfateNeomycin
Neostigmine Methylsulfate
Methylsulfate
Neostigmine
Netilmicin
Netilmicin
NiacinNiacinNiacinamide Injection
Injection
Niacinamide
Nicotinamide
Nicotinamide
Nikethamide
Nikethamide
Nine Vitamin
Vitamin Injection
Injection
Nine
Nitrofurantoin
Nitrofurantoin
Nitroglycerin
Nitroglycerin
Nitroprusside Sodium
SodiumNitroprusside
Norepinephrine
bitartrate
Norepinephrine bitartrate
Novobiocin forfor Injection
Injection
Novobiocin

40.00 mgmg
0.13 +
i40.00
0.13
1.67
3.00
mg
3.00 mg
1.67
0.43
11.60
mg
0.43 mg
11.60
0.01 mgmg
500.000.01
500.003.80 mg1.30 +
+
mg
3.80
1.30
0.04 mgmg
125.000.04
125.001.25 ++
4.00 mg
mg
4.00
1.25
1.43 mg3.50 ++
mg
1.43
3.50
3.50 ++
1.43 mg
mg
1.43
3.50
7.14
0.70 mg
mg
0.70
7.14
0.90
ml:25%
sol
5.56
0.90 011:25% sol 5.56
1.00 mL
mL
5.00
1.00
5.00
2.50
2.00
mg
2.50 mg
2.00
50.00 ug
0.10 ++
50.00 ug
0.10
1.40 mcg
3.67
1.40 meg
3.67
0.06 mg83.40 +
0.06 mg83.40 +
7.10 nig
0.70
7.10 mg
0.70

4747

B-70

ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

-O-0-

Opium
Opium Alkaloids
Alkaloids HCI
HCIOrphenadrine
CitrateOrphenadrine Citrate Injection
Injection
OuabainOuabainOxacillin
Oxacillin Sodium
Sodium
Oxymorphone
Oxymorphone HCIHCIOxytetracycline
Oxytetracycline
Oxy tocin
Oxytocin

0.28
0.28 mgmg
0.86 mg0.86
mg0.007 mg
0.007
mg
25.00
25.00 mgmg
0.021
0.021 mgmg
12.50
12.50 mgmg
0.14
0.14 units
units

17.86
17.86
5.80
5.80 ++
714.29714.290.20
0.20 ++
238.10+238.10 +0.40+
0.40 +
35.70
35.70·-P-~P-

Pancuronium
Bromide0.10 mg
Pancuronium Bromide
0.10
mg
PapaverineHCl1.70 mg
Papaverine Hel
1.70
mgParaldehyde
0.15ml,
Paraldehyde
0.15 mL
Parathyroid
Hormone0.57 units
Parathyroid Hormone
.
0.57
units
Penicillin G Benzathine Suspension
50,OOO.OOunits
Penicillin G Benzathine Suspension
50,000.00 units
Penicillin G Potassium
50,OOO.OOunits
Penicillin G Potassium
50,000.00
units
Penicillin G Procaine
Penicillin G Procaine
and Suspension
50,OOO.OOunits
and Suspension
Penicillin
G Procaine, Dihydrostreptomycin 50,000.00 units
Penicillin
G Procaine, Dihydrostreptomycin
Sulfate, Chlorpheniramine
Maleate andSulfate,Chlorpheniramine
Dexamethasone
Suspension Maleate and--.-- units
Dexamethasone
Suspension--.-- units
Penicillin
G Procaine,
Dihydrostreptomycin
Penicillin
G Procaine, Dihydrostreptomycin
Sulfate, Prednisolone
Suspension--.-- units
Sulfate,Prednisolone
Suspension-- .•• units
Penicillin G Sodium
50,OOO.OO
units
Penicillin
G
Sodium50,000.00
Pentagastrin
0.006unitsmg
Pentagastrin Isethionate
0.006mgmg
Pentamidine
4.00
Pentamidine
Isethionate
4.00 mgmg
Pentobarbital Sodium Injection
6.00
Pentobarbital
SodiumInjection
6.00
mg
Pentazocine Lactate Injection
0.86 mg
Pentazocine Lactate Injection
0.86 mg
mg
Perphenazine
0.14Perphenazine
0.14
mg
Phenobarbital Sodium Injection
20.00 mg
Phenobarbital Sodium Injection
20.00
mg
0.09 mg
Phenolsulfonphthalein
Phenolsulfonphthalein
0.09
mg
Phentolamine Mesylate
0.86
Phentolamine Mesylate
0.86
mg
7.14 mg
Pentylenetetrazol
Pentylenetetrazol
7.14
mg
0.20 mg
Phenylephrine HCI
Phenylephrine
HCIInjection
0.20 mg
20.00
Phenytoin Sodium
Phenytoin
SodiumInjection
20.00
0.06 mg
Physostigmine Salicylate
Physostigmine
0.06
0.36 mg
Phytonadione Salicylate
Phytonadione
0.36 mg
75.00
Piperacillin Sodium
4.30 mg
Piperacillin
75.00
Piperocaine SodiumHCl4.30 mg

Piperocaine HCI

48
48

B-71
I

I

50.00
50.00
2.90 +
2.90 +
33.33
33.33
8.80
8.80
0.01/100 +0.0l/100 +0.01/100 +0.0l/100 +0.01/100 +0.0l/100 +0.01/100 f

0.0l/100 +0.01/100 +0.0l/100 ++0.01/100
0.0l/100 +833.00833.001.25
1.25
0.83 ++
0.83
5.80 +
5.80 +
35.70-i35.70+0.30 -i0.30 +
55.60
55.60
5.80 +
5.80
0.70 +
0.70
25.00 +
25.00+
0.30 +
0.30
83.40 +
83.40
14.00 +
14.00
0.07 +
l0.07
1.16 +
1.16

ICCVAM Test Method Evaluation Report: Appendix B5

Plasma Protein
Protein Fraction
Fraction (5%)(5%)
PlasmaPlicamycin forfor Injection
Injection
Plicamycin
Phosphate
Polyestradiol Phosphate
Polyestradiol
Polymyxin BB Sulfate
SulfatePolymyxin*Polymyxin BB Sulfate
Sulfate*Polymyxin

May 2008

10.00
10.00 mL
mL0.05
mg0.05 mg
1.10
1.10 mgmg1666.70
units
1666.70 units
714.00714.00 units
units

Posterior Pituitary
Pituitary Injection
Injection
Posterior
Potassium
Potassium Acetate
Acetate Injection
Injection
Potassium Chloride
Chloride
Potassium
Potassium
Chloride,Lactated Ringers
Ringers
Potassium Chloride,Lactated
andand Dextrose
Dextrose Injection
Injection
Potassium
Potassium Phosphate
Phosphate Injection
Injection
Potassium
Potassium Phosphate
Phosphate inin Dextrose
Dextrose
Potassium
Potassium Phos.,
Phos., LactatedLactated Ringers
Ringers
Pralidoxine
Pralidoxine Chloride
Chloride
Prednisolone
Prednisolone Acetate
Acetate Suspension
Suspension
Prednisolone
Prednisolone Acetate
Acetate andandPrednisolone
Prednisolone Sodium
Sodium Phosphate
Phosphate
Suspension
Suspension

0.29
0.29 units
units
0.57
0.57 mEq
mEq
0.57
mEq
0.57 mEq

0.50
0.50
100.00100.00 ##
4.55
4.55
0.003
0.003
0.03/100
0.03/100
units
units
17.00
17.00 ++
8.80
8.80 ++
8.80
8.80 ++-.--.-- mL
mL
4.43
4.43 mgmg
10.00
mL
10.00 mL
10.00
10.00 mL
mL
40.00
mg
40.00 mg
0.16
0.16 mgmg

0.50
0.50 ++
1.10
1.10 ++
0.50
0.50
0.50
0.50
0.10
0.10 ++
31.25
31.25

1.14 mg
1.14
mg
Pred.
Pred. Acct.
Acet.
1.00 mg1.00
mg0.86 mg
0.86
mg
0.57
mg
0.57 mg5.70 mgmg
5.70
5.70 mg
mg
5.70
8.60
mg
8.60 mg-.-- mgmg-.--.-mg-.-- mg

4.39
4.39

Prednisolone Sodium
Sodium Phosphate
Prednisolone
Phosphate
Prednisolone
Sodium
Succinate
Prednisolone Sodium Succinate
Prednisolone Tebutate
Tebutate Suspension
Suspension
Prednisolone
Prilocaine
HCI
Prilocaine HCIPrilocaine Hel
HCl andand Epinephrine
Epinephrine
Prilocaine
Procaine
HCI
Procaine HCIProcaine HCIHCl && Epinephrine
Epinephrine
Procaine
Procaine&
Phenylephrine
HCl
Procaine & Phenylephrine HCIProcaine, Tetracaine
Tetracaine &&Procaine,
Levonordefrin
Injection- .-- mg
Levonordefrin Injection-.-- mg
Propofol
12.00 mgmg
Propofol
12.00
14.30
mg
Procainamide
HCI
Injection
Procainamide Hel Injection
14.30 mg
0.28 mg
mg
Prochlorperazine Edisylate
Edisylate && Mesylate
Mesylate
Prochlorperazine
0.28
1.43
mg
Progesterone
Aqueous&Suspension
Progesterone Aqueous & Suspension
1.43 mg2.80 mg
Promazine
HCI
Injection
Promazine HCI Injection
2.80 mg
1.00 mg
Promethazine HCIHCIPromethazine
1.00 mg
0.43
mgPropantheline Bromide
BromidePropantheline
0.43 mg1.10 mg
Propiomazine HCIHCl Injection
Injection
Propiomazine
1.10 mg
Propoxycaine,Procaine HCIHCI && Levonordefrin
Levonordefrin 6.60
6.60 mgmg
Propoxycaine,Procaine
Propoxycaine,Procaine
HCI&
Propoxycaine,Procaine Hel &6.60 mg
Norepinephrine Bitartrate
Bitartrate
Norepinephrine
6.60 mg
49

49

B-72

5.00
5.00 -t+5.80+
5.80 +
8.80+
8.80 +
0.90+
0.90 +
0.90 +
0.90 +
0.60 +
0.60 +
0.60 +
0.60 +
0.60 +

0.60 +

0.60 +

0.60 +
0.42
0.42
0.35 +
0.35 +
17.90 +
17.90
3.50 +
3.50
1.80 +
1.80
5.00 ++
5.00 +
11.60+
11.60 +
4.60 +
4.60
0.80 ++
0.80 +

0.80 +

0.80 +ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

Propranolol HCIHCI Injection
Injection
Propranolol
Protamine
SulfateInjection
Protamine Sulfate Injection
Protein Hydrolysate
Hydrolysate Injection
Injection
ProteinProthrombin
Complex
Prothrombin Complex
Protirelin
Protirelin
Pyridostigmine Bromide
Bromide
Pycidostigmine
Pyridoxine
HCI
Pyridoxine HCI

0.09 mgmg
0.09
mg0.71 mgmL
10.00 mL
50.00 units
units
50.00
7.00 mcg
mcg
7.00
0.29 mgmg
14.29 mg

55.60
55.60 ++
7.04
7.04 ++
0.50
0.50 i-+
0.10
0.10
0.70
0.70
17.00
17.00 ++
0.40
0.40 ++-Q8.608.60 mg
8.60 mg

Quinidine Sulfate
SulfateQuinidine
Quinidine Gluconate
Quinidine
Gluconate

0.60
0.60 ++
0.60
0.60-R-RRanitidine HCIHCIRanitidine
Reserpine
Reserpine
Riboflavin
Riboflavin
Rifampinfor Injection
Injection
Rifampin for
Ringer's
Injection
Ringer’s Injection
Ringer's
Irrigation
Ringer’s Irrigation
Ringer’s in Dextrose
Ringer's
Dextrose
Ringer’s - Lactated
Injection
Ringer's
Lactated Injection
Ringer’s - Lactated
Ringer's
Lactated in Dextrose
Dextrose
Ritodrine HCI
HCl
Ritodrine
Rolitetracycline forfor Injection
Injection
Rolitetracycline
Rolitetracycline Nitrate
Rolitetracycline
Nitrate&*-"".-'"

0.07 mg0.70 mg-.-- mg-.-mg
10.00 mL
10.00
mL
10.00
mL
10.00 mL
10.00 mL
mL
10.00
10.00
mL
10.00 mL
10.00
mL
10.00 mL“*-.
... ...
5.00
mg
5.00 mg
5.00 mg
mg
5.00

-

7.00
7.00
71.50
+
71.50 +
7.10+
7.10 +
0.20 ++
0.20
0.50 +
+
0.50
0.50
+
0.50 +
0.50
0.50
0.50 +
+
0.50
0.50
0.50
0.50 +
+
0.50
1.00
f
1.00 +
1.00
LOO-S-S0.26 mg
mg
0.26
1.00
unit
1.00 unit
0.29 mg
mg
0.29
0.009
mg
0.009 mg5.50 mg
mg
5.50
1.43
ug
1.43 ug
7.00 mL
mL
7.00
0.02
mcg
0.02 mcg
10.00 mg
mg
10.00
1.29
mEq
1.29 mEq
3.57
mg
3.57 mg

Saralasin Acetate
AcetateSaralasin
Secretin
Secretin
Scopolamine Butylbromide
Butylbromide
Scopolamine
Scopolamine HBr
HBr
Scopolamine
Secobarbital
SodiumInjection
Secobarbital Sodium Injection
Selenious Acid
Acid (Selenium)(Selenium)
Selenious
Selenomethionine
Se75 Injection
Injection
Selenomethionine Se75
Sincalide
Sincalide
Sisomicin Sulfate
SulfateSisomicin
Sodium Acetate
AcetateSodium
SodiumAscorbate
Sodium Ascorbate

50

50

B-73

19.20
19.20
5.00
5.00
17.24
17.24
555.60+555.60 +0.90 +
+
0.90
3.50
+
3.50 +
25.00+
25.00 +
250.00250.000.50 +
0.50 +
3.90 +
3.90
1.40 +
1.40

ICCVAM Test Method Evaluation Report: Appendix B5

Sodium Bicarbonate
Bicarbonate
Sodium
Sodium
Sodium Chloride
Chloride 0.450.9%
0.45-0.9%
Sodium
Sodium Chloride
Chloride 33- 24.3%
24.3%
Sodium
Sodium Chloride
Chloride -- Bacteriostatic
Bacteriostatic
Sodium
Sodium Chloride
Chloride 4.5%4.5%- Lactose
Lactose 3%
3%
Sodium
Sodium Chloride
Chloride Irrigation
Irrigation
SodiumCitrateSodium Citrate
Sodium
Sodium Iodide
IodideSodium
Sodium Lactate
LactateSodium
Phosphate
Sodium Phosphate Injection
Injection
Sodium
Sodium Phosphate
Phosphate P32 Solution
Solution
SodiumSalicylate
Sodium Salicylate
Sodium
Sodium Tetradecyl
Tetradecyl Sulfate
SulfateSodiumThiosalicylate
Sodium Thiosalicylate
Sodium
Sodium Thiosulfate
Thiosulfate
Somatremfor Injection
Injection
Somatrem forSomatropin& Recombinant
Recombinant
Somatropin -- Pituitary
Pituitary &SoybeanOil
Emulsion
Soybean Oil Emulsion
Spectinomycin
Spectinomycin HCl
HCIStreptokinase
Streptokinase
Streptokinase-Streptodornase
Streptokinase-Streptodornase (Local)(Local)Streptokinase-Streptodornase0
Streptokinase-Streptodornase (Th1)
Streptomycin
SulfateStreptomycin Sulfate
Streptozocin
Streptozocin
Succinylcholine
Chloride
Succinylcholine Chloride
Sufentanil
Sufentanil citrate
citrateInvertSugar
Invert Sugar
InvertSugar inSodium Chloride
Chloride
Invert Sugar
in Sodium
Sulbactam
SodiumSulbactam Sodium
Sulfadiazine Sodium
SodiumSulfadiazine
Sulfamethoxazole& Trimethoprim
Trimethoprim
Sulfamethoxazole &Sulfisoxazole
Diolamine
Injection
Sulfisoxazole Diolamine Injection
Sulfobromophthalein
Sulfobromophthalein

May 2008

1.00
5.00
1.00 mEq
mEq
5.00 ++
10.00
0.50
10.00 mL
mL
0.50 ++
1.40
3.57
1.40 mL
mL
3.57 ++
5.00
1.00
5.00 mL
mL
1.00 f+
10.00
mL
0.50
10.00 mL
0.50
10.00
0.50
10.00 mL
mL
0.50 ++
2.50
mEq
2.00
2.50 mEq
2.00
14.30
0.35
14.30 mg
mg
0.35
2.40
mEq
2.00
2.40 mEq
2.00 ++
4.00
1.10
4.00 mgmg
1.10 ++
7.00
mL
25.00
7.00 mL
25.00 ++
9.30
mg
0.54
9.30 mg
0.54
0.14
mL
35.71
0.14 mL
35.71
2.10
2.38
2.10 mgmg
2.38
167.000.03
167.00 mg
mg
0.03 ++
0..25
Iu
20.00
0.25 IU
20.00
0.20
25.00
0.20 ru
IU
25.00
3.13
1.60
3.13 mL
mL
1.60
57.00
0.09
57.00 mgmg
0.09 ++
0.02/10021428.00
IU
21428.00 IU
0.02/100
3000.00 units
0.002
3000.00
units
0.002
1000.00
0.005
1000.00 units
units
0.005
0.25
20.00
20.00 mgmg
0.25 ++
0.13
38.60
38.60 mgmg
0.13 ##
2.00
+
2.50
mg
2.50 mg2.00 +
100.000.05 mg0.05
mg100.00
10.00
mL
0.50
+
10.00 mL
0.50 +
10.00 mL
0.50
10.00
mL
0.50
14.30
mg
0.35
14.30 mg
0.35
50.00 mgmg
0.10 +
+
50.00
0.10
0.20
25.00
mg(sulf)
25.00 mg(sulf)
0.20
0.10
50.00 mg
0.10
50.00
mg
1.00 ++
5.00
mg
5.00 mg
1.00-T-T7.00 mL
mL
7.00
7.00 mL
mL
7.00
7.00
mL
7.00 mL
7.00 mL
7.00 mL
7.00 mL
7.00 mL

Technetium Tc99m
Tc99m Albumin
Albumin AggregatedAggregatedTechnetium
Technetium Tc99m
Tc99m Antimony
Antimony Trisulfate
Trisulfate
Technetium
Technetium Tc99m
Tc99m Dsofenin
DsofeninTechnetium
Technetium
Tc99m
Etidronate
Technetium Tc99m Etidronate
Technetium
Tc99m
Ferpentetate
Technetium Tc99m Ferpentetate
51
51

B-74

25.00 +
25.00 +
25.00
25.00
25.00 +
25.00 +
25.00 +
25.00 +
25.00 +
25.00 +ICCVAM Test Method Evaluation Report: Appendix B5

May 2008

Technetium Tc99m
Tc99m Gluceptate
Technetium
Gluceptate
7.00 mL
mL
Technetium
Tc99m Human
Human Serum
Serum Albumin
Technetium Tc99m
Albumin 7.00 mL
mLTechnetium
Tc99m Lidofenin
Lidofenin
Technetium Tc99m
mL
7.00 mL
Technetium Tc99m
Tc99m Mebrofenin
Technetium
MebrofeninmL
7.00 mL
Technetium
Tc99m Medronate
Technetium Tc99m
Medronate7.00 mL
mL
Technetium
Tc99m
Oxidronate
Technetium Tc99m Oxidronate
7.00 mL
mL
Technetium
Technetium Tc99m
Tc99m Pentetate
Pentetate7.00 mL
mL
Technetium
Technetium Tc99m
Tc99m Sodium
Sodium Pertechnetate
Pertechnetate7.00 mL
mL
Technetium
7.00
mL
Technetium Tc99m
Tc99m Pyrophosphate
Pyrophosphate7.00 mL
Technetium(Pyre- andand trimeta-)
trimeta-)
Technetium Tc99m
Tc99m (pyroPhosphates
7.00 mL
Phosphates
mL
Technetium
TcPPm
Succimer
7.00 mL
Technetium Tc99m Succimer
7.00
mLTechnetium Tc99m
Tc99m Sulfur
7.00
mLTechnetium
Sulfur Colloid
Colloid
7.00 mL
Terbutaline Sulfate
Sulfate0.004
Terbutaline
0.004 mgmg
Teriparatide Acetate
5.00
units
Teriparatide
Acetate5.00 units
Testolactone Suspension
Suspension
1.43 mgmg
Testolactone
1.43
Testosterone(aqueous suspension)
suspe,nsion)
1.43
mg
Testosterone (aqueous
1.43 mg“Tetracaine
Hydrochloride
0.29
mg*Tetracaine Hydrochloride
0.29 mg*Tetracaine HCIHCI and0.20
mg
and Dextrose
Dextrose, *Tetracaine
0.20 mg
Tetracycline
HCI
10.00
mg
Tetracycline HCI
10.00 mg
Tetracycline Phosphate
Complex5.00 mg
Tetracycline
Phosphate Complex
5.00
mg
Thallus
Chloride
Tl201
Injection
7.00 mL
Thallus Chloride T1201 Injection
mL
7.00
Theophylline
5.00 mgand Dextrose
Dextrose
Tbeophylline and5.00
mg
ThiamineHCl
1.43 mg
Thiamine HCI
1.43
mg
Thiamylal
Sodium5.00
mg
Thiamylal Sodium
5.00 mg
Thiethylperazine
Maleate
0.14
Thiethylperazine Maleate
0.14 mgmg
Thiopental
Sodium5.00
mg
Thiopental Sodium
5.,00 mg
Thiotepa
for
Injection
0.80
mg
Thiotepa for Injection
0.80 mg
Thiothixene
HCI
Injection
0.057
mg
Thiothixene HCI Injection
0.057 mg
Thyrotropinfor
Injection
0.14
Iu
Thyrotropin for Injection
0.14 IV
Ticarciilin Disodium
100.00 mg
Ticarcillin
Disodium
100.00mg
75.00
mg
Ticarcillin
DisodiumandClavulan&eTicarcillin Disodium and Clavulanate
75.00 mg
Tobramycin Sulfate
2.50 mgmg
Tobramycin
Sulfate
2.50
6.00
mg’
Tolazoline
HCI
6.00 mg
Tolazoline Hel
14.20 mgmg
Tolbutamide
Sodium
14.20
Tolbutamide Sodium
10.00
mg
Tranexamic
10.00 mg
Tranexamic Acid
Acid
1.14 mgmg
Triamcinolone
Acetate Suspension
Suspension
1.14
Triamcinolone Acetate
1.14
Triamcinolone Acetonide
Acetonide
1.14 mgmg
Triamcinolone
0.70mg
Triamcinolone
Diacetate
Suspension
0.70
mg
Triamcinolone Diacetate Suspension
0.29
mg
Triamcinolone
Hexacetonide
Suspension
0.29 mg
Triamcinolone Hexacetonide Suspension
mg
3.00
Tridihexethyl
Chloride
3.00 mg
Tridihexethyl Chloride
52
52

B-75

--...-------------1

25.00+
25.00 +
25.00+
25.00 +
25.00
25.00
25.00
25.00
+
25.00 +
25.00
25.00 +
+
25.00
25.00
25.00 ++
25.00
25.00 ++
25.00
25.00 ++
25.00+
25.00 +
25.00 ++
25.00
25.00 ++
25.00
1250.00 ++1250.00
1.00
1.00
3.50
3.50
+
3.50
3.50 +
0.70
0.70 ++
1.00 ++
1.00
0.50
0.50 ++
1.00 ++
1.00
25.00
+
25.00 +
4.00
J.OO

3.50
3.50
1.00 ++
1.00
35.80
35.80 ++
1.00 ++
1.00
6.20
6.20 ##
88.00+
88.00 +
36.00
36.00
0.05 ++
0.05
0.07 ++
0.07
2.00 ++
2.00
0.80 +
0.80 +
0.35+
0.35 +
0.50
0.50
4.40 +
4.40 +
4.39
4.39
7.10 +
7.10 +
17.20 +
17.20 +
1.70 +
1.70 +.

ICCVAM Test Method Evaluation Report: Appendix B5

Triethylenethiophosphoramide
Triethylenethiophosphoramide
Triethylperazine
MaleateTriethylperazine Maleate
TrifluoperazineHCI
HCIInjection
Injection
Trifluoperazine
TriflupromazineHCI
HCIInjection
Injection
Triflupromazine
Trimethaphan
Camsylate
Trimethaphan Camsylate
TrimethobenzamideHCI
HCI
Trimethobenzamide
Tromethamine
Tromethamine
TubocurarineChloride
Chloride
Tubocurarine

May 2008

0.80
mg
0.80mgmg
0.43
0.43 mg0.029
mg0.029mg0.86
mg0.86 mg
5.00
5.00mgmg2.80
mg2.80 mg154.00mg
154.00mg0.50
mg0.50 mg

6.25
6.25
11.63
11.63
172.00172.00++5.80
5.80++
1.00
1.00++
1.80
1.80++
0.03
0.03++
10.00
10.00++

-U-u-

Urea
Urea
Urofollitropine
Urofollitropine
Uro}s.inase
Urokinase1500.00
1500.00mg
mg
1.06
units
1.06 units
4,400.oo
4,400.00 IuIU

0.003
0.003 ++
4.70
4.70
0.002
0.002

-V-V-

Vancomycin HCl
Hel
Vancomycin
Vasopressin
Vasopressin
Vecuronium
Vecuronium Bromide
BromideVerapamil
Verapamil Hydrochloride
Hydrochloride
Vidarabine
Vidarabine forfor Injection
Injection
Vinblastine
Sulfate for
Injection
Vinblastine Sulfate
for Injection
Vincristine
Sulfatefor
Injection
Vincristine Sulfate for Injection
Viomycin Sulfate
SulfateViomycin
Vitamin A
Vitamin

15.00
15.00 mg
mg
0.14
units
0.14 units0.10
mg0.10 mg0.30
mg0.30 mg
10.00
10.00 mgmg0.50
mg0.50 mg
0.05 mg
0.05
mg
14.30
mg
14.30 mg
714.30 IV
IU
714.30-w-w-

Warfarin Sodium
Sodium forfor Injection
Injection
Warfarin
Water forfor Injection
Injection andand Sterile
Sterile WFI
WFI
Water
Bacteriostatic
WFI
Bacteriostatic WFISterile Water
Water forfor Inhalation
Inhalation
SterileSterile Water
Water forfor Irrigation
Irrigation
Sterile0.33 +
0.33 +
35.70
35.70
50.00
50.00
16.70 +
16.70 +
0.50 +
0.50 +
10.00 #+
10.00 #+
100.00 #100.00 #
0.35
0.35
0.007
0.007

.

0.21 mg
0.21 mg-.--.--.--

--

.

-.--

-.--

-

.

mm

-,,--

24.00 +
24.00
0.25 ++
0.25
0.50 ++
0.50
0.50 ++
0.50
0.25 ++
0.25 +-XYZ--XYZXenon Xe133
Xe133 Injection
Injection
Xenon*Ytterbium
Yb169
Pentetate Injection
Injection"'Ytterbium Yb169 Pentetate
Zinc Chloride
Chloride Injection
Injection
Zinc
Zinc
SulfateInjection
Zinc Sulfate Injection

2.00 mL
2.00
2.50 mL
mL
2.50
0.20mL
mg Zn
0.20
mg
Zn
0.20 mgZn
0.20 mg Zn
53
53

B-76

87.50 +
87.50
5.60 ++
5.60
25.00 ++
25.00
25.00 ++
25.00 +IICCVAM Test Method Evaluation Report: Appendix B5

May 2008(*) -- Intrathecal
Intrathecal Injections
Injections(*)(+) -- USP
USP Limit
Limit
(+)
NOTE:
NOTE: The
The limit
limit formula
formula forfor radiopharmaceuticals
radiopharmaceuticals isis 175/V
175/V except
except for
for
intrathecally
14N forfor intrathecal
intrathecal products.
products. VV
intrathecally administeredadministered products
products 14/V
equalsequals thethe maximum
maximum recommended
recommended dose
dose (listed(listed inin thethe dose
dose column),
column), inin
mL,
at
the
expiration
date
or
time.
mL, at the expiration date or time.
(#) -- Drug
Drug Administered
Administered on
on aa perper Square
Square Meter
Meter ofof Body
Body Surface
Surface(#)
Limit calculated
calculated according
according toto thethe following
following formula:formula:
Limit
>Ie
1.80
sq.
m.)/70
Kg)
5
EU/Kg
/
«dose
*
1.80
sq.
m&70
Kg)
5 EU/Kg / ((doseReferences:
References:
and Comparisons,Comparisons, Editors
Editors E.
E. Kastrup
Kastrup andand J.
J. Boyd,
Boyd,
Facts and
Facts
Facts
and
Comparisons,
Inc.
Facts and Comparisons, Inc.
UnitedUnited States
States Pharmacopeia
Pharmacopeia Dispensing
Dispensing Information
Information
1990,
1990, United
United States
States Pharmacopeia
Pharmacopeia Convention,
Convention, Inc.
Inc.

54
54

B-77

ICCVAM Test Method Evaluation Report: Appendix B5[This Page Intentionally Left Blank]

B-78

May 2008

ICCVAM Test Method Evaluation Report: Appendix C

May 2008

Appendix C
ICCVAM Recommended Test Method Protocols
C1
C2
C3
C4
C5

The Human Whole Blood (WB)/Interleukin (IL)-1β
In Vitro Pyrogen Test...........................................................................................C-3
The Human WB/IL-1β In Vitro Pyrogen Test: Application of
Cryopreserved (Cryo) Human WB...................................................................C-25
The Human WB/IL-6 In Vitro Pyrogen Test ....................................................C-47
The Human Peripheral Blood Mononuclear Cell (PBMC)/IL-6
In Vitro Pyrogen Test.........................................................................................C-71
The Monocytoid Cell Line Mono Mac 6 (MM6)/IL-6
In Vitro Pyrogen Test.........................................................................................C-95

C-1

ICCVAM Test Method Evaluation Report: Appendix C[This Page Intentionally Left Blank]

C-2

May 2008

ICCVAM Test Method Evaluation Report: Appendix C1

May 2008

Appendix C1
The Human Whole Blood (WB)/Interleukin (IL)-1β In Vitro Pyrogen Test

C-3

ICCVAM Test Method Evaluation Report: Appendix C1[This Page Intentionally Left Blank]

C-4

May 2008

ICCVAM Test Method Evaluation Report: Appendix C1

May 2008

ICCVAM Final Recommended Protocol for Future Studies Using the Human Whole
Blood (WB)/Interleukin (IL)-1β In Vitro Pyrogen Test
PREFACEThis protocol is for the detection of Gram-negative endotoxin, a pyrogen, in parenteral drugs,
as indicated by the release of IL-1β from monocytoid cells in human whole blood (WB). This
protocol is based on information obtained from 1) the European Centre for the Validation of
Alternative Methods (ECVAM)1 WB/IL-1β Background Review Document (BRD) presented
in Appendix A of the Interagency Coordinating Committee on the Validation of Alternative
Methods (ICCVAM) BRD (available at
http://iccvam.niehs.nih.gov/methods/pyrogen/pyr_brd.htm), and 2) information provided to
the National Toxicology Program (NTP) Interagency Center for the Validation of Alternative
Toxicological Methods (NICEATM) by Dr. Thomas Hartung, Head of ECVAM. The
ICCVAM BRD includes the ECVAM Standard Operating Procedures (SOPs) for the
WB/IL-1β test (could be referred to as Monocyte Activation Test), which is first describedby Hartung and Wendel (1996). A table of comparison between the ICCVAM recommended
protocol and the ECVAM SOPs is provided in Table 1.
Users should contact the relevant regulatory authority for guidance when using thisICCVAM recommended protocol to demonstrate product specific validation, and any
deviations from this protocol should be accompanied by scientifically justified rationale.
Future studies using the WB/IL-1β pyrogen test may include further characterization of theusefulness or limitations of the assay for regulatory decision-making. Users should be awarethat this protocol might be revised based on additional optimization and/or validation studies.
ICCVAM recommends that test method users routinely consult the ICCVAM/NICEATM
website (http://iccvam.niehs.nih.gov) to ensure that the most current protocol is used.

1

ECVAM is a unit of the Institute for Health and Consumer Protection at the European Commission's Joint
Research Centre.
C-5

Table 1

Comparison of ICCVAM Recommended Protocol with the ECVAM SOPs for the WB/IL-1β
Pyrogen Test

Protocol Component
Test Substance
Number of Blood Donors
Decision Criteria forInterference
Incubation Plate(The number of samples orcontrols measured inquadruplicate)

ELISA Plate

Assay Acceptability
Criteria

ECVAM SOP1

ECVAM Validation SOP1

Test neat or at minimal
dilution that produces nointerference

Test at MVD

Minimum of 1

Minimum of 1

Mean OD of PPC is 50% to200% of 1.0 EU/mL EC
NSC (1) in triplicate
EC (5)in triplicate

PPC (0)NPC (0)LTAC (1) in triplicate

Mean OD of PPC is 50% to200% of 1.0 EU/mL EC
NSC (1)
EC (2)TS (3) x EC (5) spikes = 15
TS
PPC (3) = 3 TS
NPC (3) = 3 TS
LTAC (0)Not included

Not included

Outliers rejected using
Dixon's test6
Not included

Outliers rejected using
Dixon's test6
Not included

Not included

Not included

Not included

Not included

ICCVAM Protocol
Test neat or in serial
dilutions that produce nointerference, not to exceed
the MVD
Minimum of 3(independent or pooled)
Mean OD2 of PPC is 50%
to 200% of 1.0 EU/mL EC
NSC (1)EC (5)
TS (14)

TS (25) in triplicate

PPC3 (0)NPC3 (0)LTAC4 (0)Includes seven point IL-1β
SC and blank in duplicate
Outliers rejected using
Dixon's test
Mean OD of NSC ≤0.15
Quadratic function of IL-1β
SC r ≥0.955
EC SC produces OD values
that ascend in a sigmoidal
concentration response

Mean OD of 0.5 EU/mL EC≥ 1.6x Mean OD of NSC
Decision Criteria for
Endotoxin concentration TS
OD TS > OD 0.5 EU/mL
OD TS > OD 0.5 EU/mL EC
Pyrogenicity> ELC7 TS
EC
Abbreviations: EC = Endotoxin control; ELISA = Enzyme-linked immunosorbent assay; EU = Endotoxin units; IL-1β = Interleukin-1β;
LTAC = Lipoteichoic acid (LTA) control; MVD = Maximum valid dilution; NPC = Negative product control; NSC = Negative saline
control; OD = Optical density; PPC = Positive product control; SC = Standard curve; SOP = Standard operating procedure;
TS = Test substance; WB = Whole blood
Not included

Mean OD of 0.5 EU/mL EC≥ 1.6x Mean OD of NSC

1

ECVAM WB/IL-1β SOPs are presented in Appendix A of the ICCVAM BRD (available at http://iccvam.niehs.nih.gov/methods/pyrogen/pyr_brd.htm).
Mean OD values are corrected (i.e., reference filter reading, if applicable, and NSC are subtracted).
3
In the ICCVAM WB/IL-1β protocol, PPC and NPC are assessed in the interference test described in Section 4.2, which is performed prior to the ELISA. In the
ECVAM SOP, PPC and NPC were only included in the ECVAM validation study.
4
LTAC was only included in the ECVAM SOP.
5
Correlation coefficient (r), an estimate of the correlation of x and y values in a series of n measurements.
6
Included in the ECVAM Trial data report presented in Appendix D of the ICCVAM BRD.
7
Where unknown, the ELC is calculated (see Section 12.2).
2

ICCVAM Test Method Evaluation Report: Appendix C1

1.0

May 2008

PURPOSE AND APPLICABILITY

The purpose of this protocol is to describe the procedures used to evaluate the presence ofGram-negative endotoxin, a pyrogen, in parenteral drugs. The presence of Gram-negative
endotoxin is detected by its ability to induce the release of interleukin (IL)-1β frommonocytoid cells in whole blood (WB). The concentration of IL-1β released by incubation of
WB cells with a test substance or controls (i.e., positive and negative) is quantified using an
enzyme-linked immunosorbent assay (ELISA) that includes monoclonal or polyclonal
antibodies specific for IL-1β. The amount of pyrogen present is determined by comparing thevalues of endotoxin equivalents produced by WB cells exposed to the test substance to those
exposed to an internationally harmonized Reference Standard Endotoxin (RSE)1 or anequivalent standard expressed in Endotoxin Units (EU)/mL. A test substance is consideredpyrogenic if the endotoxin concentration of the test substance exceeds the Endotoxin Limit
Concentration (ELC) for the test substance.
The relevance and reliability of this test method to detect non-endotoxin pyrogens have notbeen demonstrated in a formal validation study, although data are available in the literature tosuggest that this assay has the potential to serve this purpose.
2.0

SAFETY AND OPERATING PRECAUTIONS

All procedures that use human blood-derived materials should follow national /international
procedures for handling blood potentially contaminated with pathogens. An example of suchguidelines is the Universal Precautions available at
http://www.niehs.nih.gov/odhsb/biosafe/univers.htm. For non-human blood procedures (e.g.,
ELISAs), standard laboratory precautions are recommended including the use of laboratory
coats, eye protection, and gloves. If necessary, additional precautions required for specificchemicals will be identified in the Material Safety Data Sheet (MSDS).
The stop solution used in the ELISA kit is acidic and corrosive and should be handled withthe proper personal protective devices. If this reagent comes into contact with skin or eyes,
wash thoroughly with water. Seek medical attention, if necessary.
Tetramethylbenzidine (TMB) solution contains a hydrogen peroxide substrate and 3, 3', 5, 5'TMB. This reagent is a strong oxidizing agent and a suspected mutagen. Appropriate
personal protection should be used to prevent bodily contact.
Bacterial endotoxin is a toxic agent (i.e., can induce sepsis, shock, vascular damage,antigenic response) and should be handled with care. Skin cuts should be covered andappropriate personal protective devices should be worn. In case of contact with endotoxin,
immediately flush eyes or skin with water for at least 15 minutes (min). If inhaled, remove
the affected individual from the area and provide oxygen and/or artificial respiration asneeded. Skin absorption, ingestion, or inhalation may produce fever, headache, andhypotension.
1

RSEs are internationally harmonized reference standards (e.g., WHO-lipopolysaccharide [LPS] 94/580
Escherichia coli [E. coli] O113:H10:K-; United States Pharmacopeia [USP] RSE E. coli LPS Lot G3E069; USP
RSE E. coli Lot G; FDA E. coli Lot EC6). Equivalent endotoxins include commercially available E. coliderived LPS Control Standard Endotoxin (CSE) or other E. coli LPS preparations that have been calibrated with
an appropriate RSE.
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ICCVAM Test Method Evaluation Report: Appendix C1

3.0

MATERIALS, EQUIPMENT, AND SUPPLIES

3.1

Blood Donor Eligibility

May 2008

Monocytoid cells from WB are the source of cytokine production in the WB/IL-1β test
method as described by Hartung and Wendel (1996) and Schindler et al. (2006). In the
United States (U.S.), the collection of blood and blood components for transfusion andfurther manufacture (including the use of resulting monocytes in a licensed test) is currently
regulated under Section 351 of the Public Health Service (PHS) Act (U.S. Code [U.S.C.],
Title 42, Chapter 6A) and/or the Federal Food Drug and Cosmetic Act (U.S.C., Title 21,
Chapter 9), both of which require compliance with Current Good Manufacturing Practice(cGMP) regulations (21 CFR Parts 210, 211 and 600-6402).
These regulations and the associated FDA guidance's provide an important resource for
information regarding the currently accepted practice for blood manufacture and collection(including donor screening) (http://www.fda.gov/cber/blood.htm). Specifically, guidance
regarding donor screening questionnaires and links to currently acceptable questionnaires canbe found at http://www.fda.gov/cber/gdlns/donorhistques.htm#iv. If you have questions
regarding the applicability of blood regulations to your specific situation3, it is recommended
that you e-mail the Manufacturers Assistance and Technical Training (MATT) Branchestablished by FDA at matt@cber.fda.gov for advice.
Any participating blood establishment should address how unused components of blood
donations will be accounted for and ultimately destroyed, and if the establishment will store
the blood preparation, describe the storage procedures to be followed.
3.2

Equipment and Supplies

For all steps in the protocol, excluding the ELISA procedure, the materials that will be inclose contact with samples and/or blood cells (e.g., pipet tips, containers, solutions) should besterile and pyrogen-free.
3.2.1

Blood Incubation

3.2.1.1

Equipment• Centrifuge•

Hood; Bio-safety, laminar flow (recommended)•

Incubator; cell culture (37±1°C + 5% CO2)

2

The requirements for WB can be found at 21 CFR 640.1 et seq. In addition, there are specific regulations
applicable to red blood cells, platelets, and other blood components. See, for example, 21 CFR 640.10-640.27.
Other regulations applicable to the manufacture of blood and blood components include 21 CFR Part 606, thecGMP requirements for blood and blood components, 21 CFR 610.40, the requirements for testing of WB
donations, and 21 CFR 640.3, the requirements for determining the suitability of the donor. Blood that enters
into U.S. interstate commerce should be tested for antibodies to HIV 1/2, HCV, HTLV I and II, HBc, HBsAg
and RPR, WNV and Chagas.
3
The collection of blood for research and development purposes or as a component of an in vitro test (that is not
subject to licensure) may potentially not be required to adhere to the FDA regulations outlined above.
C-9

ICCVAM Test Method Evaluation Report: Appendix C1

3.2.1.2•

Pipetter; multichannel (8- or 12-channel)•

Pipetters; single-channel adjustable (20 and 200 µL)•

Repeating pipetter•

Vortex mixer

Consumables• Centrifuge tubes; polystyrene (15 and 50 mL)•

Combitips; repeating pipetter (1.0 and 2.5 mL)•

Needle set; multifly, pyrogen-free, 19 mm, 21 gauge•

Plates; microtiter, 96-well, polystyrene, tissue culture•

Pyrogen-free saline (PFS)•

Reaction tubes; polystyrene (1.5 mL)•

Reservoirs; for blood collection•

Tips; pipetter, sterile, pyrogen-free (20 and 200 µL)

3.2.2

ELISA

3.2.2.1

Equipment• Microplate mixer

3.2.2.2

4

May 2008•

Microplate reader (450 nm with an optional reference filter in the range of
600-690 nm4)•

Microplate washer (optional)•

Multichannel pipetter

Consumables• Container; storage, plastic•

Deionized water; nonsterile•

Plates; microtiter, 96-well, polystyrene•

Pyrogen-free water (PFW)•Reservoirs; fluid

The TMB chromagen is measured at OD450. However, the use of an IL-1β ELISA kit with a chromagen otherthan TMB is acceptable. The ELISA should be measured at a wavelength appropriate for the specificchromagen used.
C-10

ICCVAM Test Method Evaluation Report: Appendix C1•

Tips; pipetter, nonsterile•

Tubes; polystyrene (12 mL)

May 2008

3.2.2.3 ELISA Kit
An ELISA that measures IL-1β release is used. A variety of IL-1β ELISA kits arecommercially available and the IL-1β ELISA procedure outlined in this protocol is intended
to serve as an example for using an ELISA kit. The IL-1β ELISA should be calibrated using
an IL-1β international reference standard (e.g., World Health Organization [WHO] 86/680)
prior to use. The IL-1β cytokine assay kits do not provide the RSE or endotoxin equivalent;
therefore, this reagent must be purchased separately. Results obtained using these products
are subject to the assay acceptability and decision criteria described in Sections 8.0 and 9.0.
IL-1β ELISA kit components may include the following:

3.3•

ELISA plates coated with anti-human IL-1β capture antibody; monoclonal or
polyclonal•

Buffered wash solution•

Dilution buffer•

Enzyme-labeled detection antibody•

Human IL-1β reference standard

•

PFS

•

Stop solution•

TMB5/substrate solution

Chemicals•

3.4

Endotoxin (e.g., WHO-lipopolysaccharide [LPS] 94/580 Escherichia coli [E.
coli] O113:H10:K-; United States Pharmacopeia [USP] RSE E. coli LPS Lot
G3E069; USP RSE E. coli Lot G; FDA E. coli Lot EC6)

Solutions

ELISA solutions are listed in Section 3.2.
4.0

ASSAY PREPARATION

All test substances, endotoxin, and endotoxin-spiked solutions should be stored as specified
in the manufacturer's instructions. The collection of WB is outlined in Section 6.1.
4.1

Endotoxin Standard Curve

An internationally harmonized RSE or equivalent is used to generate the endotoxin standard
curve. The use of any other E. coli LPS requires calibration against a RSE using the
WB/IL-1β pyrogen test. A standard endotoxin curve consisting of a Negative Saline Control

5

The use of an IL-1β ELISA kit with a chromagen other than TMB is acceptable.
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ICCVAM Test Method Evaluation Report: Appendix C1

May 2008(NSC) and five RSE concentrations (0.25, 0.50, 1.0, 2.5, and 5.0 EU/mL) are included in the
incubation step (refer to Table 4-1) and then transferred to the ELISA plate. To prepare theendotoxin standard curve, first obtain a 2000 EU/mL stock solution by addition of PFW tothe lyophilized content of the stock vial by following the instructions provided by the
manufacturer (e.g., 5 mL of PFW is added to a vial containing 10,000 EU). To reconstitutethe endotoxin, the stock vial should be vortexed vigorously for at least 30 min or sonicated ina bath sonicator for at least 5 min. Subsequent dilutions should be vortexed vigorously
immediately prior to use. The stock solution is stable for not more 14 days when stored at 2
to 8°C or for up to 6 months when kept in a -20°C freezer. An endotoxin standard curve isprepared as described in Table 4-1 by making serial dilutions of the stock solution in PFS
with vigorous vortexing at each dilution step. Dilutions should not be stored, because diluteendotoxin solutions are not as stable as concentrated solutions due to loss of activity byadsorption, in the absence of supporting data to the contrary.
Table 4-1

Preparation of Endotoxin Standard Curve

Stock Endotoxin
EU/mL1µL of Stock
EndotoxinµL of PFS

Endotoxin
Concentration
EU/mL
504
5.0
2.5
1.0
0.50
0.25
0

20002,3
50
1950
50
100
900
5.0
500
500
2.5
500
500
1.0
500
500
0.50
500
500
0
0
1000
Abbreviations: EU = Endotoxin units; PFS = Pyrogen-free saline
Each stock tube should be vortexed prior to its use to make the subsequent dilution.
1
To reconstitute the endotoxin, the stock vial should be vortexed vigorously for at least 30 min or sonicated in abath sonicator for at least 5 min. Subsequent dilutions should be vortexed vigorously immediately prior to use.
2
A 2000 EU/mL stock solution of endotoxin is prepared according to the manufacturer's instructions.
3
The stock solution is stable for not more 14 days when stored at 2 to 8°C or for up to 6 months when kept in a 20°C freezer.
4This concentration is not used in the assay.

4.2

Interference Test

For every test substance lot, interference testing must be performed to check for interference
between the test substance and the cell system and/or ELISA. The purpose of the interference
test is to determine whether the test substance (or specific lot of test substance) has an effecton cytokine release.
4.2.1
Interference with the Cell System
All test substances must be labeled as pyrogen-free (i.e., endotoxin levels at an acceptablelevel prior to release by the manufacturer) to ensure that exogenous levels of endotoxin do
not affect the experimental outcome. Liquid test substances should be diluted in PFS. Solid
test substances should be prepared as solutions in PFS or, if insoluble in saline, dissolved indimethyl sulfoxide (DMSO) and then diluted up to 0.5% (v/v) with PFS, provided that thisconcentration of DMSO does not interfere with the assay. To ensure a valid test, a test
substance cannot be diluted beyond its Maximum Valid Dilution (MVD) (refer to Section
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ICCVAM Test Method Evaluation Report: Appendix C1

May 2008

12.3). The calculation of the MVD is dependent on the ELC for a test substance. The ELC
can be calculated by dividing the threshold human pyrogenic dose by the maximum
recommended human dose in a single hour period (see Section 12.2) (USP 2007; FDA
1987). Furthermore, test substances should not be tested at concentrations that are cytotoxic
to blood cells.
4.2.1.1 Reference Endotoxin for Spiking Test Substances
The WHO-LPS 94/580 [E. coli O113:H10:K-] or equivalent internationally harmonized RSE
is recommended for preparation of the endotoxin-spike solution and the endotoxin standard
curve (see Section 4.1).
4.2.1.2 Spiking Test Substances with Endotoxin
Non-spiked and endotoxin-spiked test substances are prepared in quadruplicate and an invitro pyrogen test is performed. A fixed concentration of the RSE (i.e., 1.0 EU/mL or aconcentration equal to or near the middle of the endotoxin standard curve) is added to theundiluted test substance (or in serial two-fold dilutions, not to exceed the MVD). An
illustrative example of endotoxin-spiking solutions is shown in Table 4-2. For non-spiked
solutions, 200 µL of PFS is added to a well followed by 20 µL of the test substance (i.e.,equivalent to the negative product control [NPC]) and 20 µL of WB. Endotoxin-spiked
solutions are prepared by adding 180 µL of PFS to each well followed by 20 µL of the test
substance, and 20 µL of WB. Then, 20 µL of an endotoxin-spike solution (1.0 EU/mL) (i.e.,equivalent to the positive product control [PPC]) is added to each well. The contents of thewells are mixed and incubated as outlined in Section 6.1.3, Steps 6-8. An ELISA is thenperformed as outlined in Section 6.2, without the IL-1β standard curve.
Table 4-2

Preparation of Endotoxin-Spiked and Non-Spiked Solutions for
Determination of Test Substance Interference
Sample Addition

SpikedNon-spikedµL/well

PFS
Endotoxin-spike solution2
Test substance (neat and each serial dilution)
WB
Total3

180
20
20
20
240

1

200
0
20
20
240

Abbreviations: PFS = Pyrogen-free saline; WB = Whole blood1
n=4 replicates each2
Endotoxin concentration is 1.0 EU/mL in PFS.
3
A total volume of 240 µL per well is used for the incubation.

The optical density (OD) values of the endotoxin-spiked and non-spiked test substances arecalibrated against the endotoxin calibration curve. The resulting EU value of the non-spiked
test substance is subtracted from the corresponding EU value of the endotoxin-spiked test
substance at each dilution. The spike recovery for each sample dilution is calculated as apercentage by setting the theoretical value (i.e., endotoxin-spike concentration of 1.0 EU/mL)
at 100%. For example, consider the following interference test results in Table 4-3:
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ICCVAM Test Method Evaluation Report: Appendix C1

Table 4-3

May 2008

Example of Interference Data Used to Determine
Sample Dilution

Sample Dilution
None
1:2
1:4
1:8

% Recovery of Endotoxin Control
25
49
90
110

If a spike recovery between 50% and 200% is obtained, then no interference of the test
substance with either the cell system or the ELISA is demonstrated (i.e., the test substancedoes not increase or decrease the concentration of IL-1β relative to the endotoxin spike). The
lowest dilution (i.e., highest concentration) of a test substance that yields an endotoxin-spikerecovery between 50% and 200% is determined. The test substance is then diluted in serial
two-fold dilutions beginning at this dilution, not to exceed the MVD, for use in the assay.
Based on the results illustrated in Table 4-3, the initial dilution of the test substance would
be 1:4 (i.e., the lowest dilution between 50% and 200% of the 1.0 EU/mL EC).
4.2.2
Interference at the MVD
If the data obtained from the experiment in Section 4.2.1 suggests the presence ofinterference at the MVD, then consideration should be given for using another validated
pyrogen test method.
5.0

CONTROLS

5.1

Benchmark Controls

Benchmark controls may be used to demonstrate that the test method is functioning properly,
or to evaluate the relative pyrogenic potential of chemicals (e.g., parenteral pharmaceuticals,
medical device eluates) of a specific class or a specific range of responses, or for evaluating
the relative pyrogenic potential of a test substance. Appropriate benchmark controls shouldhave the following properties:

5.2•

consistent and reliable source(s) for the chemicals (e.g., parenteralpharmaceuticals, medical device eluates)•

structural and functional similarities to the class of substance being tested•

known physical/chemical characteristics•

supporting data on known effects in animal models•

known potency in the range of response

Endotoxin Control

The EC (i.e., WB incubated with an internationally harmonized RSE) serves as the positive
control in each experiment. The results should be compared to historical values to insure thatit provides a known level of cytokine release relative to the NSC.

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ICCVAM Test Method Evaluation Report: Appendix C1

5.3

May 2008

Negative Saline Control

The NSC (i.e., WB incubated with PFS instead of the test substance) is included in eachexperiment in order to detect nonspecific changes in the test system, as well as to provide abaseline for the assay endpoints.
5.4

Solvent Control

Solvent controls are recommended to demonstrate that the solvent is not interfering with thetest system when solvents other than PFS are used to dissolve test substances.
6.0

EXPERIMENTAL DESIGN

6.1

Incubation with Test Samples and Measurement of IL-1β Release

6.1.1
Collection of Human Blood
Human volunteers that have met the donor eligibility criteria described in Section 3.1 areused as the source of WB. All components of the blood collection system (e.g., syringes,
tubes, connecting lines) must be sterile and pyrogen-free. WB is drawn by venipuncture6
from the medial cubital or cephalic vein of either the right or left arm and collected in asterile container that contains anticoagulant solution (e.g., heparin). The total volume ofblood collected per donor (i.e., up to 500 mL) will be dictated by experimental design anddetermined by the test method user. WB should be stored at room temperature (RT) and must
be used within 4 hr. All subsequent handling of WB should be performed in a laminar flowhood using sterile technique to prevent contamination.
Prior to use in the assay, an equal volume of WB from multiple individual donors should bepooled7.
6.1.2
Incubation Plate
Test substances are prepared at a level of dilution that did not show interference with the test
system, provided that this dilution does not exceed the MVD. Each incubation plate canaccommodate an endotoxin standard curve, a NSC, and 14 test samples (see Table 6-1).

6

WB is obtained using Universal Precautions (e.g., latex gloves, labcoats, safety glasses) and sterile equipment(e.g., syringes, needles, collection tubes) within a hospital or clinical setting by qualified and adequately trained
personnel (i.e., registered nurse, licensed phlebotomist, or medical doctor).
7
Multiple donors (i.e., a minimum of three) should meet the acceptability criteria as outlined in Section 8.0
either as a pool of multiple individual donors or as multiple individual donors tested independently.
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ICCVAM Test Method Evaluation Report: Appendix C1

Table 6-1
Numberof Wells

May 2008

Overview of Incubation Plate Preparation in the WB/IL-1β Pyrogen Test

Sample

PFS

EC

Test
SampleWB

Mix thesamples;incubate for
10 to 24 hr at
37±1°C in ahumidified
atmospherewith 5%CO2.

Mix thesamples;
immediatelytransfer to an
ELISA plate3
and runELISA orstore plate ina -20°C or-80°C freezer.

µL
EC
200
20
0
20
NSC
220
0
0
20
Test
562
samples200
020
20(1-14)
Abbreviations: EC = Endotoxin control; IL-1β = Interleukin-1β; NSC = Negative saline control; PFS =Pyrogen-free saline; WB = Whole blood
1
Five EC concentrations (0.25, 0.50, 1.0, 2.5, and 5.0 EU/mL) in quadruplicate
2
14 test samples (n=4) per plate
3
An IL-1β standard curve is prepared in Columns 11 and 12 on the ELISA plate (see Table 6-3). Therefore, 80
wells are available for test samples and controls on the incubation plate.
201
4

6.1.3
Incubation Assay for IL-1β Release
Test substances should be vortexed vigorously for at least 30 min or sonicated in a bathsonicator for at least 5 min prior to use in the assay. Test substances should be prepared inserial two-fold dilutions beginning at a level of dilution that did not show interference withthe test system (see Section 4.2) in as many subsequent dilutions that are necessary to bewithin the linear range of the endotoxin standard curve, not to exceed the MVD. Blood
samples are prepared in a microtiter plate using a laminar flow hood. All consumables andsolutions must be sterile and pyrogen-free. Each plate should be labeled appropriately with apermanent marker. An overview of the incubation plate preparation is shown in Table 6-1.
The incubation procedure is outlined below:
Step 1. Refer to the incubation plate template presented in Table 6-2.
Step 2. Using a pipetter, transfer 200 µL of PFS into each well.
Step 3. Transfer 20 µL of test sample or 20 µL of PFS for the NSC into theappropriate wells as indicated in the template.
Step 4. Transfer 20 µL of the EC (standard curve) in quadruplicate into theappropriate wells according to the template.
Step 5. Transfer 20 µL of WB into each well and mix by gently swirling the plate.
Step 6. Mix the contents of the wells thoroughly by gently pipetting up and down
five times using a multichannel pipetter, changing the tips between each row to
avoid cross-contamination.
Step 7. Place the covered plate in a tissue culture incubator for 10 to 24 hr at
37±1°C in a humidified atmosphere containing 5% CO2.

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ICCVAM Test Method Evaluation Report: Appendix C1

May 2008

Step 8. Prior to transferring the test samples onto the ELISA plate, mix thecontents of the wells by pipetting up and down three times using a multichannel
pipetter, changing the tips between each row to avoid cross-contamination.
Note: The aliquots may be tested immediately in the ELISA or stored in a -20°C or-80°C freezer for testing at a later time. After transfer to the ELISA plate, freeze theremaining aliquots in a -20°C or -80°C freezer for subsequent experiments, if
necessary.
Table 6-2
1

Incubation Plate - Sample and Control Template
2

3

4

5

6

7

8

9

10

11

12

1

EC
EC
EC
EC
TS3
TS3
TS3
TS3
TS11 TS11
Void3
5.0
5.0
5.0
5.0
EC
EC
EC
EC
B
TS4
TS4
TS4
TS4
TS11 TS11
Void
2.5
2.5
2.5
2.5
EC
EC
EC
EC
C
TS5
TS5
TS5
TS5
TS12 TS12
Void
1.0
1.0
1.0
1.0
EC
EC
EC
EC
D
TS6
TS6
TS6
TS6
TS12 TS12
Void0.50 0.50
0.50
0.50
EC
EC
EC
EC
E
TS7
TS7
TS7
TS7
TS13 TS13
Void0.25 0.25
0.25
0.25
F NSC NSC NSC
NSC
TS8
TS8
TS8
TS8
TS13 TS13
Void
G TS12 TS1
TS1
TS1
TS9
TS9
TS9
TS9
TS14 TS14
Void
H
TS2 TS2
TS2
TS2
TS10
TS10
TS10
TS10
TS14 TS14
Void
Abbreviations: EC = Endotoxin control; NSC = Negative saline control; TS = Test substance1
EC value (e.g., EC 5.0) represents the endotoxin concentration in EU/mL.
2
TS number (e.g., TS1) represents an arbitrary sequence for individual test substances.
3
Columns 11 and 12 are reserved for the IL-1β standard curve on the ELISA plate (see Table 6-3).
A

6.2

Void
Void
Void
Void
Void
Void
Void
Void

ELISA to Measure IL-1β Release

6.2.1
IL-1β Standard Curve
An IL-1β standard, supplied with the ELISA kit, is used. IL-1β standards are typicallysupplied in lyophilized form and should be reconstituted according to the manufacturer's
instructions. The stock solution should be diluted in PFS to the following concentrations: 0,
62.5, 125, 250, 500, 1000, 2000, and 4000 pg/mL. Each well on the ELISA plate will receive
100 µL of an IL-1β blank or standard.
6.2.2
ELISA
The manufacturer's instructions provided with the ELISA kit should be followed and atypical experimental design is outlined below. The ELISA should be carried out at RT and
therefore all components must be at RT prior to use. Frozen specimens should not be thawedby heating them in a water bath. A sample ELISA plate template is shown in Table 6-3,
which includes a five-point EC standard curve, an eight-point IL-1β standard curve (0 to4000 pg/mL), and available wells for up to 14 test substances and a NSC each inquadruplicate. The EC standard curve, the NSC, and the test sample supernatants aretransferred directly from the incubation plate. The IL-1β standard curve is prepared as
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ICCVAM Test Method Evaluation Report: Appendix C1

May 2008

described in Section 6.2.1. An overview of the ELISA plate preparation is shown in Table
6-4.
Step 1. Add 100 µL of enzyme-labeled detection antibody to each well.
Step 2. After pipetting up and down three times to mix the supernatant, transfer
100 µL from each well of the Incubation Plate (A1-10; H1-10) to the ELISA plate.
Step 3. Add 100 µL of each IL-1β standard (0 to 4000 pg/mL) into the respective
wells on the ELISA plate.
Step 4. Cover the microtiter plate(s) with adhesive film and incubate for 90 min ona microplate mixer at 350-400 rpm at RT.
Step 5. Decant and wash each well five to six times with 300 µL Buffered Wash
Solution per well and then rinse three times with deionized water. Place the plates
upside down and tap to remove the wash solution.
Step 6. Add 200 µL of TMB/Substrate Solution to each well and incubate at RT in
the dark for 10 to 15 min. If necessary, decrease the incubation time.
Step 7. Add 50 µL of Stop Solution to each well.
Step 8. Tap the plate gently after the addition of Stop Solution to aid in mixing.
Step 9. Read the OD450 within 15 min of adding the Stop Solution. Measurementwith a reference wavelength of 600-690 nm is recommended.8

8

The TMB chromagen is measured at OD450. However, the use of an IL-1β ELISA kit with a chromagen otherthan TMB is acceptable. The ELISA should be measured at a wavelength appropriate for the specificchromagen used.
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ICCVAM Test Method Evaluation Report: Appendix C1

Table 6-3

ELISA Plate - Sample and Control Template

1

2

3

4

EC1
5.0
EC
2.5
EC
1.0
EC
0.50
EC
0.25

EC
5.0
EC
2.5
EC
1.0
EC
0.50
EC
0.25

EC
5.0
EC
2.5
EC
1.0
EC
0.50
EC
0.25

EC
5.0
EC
2.5
EC
1.0
EC
0.50
EC
0.25

F

NSC

NSC

NSC

G

TS12

TS1

H

TS2

TS2

A
B
C
D
E

May 2008

5

6

7

8

9

10

TS3

TS3

TS3

TS3

TS11

TS11

TS4

TS4

TS4

TS4

TS11

TS11

TS5

TS5

TS5

TS5

TS12

TS12

TS6

TS6

TS6

TS6

TS12

TS12

TS7

TS7

TS7

TS7

TS13

TS13

NSC

TS8

TS8

TS8

TS8

TS13

TS13

TS1

TS1

TS9

TS9

TS9

TS9

TS14

TS14

TS2

TS2

TS10

TS10

TS10

TS10

TS14

TS14

11

12

IL-1β3
0
IL-1β
62.5
IL-1β
125
IL-1β
250
IL-1β
500
IL-1β
1000
IL-1β
2000
IL-1β
4000

IL-1β
0
IL-1β
62.5
IL-1β
125
IL-1β
250
IL-1β
500
IL-1β
1000
IL-1β
2000
IL-1β
4000

Abbreviations: EC = Endotoxin control; NSC = Negative saline control; TS = Test substance1
EC value (e.g., EC 5.0) represents the endotoxin concentration in EU/mL.
2
TS number (e.g., TS1) represents an arbitrary sequence for individual test substances.
3
IL-1β values in columns 11 and 12 are in pg/mL.

Table 6-4

Overview of ELISA Procedure

Decantandwash
Material
Enzymeeachwell TMB/Substratetransferlabeledthree
fromSolution
Incubate
Antibody
times
Incubation
(µL)
90
min
(µL)
with 300
Plate (µ L)
on a
µL
plate
Bufferedmixer atWash
350 toSolution
400 rpm
and threeat RT.
100
100
100
200
times
with
deionized
water.
Abbreviations: OD450 = Optical density at 450 nm; RT = Room temperature
IL-1βstandard(0 to
4000
pg/mL)(µL)

C-19

Incubatefor lessthan 15
min at
RT in
dark.

Stop
Solution(µL)

50

Read eachwell at
OD450 witha 600 to690 nmreference
filter.

ICCVAM Test Method Evaluation Report: Appendix C1

7.0

EVALUATION OF TEST RESULTS

7.1

OD Measurements

May 2008

The OD of each well is obtained by reading the samples in a standard microplate
spectrophotometer (i.e., plate reader) using a visible light wavelength of 450 nm (OD450) witha 600 to 690 nm reference filter (recommended)9. OD values are used to determine assay
acceptability and in the decision criteria for pyrogen detection (see Sections 8.0 and 9.0).
8.0

CRITERIA FOR AN ACCEPTABLE TESTAn EC (five-point standard curve) and a NSC should be included in each experiment. An
IL-1β standard curve should be included in each ELISA as shown in the template presented
in Table 6-3. An assay is considered acceptable only if the following minimum criteria aremet:•

The quadratic function of the IL-1β standard curve produces an r ≥ 0.9510 and
the OD of the blank control is below 0.15.

•

The endotoxin standard curve produces OD values that ascend in a sigmoidal
concentration response.

An outlying observation that represents either a pool of multiple independent donors or asingle individual donor may be excluded if there is confirmation that the accuracy of the
medical information provided by an individual donor is suspect, or if the aberrant response isidentified using acceptable statistical methodology (e.g., Dixon's test [Dixon 1950; Barnett etal. 1994], Grubbs' test [Barnett et al. 1994; Grubbs 1969; Iglewicz and Houghlin 1993]).
9.0

DATA INTERPRETATION/DECISION CRITERIA

9.1

Decision Criteria for Pyrogen Detection

A test substance is considered pyrogenic when the endotoxin concentration of the test
substance exceeds the ELC for the test sample. The ELC can be calculated as shown in
Section 12.2.
10.0

STUDY REPORTThe test report should include the following information:
Test Substances and Control Substances•

Name of test substance•Purity and composition of the substance or preparation•

Physicochemical properties (e.g., physical state, water solubility)

9

The TMB chromagen is measured at OD450. However, the use of an IL-1β ELISA kit with a chromagen otherthan TMB is acceptable. The ELISA should be measured at a wavelength appropriate for the specificchromagen used.
10
Correlation coefficient (r), an estimate of the correlation of x and y values in a series of n measurements.
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ICCVAM Test Method Evaluation Report: Appendix C1

May 2008•

Quality assurance data•

Treatment of the test/control substances prior to testing (e.g., vortexing,
sonication, warming, resuspension solvent)Justification of the In Vitro Test Method and Protocol Used
Test Method Integrity•

The procedure used to ensure the integrity (i.e., accuracy and reliability) of thetest method over time•

If the test method employs proprietary components, documentation on theprocedure used to ensure their integrity from “lot-to-lot” and over time•

The procedures that the user may employ to verify the integrity of the
proprietary components

Criteria for an Acceptable Test•

Acceptable concurrent positive control ranges based on historical data•

Acceptable negative control data

Test Conditions•

Cell system used•

Calibration information for the spectrophotometer used to read the ELISA•

Details of test procedure•

Description of any modifications of the test procedure•

Reference to historical data of the model•

Description of evaluation criteria used•

Tabulation of data from individual test samples

Results
Description of Other Effects Observed
Discussion of the Results
Conclusion
A Quality Assurance Statement for Good Laboratory Practice (GLP)-Compliant Studies•

This statement should indicate all inspections made during the study and thedates any results were reported to the Study Director. This statement should
also confirm that the final report reflects the raw data.

If GLP-compliant studies are performed, then additional reporting requirements provided in
the relevant guidelines (e.g., OECD 1998; EPA 2003a, 2003b; FDA 2003) should be
followed.

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ICCVAM Test Method Evaluation Report: Appendix C1

11.0

May 2008

REFERENCES

Barnett V, Lewis T. 1994. Outliers in Statistical Data. In: Wiley Series in Probability andMathematical Statistics. Applied Probability and Statistics. 3rd Ed. New York: John Wiley &Sons.
Dixon WJ. 1950. Analysis of extreme values. Annals of Mathematical Statistics 21:488-506.
EPA. 2003a. Good Laboratory Practice Standards. Toxic Substances Control Act. 40 CFR
792.
EPA. 2003b. Good Laboratory Practice Standards. Federal Insecticide, Fungicide, andRodenticide Act. 40 CFR 160.
FDA. 1987. Guideline on Validation of the Limulus Amebocyte Lysate Test as an
End-product Endotoxin Test for Human and Animal Parenteral Drugs, Biological Products,
and Medical Devices. Rockville, MD:U.S. Department of Health and Human Services(DHHS), Food and Drug Administration (FDA).
FDA. 2003. Good Laboratory Practices for Nonclinical Laboratory Studies. 21 CFR 58.
Grubbs FE. 1969. Procedures for detecting outlying observations in samples. Technometrics
11(1):1-21.
Hartung T, Wendel A. 1996. Detection of pyrogens using human whole blood. In Vitro
Toxicol. 9(4):353-359.
Iglewicz B, Houghlin DC. 1993. How to detect and handle outliers. In: ASQC Basic
Reference in Quality Control. Vol. 14. Milwaukee, WI: ASQ Quality Press.
Organization for Economic Cooperation and Development (OECD). 1998. OECD Series on
Principle of Good Laboratory Practice and Compliance Monitoring. No. 1. OECD Principles
of Good Laboratory Practice (as revised in 1997). Organisation for Economic Co-operation
and Development (OECD), ENV/MC/CHEM (98)17. Paris: OECD.
Schindler S, Spreitzer I, Löschner B, Hoffmann S, Hennes K, Halder M, Brügger P, Frey E,
Hartung T, Montag T. 2006. International validation of pyrogen tests based on cryopreserved
human primary blood cells. J Immunol Methods 316:42-51.
USP. 2007. The U.S. Pharmacopeia. USP30 NF25<85>. Ed. The U.S. Pharmacopeial
Convention. Rockville, MD:The U.S. Pharmacopeial Convention.

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ICCVAM Test Method Evaluation Report: Appendix C1

12.0
12.1

May 2008

TERMINOLOGY AND FORMULA
Assay Sensitivity (λ)1

The variable λ is defined as the labeled sensitivity (in EU/mL) of the LAL Reagent in
endpoint assays (e.g., the BET gel-clot technique). For kinetic BET assays, λ is the lowest
point used in the endotoxin standard curve.
12.2

Endotoxin Limit Concentration (ELC)1,2

The ELC for parenteral drugs is expressed in Endotoxin Units (EU) per volume (mL) or
weight (mg). The ELC is equal to K/M, where:
K is the threshold human pyrogenic dose of endotoxin (EU) per body weight (kg). K is equal
to 5.0 EU/kg for intravenous administration. For intrathecal administration, K is equal to 0.2
EU/kg (see also Section 12.5).
M is the rabbit test dose or the maximum recommended human dose of product (mL or mg)
per body weight (kg) in a single hour period (see also Section 12.8).
For example, if a non-intrathecal product were used at an hourly dose of 10 mL per patient,then the ELC would be 0.50 EU/mL.
12.3

Maximum Valid Dilution (MVD)1,2

The MVD is the maximum allowable dilution of a test substance at which the endotoxin limit
can be determined. The calculation of the MVD is dependent on the ELC for a test substance.
When the ELC is known, the MVD is1:
MVD = (ELC x Product Potency [PP])/λAs an example, for Cyclophosphamide Injection, the ELC is 0.17 EU/mg, PP is 20
mg/mL, and the assay sensitivity is 0.065 EU/mL. The calculated MVD would be 1:52.3 or
1:52. The test substance can be diluted no more than 1:52 prior to testing.
If the ELC is not known, the MVD is1:
MVD = PP/Minimum Valid Concentration (MVC)
where, MVC = (λ x M)/K
where, M is the maximum human dose
As an example, for Cyclophosphamide Injection, the PP is 20 mg/mL, M is 30 mg/kg,
and assay sensitivity is 0.065 EU/mL. The calculated MVC is 0.390 mg/mL and the MVD is
1:51.2 or 1:51. The test substance can be diluted no more than 1:51 in the assay prior to
testing.
12.4

Negative Product Control (NPC)

For interference testing, the NPC is a test sample to which pyrogen-free saline (PFS) is
added. The NPC is the baseline for determination of cytokine release relative to theendotoxin-spiked PPC.
1
2

From FDA (1987)
From USP (2007)
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ICCVAM Test Method Evaluation Report: Appendix C1

12.5

May 2008

Parenteral Threshold Pyrogen Dose (K)1,2

The value K is defined as the threshold human pyrogenic dose of endotoxin (EU) per bodyweight (kg). K is equal to 5.0 EU/kg for parenteral drugs except those administeredintrathecally; 0.2 EU/kg for intrathecal drugs.
12.6

Positive Product Control (PPC)

For interference testing, the PPC is a test substance spiked with the control standard
endotoxin (i.e., 0.5 EU/mL or an amount of endotoxin equal to that which produces ½ themaximal increase in optical density (OD) from the endotoxin standard curve) to insure thatthe test system is capable of endotoxin detection in the product as diluted in the assay.
12.7

Product Potency (PP)1,2

The test sample concentration expressed as mg/mL or mL/mL.
12.8

Rabbit Pyrogen Test (RPT) Dose or Maximum Human Dose (M)1,2

The variable M is equal to the rabbit test dose or the maximum recommended human dose of
product per kg of body weight in a single hour period. M is expressed in mg/kg or mL/kg andvaries with the test substance. For radiopharmaceuticals, M equals the rabbit dose or
maximum human dose/kg at the product expiration date or time. Use 70 kg as the weight of
the average human when calculating the maximum human dose per kg. If the pediatricdose/kg is higher than the adult dose, then it shall be the dose used in the formula.

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ICCVAM Test Method Evaluation Report: Appendix C2

May 2008

Appendix C2
The Human WB/IL-1β In Vitro Pyrogen Test: Application of Cryopreserved (Cryo)
Human WB

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ICCVAM Test Method Evaluation Report: Appendix C2[This Page Intentionally Left Blank]

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May 2008

ICCVAM Test Method Evaluation Report: Appendix C2

May 2008

ICCVAM Final Recommended Protocol for Future Studies Using the Cryopreserved(Cryo) Whole Blood (WB)/Interleukin (IL)-1β In Vitro Pyrogen Test
PREFACEThis protocol is for the detection of Gram-negative endotoxin, a pyrogen, in parenteral drugs,
as indicated by the release of IL-1β from monocytoid cells in human whole blood (WB) thathave been cryopreserved (Cryo). This protocol is based on information obtained from 1) theEuropean Centre for the Validation of Alternative Methods (ECVAM)1 Cryo WB/IL-1β
Background Review Document (BRD) presented in Appendix A of the Interagency
Coordinating Committee on the Validation of Alternative Methods (ICCVAM) BRD(available at http://iccvam.niehs.nih.gov/methods/pyrogen/pyr_brd.htm), and 2) information
provided to the National Toxicology Program (NTP) Interagency Center for the Evaluationof Alternative Toxicological Methods (NICEATM) by Dr. Thomas Hartung, Head of
ECVAM. The ICCVAM BRD includes the ECVAM Standard Operating Procedure (SOP)for the Cryo WB/IL-1β test (could be referred to as Monocyte Activation Test), which isbased on the Cryo WB/IL-1β method first described by Schindler et al. (2004). A table ofcomparison between the ICCVAM recommended protocol and the ECVAM SOP is provided
in Table 1.
Users should contact the relevant regulatory authority for guidance when using thisICCVAM recommended protocol to demonstrate product specific validation, and any
deviations from this protocol should be accompanied by scientifically justified rationale.
Future studies using the Cryo WB/IL-1β pyrogen test may include further characterization of
the usefulness or limitations of the assay for regulatory decision-making. Users should beaware that this protocol might be revised based on additional optimization and/or validation
studies. ICCVAM recommends that test method users routinely consult the
ICCVAM/NICEATM website (http://iccvam.niehs.nih.gov) to ensure that the most current
protocol is used.

1

ECVAM is a unit of the Institute for Health and Consumer Protection at the European Commission's Joint
Research Centre.
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ICCVAM Test Method Evaluation Report: Appendix C2Table 1

May 2008

Comparison of ICCVAM Recommended Protocol with the ECVAM SOP
for the Cryo WB/IL-1β Pyrogen Test

Protocol Component
Test Substance
Number of Blood Donors
Decision Criteria forInterference

ICCVAM Protocol
Test neat or in serial dilutions that
produce no interference, not toexceed the MVD
Minimum of 3 (independent orpooled)Mean OD3 of PPC is 50% to 200%
of 0.5 EU/mL EC
Not included

Incubation Plate(The number of samples orcontrols measured inquadruplicate)
ELISA Plate

Assay Acceptability Criteria

NSC (1)EC (5)
TS (14)
PPC4 (0)NPC4 (0)Includes seven point IL-1β SC andblank in duplicate
Mean OD of NSC ≤0.15
Quadratic function of IL-1β SC r
≥0.955EC SC produces OD values thatascend in a sigmoidal
concentration response
Not included
Not included

ECVAM Catch-Up Validation SOP1
Test at MVD
5 (pooled)2
Mean OD of PPC is 50%
to 200% of 0.5 EU/mL EC
Mean OD of PPC ≥ 1.6x
Mean OD of NPC
NSC (1)
EC (2)TS (3) x EC (5) spikes = 15 TS
PPC (3) = 3 TS
NPC (3) = 3 TS
Not included
Mean OD of NSC ≤100 m OD
Not included
Not included
Mean OD of 0.5 EU/mL EC ≥ 1.6x
Mean OD of NSC
If one OD of 1.0 EU/mL EC > Max,ELISA may be repeated using reduced
incubation time
Outliers rejected using Dixon's test6

Outliers rejected using Dixon's test
Decision Criteria for
Endotoxin concentration
OD TS > OD 0.5 EU/mL EC6
Pyrogenicity
TS > ELC7 TS
Abbreviations: Cryo = Cryopreserved; EC = Endotoxin control; ELISA = Enzyme-linked immunosorbent
assay; EU = Endotoxin units; IL-1β = Interleukin-1β; MVD = Maximum valid dilution; NPC = Negative
product control; NSC = Negative saline control; OD = Optical density; PPC = Positive product control; SC =Standard curve; SOP = Standard operating procedure; TS = Test substance; WB = Whole blood
1
ECVAM Cryo WB/IL-1β catch-up validation SOP is presented in Appendix A of the ICCVAM BRD(available at http://iccvam.niehs.nih.gov/methods/pyrogen/pyr_brd.htm).
2
Samples are collected from five donors and pooled prior to cryopreservation.
3
Mean OD values are corrected (i.e., reference filter reading, if applicable, and NSC are subtracted).
4
In the ICCVAM Cryo WB/IL-1β protocol, PPC and NPC are assessed in the interference test described in
Section 4.2, which is performed prior to the ELISA.
5
Correlation coefficient (r), an estimate of the correlation of x and y values in a series of n measurements.
6
Included in the ECVAM Trial data report presented in Appendix D of the ICCVAM BRD.
7
Where unknown, the ELC is calculated (see Section 12.2).

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ICCVAM Test Method Evaluation Report: Appendix C2

1.0

May 2008

PURPOSE AND APPLICABILITY

The purpose of this protocol is to describe the procedures used to evaluate the presence ofGram-negative endotoxin, a pyrogen, in parenteral drugs. The presence of Gram-negative
endotoxin is detected by its ability to induce the release of interleukin (IL)-1β frommonocytoid cells in whole blood (WB) that have been cryopreserved (Cryo). The
concentration of IL-1β released by incubation of Cryo WB cells with a test substance orcontrols (i.e., positive and negative) is quantified using an enzyme-linked immunosorbent
assay (ELISA) that includes monoclonal or polyclonal antibodies specific for IL-1β. The
amount of pyrogen present is determined by comparing the values of endotoxin equivalents
produced by Cryo WB cells exposed to the test substance to those exposed to aninternationally harmonized Reference Standard Endotoxin (RSE)1 or an equivalent standard
expressed in Endotoxin Units (EU)/mL. A test substance is considered pyrogenic if theendotoxin concentration of the test substance exceeds the Endotoxin Limit Concentration(ELC) for the test substance.
The relevance and reliability of this test method to detect non-endotoxin pyrogens have notbeen demonstrated in a formal validation study, although data are available in the literature tosuggest that this assay has the potential to serve this purpose.
2.0

SAFETY AND OPERATING PRECAUTIONS

All procedures that use human blood-derived materials should follow national/international
procedures for handling blood potentially contaminated with pathogens. An example of suchguidelines is the Universal Precautions available at
http://www.niehs.nih.gov/odhsb/biosafe/univers.htm. For non-human blood procedures (e.g.,
ELISAs), standard laboratory precautions are recommended including the use of laboratory
coats, eye protection, and gloves. If necessary, additional precautions required for specificchemicals will be identified in the Material Safety Data Sheet (MSDS).
The stop solution used in the ELISA kit is acidic and corrosive and should be handled withthe proper personal protective devices. If this reagent comes into contact with skin or eyes,
wash thoroughly with water. Seek medical attention, if necessary.
Tetramethylbenzidine (TMB) solution contains a hydrogen peroxide substrate and 3, 3', 5,5'-TMB. This reagent is a strong oxidizing agent and a suspected mutagen. Appropriate
personal protection should be used to prevent bodily contact.
Bacterial endotoxin is a toxic agent (i.e., can induce sepsis, shock, vascular damage,antigenic response) and should be handled with care. Skin cuts should be covered andappropriate personal protective devices should be worn. In case of contact with endotoxin,
immediately flush eyes or skin with water for at least 15 minutes (min). If inhaled, remove
the affected individual from the area and provide oxygen and/or artificial respiration as

1

RSEs are internationally harmonized reference standards (e.g., WHO-lipopolysaccharide [LPS] 94/580
Escherichia coli [E. coli] O113:H10:K-; United States Pharmacopeia [USP] RSE E. coli LPS Lot G3E069; USP
RSE E. coli Lot G; FDA E. coli Lot EC6). Equivalent endotoxins include commercially available E. coliderived LPS Control Standard Endotoxin (CSE) or other E. coli LPS preparations that have been calibrated with
an appropriate RSE.
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ICCVAM Test Method Evaluation Report: Appendix C2

May 2008

needed. Skin absorption, ingestion, or inhalation may produce fever, headache, andhypotension.
3.0

MATERIALS, EQUIPMENT, AND SUPPLIES

3.1

Blood Donor Eligibility

Monocytoid cells from WB are the primary source of cytokine production in the Cryo
WB/IL-1β test method as described by Hartung and Wendel (1999) and Schindler et al.
(2004, 2006). In the United States (U.S.), the collection of blood and blood components fortransfusion and further manufacture (including the use of resulting monocytes in a licensed
test) is currently regulated under Section 351 of the Public Health Service (PHS) Act (U.S.
Code [U.S.C.], Title 42, Chapter 6A) and/or the Federal Food Drug and Cosmetic Act(U.S.C., Title 21, Chapter 9), both of which require compliance with Current Good
Manufacturing Practice (cGMP) regulations (21 CFR Parts 210, 211 and 600-6402).
These regulations and the associated FDA guidance's provide an important resource for
information regarding the currently accepted practice for blood manufacture and collection(including donor screening) (http://www.fda.gov/cber/blood.htm). Specifically, guidance
regarding donor screening questionnaires and links to currently acceptable questionnaires canbe found at http://www.fda.gov/cber/gdlns/donorhistques.htm#iv. If you have questions
regarding the applicability of blood regulations to your specific situation3, it is recommended
that you e-mail the Manufacturers Assistance and Technical Training (MATT) Branchestablished by FDA at matt@cber.fda.gov for advice.
Any participating blood establishment should address how unused components of blood
donations will be accounted for and ultimately destroyed, and if the establishment will store
the blood preparation, describe the storage procedures to be followed.
3.2

Equipment and Supplies

For all steps in the protocol, excluding the ELISA, the materials that will be in close contact
with samples and/or blood cells (e.g., pipet tips, containers, and solutions) should be sterileand pyrogen-free.
3.2.1

Blood Incubation

3.2.1.1

Equipment• Centrifuge

2

The requirements for WB can be found at 21 CFR 640.1 et seq. In addition, there are specific regulations
applicable to red blood cells, platelets, and other blood components. See, for example, 21 CFR 640.10-640.27.
Other regulations applicable to the manufacture of blood and blood components include 21 CFR Part 606, thecGMP requirements for blood and blood components, 21 CFR 610.40, the requirements for testing of WB
donations, and 21 CFR 640.3, the requirements for determining the suitability of the donor. Blood that enters
into U.S. interstate commerce should be tested for antibodies to HIV 1/2, HCV, HTLV I and II, HBc, HBsAg
and RPR, WNV and Chagas.
3
The collection of blood for research and development purposes or as a component of an in vitro test (that is not
subject to licensure) may potentially not be required to adhere to the FDA regulations outlined above.
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ICCVAM Test Method Evaluation Report: Appendix C2

3.2.1.2•

Hood; Bio-safety, laminar flow (recommended)•

Incubator; cell culture (37±1°C + 5% CO2)•

Pipetter; multichannel (8- or 12-channel)•

Pipetters; single-channel adjustable (20 and 200 µL)•

Repeating pipetter•

Vortex mixer

May 2008

Consumables• Centrifuge tubes; polystyrene (15 and 50 mL)•

Combitips; repeating pipetter (1.0 and 2.5 mL)•

Needle set; multifly, pyrogen-free, 19 mm, 21 gauge•

Plates; microtiter, 96-well, polystyrene, tissue culture•

Pyrogen-free saline (PFS)•

Reaction tubes; polystyrene (1.5 mL)•

Reservoirs; for fluid collection•

RPMI-1640 cell culture medium•

Tips; pipetter, sterile, pyrogen-free (20 and 200 µL)

3.2.2 ELISA
3.2.2.1

3.2.2.2

4

Equipment• Microplate mixer•

Microplate reader (450 nm with an optional reference filter in the range of
600-690 nm4)•

Microplate washer (optional)•

Multichannel pipetter

Consumables• Container; storage, plastic•

Deionized water; nonsterile•

Plates; microtiter, 96-well, polystyrene•

Pyrogen-free water (PFW)•Reservoirs; fluid•

Tips; pipetter, nonsterile

The TMB chromagen is measured at OD450. However, the use of an IL-1β ELISA kit with a chromagen otherthan TMB is acceptable. The ELISA should be measured at a wavelength appropriate for the specificchromagen used.
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ICCVAM Test Method Evaluation Report: Appendix C2•

May 2008

Tubes; polystyrene (12mL)

3.2.2.3 ELISA Kit
An ELISA that measures IL-1β release is used. A variety of IL-1β ELISA kits arecommercially available and the IL-1β ELISA procedure outlined in this protocol is intended
to serve as an example for using an ELISA kit. The IL-1β ELISA should be calibrated using
an international reference standard (e.g., World Health Organization [WHO] 86/680) prior touse. The IL-1β cytokine assay kits do not provide the RSE or endotoxin equivalent;
therefore, this reagent must be purchased separately. Results obtained using these products
are subject to the assay acceptability and decision criteria described in Sections 8.0 and 9.0.
IL-1β ELISA kit components may include the following:

3.3•

ELISA plates coated with anti-human IL-1β capture antibody; monoclonal or
polyclonal•

Buffered wash solution•

Dilution buffer•

Enzyme-labeled detection antibody•

Human IL-1β reference standard

•

PFS

•

Stop solution•

TMB5/substrate solution

Chemicals•

3.4

Solutions•

4.0

Endotoxin (e.g., WHO-lipopolysaccharide [LPS] 94/580 Escherichia coli [E.
coli] O113:H10:K-; United States Pharmacopeia [USP] RSE E. coli LPS Lot
G3E069; USP RSE E. coli Lot G; FDA E. coli Lot EC6)

RPMI-1640 cell culture medium

ASSAY PREPARATION

All test substances, endotoxin, and endotoxin-spiked solutions should be stored as specified
in the manufacturer's instructions. The collection of WB and the procedure forcryopreservation of WB is outlined in Section 6.1.
4.1

Endotoxin Standard Curve

An internationally harmonized RSE or equivalent is used to generate the endotoxin standard
curve. The use of any other E. coli LPS requires calibration against a RSE using the Cryo
WB/IL-1β pyrogen test. A standard endotoxin curve consisting of a Negative Saline Control(NSC) and five RSE concentrations (0.25, 0.50, 1.0, 2.5, and 5.0 EU/mL) are included in the5

The use of an IL-1β ELISA kit with a chromagen other than TMB is acceptable.
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ICCVAM Test Method Evaluation Report: Appendix C2

May 2008

incubation step (refer to Table 4-1) and then transferred to the ELISA plate. To prepare theendotoxin standard curve, first obtain a 2000 EU/mL stock solution by addition of PFW tothe lyophilized content of the stock vial by following the instructions provided by the
manufacturer (i.e., 5 mL of PFW is added to a vial containing 10,000 EU). To reconstitutethe endotoxin, the stock vial should be vortexed vigorously for at least 30 min or sonicated ina bath sonicator for at least 5 min. Subsequent dilutions should be vortexed vigorously
immediately prior to use. The stock solution is stable for not more 14 days when stored at 2
to 8°C or for up to 6 months when kept in a -20°C freezer. An endotoxin standard curve isprepared as described in Table 4-1 by making serial dilutions of the stock solution in PFS
with vigorous vortexing at each dilution step. Dilutions should not be stored, because diluteendotoxin solutions are not as stable as concentrated solutions due to loss of activity byadsorption, in the absence of supporting data to the contrary.
Table 4-1

Preparation of Endotoxin Standard Curve

Endotoxin
Concentration
EU/mL
20002,3
50
1950
504
50
100
900
5.0
5.0
500
500
2.5
2.5
400
600
1.0
1.0
500
500
0.50
0.50
500
500
0.25
0
0
1000
0
Abbreviations: EU = Endotoxin units; PFS = Pyrogen-free saline1
To reconstitute the endotoxin, the stock vial should be vortexed vigorously for at least 30 min or sonicated in abath sonicator for at least 5 min. Subsequent dilutions should be vortexed vigorously immediately prior to use.
2
A 2000 EU/mL stock solution of endotoxin is prepared according to the manufacturer's instructions.
3
The stock solution is stable for not more 14 days when stored at 2 to 8°C or for up to 6 months when kept in a
-20°C freezer.
4This concentration is not used in the assay.
Stock Endotoxin
EU/mL1

4.2µL of Stock
EndotoxinµL of PFSInterference Test

For every test substance lot, interference testing must be performed to check for interference
between the test substance and the cell system and/or ELISA. The purpose of the interference
test is to determine whether the test substance (or specific lot of test substance) has an effecton cytokine release.
4.2.1
Interference with the Cell System
All test substances must be labeled as pyrogen-free (i.e., endotoxin levels at an acceptablelevel prior to release by the manufacturer) to ensure that exogenous levels of endotoxin do
not affect the experimental outcome. Liquid test substances should be diluted in PFS. Solid
test substances should be prepared as solutions in PFS or, if insoluble in saline, dissolved indimethyl sulfoxide (DMSO) and then diluted up to 0.5% (v/v) with PFS, provided that thisconcentration of DMSO does not interfere with the assay. To ensure a valid test, a test
substance cannot be diluted beyond its Maximum Valid Dilution (MVD) (refer to Section
12.3). The calculation of the MVD is dependent on the ELC for a test substance. The ELC
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ICCVAM Test Method Evaluation Report: Appendix C2

May 2008

can be calculated by dividing the threshold human pyrogenic dose by the maximum
recommended human dose in a single hour period (see Section 12.2) (USP 2007; FDA
1987). Furthermore, test substances should not be tested at concentrations that are cytotoxic
to blood cells.
4.2.1.1 Reference Endotoxin for Spiking Test Substances
The WHO-LPS 94/580 [E. coli O113:H10:K-] or equivalent internationally harmonized RSE
is recommended for preparation of the endotoxin-spike solution and the endotoxin standard
curve (see Section 4.1).
4.2.1.2 Spiking Test Substances with Endotoxin
Non-spiked and endotoxin-spiked test substances are prepared in quadruplicate and an invitro pyrogen test is performed. A fixed concentration of the RSE (i.e., 1.0 EU/mL or aconcentration equal to or near the middle of the endotoxin standard curve) is added to theundiluted test substance (or in serial two-fold dilutions, not to exceed the MVD). An
illustrative example of endotoxin-spiking solutions is shown in Table 4-2. For non-spiked
solutions, 200 µL of RPMI is added to a well followed by 20 µL of the test substance (i.e.,equivalent to the negative product control [NPC]) and 20 µL of Cryo WB (Section 6.1).
Endotoxin-spiked solutions are prepared by adding 180 µL of RPMI to each well followed
by 20 µL of the test substance and 20 µL of Cryo WB. Then, 20 µL of an endotoxin-spikesolution (1.0 EU/mL) (i.e., equivalent to the positive product control [PPC]) is added to eachwell. The contents of the wells are mixed and incubated as outlined in Section 6.1.5, Steps
6-9. An ELISA is then performed as outlined in Section 6.2, without the IL-1β standard
curve.
Table 4-2

Preparation of Endotoxin-Spiked and Non-Spiked Solutions for
Determination of Test Substance Interference
SpikedSample Addition

Non-spiked1µL/well
RPMI
Endotoxin-spike solution2
Test substance (neat and each serial dilution)
Cryo WB
Total3Abbreviations: Cryo = Cryopreserved; WB = Whole blood1
n=4 replicates each2
Endotoxin concentration is 1.0 EU/mL in RPMI.
3
A total volume of 240 µL per well is used for the incubation.

180
20
20
20
240

200
0
20
20
240

The optical density (OD) values of the endotoxin-spiked and non-spiked test substances arecalibrated against the endotoxin calibration curve. The resulting EU value of the non-spiked
test substance is subtracted from the corresponding EU value of the endotoxin-spiked test
substance at each dilution. The spike recovery for each sample dilution is calculated as apercentage by setting the theoretical value (i.e., endotoxin-spike concentration of 1.0 EU/mL)
at 100%. For example, consider the following interference test results in Table 4-3:

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ICCVAM Test Method Evaluation Report: Appendix C2Table 4-3

May 2008

Example of Interference Data Used to Determine Sample Dilution

Sample Dilution
None
1:2
1:4
1:8

% Recovery of Endotoxin Control
25
49
90
110

If a spike recovery between 50% and 200% is obtained, then no interference of the test
substance with either the cell system or the ELISA is demonstrated (i.e., the test substancedoes not increase or decrease the concentration of IL-1β relative to the endotoxin spike). The
lowest dilution (i.e., highest concentration) of a test substance that yields an endotoxin-spikerecovery between 50% and 200% is determined. The test substance is then diluted in serial
two-fold dilutions beginning at this dilution, not to exceed the MVD, for use in the assay.
Based on the results illustrated in Table 4-3, the initial dilution of the test substance would
be 1:4 (i.e., the lowest dilution between 50% and 200% of the 1.0 EU/mL EC).
4.2.2
Interference at the MVD
If the data obtained from the experiment in Section 4.2.1 suggests the presence ofinterference at the MVD, then consideration should be given for using another validated
pyrogen test method.
5.0

CONTROLS

5.1

Benchmark Controls

Benchmark controls may be used to demonstrate that the test method is functioning properly,
or to evaluate the relative pyrogenic potential of chemicals (e.g., parenteral pharmaceuticals,
medical device eluates) of a specific class or a specific range of responses, or for evaluating
the relative pyrogenic potential of a test substance. Appropriate benchmark controls shouldhave the following properties:

5.2•

consistent and reliable source(s) for the chemicals (e.g., parenteralpharmaceuticals, medical device eluates)•

structural and functional similarities to the class of substance being tested•

known physical/chemical characteristics•

supporting data on known effects in animal models•

known potency in the range of response

Endotoxin Control

The EC (i.e., WB incubated with an internationally harmonized RSE) serves as the positive
control in each experiment. The results should be compared to historical values to insure thatit provides a known level of cytokine release relative to the NSC.

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ICCVAM Test Method Evaluation Report: Appendix C2

5.3

May 2008

Negative Saline Control

The NSC (i.e., Cryo WB incubated with PFS instead of the test substance) is included in eachexperiment in order to detect nonspecific changes in the test system, as well as to provide abaseline for the assay endpoints.
5.4

Solvent Control

Solvent controls are recommended to demonstrate that the solvent is not interfering with thetest system when solvents other than PFS are used to dissolve test substances.
6.0

EXPERIMENTAL DESIGN

6.1

Incubation with Test Samples and Measurement of IL-1β Release

6.1.1
Collection of Human Blood
Human volunteers that have met the donor eligibility criteria described in Section 3.1 areused as the source of WB. All components of the blood collection system (e.g., syringes,
tubes, connecting lines) must be sterile and pyrogen-free. WB is drawn by venipuncture6
from the medial cubital or cephalic vein of either the right or left arm and collected in asterile container that contains anticoagulant solution (e.g., heparin). The total volume ofblood collected per donor (i.e., up to 500 mL) will be dictated by experimental design anddetermined by the test method user. All subsequent handling of WB should be performed in alaminar flow hood using sterile technique to prevent contamination.
6.1.2
Cryopreservation Procedure
The two methods available for cryopreservation of blood are 1) the PEI method developed at
the Paul Ehrlich Institute (Langen, Germany) and 2) the Konstanz method developed at the
University of Konstanz (Konstanz, Germany).
6.1.3
PEI Method of Cryopreservation
In the PEI method (Schindler et al. 2006), an equal volume of WB from multiple independent
donors is pooled7 and frozen in a cryoprotective phosphate buffer (Sorensen's) containing
20% (v/v) pyrogen-free, clinical-grade DMSO. The tubes can be stored in a -80°C freezer orin the vapor phase of liquid nitrogen until used.
6.1.3.1 Konstanz Method of Cryopreservation
In the Konstanz method (Schindler et al. 2004), pyrogen-free, clinical grade DMSO is added
to WB of individual donors at a final concentration of 10% (v/v). An equal volume of WB
from multiple independent donors is pooled7 and frozen in a computer-controlled freezer
using several cycles of programmed freezing down to -120°C. Tubes of WB are thenremoved from the instrument and stored in the vapor phase of liquid nitrogen until used.

6

WB is obtained using Universal Precautions (e.g., latex gloves, labcoats, safety glasses) and sterile equipment(e.g., syringes, needles, collection tubes) within a hospital or clinical setting by qualified and adequately trained
personnel (i.e., registered nurse, licensed phlebotomist, or medical doctor).
7
Multiple donors (i.e., a minimum of three) should meet the acceptability criteria as outlined in Section 8.0
either as a pool of multiple individual donors or as multiple individual donors tested independently.
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ICCVAM Test Method Evaluation Report: Appendix C2

May 2008

6.1.3.2 Thawing Procedure
The tubes are thawed in an incubator at 37±1°C for 15 min. Prior to use in the assay, thepooled7 WB cells should be examined under a microscope to determine that the morphologyof the cells is consistent with the appearance of cells that previously yielded acceptable
results. The results of this examination should be included in the study report.
6.1.4
Incubation Plate
Test substances should be vortexed vigorously for at least 30 min or sonicated in a bathsonicator for at least 5 min prior to use in the assay. Test substances should be prepared inserial two-fold dilutions beginning at a level of dilution that did not show interference withthe test system (see Section 4.2) in as many subsequent dilutions that are necessary to bewithin the linear range of the endotoxin standard curve, not to exceed the MVD. Each
incubation plate can accommodate an endotoxin standard curve, a NSC, and 14 test samples(see Table 6-1).
Table 6-1

Numberof Wells
20
42

Overview of Incubation Plate Preparation in the Cryo WB/IL-1β
Pyrogen Test (PEI Method)

SampleRPMI

Test
SampleECµL

1Cryo WB

Mix thesamples;incubate for
10 to 24 hr at
37±1°C in ahumidified
atmospherewith 5%CO2.

Mix thesamples;
immediatelytransfer to an
ELISA plate5
and runELISA orstore plate ina -20°C or-80°C freezer.

EC
180
20
0
40
NSC
180
0
03
40
Test
samples
564
180
0
20
40
(1-14)
Abbreviations: Cryo = Cryopreserved; EC = Endotoxin control; IL-1β = Interleukin-1β; NSC = Negative saline
control; PEI = Paul Ehrlich Institute; PFS = Pyrogen-free saline; WB = Whole blood
1
For the Konstanz method of cryopreservation, 20 µL of Cryo WB is used and the volume of RPMI is adjusted
to 200 µL.
2Five EC concentrations (0.25, 0.50, 1.0, 2.5, 5.0 EU/mL) in quadruplicate
320 µL of PFS is added instead of the test sample.
4
14 test samples (n=4) per plate
5
An IL-1β standard curve is prepared in Columns 11 and 12 on the ELISA plate (see Table 6-3). Therefore, 80
wells are available for test samples and controls on the incubation plate.

6.1.5
Incubation Assay for IL-1β Release
Cryo WB is prepared in a microtiter plate using a laminar flow hood (refer to Section 6.1.1).
All consumables and solutions must be sterile and pyrogen-free. Each plate should be labeledappropriately with a permanent marker. An overview of the incubation plate preparation isshown in Table 6-1. The incubation procedure is outlined below:
Step 1. Refer to the incubation plate template presented in Table 6-2.
Step 2. Using a pipetter, transfer either 180 or 200 µL of RPMI into each well (forthe PEI or Konstanz method of cryopreservation, respectively – refer to Step 5
below).
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ICCVAM Test Method Evaluation Report: Appendix C2

May 2008

Step 3. Transfer 20 µL of test sample or 20 µL of PFS for the NSC into theappropriate wells as indicated in the template.
Step 4. Transfer 20 µL of the EC (standard curve) in quadruplicate into theappropriate wells according to the template.
Step 5. Transfer either 40 or 20 µL of Cryo WB (for the PEI or Konstanz methodof cryopreservation, respectively) into each well and mix by gently swirling theplate.
Step 6. Mix the contents of the wells thoroughly by gently pipetting up and down
five times using a multichannel pipetter, changing the tips between each row inorder to avoid cross-contamination.
Step 7. Place the covered plate in a tissue culture incubator for 10 to 24 hr at
37±1°C in a humidified atmosphere containing 5% CO2.
Step 8. If using the Konstanz method, freeze the plate in a -20°C or -80°C freezer
until the contents of the well are completely frozen and then, thaw the plate at RT
or in a water bath not exceeding 37±1°C.
Step 9. Prior to transferring the test samples onto the ELISA plate, mix thecontents of the wells by pipetting up and down three times using a multichannel
pipetter, changing the tips between each row in order to avoid cross-contamination.
Note: The aliquots may be tested immediately in the ELISA or stored in a -20°C or-80°C freezer for testing at a later time. After transfer to the ELISA plate, freeze theremaining aliquots in a -20°C or -80°C freezer for subsequent experiments, if
necessary.
Table 6-2
1

Incubation Plate - Sample and Control Template
2

3

4

5

6

7

8

9

10

11

12

1

EC
EC
EC
EC
TS3
TS3
TS3
TS3
TS11 TS11
Void3
5.0
5.0
5.0
5.0
EC
EC
EC
EC
B
TS4
TS4
TS4
TS4
TS11 TS11
Void
2.5
2.5
2.5
2.5
EC
EC
EC
EC
C
TS5
TS5
TS5
TS5
TS12 TS12
Void
1.0
1.0
1.0
1.0
EC
EC
EC
EC
D
TS6
TS6
TS6
TS6
TS12 TS12
Void0.50 0.50 0.50
0.50
EC
EC
EC
EC
E
TS7
TS7
TS7
TS7
TS13 TS13
Void0.25 0.25 0.25
0.25
F NSC NSC NSC
NSC
TS8
TS8
TS8
TS8
TS13 TS13
Void
G TS12 TS1
TS1
TS1
TS9
TS9
TS9
TS9
TS14 TS14
Void
H
TS2 TS2
TS2
TS2
TS10
TS10
TS10
TS10
TS14 TS14
Void
Abbreviations: EC = Endotoxin control; NSC = Negative saline control; TS = Test substance1
EC value (e.g., EC 5.0) represents the endotoxin concentration in EU/mL.
2
TS number (e.g., TS1) represents an arbitrary sequence for individual test substances.
3
Columns 11 and 12 are reserved for the IL-1β standard curve on the ELISA plate (see Table 6-3).
A

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Void
Void
Void
Void
Void
Void
Void
Void

ICCVAM Test Method Evaluation Report: Appendix C2

6.2

May 2008

ELISA to Measure IL-1β Release

6.2.1
IL-1β Standard Curve
An IL-1β standard, supplied with the ELISA kit, is used. IL-1β standards are typicallysupplied in lyophilized form and should be reconstituted according to the manufacturer's
instructions. The stock solution should be diluted in RPMI to the following concentrations: 0,
62.5, 125, 250, 500, 1000, 2000, and 4000 pg/mL. Each well on the ELISA plate will receive
100 µL of an IL-1β blank or standard.
6.2.2
ELISA
The manufacturer's instructions provided with the ELISA kit should be followed and atypical experimental design is outlined below. The ELISA should be carried out at RT and
therefore all components must be at RT prior to use. Frozen specimens should not be thawedby heating them in a water bath. A suggested ELISA plate template is shown in Table 6-3,
which includes a five-point EC standard curve, an eight-point IL-1β standard curve (0 to4000 pg/mL), and available wells for up to 14 test substances and a NSC each inquadruplicate. The EC standard curve, the NSC, and the test sample supernatants aretransferred directly from the incubation plate. The IL-1β standard curve is prepared as
described in Section 6.2.1. An overview of the ELISA plate preparation is shown in Table 64.
Step 1. Add 100 µL of enzyme-labeled detection antibody to each well.
Step 2. After pipetting up and down three times to mix the supernatant, transfer
100 µL from each well of the Incubation Plate (A1-10; H1-10) to the ELISA plate.
Step 3. Add 100 µL of each IL-1β standard (0 to 4000 pg/mL) into the respective
wells on the ELISA plate.
Step 4. Cover the microtiter plate(s) with adhesive film and incubate for 90 min ona microplate mixer at 350-400 rpm at RT.
Step 5. Decant and wash each well three times with 300 µL Buffered Wash
Solution and then rinse three times with deionized water. Place the plates upside
down and tap to remove water.
Step 6. Add 200 µL of TMB/Substrate Solution to each well and incubate at RT in
the dark for 15 min. If necessary, decrease the incubation time.
Step 7. Add 50 µL of Stop Solution to each well.
Step 8. Tap the plate gently after the addition of Stop Solution to aid in mixing.
Step 9. Read the OD450 within 15 min of adding the Stop Solution. Measurementwith a reference wavelength of 600-690 nm is recommended.8

8

The TMB chromagen is measured at OD450. However, the use of an IL-1β ELISA kit with a chromagen otherthan TMB is acceptable. The ELISA should be measured at a wavelength appropriate for the specificchromagen used.
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ICCVAM Test Method Evaluation Report: Appendix C2Table 6-3

ELISA Plate - Sample and Control Template

1

2

3

4

EC1
5.0
EC
2.5
EC
1.0
EC
0.50
EC
0.25

EC
5.0
EC
2.5
EC
1.0
EC
0.50
EC
0.25

EC
5.0
EC
2.5
EC
1.0
EC
0.50
EC
0.25

EC
5.0
EC
2.5
EC
1.0
EC
0.50
EC
0.25

F

NSC

NSC

NSC

G

TS12

TS1

H

TS2

TS2

A
B
C
D
E

May 2008

5

6

7

8

9

10

TS3

TS3

TS3

TS3

TS11

TS11

TS4

TS4

TS4

TS4

TS11

TS11

TS5

TS5

TS5

TS5

TS12

TS12

TS6

TS6

TS6

TS6

TS12

TS12

TS7

TS7

TS7

TS7

TS13

TS13

NSC

TS8

TS8

TS8

TS8

TS13

TS13

TS1

TS1

TS9

TS9

TS9

TS9

TS14

TS14

TS2

TS2

TS10

TS10

TS10

TS10

TS14

TS14

11

12

IL-1β3
0
IL-1β
62.5
IL-1β
125
IL-1β
250
IL-1β
500
IL-1β
1000
IL-1β
2000
IL-1β
4000

IL-1β
0
IL-1β
62.5
IL-1β
125
IL-1β
250
IL-1β
500
IL-1β
1000
IL-1β
2000
IL-1β
4000

Abbreviations: EC = Endotoxin control; NSC = Negative saline control; TS = Test substance1
EC value (e.g., EC 5.0) represents the endotoxin concentration in EU/mL.
2
TS number (e.g., TS1) represents an arbitrary sequence for individual test substances.
3
IL-1β values in columns 11 and 12 are in pg/mL.

Table 6-4

Overview of ELISA Procedure

Decant andwash each
Material
IL-1β
Enzymewell threetransfer
TMB/Substrate
standardlabeledtimes with
Incubatefrom
Solution(0 to 4000
Antibody
90 min on
300 µL
Incubation(µL)
pg/mL)(µL)
a plateBuffered
Plate (µ L)(µL)
mixer atWash
350 to 400 Solution andrpm at RT.
three times
with
100
100
100
200
deionized
water.
Abbreviations: OD450 = Optical density at 450 nm; RT = Room temperature

7.0

EVALUATION OF TEST RESULTS

7.1

OD Measurements

Incubatefor lessthan 15
min at RT
in dark.

Stop
Solution(µL)

50

Read eachwell at
OD450
with a 600
to 690 nm
reference
filter.

The OD of each well is obtained by reading the samples in a standard microplate
spectrophotometer (i.e., plate reader) using a visible light wavelength of 450 nm (OD450) with

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ICCVAM Test Method Evaluation Report: Appendix C2

May 2008

a 600 to 690 nm reference filter (recommended)9. OD values are used to determine assay
acceptability and in the decision criteria for pyrogen detection (see Sections 8.0 and 9.0).
8.0

CRITERIA FOR AN ACCEPTABLE TESTAn EC (five-point standard curve) and a NSC should be included in each experiment. An
IL-1β standard curve should be included in each ELISA as shown in the template presented
in Table 6-3. An assay is considered acceptable only if the following minimum criteria aremet:•

The quadratic function of the IL-1β standard curve produces an r ≥ 0.9510 and
the OD of the blank control is below 0.15.

•

The endotoxin standard curve produces OD values that ascend in a sigmoidal
concentration response.

An outlying observation that represents either a pool of multiple independent donors or asingle individual donor may be excluded if there is confirmation that the accuracy of the
medical information provided by an individual donor is suspect, or if the aberrant response isidentified using acceptable statistical methodology (e.g., Dixon's test [Dixon 1950; Barnett etal. 1984], Grubbs' test [Barnett et al. 1994; Grubbs 1969; Iglewicz and Houghlin 1993]).
9.0

DATA INTERPRETATION/DECISION CRITERIA

9.1

Decision Criteria for Pyrogen Detection

A test substance is considered pyrogenic when the endotoxin concentration of the test
substance exceeds the ELC for the test sample. The ELC can be calculated as shown in
Section 12.2.
10.0

STUDY REPORTThe test report should include the following information:
Test Substances and Control Substances•

Name of test substance•Purity and composition of the substance or preparation•

Physicochemical properties (e.g., physical state, water solubility)•

Quality assurance data•

Treatment of the test/control substances prior to testing (e.g., vortexing,
sonication, warming, and resuspension solvent)Justification of the In Vitro Test Method and Protocol Used
Test Method Integrity
9

The TMB chromagen is measured at OD450. However, the use of an IL-1β ELISA kit with a chromagen otherthan TMB is acceptable. The ELISA should be measured at a wavelength appropriate for the specificchromagen used.
10
Correlation coefficient (r), an estimate of the correlation of x and y values in a series of n measurements.
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ICCVAM Test Method Evaluation Report: Appendix C2

May 2008•

The procedure used to ensure the integrity (i.e., accuracy and reliability) of thetest method over time•

If the test method employs proprietary components, documentation on theprocedure used to ensure their integrity from “lot-to-lot” and over time•

The procedures that the user may employ to verify the integrity of the
proprietary components

Criteria for an Acceptable Test•

Acceptable concurrent positive control ranges based on historical data•

Acceptable negative control data

Test Conditions•

Cell system used•

Calibration information for the spectrophotometer used to read the ELISA•

Details of test procedure•

Description of any modifications of the test procedure•

Reference to historical data of the model•

Description of evaluation criteria used•

Tabulation of data from individual test samples

Results
Description of Other Effects Observed
Discussion of the Results
Conclusion
A Quality Assurance Statement for Good Laboratory Practice (GLP)-Compliant Studies•

This statement should indicate all inspections made during the study and thedates any results were reported to the Study Director. This statement should
also confirm that the final report reflects the raw data.

If GLP-compliant studies are performed, then additional reporting requirements provided in
the relevant guidelines (e.g., OECD 1998; EPA 2003a, 2003b; FDA 2003) should be
followed.
11.0

REFERENCES

Barnett V, Lewis T. 1994. Outliers in Statistical Data. In: Wiley Series in Probability andMathematical Statistics. Applied Probability and Statistics. 3rd Ed. New York: John Wiley &Sons.
Dixon WJ. 1950. Analysis of extreme values. Annals of Mathematical Statistics 21:488-506.
EPA. 2003a. Good Laboratory Practice Standards. Toxic Substances Control Act. 40 CFR
792.
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ICCVAM Test Method Evaluation Report: Appendix C2

May 2008

EPA. 2003b. Good Laboratory Practice Standards. Federal Insecticide, Fungicide, andRodenticide Act. 40 CFR 160.
FDA. 1987. Guideline on Validation of the Limulus Amebocyte Lysate Test as an
End-product Endotoxin Test for Human and Animal Parenteral Drugs, Biological Products,
and Medical Devices. Rockville, MD:U.S. Department of Health and Human Services(DHHS), Food and Drug Administration (FDA).
FDA. 2003. Good Laboratory Practices for Nonclinical Laboratory Studies. 21 CFR 58.
Grubbs FE. 1969. Procedures for detecting outlying observations in samples. Technometrics
11(1):1-21.
Hartung T, Wendel A. 1999. Detection of pyrogens using human whole blood. In Vitro
Toxicol. 9(4):353-359.
Iglewicz B, Houghlin DC. 1993. How to detect and handle outliers. In: ASQC Basic
Reference in Quality Control. Vol. 14. Milwaukee, WI: ASQ Quality Press.
Organization for Economic Cooperation and Development (OECD). 1998. OECD Series on
Principle of Good Laboratory Practice and Compliance Monitoring. No. 1. OECD Principles
of Good Laboratory Practice (as revised in 1997). Organisation for Economic Co-operation
and Development (OECD), ENV/MC/CHEM (98)17. Paris: OECD.
Schindler S, Asmus S, von Aulock S, Wendel A, Hartung T, Fennrich S. 2004.
Cryopreservation of human whole blood for pyrogenicity testing. J Immunol Methods
294:89-100.
Schindler S, Spreitzer I, Löschner B, Hoffmann S, Hennes K, Halder M, Brügger P, Frey E,
Hartung T, Montag T. 2006. International validation of pyrogen tests based on cryopreserved
human primary blood cells. J Immunol Methods 316:42-51.
USP. 2007. The U.S. Pharmacopeia. USP30 NF25<85>. Ed. The U.S. Pharmacopeial
Convention. Rockville, MD:The U.S. Pharmacopeial Convention.
12.0
12.1

TERMINOLOGY AND FORMULA
Assay Sensitivity (λ)11

The variable λ is defined as the labeled sensitivity (in EU/mL) of the LAL Reagent in
endpoint assays (e.g., the BET gel-clot technique). For kinetic BET assays, λ is the lowest
point used in the endotoxin standard curve.
12.2

Endotoxin Limit Concentration (ELC)11,12

The ELC for parenteral drugs is expressed in Endotoxin Units (EU) per volume (mL) or
weight (mg). The ELC is equal to K/M, where:

11

From FDA (1987)
From USP (2007)

12

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ICCVAM Test Method Evaluation Report: Appendix C2

May 2008

K is the threshold human pyrogenic dose of endotoxin (EU) per body weight (kg). K is equal
to 5.0 EU/kg for intravenous administration. For intrathecal administration, K is equal to 0.2
EU/kg (see also Section 12.5).
M is the rabbit test dose or the maximum recommended human dose of product (mL or mg)
per body weight (kg) in a single hour period (see also Section 12.8).
For example, if a non-intrathecal product were used at an hourly dose of 10 mL per patient,then the ELC would be 0.50 EU/mL.
12.3

Maximum Valid Dilution (MVD)11,12

The MVD is the maximum allowable dilution of a test substance at which the endotoxin limit
can be determined. The calculation of the MVD is dependent on the ELC for a test substance.
When the ELC is known, the MVD is11:
MVD = (ELC x Product Potency [PP])/λAs an example, for Cyclophosphamide Injection, the ELC is 0.17 EU/mg, PP is 20
mg/mL, and the assay sensitivity is 0.065 EU/mL. The calculated MVD would be 1:52.3 or
1:52. The test substance can be diluted no more than 1:52 prior to testing.
If the ELC is not known, the MVD is11:
MVD = PP/Minimum Valid Concentration (MVC)
where, MVC = (λ x M)/K
where, M is the maximum human dose
As an example, for Cyclophosphamide Injection, the PP is 20 mg/mL, M is 30 mg/kg,
and assay sensitivity is 0.065 EU/mL. The calculated MVC is 0.390 mg/mL and the MVD is
1:51.2 or 1:51. The test substance can be diluted no more than 1:51 in the assay prior to
testing.
12.4

Negative Product Control (NPC)

For interference testing, the NPC is a test sample to which pyrogen-free saline (PFS) is
added. The NPC is the baseline for determination of cytokine release relative to theendotoxin-spiked PPC.
12.5

Parenteral Threshold Pyrogen Dose (K)11,12

The value K is defined as the threshold human pyrogenic dose of endotoxin (EU) per bodyweight (kg). K is equal to 5.0 EU/kg for parenteral drugs except those administeredintrathecally; 0.2 EU/kg for intrathecal drugs.
12.6

Positive Product Control (PPC)

For interference testing, the PPC is a test substance spiked with the control standard
endotoxin (i.e., 0.5 EU/mL or an amount of endotoxin equal to that which produces ½ themaximal increase in optical density (OD) from the endotoxin standard curve) to insure thatthe test system is capable of endotoxin detection in the product as diluted in the assay.
12.7

Product Potency (PP)11,12

The test sample concentration expressed as mg/mL or mL/mL.
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ICCVAM Test Method Evaluation Report: Appendix C2

12.8

May 2008

Rabbit Pyrogen Test (RPT) Dose or Maximum Human Dose (M)11,12

The variable M is equal to the rabbit test dose or the maximum recommended human dose of
product per kg of body weight in a single hour period. M is expressed in mg/kg or mL/kg andvaries with the test substance. For radiopharmaceuticals, M equals the rabbit dose or
maximum human dose/kg at the product expiration date or time. Use 70 kg as the weight of
the average human when calculating the maximum human dose per kg. If the pediatricdose/kg is higher than the adult dose, then it shall be the dose used in the formula.

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May 2008

ICCVAM Test Method Evaluation Report: Appendix C3Appendix C3
The Human WB/IL-6 In Vitro Pyrogen Test

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May 2008

ICCVAM Test Method Evaluation Report: Appendix C3

May 2008

ICCVAM Final Recommended Protocol for Future Studies Using the Human Whole
Blood (WB)/Interleukin (IL)-6 In Vitro Pyrogen Test
PREFACEThis protocol is for the detection of Gram-negative endotoxin, a pyrogen, in parenteral drugs,
as indicated by the release of IL-6 from monocytoid cells in human whole blood (WB). This
protocol is based on information obtained from 1) the European Centre for the Validation of
Alternative Methods (ECVAM)1 WB/IL-6 Background Review Document (BRD) presented
in Appendix A of the Interagency Coordinating Committee on the Validation of Alternative
Methods (ICCVAM) BRD (available at
http://iccvam.niehs.nih.gov/methods/pyrogen/pyr_brd.htm), and 2) information provided to
the National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative
Toxicological Methods (NICEATM) by Dr. Thomas Hartung, Head of ECVAM. The
ICCVAM BRD includes the ECVAM Standard Operating Procedure (SOP) for the WB/IL-6
test (could be referred to as Monocyte Activation Test), which is based on the WB/IL-6
method first described by Pool et al. (1998). A table of comparison between the ICCVAM
recommended protocol and the ECVAM SOP is provided in Table 1.
Users should contact the relevant regulatory authority for guidance when using thisICCVAM recommended protocol to demonstrate product specific validation, and any
deviations from this protocol should be accompanied by scientifically justified rationale.
Future studies using the WB/IL-6 pyrogen test may include further characterization of theusefulness or limitations of the assay for regulatory decision-making. Users should be awarethat this protocol might be revised based on additional optimization and/or validation studies.
ICCVAM recommends that test method users routinely consult the ICCVAM/NICEATM
website (http://iccvam.niehs.nih.gov) to ensure that the most current protocol is used.

1

ECVAM is a unit of the Institute for Health and Consumer Protection at the European Commission's Joint
Research Centre.
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May 2008

Table 1

Comparison of ICCVAM Recommended Protocol with the ECVAM SOP for the WB/IL-6 Pyrogen Test

ECVAM SOP1
Test neat or at minimal dilution that produces no
Test Substanceinterference
Number of Blood Donors
Minimum of 3 (independent)
Mean OD of PPC is 50% to 200% of 1.0 EU/mL
Decision Criteria for InterferenceEC
NSC (1)EC (5)
Incubation Plate for ELISA(The number of samples or controls
TS (14)
measured in quadruplicate)
PPC (0)NPC (0)Includes seven point IL-6 SC and blank in
ELISA Plate
duplicate
NSC < 200 pg/mL IL-6
EC SC satisfies ICH Harmonized Tripartite
Quadratic function of IL-6 SC r ≥0.954
Guideline: Validation of Analytical Procedures
Methodology; ICH Q2B, Nov 1996
Wilcoxon rank-sum test used to show that at
Assay Acceptability Criteria
EC SC produces OD values that ascend in aleast 3 of 4 replicates at each increasing EC
sigmoidal concentration response
concentration are higher relative to the next
lowest concentration
High responder blood donors (i.e., >200 pg/mL IL- High responder blood donors (i.e., >200 pg/mL6) may be excludedIL-6) may be excluded
Outliers rejected using Dixon's test
Outliers rejected using Dixon's test 5
Endotoxin concentration TS > ELC TS
OR
Decision Criteria for Pyrogenicity
Endotoxin concentration TS > ELC6 TS
Limit test is run to determine whether or not a
TS after correction and dilution contains < 0.5
EU/mL of endotoxin
Abbreviations: EC = Endotoxin control; ELC = Endotoxin limit concentration; ELISA = Enzyme-linked immunosorbent assay; EU = Endotoxin units; IL-6 =Interleukin-6; MVD = Maximum valid dilution; NSC = Negative saline control; OD = Optical density; PPC = Positive product control; SC = Standard curve; TS= SOP = Standard operating procedure; Test substance; WB = Whole blood
1
ECVAM WB/IL-6 SOP is presented in Appendix A of the ICCVAM BRD (available at http://iccvam.niehs.nih.gov/methods/pyrogen/pyr_brd.htm).
2
Mean OD values are corrected (i.e., reference filter reading, if applicable, and NSC are subtracted).
3
In the ICCVAM WB/IL-6 protocol, PPC and NPC are assessed in the interference test described in Section 4.2, which is performed prior to the ELISA. In the
ECVAM SOP, PPC and NPC were only included in the ECVAM validation study.
Protocol Component

ICCVAM Protocol
Test neat or in serial dilutions that produce nointerference, not to exceed the MVD
Minimum of 3 (independent or pooled)
Mean OD2 of PPC is 50% to 200% of 1.0 EU/mL
EC
NSC (1)EC (5)
TS (14)
PPC3 (0)NPC3 (0)Includes seven point IL-6 SC and blank in
duplicate
Mean OD of NSC ≤0.15

4Correlation coefficient (r), an estimate of the correlation of x and y values in a series of n measurements.
Included in the ECVAM Trial data report presented in Appendix D of the ICCVAM BRD.
6
Where unknown, the ELC is calculated (see Section 12.2)
5

ICCVAM Test Method Evaluation Report: Appendix C3

1.0

May 2008

PURPOSE AND APPLICABILITY

The purpose of this protocol is to describe the procedures used to evaluate the presence of Gramnegative endotoxin, a pyrogen, in parenteral drugs. The presence of Gram-negative endotoxin is
detected by its ability to induce the release of interleukin (IL)-6 from monocytoid cells in whole
blood (WB). The concentration of IL-6 released by incubation of WB with a test substance orcontrols (i.e., positive and negative) is quantified using an enzyme-linked immunosorbent assay(ELISA) that includes monoclonal or polyclonal antibodies specific for IL-6. The amount ofpyrogen present is determined by comparing the values of endotoxin equivalents produced by
WB cells exposed to the test substance to those exposed to an internationally harmonized
Reference Standard Endotoxin (RSE)1 or an equivalent standard expressed in Endotoxin Units(EU)/mL. A test substance is considered pyrogenic if the endotoxin concentration of the test
substance exceeds the Endotoxin Limit Concentration (ELC) for the test substance.
The relevance and reliability of this test method to detect non-endotoxin pyrogens have not been
demonstrated in a formal validation study, although data are available in the literature to suggestthat this assay has the potential to serve this purpose.
2.0

SAFETY AND OPERATING PRECAUTIONS

All procedures that use human blood-derived materials should follow national/international
procedures for handling blood potentially contaminated with pathogens. An example of suchguidelines is the Universal Precautions available at
http://www.niehs.nih.gov/odhsb/biosafe/univers.htm. For non-human blood procedures (e.g.,
ELISAs), standard laboratory precautions are recommended including the use of laboratory
coats, eye protection, and gloves. If necessary, additional precautions required for specificchemicals will be identified in the Material Safety Data Sheet (MSDS).
The stop solution used in the ELISA kit is acidic and corrosive and should be handled with theproper personal protective devices. If this reagent comes into contact with skin or eyes, wash
thoroughly with water. Seek medical attention, if necessary.
Tetramethylbenzidine (TMB) solution contains a hydrogen peroxide substrate and 3, 3', 5,5'-TMB. This reagent is a strong oxidizing agent and a suspected mutagen. Appropriate personal
protection should be used to prevent bodily contact.
Bacterial endotoxin is a toxic agent (i.e., can induce sepsis, shock, vascular damage, antigenic
response) and should be handled with care. Skin cuts should be covered and appropriate personal
protective devices should be worn. In case of contact with endotoxin, immediately flush eyes orskin with water for at least 15 minutes (min). If inhaled, remove the affected individual from thearea and provide oxygen and/or artificial respiration as needed. Skin absorption, ingestion, or
inhalation may produce fever, headache, and hypotension.

1

RSEs are internationally-harmonized reference standards (e.g., WHO-lipopolysaccharide [LPS] 94/580 Escherichia
coli [E. coli] O113:H10:K-; United States Pharmacopeia [USP] RSE E. coli LPS Lot G3E069; USP RSE E. coli Lot
G; FDA E. coli Lot EC6). Equivalent endotoxins include commercially available E. coli-derived LPS Control
Standard Endotoxin (CSE) or other E. coli LPS preparations that have been calibrated with an appropriate RSE.
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May 2008

3.0

MATERIALS, EQUIPMENT, AND SUPPLIES

3.1

Blood Donor Eligibility

Monocytoid cells from fresh WB are the source of cytokine production in the WB/IL-6 test
method as described by Hartung and Wendel (1996), Pool et al. (1998), and Schindler et al.
(2006). In the United States (U.S.), the collection of blood and blood components for transfusionand further manufacture (including the use of resulting monocytes in a licensed test) is currently
regulated under Section 351 of the Public Health Service (PHS) Act (U.S. Code [U.S.C.], Title
42, Chapter 6A) and/or the Federal Food Drug and Cosmetic Act (U.S.C., Title 21, Chapter 9),
both of which require compliance with Current Good Manufacturing Practice (cGMP)
regulations (21 CFR Parts 210, 211 and 600-6402).
These regulations and the associated FDA guidance's provide an important resource for
information regarding the currently accepted practice for blood manufacture and collection(including donor screening) (http://www.fda.gov/cber/blood.htm). Specifically, guidance
regarding donor screening questionnaires and links to currently acceptable questionnaires can befound at http://fda.gov/cber/gdlns/donorshitques.htm#iv. If you have any questions regarding theapplication of blood regulations to your specific situation3, it is recommended that you e-mail the
Manufacturers Assistance and Technical Training (MATT) Branch established by FDA at
matt@cber.fda.gov for advice.
Any participating blood establishment should address how the unused components of blood
donations will be accounted for and ultimately destroyed, and if the establishment will store theblood preparation, describe the storage procedures to be followed.
3.2

Equipment and Supplies

For all steps in the protocol, excluding the ELISA procedure, the materials that will be in close
contact with samples and/or blood cells (e.g., pipet tips, containers, solutions) should be sterileand pyrogen-free.
3.2.1 Blood Incubation
3.2.1.1

Equipment• Centrifuge•

Hood; Bio-safety, laminar flow (recommended)•

Incubator; cell culture (37±1°C + 5% CO2)

2

The requirements for WB can be found at 21 CFR 640.1 et seq. In addition, there are specific regulations applicable
to red blood cells, platelets, and other blood components. See, for example, 21 CFR 640.10-640.27. Other
regulations applicable to the manufacture of blood and blood components include 21 CFR Part 606, the cGMP
requirements for blood and blood components, 21 CFR 610.40, the requirements for testing of WB donations, and
21 CFR 640.3, the requirements for determining the suitability of the donor. Blood that enters into U.S. interstate
commerce should be tested for antibodies to HIV 1/2, HCV, HTLV I and II, HBc, HBsAg and RPR, WNV and
Chagas.
3
The collection of blood for research and development purposes or as a component of an in vitro test (that is not
subject to licensure) may potentially not be required to adhere to the FDA regulations outlined above.
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3.2.1.2•

Pipetter, multichannel (8- or 12-channel)•

Pipetters, single-channel adjustable (20 and 200 µL)•

Repeating pipetter•

Vortex mixer

Consumables• Centrifuge tubes; polystyrene (15 and 50 mL)•

Combitips; repeating pipetter (1.0 and 2.5 mL)•

Needle set; multifly, pyrogen-free, 19 mm, 21 gauge•

Plates; microtiter, 96-well, polystyrene, tissue culture•

Pyrogen-free saline (PFS)•

Reaction tubes; polystyrene (1.5 mL)•

Reservoirs; for blood collection•

Tips; pipetter, sterile, pyrogen-free (20 and 200 µL)

3.2.2

ELISA

3.2.2.1

Equipment• Microplate mixer

3.2.2.2

May 2008•

Microplate reader (450 nm with an optional reference filter in the range of 540590 nm)4•

Microplate washer (optional)•

Multichannel pipetter

Consumables• Container; storage, plastic•

Deionized water; nonsterile•

Plates; microtiter, 96-well, polystyrene•

Pyrogen-free water (PFW)•Reservoirs; fluid•

Tips; pipetter, nonsterile•

Tubes; polystyrene (12 mL)

4

The TMB chromagen is measured at OD450. However, the use of an IL-1β ELISA kit with a chromagen other than
TMB is acceptable. The ELISA should be measured at a wavelength appropriate for the specific chromagen used.
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May 2008

3.2.2.3 ELISA Kit
An ELISA that measures IL-6 release is used. A variety of IL-6 ELISA kits are commerciallyavailable and the IL-6 ELISA procedure outlined in this protocol is intended to serve as an
example for using an ELISA kit. The IL-6 ELISA should be calibrated using an IL-6
international reference standard (e.g., World Health Organization [WHO] 89/548) prior to use.
The IL-6 cytokine assay kits do not provide the RSE or endotoxin equivalent; therefore thisreagent must be purchased separately. Results obtained using these products are subject to theassay acceptability and decision criteria described in Sections 8.0 and 9.0. IL-6 ELISA kit
components may include the following:

3.3•

ELISA plates coated with anti-human IL-6 capture antibody; monoclonal or
polyclonal•

Buffered wash solution•

Dilution buffer•

Enzyme-labeled detection antibody•

Human IL-6 reference standard

•

PFS

•

Stop solution•

TMB5/substrate solution

Chemicals•

3.4

Endotoxin (e.g., WHO-lipopolysaccharide [LPS] 94/580 Escherichia coli [E. coli]
O113:H10:K-; United States Pharmacopeia [USP] RSE E. coli LPS Lot G3E069;
USP RSE E. coli Lot G; FDA E. coli Lot EC6)

Solutions

ELISA solutions are listed in Section 3.2.
4.0

ASSAY PREPARATION

All test substances, endotoxin, and endotoxin-spiked solutions should be stored as specified in
the manufacturer's instructions. The collection of WB is outlined in Section 6.1.
4.1

Endotoxin Standard Curve

An internationally harmonized RSE or equivalent is used to generate the endotoxin standard
curve. The use of any other E. coli LPS requires calibration against a RSE using the WB/IL-6
pyrogen test. A standard endotoxin curve consisting of a Negative Saline Control (NSC) and five
RSE concentrations (0.125, 0.25, 0.50, 1.0, and 2.0 EU/mL) are included in the incubation step(refer to Table 4-1) and then transferred to the ELISA plate. To prepare the endotoxin standard
curve, first obtain a 2000 EU/mL stock solution by addition of PFW to the lyophilized content of
5

The use of an IL-6 ELISA kit with a chromagen other than TMB is acceptable.
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May 2008

the stock vial by following the instructions provided by the manufacturer (e.g., 5 mL of PFW is
added to a vial containing 10,000 EU). To reconstitute the endotoxin, the stock vial should bevortexed vigorously for at least 30 min or sonicated in a bath sonicator for at least 5 min.
Subsequent dilutions should be vortexed vigorously immediately prior to use. The stock solution
is stable for not more 14 days when stored at 2 to 8°C or for up to 6 months when kept in a -20°C
freezer. An endotoxin standard curve is prepared as described in Table 4-1 by making serial
dilutions of the stock solution in PFS with vigorous vortexing at each dilution step. Dilutions
should not be stored, because dilute endotoxin solutions are not as stable as concentrated
solutions due to loss of activity by adsorption, in the absence of supporting data to the contrary.
Table 4-1

Preparation of Endotoxin Standard Curve

Stock Endotoxin
EU/mL1µL of Stock
EndotoxinµL of PFS

Endotoxin
Concentrationin Tube
EU/mL
204
2.0
1.0
0.50
0.25
0.125
0

20002,3
20
1980
20
100
900
2.0
500
500
1.0
500
500
0.50
500
500
0.25
500
500
0
0
1000
Abbreviations: EU = Endotoxin units; PFS = Pyrogen-free saline
Each stock tube should be vortexed prior to its use to make the subsequent dilution.
1
To reconstitute the endotoxin, the stock vial should be vortexed vigorously for at least 30 min or sonicated in a bathsonicator for at least 5 min. Subsequent dilutions should be vortexed vigorously immediately prior to use.
2
A 2000 EU/mL stock solution of endotoxin is prepared according to the manufacturer's instructions.
3
The stock solution is stable for not more 14 days when stored at 2 to 8°C or for up to 6 months when kept in a 20°C freezer.
4This concentration is not used in the assay.

4.2

Interference Test

For every test substance lot, interference testing must be performed to check for interference
between the test substance and the cell system and/or ELISA. The purpose of the interference
test is to determine whether the test substance (or specific lot of test substance) has an effect on
cytokine release.
4.2.1
Interference with the Cell System
All test substances must be labeled as pyrogen-free (i.e., endotoxin levels at an acceptable level
prior to release by the manufacturer) to ensure that exogenous levels of endotoxin do not affectthe experimental outcome. Liquid test substances should be diluted in PFS. Solid test substances
should be prepared as solutions in PFS or, if insoluble in saline, dissolved in dimethyl sulfoxide(DMSO) and then diluted up to 0.5% (v/v) with PFS, provided that this concentration of DMSO
does not interfere with the assay. To ensure a valid test, a test substance cannot be diluted
beyond its Maximum Valid Dilution (MVD) (refer to Section 12.3). The calculation of the MVD

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May 2008

is dependent on the ELC for a test substance. The ELC can be calculated by by dividing thethreshold human pyrogenic dose by the maximum recommended human dose in a single hour
period (see Section 12.2) (USP 2007; FDA 1987). Furthermore, test substances should not be
tested at concentrations that are cytotoxic to blood cells.
4.2.1.1 Reference Endotoxin for Spiking Test Substances
The WHO-LPS 94/580 [E. coli O113:H10:K-] or equivalent internationally harmonized RSE isrecommended for preparation of the endotoxin-spike solution and the endotoxin standard curve(see Section 4.1).
4.2.1.2 Spiking Test Substances with Endotoxin
Non-spiked and endotoxin-spiked test substances are prepared in quadruplicate and an in vitro
pyrogen test is performed. A fixed concentration of the RSE (i.e., 1.0 EU/mL or a concentration
equal to or near the middle of the endotoxin standard curve) is added to the undiluted test
substance (or in serial two-fold dilutions, not to exceed the MVD). An illustrative example ofendotoxin-spiking solutions is shown in Table 4-2. For non-spiked solutions, 50 µL of PFS is
added to a well followed by 50 µL of WB and mixed by inversion. Then, 50 µL of the test
substance (i.e., equivalent to the negative product control [NPC]) is added followed by 100 µL of
PFS and the well contents are mixed. Endotoxin-spiked solutions are prepared by adding 50 µL
of PFS to each well followed by 50 µL of WB and mixed by inversion. Then, 50 µL of the test
substance, 50 µL of an endotoxin-spike solution (1.0 EU/mL), and 50 µL of PFS (i.e., equivalent
to the positive product control [PPC]) are added to each well. The contents of the wells are
mixed and incubated as outlined in Section 6.1.3, Steps 6-8. An ELISA is then performed as
outlined in Section 6.2, without the IL-6 standard curve.
Table 4-2

Preparation of Endotoxin-Spiked and Non-Spiked Solutions for
Determination of Test Substance Interference
SpikedSample Addition

Non-spiked1µL/well
PFS (total volume added)
1002
1502
3
Endotoxin-spike solution
50
0
Test substance (neat and each serial dilution)
50
50
WB
50
50
Total4250
250
Abbreviations: PFS = Pyrogen-free saline; WB = Whole blood1
n=4 replicates each2
50 µL of WB and 50 µL of PFS are added to each well and mixed by inversion prior to the addition of theremaining components and volume of PFS.
3
Endotoxin concentration is 1.0 EU/mL in PFS.
4
A total volume of 250 µL per well is used for the incubation.

The optical density (OD) values of the endotoxin-spiked and non-spiked test substances arecalibrated against the endotoxin calibration curve. The resulting EU value of the non-spiked test
substance is subtracted from the corresponding EU value of the endotoxin-spiked test substanceat each dilution. The spike recovery for each sample dilution is calculated as a percentage by
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May 2008

setting the theoretical value (i.e., endotoxin-spike concentration of 1.0 EU/mL) at 100%. For
example, consider the following interference test results in Table 4-3:

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Table 4-3

May 2008

Example of Interference Data Used to Determine Sample Dilution
Sample Dilution
None
1:2
1:4
1:8

% Recovery of Endotoxin Control
25
49
90
110

If a spike recovery between 50% and 200% is obtained, then no interference of the test substancewith either the cell system or the ELISA is demonstrated (i.e., the test substance does notincrease or decrease the concentration of IL-6 relative to the endotoxin spike). The lowest
dilution (i.e., highest concentration) of a test substance that yields an endotoxin-spike recovery
between 50% and 200% is determined. The test substance is then diluted in serial two-folddilutions beginning at this dilution, not exceed the MVD for use in the assay. Based on the
results illustrated in Table 4-3, the initial dilution of the test substance would be 1:4 (i.e., thelowest dilution between 50% and 200% of the 1.0 EU/mL EC).
4.2.2
Interference at the MVD
If the data obtained from the experiment in Section 4.2.1 suggests the presence of interference at
the MVD, then consideration should be given for using another validated pyrogen test method.
5.0

CONTROLS

5.1

Benchmark Controls

Benchmark controls may be used to demonstrate that the test method is functioning properly, or
to evaluate the relative pyrogenic potential of chemicals (e.g., parenteral pharmaceuticals,
medical device eluates) of a specific class or a specific range of responses, or for evaluating therelative pyrogenic potential of a test substance. Appropriate benchmark controls should have thefollowing properties:

5.2•

consistent and reliable source(s) for the chemicals (e.g., parenteralpharmaceuticals, medical device eluates)•

structural and functional similarities to the class of substance being tested•

known physical/chemical characteristics•

supporting data on known effects in animal models•

known potency in the range of response

Endotoxin Control

The EC (i.e., WB incubated with an internationally harmonized RSE) serves as the positive
control in each experiment. The results should be compared to historical values to insure that itprovides a known level of cytokine release relative to the NSC.

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5.3

May 2008

Negative Saline Control

The NSC (i.e., WB incubated with PFS instead of the test substance) is included in eachexperiment in order to detect nonspecific changes in the test system, as well as to provide abaseline for the assay endpoints.
5.4

Solvent Control

Solvent controls are recommended to demonstrate that the solvent is not interfering with the test
system when solvents other than PFS are used to dissolve test substances.
6.0

EXPERIMENTAL DESIGN

6.1

Incubation with Test Samples and Measurement of IL-6 Release

6.1.1
Collection of Human Blood
Human volunteers that have met the donor eligibility criteria described in Section 3.1 are used as
the source of WB. All components of the blood collection system (e.g., syringes, tubes,
connecting lines) must be sterile and pyrogen-free. WB is drawn by venipuncture6 from themedial cubital or cephalic vein of either the right or left arm and collected in a sterile containerthat contains anticoagulant solution (e.g., heparin). The total volume of blood collected per donor(i.e., up to 500 mL) will be dictated by experimental design and determined by the test method
user. WB should be stored at room temperature (RT) and must be used within 4 hr7. All
subsequent handling of WB should be performed in a laminar flow hood using sterile techniqueto prevent contamination.
Prior to use in the assay, an equal volume of WB from multiple individual donors should bepooled8.
6.1.2
Incubation Plate
Test substances are prepared at a level of dilution that did not show interference with the test
system, provided that this dilution does not exceed the MVD. Each incubation plate canaccommodate an endotoxin standard curve, a NSC, and 14 test substances (see Table 6-1).

6

WB is obtained using Universal Precautions (e.g., latex gloves, labcoats, safety glasses) and sterile equipment (e.g.,syringes, needles, collection tubes) within a hospital or clinical setting by qualified and adequately trained personnel(i.e., registered nurse, licensed phlebotomist, or medical doctor).
7
Although the ECVAM SOP did not describe the use of cryopreserved WB for the WB/IL-6 test method, the use of
cryopreserved WB with the WB/IL-1 test method was outlined and this methodology may also be appropriate for the
WB/IL-6 test method, but this has yet to be demonstrated.
8
Multiple donors (i.e., a minimum of three) should meet the acceptability criteria as outlined in Section 8.0 either asa pool of multiple individual donors or as multiple individual donors tested independently.
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Table 6-1

May 2008

Overview of Incubation Plate Preparation in the WB/IL-6 Pyrogen Test

Numberof Wells

Sample

PFS

EC

Test
SampleWB

Mix thesamples;incubate for
16 to 24 hr at
37±1°C in ahumidified
atmospherewith 5%CO2.

Mix thesamples;
immediatelytransfer to an
ELISA plate3
and runELISA.

µL
EC
100
50
0
50
NSC
150
0
0
50
Test
562
samples
100
050
50(1-14)
Abbreviations: EC = Endotoxin control; IL-6 = Interleukin-6; NSC = Negative saline control; PFS = Pyrogen-freesaline WB = Whole blood
1
Five EC concentrations (0.125, 0.25, 0.50, 1.0, and 2.0 EU/mL) in quadruplicate
2
14 test samples (n=4 each) per plate
3
An IL-6 standard curve is prepared in Columns 11 and 12 on the ELISA plate (see Table 6-3). Therefore, 80 wells
are available for test samples and controls on the incubation plate.
201
4

6.1.3
Incubation Assay for IL-6 Release
Test substances should be vortexed vigorously for at least 30 min or sonicated in a bath sonicator
for at least 5 min prior to use in the assay. Test substances should be prepared in serial two-folddilutions beginning at a level of dilution that did not show interference with the test system (seeSection 4.2) in as many subsequent dilutions that are necessary to be within the linear range ofthe endotoxin standard curve, not to exceed the MVD. Blood samples are prepared in a
microtiter plate using a laminar flow hood. All consumables and solutions must be sterile andpyrogen-free. Each plate should be labeled appropriately with a permanent marker. An overview
of the incubation plate preparation is shown in Table 6-1. The incubation procedure is outlined
below:•

Step 1. Refer to the incubation plate template presented in Table 6-2.

•

Step 2. Using a pipetter, transfer 100 µL of PFS into each well.

•

Step 3. Transfer 50 µL of test sample or 50 µL of PFS for the NSC into theappropriate wells as indicated in the template.

•

Step 4. Transfer 50 µL of the EC (standard curve) in quadruplicate into theappropriate wells according to the template.

•

Step 5. Transfer 50 µL of WB into each well and mix by gently swirling the plate.

•

Step 6. Mix the contents of the wells thoroughly by gently pipetting up and down
several times using a multichannel pipetter, changing the tips between each row inorder to avoid cross-contamination.

•

Step 7. Place the covered plate in a tissue culture incubator for 16 to 24 hr at
37±1°C in a humidified atmosphere containing 5% CO2.

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ICCVAM Test Method Evaluation Report: Appendix C3•

Table 6-2

May 2008

Step 8. Prior to transferring the test samples to the ELISA plate, mix the contentsof the wells by pipetting up and down using a multichannel pipetter, changing thetips between each row in order to avoid cross-contamination.
Incubation Plate - Sample and Control Template

1
2
3
4
5
6
7
8
9
10
11
EC1
EC
EC
EC
A
TS3
TS3
TS3
TS3
TS11 TS11
Void3
2.0
2.0
2.0
2.0
EC
EC
EC
EC
B
TS4
TS4
TS4
TS4
TS11 TS11
Void
1.0
1.0
1.0
1.0
EC
EC
EC
EC
C
TS5
TS5
TS5
TS5
TS12 TS12
Void
0.50
0.50
0.50
0.50
EC
EC
EC
EC
D
TS6
TS6
TS6
TS6
TS12 TS12
Void
0.25
0.25
0.25
0.25
EC
EC
EC
EC
E
TS7
TS7
TS7
TS7
TS13 TS13
Void
0.125 0.125 0.125
0.125
F
NSC NSC
NSC
NSC
TS8
TS8
TS8
TS8
TS13 TS13
Void
G
TS12
TS1
TS1
TS1
TS9
TS9
TS9
TS9
TS14 TS14
Void
H
TS2
TS2
TS2
TS2
TS10
TS10
TS10
TS10
TS14 TS14
Void
Abbreviations: EC = Endotoxin control; NSC = Negative saline control; TS = Test substance1
EC value (e.g., EC 2.0) represents the endotoxin concentration in EU/mL.
2
TS number (e.g., TS 1) represents an arbitrary sequence for individual test substances.
3
Columns 11 and 12 are reserved for the IL-6 standard curve on the ELISA plate (see Table 6-3).

6.2

12
Void
Void
Void
Void
Void
Void
Void
Void

ELISA to Measure IL-6 Release

6.2.1
IL-6 Standard Curve
An IL-6 standard, supplied with the ELISA kit, is used. IL-6 standards are typically supplied inlyophilized form and should be reconstituted according to the manufacturer's instructions. The
stock solution should be diluted in PFS to the following concentrations: 0, 62.5, 125, 250, 500,
1000, 2000, and 4000 pg/mL in volumes of at least 500 µL. Each well on the ELISA plate will
receive 50 µL of an IL-6 blank or standard.
6.2.2
ELISA
The manufacturer's instructions provided with the ELISA kit should be followed and a typicalexperimental design is outlined below. The ELISA should be carried out at RT and therefore allcomponents must be at RT prior to use. Frozen specimens should not be thawed by heating themin a water bath. A suggested ELISA plate template is shown in Table 6-3, which includes a fivepoint EC standard curve, an eight-point IL-6 standard curve (0 to 4000 pg/mL), and available
wells for up to 14 test substances and a NSC each in quadruplicate. The EC standard curve, theNSC, and the test sample supernatants are transferred directly from the incubation plate. The IL6 standard curve is prepared as described in Section 6.2.1. An overview of the ELISA plate
preparation is shown in Table 6-4.
Step 1. After pipetting up and down three times to mix the supernatant, transfer 50 µL
from each well of the Incubation Plate (A1-10; H1-10) to the ELISA plate.

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ICCVAM Test Method Evaluation Report: Appendix C3

May 2008

Step 2. Add 50 µL of each IL-6 standard (0 to 4000 pg/mL) into the respective wellson the ELISA plate.
Step 3. Add 200 µL of the enzyme-labeled detection antibody (neat as supplied, or
diluted, if necessary) to each of the wells.
Step 4. Cover the microtiter plate(s) with adhesive film and incubate for 2 to 3 hr at
RT.
Step 5. Decant and wash each well three times with 300 µL Buffered Wash Solution
and then rinse three times with deionized water. Place the plates upside down and tap
to remove water.
Step 6. Add 200 µL of TMB/Substrate Solution to each well and incubate at RT in thedark for 15 min. If necessary, decrease the incubation time.
Step 7. Add 50 µL of Stop Solution to each well.
Step 8. Tap the plate gently after the addition of Stop Solution to aid in mixing.
Step 9. Read the OD450 within 15 min of adding the Stop Solution. Measurement witha reference wavelength of 540 to 590 nm is recommended.9
Table 6-3

ELISA Plate - Sample and Control Template

1

2

3

4

EC1
2.0
EC
1.0
EC
0.50
EC
0.25
EC
0.125

EC
2.0
EC
1.0
EC
0.50
EC
0.25
EC
0.125

EC
2.0
EC
1.0
EC
0.50
EC
0.25
EC
0.125

EC
2.0
EC
1.0
EC
0.50
EC
0.25
EC
0.125

F

NSC

NSC

NSC

G

TS12

TS1

H

TS2

TS2

A
B
C
D
E

5

6

7

8

9

10

TS3

TS3

TS3

TS3

TS11

TS11

TS4

TS4

TS4

TS4

TS11

TS11

TS5

TS5

TS5

TS5

TS12

TS12

TS6

TS6

TS6

TS6

TS12

TS12

TS7

TS7

TS7

TS7

TS13

TS13

NSC

TS8

TS8

TS8

TS8

TS13

TS13

TS1

TS1

TS9

TS9

TS9

TS9

TS14

TS14

TS2

TS2

TS10

TS10

TS10

TS10

TS14

TS14

11

12

IL-63
0
IL-6
62.5
IL-6
125
IL-6
250
IL-6
500
IL-6
1000
IL-6
2000
IL-6
4000

IL-6
0
IL-6
62.5
IL-6
125
IL-6
250
IL-6
500
IL-6
1000
IL-6
2000
IL-6
4000

Abbreviations: EC = Endotoxin control; NSC = Negative saline control; TS = Test substance1
EC value (e.g., EC 2.0) represents the endotoxin concentration in EU/mL.
2
TS number (e.g., TS1) represents an arbitrary sequence for individual test substances.
3
IL-6 values in columns 11 and 12 are in pg/mL.

9

The TMB chromagen is measured at OD450. However, the use of an IL-1β ELISA kit with a chromagen other than
TMB is acceptable. The ELISA should be measured at a wavelength appropriate for the specific chromagen used.
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ICCVAM Test Method Evaluation Report: Appendix C3

Table 6-4
MaterialtransferfromIncubation
Plate

May 2008

Overview of ELISA Procedure

(µL)

IL-6
standard(0 to
4000
pg/mL)
(µL)

50

50

Enzymelabeled
Antibody(µL)

200

Cover theIncubation
Plate andincubatefor 2 to 3
hr at RT.

Decantand washeach well
three
times
with 300µL
Buffered
Wash
Solutionand three
times
with
deionized
water.

TMB/Substrate
Solution
(µL)

200

Stop
Solution
Incubatefor lessthan 15
min at
RT in
the
dark.

(µL)

50

Read
each wellat OD450with a
540 to590 nmreference
filter.

Abbreviations: OD450 = Optical density at 450 nm; RT = Room temperature

7.0

EVALUATION OF TEST METHODS

7.1

OD Measurements

The OD of each well is obtained by reading the samples in a standard microplate
spectrophotometer (i.e., plate reader) using a visible light wavelength of 450 nm (OD450) with a
540 to 590 nm reference filter (recommended)10. OD values are used to determine assay
acceptability and in the decision criteria for pyrogen detection (see Sections 8.0 and 9.0).
8.0

CRITERIA FOR AN ACCEPTABLE TESTAn EC (five-point standard curve) and a NSC should be included in each experiment. An IL-6
standard curve should be included in each ELISA as shown in the template presented in Table 63. An assay is considered acceptable only if the following minimum criteria are met:•

The quadratic function of the IL-6 standard curve produces an r ≥0.9511 and the
OD of the blank control is below 0.15.

•

The endotoxin standard curve produces OD values that ascend in a sigmoidal
concentration response.

Blood donors (or a pool of blood donors) are considered to be high responders if theirconcentration of IL-6 is greater than 200 pg/mL. High responders should be excluded fromanalysis. The preparation being examined is required to pass the test with blood donations fromat least three different donors (i.e., either as a pool of three individual donors or as three
individual donors tested independently).
10

The TMB chromagen is measured at OD450. However, the use of an IL-1β ELISA kit with a chromagen other than
TMB is acceptable. The ELISA should be measured at a wavelength appropriate for the specific chromagen used.
11
Correlation coefficient (r), an estimate of the correlation of x and y values in a series of n measurements.

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May 2008

An outlying observation that represents either a pool of multiple independent donors or a single
individual donor may be excluded if there is confirmation that the accuracy of the medical
information provided by an individual donor is suspect, or if the aberrant response is identified
using acceptable statistical methodology (e.g., Dixon's test [Dixon 1950; Barnett and Lewis
1994], Grubbs' test [Barnett and Lewis 1994; Grubbs 1969; Iglewicz and Houghlin 1993]).
9.0

DATA INTERPRETATION/DECISION CRITERIA

9.1

Decision Criteria for Pyrogen Detection

A test substance is considered pyrogenic when the endotoxin concentration of the test substanceexceeds the ELC for the test sample. The ELC can be calculated as shown in Section 12.2.
10.0

STUDY REPORTThe test report should include the following information:
Test Substances and Control Substances•

Name of test substance•Purity and composition of the substance or preparation•

Physicochemical properties (e.g., physical state, water solubility)•

Quality assurance data•

Treatment of the test/control substances prior to testing (e.g., vortexing,
sonication, warming, resuspension solvent)Justification of the Test Method and the Protocol Used
Test Method Integrity•

The procedure used to ensure the integrity (i.e., accuracy and reliability) of thetest method over time•

If the test method employs proprietary components, documentation on theprocedure used to ensure their integrity from “lot-to-lot” and over time•

The procedures that the user may employ to verify the integrity of the proprietarycomponents

Criteria for an Acceptable Test•

Acceptable concurrent positive control ranges based on historical data•

Acceptable negative control data

Test Conditions•

Cell system used•

Calibration information for the spectrophotometer used to read the ELISA•

Details of test procedure used

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May 2008•

Description of any modification to the test procedure•

Reference to historical data of the model•

Description of the evaluation criteria used•

Tabulation of data from individual test samples

Results
Description of Other Effects Observed
Discussion of the Results
Conclusion
A Quality Assurance Statement for Good Laboratory Practice (GLP)-Compliant Studies•

This statement should indicate all inspections made during the study and the dates
any results were reported to the Study Director. This statement should also
confirm that the final report reflects the raw data.

If GLP-compliant studies are performed, then additional reporting requirements provided in therelevant guidelines (e.g., OECD 1998; EPA 2003a, 2003b; FDA 2003) should be followed.
11.0

REFERENCES

Barnett V, Lewis T. 1994. Outliers in Statistical Data. In: Wiley Series in Probability andMathematical Statistics. Applied Probability and Statistics. 3rd Ed. New York: John Wiley &Sons.
Dixon WJ. 1950. Analysis of extreme values. Annals of Mathematical Statistics 21:488-506.
EPA. 2003a. Good Laboratory Practice Standards. Toxic Substances Control Act. 40 CFR 792.
EPA. 2003b. Good Laboratory Practice Standards. Federal Insecticide, Fungicide, andRodenticide Act. 40 CFR 160.
FDA. 1987. Guideline on Validation of the Limulus Amebocyte Lysate Test as an End-product
Endotoxin Test for Human and Animal Parenteral Drugs, Biological Products, and Medical
Devices. Rockville, MD:U.S. Department of Health and Human Services (DHHS), Food andDrug Administration (FDA).
FDA. 2003. Good Laboratory Practices for Nonclinical Laboratory Studies. 21 CFR 58.
Grubbs FE. 1969. Procedures for detecting outlying observations in samples. Technometrics
11(1):1-21.
Hartung T, Wendel A. 1996. Detection of pyrogens using human whole blood. In Vitro Toxicol
9(4):353-359.
Iglewicz B, Houghlin DC. 1993. How to detect and handle outliers. In: ASQC Basic Referencein Quality Control. Vol. 14. Milwaukee, WI: ASQ Quality Press.
Organization for Economic Co-operation and Development (OECD). 1998. OECD Series on
Principle of Good Laboratory Practice and Compliance Monitoring. No. 1. OECD Principles of

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ICCVAM Test Method Evaluation Report: Appendix C3

May 2008

Good Laboratory Practice (as revised in 1997). Organisation for Economic Co-operation andDevelopment, ENV/MC/CHEM (98)17. Paris: OECD.
Pool EJ, Johaar G, James S, Petersen I, Bouic P. 1998. The detection of pyrogens in blood
products using an ex vivo whole blood culture assay. J Immunoassay 19:95-111.
Schindler S, Spreitzer I, Löschner B, Hoffmann S, Hennes K, Halder M, Brügger P, Frey E,
Hartung T, Montag T. 2006. International validation of pyrogen tests based on cryopreserved
human primary blood cells. J Immunol Methods 316:42-51.
USP. 2007. The U.S. Pharmacopeia. USP30 NF25<85>. Ed. The U.S. Pharmacopeial
Convention. Rockville, MD:The U.S. Pharmacopeial Convention.

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ICCVAM Test Method Evaluation Report: Appendix C3

12.0
12.1

May 2008

TERMINOLOGY AND FORMULA
Assay Sensitivity (λ)1

The variable λ is defined as the labeled sensitivity (in EU/mL) of the LAL Reagent in
endpoint assays (e.g., the BET gel-clot technique). For kinetic BET assays, λ is the lowest
point used in the endotoxin standard curve.
12.2

Endotoxin Limit Concentration (ELC)1,2

The ELC for parenteral drugs is expressed in Endotoxin Units (EU) per volume (mL) or
weight (mg). The ELC is equal to K/M, where:
K is the threshold human pyrogenic dose of endotoxin (EU) per body weight (kg). K is equal
to 5.0 EU/kg for intravenous administration. For intrathecal administration, K is equal to 0.2
EU/kg (see also Section 12.5).
M is the rabbit test dose or the maximum recommended human dose of product (mL or mg)
per body weight (kg) in a single hour period (see also Section 12.8).
For example, if a non-intrathecal product were used at an hourly dose of 10 mL per patient,then the ELC would be 0.50 EU/mL.
12.3

Maximum Valid Dilution (MVD)1,2

The MVD is the maximum allowable dilution of a test substance at which the endotoxin limit
can be determined. The calculation of the MVD is dependent on the ELC for a test substance.
When the ELC is known, the MVD is1:
MVD = (ELC x Product Potency [PP])/λAs an example, for Cyclophosphamide Injection, the ELC is 0.17 EU/mg, PP is 20
mg/mL, and the assay sensitivity is 0.065 EU/mL. The calculated MVD would be 1:52.3 or
1:52. The test substance can be diluted no more than 1:52 prior to testing.
If the ELC is not known, the MVD is1:
MVD = PP/Minimum Valid Concentration (MVC)
where, MVC = (λ x M)/K
where, M is the maximum human dose
As an example, for Cyclophosphamide Injection, the PP is 20 mg/mL, M is 30 mg/kg,
and assay sensitivity is 0.065 EU/mL. The calculated MVC is 0.390 mg/mL and the MVD is
1:51.2 or 1:51. The test substance can be diluted no more than 1:51 in the assay prior to
testing.
12.4

Negative Product Control (NPC)

For interference testing, the NPC is a test sample to which pyrogen-free saline (PFS) is
added. The NPC is the baseline for determination of cytokine release relative to theendotoxin-spiked PPC.
1
2

From FDA (1987)
From USP (2007)
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ICCVAM Test Method Evaluation Report: Appendix C3

12.5

May 2008

Parenteral Threshold Pyrogen Dose (K)1,2

The value K is defined as the threshold human pyrogenic dose of endotoxin (EU) per bodyweight (kg). K is equal to 5.0 EU/kg for parenteral drugs except those administeredintrathecally; 0.2 EU/kg for intrathecal drugs.
12.6

Positive Product Control (PPC)

For interference testing, the PPC is a test substance spiked with the control standard
endotoxin (i.e., 0.5 EU/mL or an amount of endotoxin equal to that which produces ½ themaximal increase in optical density (OD) from the endotoxin standard curve) to insure thatthe test system is capable of endotoxin detection in the product as diluted in the assay.
12.7

Product Potency (PP)1,2

The test sample concentration expressed as mg/mL or mL/mL.
12.8

Rabbit Pyrogen Test (RPT) Dose or Maximum Human Dose (M)1,2

The variable M is equal to the rabbit test dose or the maximum recommended human dose of
product per kg of body weight in a single hour period. M is expressed in mg/kg or mL/kg andvaries with the test substance. For radiopharmaceuticals, M equals the rabbit dose or
maximum human dose/kg at the product expiration date or time. Use 70 kg as the weight of
the average human when calculating the maximum human dose per kg. If the pediatricdose/kg is higher than the adult dose, then it shall be the dose used in the formula.

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ICCVAM Test Method Evaluation Report: Appendix C

May 2008

Appendix C4
The Human Peripheral Blood Mononuclear Cell (PBMC)/IL-6 In Vitro Pyrogen Test

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May 2008

ICCVAM Test Method Evaluation Report: Appendix C4

May 2008

ICCVAM Final Recommended Protocol for Future Studies Using the Human
Peripheral Blood Mononuclear Cell (PBMC)/Interleukin (IL)-6 In Vitro Pyrogen Test
PREFACEThis protocol is for the detection of Gram-negative endotoxin, a pyrogen, in parenteral drugs,
as indicated by the release of IL-6 from human peripheral blood mononuclear cells (PBMCs).
This protocol is based on information obtained from 1) the European Centre for the
Validation of Alternative Methods (ECVAM)1 PBMC/IL-6 Background Review Document(BRD) presented in Appendix A of the Interagency Coordinating Committee on the
Validation of Alternative Methods (ICCVAM) BRD (available at
http://iccvam.niehs.nih.gov/methods/pyrogen/pyr_brd.htm), and 2) information provided to
the National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative
Toxicological Methods (NICEATM) by Dr. Thomas Hartung, Head of ECVAM. The
ICCVAM BRD includes the ECVAM Standard Operating Procedures (SOPs) for the
PBMC/IL-6 test (could be referred to as Monocyte Activation Test), which is based on
various methods that use human PBMCs to detect cytokine production as a measure ofpyrogen presence (Bleeker et al. 1994; Dinarello et al. 1984; Poole et al. 2003). A table ofcomparison between the ICCVAM recommended protocol and the ECVAM SOPs isprovided in Table 1.
Users should contact the relevant regulatory authority for guidance when using thisICCVAM recommended protocol to demonstrate product specific validation, and any
deviations from this protocol should be accompanied by scientifically justified rationale.
Future studies using the PBMC/IL-6 pyrogen test may include further characterization of theusefulness or limitations of the assay for regulatory decision-making. Users should be awarethat this protocol might be revised based on additional optimization and/or validation studies.
ICCVAM recommends that test method users routinely consult the ICCVAM/NICEATM
website (http://iccvam.niehs.nih.gov) to ensure that the most current protocol is used.

1

ECVAM is a unit of the Institute for Health and Consumer Protection at the European Commission's Joint
Research Centre.
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ICCVAM Test Method Evaluation Report: Appendix C4[This Page Intentionally Left Blank]

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May 2008

Table 1

Comparison of ICCVAM Recommended Protocol with the ECVAM SOPs for the PBMC/IL-6 Pyrogen Test

Protocol Component
Test Substance
Number of Blood Donors
Decision Criteria for Interference
Incubation Plate for ELISA(The number of samples or controls
measured in quadruplicate)
ELISA PlateICCVAM Protocol
Test neat or in serial dilutions that
produce no interference, not to exceed the
MVD
Minimum of 3 (independent or pooled)
Mean OD3 of PPC is 50% to 200% of 0.25
EU/mL EC
NSC (1)EC (5)
TS (14)
PPC4 (0)NPC4 (0)Includes seven point IL-6 SC and blank in
duplicate
Mean OD of NSC ≤0.15

Test at MVD

Test at MVD

Minimum of 32 (independent)
Mean OD of PPC is 50% to 200%
of 0.25 EU/mL EC
NSC (1)EC (5)TS (2) x EC (5) spikes = 10 TS
PPC (2) = 2 TS
NPC (2) = 2TS
Includes seven point IL-6 SC andblank in duplicate
Mean OD of NSC ≤0.15
Quadratic function of IL-6 SC r
≥0.95
EC SC produces OD values thatascend in a sigmoidal
concentration responseHigh responder blood donors (i.e., > 200
pg/mL IL-6) or low responder blood
donors (i.e., Mean OD of 1EU/mL EC issignificantly less than that of 1000 pg/mLIL-6) may be excluded

High responder blood donors (i.e.,> 200 pg/mL IL-6) or low
responder blood donors (i.e., Mean
OD of 1EU/mL EC is significantly
less than that of 1000 pg/mL IL-6)
may be excluded

Outliers rejected using
Dixon's test

Outliers rejected using
Dixon's test

Endotoxin concentration
TS > ELC6 TS

Endotoxin concentration
TS > ELC TS

Endotoxin concentration
TS > ELC TS7EC SC produces OD values that ascend ina sigmoidal concentration response

Decision Criteria for Pyrogenicity

ECVAM Validation SOP1

Minimum of 4 (independent)
Mean OD of PPC is 50% to200% of 0.25 EU/mL EC
NSC (1)EC (5)TS (2) x EC (5) spikes = 10 TS
PPC (2) = 2 TS
NPC (2) = 2TS
Includes seven point IL-6 SC
and blank in duplicate
Mean OD of NSC ≤0.15
Quadratic function of IL-6 SC r
≥0.95
EC SC produces OD values thatascend in a sigmoidal
concentration responseHigh responder blood donors(i.e., > 200 pg/mL IL-6) or low
responder blood donors (i.e.,
Mean OD of 1EU/mL EC issignificantly less than that of1000 pg/mL IL-6) may be
excluded
Outliers rejected using
Dixon's test

Quadratic function of IL-6 SC r ≥0.955

Assay Acceptability Criteria

ECVAM Catch-Up
Validation SOP1

Abbreviations: EC = Endotoxin control; ELC = Endotoxin limit concentration; ELISA = Enzyme-linked immunosorbent assay; EU = Endotoxin units; IL-6
= Interleukin-6; MVD = Maximum valid dilution; NSC = Negative saline control; OD = Optical density; PBMC = Peripheral blood mononuclear cell; PPC =Positive product control; SC = Standard curve; SOP = Standard operating procedure; TS = Test substance
1
ECVAM PBMC/IL-6 SOPs are presented in Appendix A of the ICCVAM BRD (available at http://iccvam.niehs.nih.gov/methods/pyrogen/pyr_brd.htm).
2
Sample are cryopreserved prior to use in the assay.
3
Mean OD values are corrected (i.e., reference filter reading, if applicable, and NSC are subtracted).
4
In the ICCVAM PBMC/IL-6 protocol, PPC and NPC are assessed in the interference test described in Section 4.2, which is performed prior to the ELISA.
5
Correlation coefficient (r), an estimate of the correlation of x and y values in a series of n measurements.
6
Where unknown, the ELC is calculated (see Section 12.2).
7
Decision criteria for individual donors were defined in the ECVAM Validation SOP for the PBMC/IL-6 test method. Test method users should refer tothese criteria if multiple donors are tested independently.

ICCVAM Test Method Evaluation Report: Appendix C4

1.0

May 2008

PURPOSE AND APPLICABILITY

The purpose of this protocol is to describe the procedures used to evaluate the presence ofGram-negative endotoxin, a pyrogen, in parenteral drugs. The presence of Gram-negative
endotoxin is detected by its ability to induce the release of interleukin (IL)-6 from humanperipheral blood mononuclear cells (PBMCs). The concentration of IL-6 released byincubation of PBMCs with a test substance or controls (i.e., positive and negative) is
quantified using an enzyme-linked immunosorbent assay (ELISA) that includes monoclonalor polyclonal antibodies specific for IL-6. The amount of pyrogen present is determined bycomparing the values of endotoxin equivalents produced by PBMCs exposed to the test
substance to those exposed to an internationally harmonized Reference Standard Endotoxin(RSE)1 or an equivalent standard expressed in Endotoxin Units (EU)/mL. A test substance isconsidered pyrogenic if the endotoxin concentration of the test substance exceeds theEndotoxin Limit Concentration (ELC) for the test substance.
The relevance and reliability of this test method to detect non-endotoxin pyrogens have notbeen demonstrated in a formal validation study, although data are available in the literature tosuggest that this assay has the potential to serve this purpose.
2.0

SAFETY AND OPERATING PRECAUTIONS

All procedures that use human blood-derived materials should follow national/international
procedures for handling blood potentially contaminated with pathogens. An example of suchguidelines is the Universal Precautions available at
http://www.niehs.nih.gov/odhsb/biosafe/univers.htm. For non-human blood procedures (e.g.,
ELISAs), standard laboratory precautions are recommended including the use of laboratory
coats, eye protection, and gloves. If necessary, additional precautions required for specificchemicals will be identified in the Material Safety Data Sheet (MSDS).
The stop solution used in the ELISA kit is acidic and corrosive and should be handled withthe proper personal protective devices. If this reagent comes into contact with skin or eyes,
wash thoroughly with water. Seek medical attention, if necessary.
Tetramethylbenzidine (TMB) solution contains a hydrogen peroxide substrate and 3, 3’, 5,5’- TMB. This reagent is a strong oxidizing agent and a suspected mutagen. Appropriate
personal protection should be used to prevent bodily contact.
Bacterial endotoxin is a toxic agent (i.e., can induce sepsis, shock, vascular damage,antigenic response) and should be handled with care. Skin cuts should be covered andappropriate personal protective devices should be worn. In case of contact with endotoxin,
immediately flush eyes or skin with water for at least 15 minutes (min). If inhaled, remove
the affected individual from the area and provide oxygen and/or artificial respiration asneeded. Skin absorption, ingestion, or inhalation may produce fever, headache, andhypotension.
1

RSEs are internationally-harmonized reference standards (e.g., WHO-lipopolysaccharide [LPS] 94/580
Escherichia coli [E. coli] O113:H10:K-; United States Pharmacopeia [USP] RSE E. coli LPS Lot G3E069; USP
RSE E. coli Lot G; FDA E. coli Lot EC6). Equivalent endotoxins include commercially available E. coliderived LPS Control Standard Endotoxin (CSE) or other E. coli LPS preparations that have been calibrated with
an appropriate RSE.
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ICCVAM Test Method Evaluation Report: Appendix C4

3.0

MATERIALS, EQUIPMENT, AND SUPPLIES

3.1

Blood Donor Eligibility

May 2008

PBMCs from fresh whole blood (WB) are the source of cells for cytokine production in the
PBMC/IL-6 test method as reported by Poole et al. (2003)2. In the United States (U.S.), thecollection of blood and blood components for transfusion and further manufacture (including
the use of resulting PBMCs in a licensed test) is currently regulated under Section 351 of the
Public Health Service (PHS) Act (U.S. Code [U.S.C.], Title 42, Chapter 6A) and/or theFederal Food Drug and Cosmetic Act (U.S.C., Title 21, Chapter 9), both of which requirecompliance with Current Good Manufacturing Practice (cGMP) regulations (21 CFR Parts
210, 211 and 600-6403).
These regulations and the associated FDA guidance's provide an important resource for
information regarding the currently accepted practice for blood manufacture and collection(including donor screening) (http://www.fda.gov/cber/blood.htm). Specifically, guidance
regarding donor screening questionnaires and links to currently acceptable questionnaires canbe found at http://www.fda.gov/cber/gdlns/donorhistques.htm#iv. If you have questions
regarding the applicability of blood regulations to your specific situation4, it is recommended
that you e-mail the Manufacturers Assistance and Technical Training (MATT) Branchestablished by FDA at matt@cber.fda.gov for advice.
Any participating blood establishment should address how unused components of blood
donations will be accounted for and ultimately destroyed, and if the establishment will store
the blood preparation, describe the storage procedures to be followed.
3.2

Equipment and Supplies

For all steps in the protocol, excluding the ELISA procedure, the materials that will be inclose contact with samples and/or blood cells (e.g., pipet tips, containers, solutions) should besterile and pyrogen-free.
3.2.1

Preparation of PBMCs

3.2.1.1

Equipment• Centrifuge•

Hood; Bio-safety, laminar flow (recommended)2

As indicated by the ECVAM Catch-Up Validation SOP for the PBMC/IL-6 test method, PBMCs that havebeen cryopreserved can also be used as the source of cells in the PBMC/IL-6 test method.
3
The requirements for WB can be found at 21 CFR 640.1 et seq. In addition, there are specific regulations
applicable to red blood cells, platelets, and other blood components. See, for example, 21 CFR 640.10-640.27.
Other regulations applicable to the manufacture of blood and blood components include 21 CFR Part 606, thecGMP requirements for blood and blood components, 21 CFR 610.40, the requirements for testing of WB
donations, and 21 CFR 640.3, the requirements for determining the suitability of the donor. Blood that enters
into U.S. interstate commerce should be tested for antibodies to HIV 1/2, HCV, HTLV I and II, HBc, HBsAg
and RPR, WNV and Chagas.
4
The collection of blood for research and development purposes or as a component of an in vitro test (that is not
subject to licensure) may potentially not be required to adhere to the FDA regulations outlined above.
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ICCVAM Test Method Evaluation Report: Appendix C4

3.2.1.2•

Incubator; cell culture (37±1°C + 5% CO2)•

Lymphoprep™

•

Pipetter; multichannel (8- or 12-channel)•

Pipetters; single-channel adjustable (20, 200, and 1000 µL)•

Repeating pipetter•

Vortex mixer

May 2008

Consumables• Centrifuge tubes; polystyrene (15 and 50 mL)•

Combitips; repeating pipetter (2.5 and 5.0 mL)•

Cryotubes; screw-cap, 2 mL•

Filters; sterile, 0.22 µm•

Needle set; multifly, pyrogen-free, 19 mm, 21 gauge•

Phosphate buffered saline (PBS); sterile•

Pipettes; serological, sterile (5, 10, and 25 mL)•

Plates; microtiter, 96-well, polystyrene, tissue culture•

Pyrogen-free saline (PFS)•

Reaction tubes; polystyrene (1.5 mL)•

Reservoirs; for blood collection•

RPMI-1640 cell culture medium (500 mL); supplemented with the following
reagents to yield RPMI-CoHuman serum albumin (HSA); 5 mL or a 1% final concentration)o

L–Glutamine; 200 mM

oPenicillin/streptomycin (10,000 IU/mL penicillin, 10 mg/mL
streptomycin)•

Syringes; sterile (100 µL and 30 mL)•

Tips; pipetter, sterile, pyrogen-free (20, 200, and 1000 µL)

3.2.2

ELISA

3.2.2.1

Equipment• Microplate mixer

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ICCVAM Test Method Evaluation Report: Appendix C4

3.2.2.2

May 2008•

Microplate reader (450 nm with an optional reference filter in the range of
540-590 nm)5•

Microplate washer (optional)•

Multichannel pipetter

Consumables• Container; storage, plastic•

Deionized water; nonsterile•

Plates; microtiter, 96-well, polystyrene•

Pyrogen-free water (PFW)•Reservoirs; fluid•

Tips; pipetter, nonsterile•

Tubes; polystyrene (12 mL)

3.2.2.3 ELISA Kit
An ELISA that measures IL-6 release is used. A variety of IL-6 ELISA kits are commerciallyavailable and the IL-6 ELISA procedure outlined in this protocol is intended to serve as an
example for using an ELISA kit. The IL-6 ELISA should be calibrated using an IL-6
international reference standard (e.g., World Health Organization [WHO] 89/548) prior touse. The IL-6 cytokine assay kits do not provide the RSE or endotoxin equivalent; therefore,
this reagent must be purchased separately. Results obtained using these products are subject
to the assay acceptability and decision criteria described in Sections 8.0 and 9.0. IL-6 ELISA
kit components may include the following:

5•

ELISA plates coated with anti-human IL-6 capture antibody; monoclonal or
polyclonal•

Buffered wash solution•

Dilution buffer•

Enzyme-labeled detection antibody•

Human IL-6 reference standard

•

PFS

•

Stop solution•

TMB6/substrate solution

The TMB chromagen is measured at OD450. However, the use of an IL-1β ELISA kit with a chromagen otherthan TMB is acceptable. The ELISA should be measured at a wavelength appropriate for the specificchromagen used.
6
The use of an IL-6 ELISA kit with a chromagen other than TMB is acceptable.
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ICCVAM Test Method Evaluation Report: Appendix C4

3.3

Chemicals•

3.4

Endotoxin (e.g., WHO-lipopolysaccharide [LPS] 94/580 Escherichia coli [E.
coli] O113:H10:K-; United States Pharmacopeia [USP] RSE E. coli LPS Lot
G3E069; USP RSE E. coli Lot G; FDA E. coli Lot EC6)

Solutions•

4.0

May 2008

RPMI-C cell culture medium; supplemented as described in Section 3.2.1.2

ASSAY PREPARATION

All test substances, endotoxin, and endotoxin-spiked solutions should be stored as specified
in the manufacturer's instructions. The collection of WB, the isolation of PBMCs from WB,
and the procedure for cryopreservation of PBMCs is outlined in Section 6.1.
4.1

Endotoxin Standard Curve

An internationally harmonized RSE or equivalent is used to generate the endotoxin standard
curve. The use of any other E. coli LPS requires calibration against a RSE using the
PBMC/IL-6 pyrogen test. A standard endotoxin curve consisting of a Negative Saline
Control (NSC) and five RSE concentrations (0.063, 0.125, 0.25, 0.50, and 1.0 EU/mL) are
included in the incubation step (refer to Table 4-1) and then transferred to the ELISA plate.
Table 4-1

Preparation of Endotoxin Standard Curve

Stock Endotoxin
EU/mL1µL of Stock
EndotoxinµL of PFS

Endotoxin
Concentration
EU/mL
204
1.0
0.50
0.25
0.125
0.063
0

20002,3
40
3960
20
100
1900
1.0
500
500
0.50
500
500
0.25
500
500
0.125
500
500
0
0
1000
Abbreviations: EU = Endotoxin units; PFS = Pyrogen-free saline
Each stock tube should be vortexed vigorously prior to its use to make the subsequent
dilution.
1
To reconstitute the endotoxin, the stock vial should be vortexed vigorously for at least 30 min or sonicated in abath sonicator for at least 5 min. Subsequent dilutions should be vortexed vigorously immediately prior to use.
2
A 2000 EU/mL stock solution of endotoxin is prepared according to the manufacturer's instructions.
3
The stock solution is stable for not more 14 days when stored at 2 to 8°C or for up to 6 months when kept in a
-20°C freezer.
4This concentration is not used in the assay.

To prepare the endotoxin standard curve, first obtain a 2000 EU/mL stock solution byaddition of PFW to the lyophilized content of the stock vial by following the instructions
provided by the manufacturer (e.g., 5 mL of PFW is added to a vial containing 10,000 EU).
To reconstitute the endotoxin, the stock vial should be vortexed vigorously for at least 30 min
or sonicated in a bath sonicator for at least 5 min. Subsequent dilutions should be vortexed
vigorously immediately prior to use. The stock solution is stable for not more 14 days when
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May 2008

stored at 2 to 8°C or for up to 6 months when kept in a -20°C freezer. An endotoxin standard
curve is prepared as described in Table 4-1 by making serial dilutions of the stock solution in
PFS with vigorous vortexing at each dilution step. Dilutions should not be stored, because
dilute endotoxin solutions are not as stable as concentrated solutions due to loss of activity byadsorption, in the absence of supporting data to the contrary.
4.2

Interference Test

For every test substance lot, interference testing must be performed to check for interference
between the test substance and the cell system and/or ELISA. The purpose of the interference
test is to determine whether the test substance (or specific lot of test substance) has an effecton cytokine release.
4.2.1
Interference with the Cell System
All test substances must be labeled as pyrogen-free (i.e., endotoxin levels at an acceptablelevel prior to release by the manufacturer) to ensure that exogenous levels of endotoxin do
not affect the experimental outcome. Liquid test substances should be diluted in PFS. Solid
test substances should be prepared as solutions in PFS or, if insoluble in saline, dissolved indimethyl sulfoxide (DMSO) and then diluted up to 0.5% (v/v) with PFS, provided that thisconcentration of DMSO does not interfere with the assay. To ensure a valid test, a test
substance cannot be diluted beyond its Maximum Valid Dilution (MVD) (refer to Section
12.3). The calculation of the MVD is dependent on the ELC for a test substance. The ELC
can be calculated by dividing the threshold human pyrogenic dose by the maximum
recommended human dose in a single hour period (see Section 12.2) (USP 2007; FDA
1987). Furthermore, test substances should not be tested at concentrations that are cytotoxic
to blood cells.
4.2.1.1 Reference Endotoxin for Spiking Test Substances
The WHO-LPS 94/580 [E. coli O113:H10:K-] or equivalent internationally harmonized RSE
is recommended for preparation of the endotoxin-spike solution and the endotoxin standard
curve (see Section 4.1).
4.2.1.2 Spiking Test Substances with Endotoxin
Non-spiked and endotoxin-spiked test substances are prepared in quadruplicate and an invitro pyrogen test is performed. A fixed concentration of the RSE (i.e., 0.25 EU/mL or aconcentration equal to or near the middle of the endotoxin standard curve) is added to theundiluted test substance (or in serial two-fold dilutions, not to exceed the MVD). An
illustrative example of endotoxin-spiking solutions is shown in Table 4-2. For non-spiked
solutions, 150 µl of RPMI-C is added to a well followed by 50 µl of the test substance (i.e.,equivalent to the negative product control [NPC]) and 50 µL of PBMCs and the well contents
are mixed. Endotoxin-spiked solutions are prepared by adding 100 µL of RPMI-C to eachwell followed by 50 µL of the test substance, and 50 µL of an endotoxin-spike solution (0.25
EU/mL) (i.e., equivalent to the positive product control [PPC]). Finally, 50 µL of PBMCs areadded to each well and the wells are mixed and incubated as outlined in Section 6.1.3, Steps
6-8. An ELISA is then performed as outlined in Section 6.2, without the IL-6 standard curve.

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ICCVAM Test Method Evaluation Report: Appendix C4Table 4-2

May 2008

Preparation of Endotoxin-Spiked and Non-Spiked Solutions for
Determination of Test Substance Interference
SpikedSample Addition
RPMI-C
Endotoxin-spike solution2
Test substance (neat and each serial dilution)
PBMCs3
Total4Abbreviations: PBMC = Peripheral blood mononuclear cells
1
n=4 replicates each2
Endotoxin concentration is 0.25 EU/mL in RPMI-C.
3
PBMCs are resuspended in RPMI-C (1 x 106 cells/mL).
4
A total volume of 250 µL per well is used for the incubation.

µL/well1

Non-spiked

100
50
50
50
250

150
0
50
50
250

The optical density (OD) values of the endotoxin-spiked and non-spiked test substances arecalibrated against the endotoxin calibration curve. The resulting EU value of the non-spiked
test substance is subtracted from the corresponding EU value of the endotoxin-spiked test
substance at each dilution. The spike recovery for each sample dilution is calculated as apercentage by setting the theoretical value (i.e., endotoxin-spike concentration of 0.25
EU/mL) at 100%. For example, consider the following interference test results in Table 4-3:
Table 4-3

Example of Interference Data Used to Determine Sample
Dilution
Sample Dilution
None
1:2
1:4
1:8

% Recovery of Endotoxin Control
25
49
90
110

If a spike recovery between 50% and 200% is obtained, then no interference of the test
substance with either the cell system or the ELISA is demonstrated (i.e., the test substancedoes not increase or decrease the concentration of IL-6 relative to the endotoxin spike). The
lowest dilution (i.e., highest concentration) of a test substance that yields an endotoxin-spikerecovery between 50% and 200% is determined. The test substance is then diluted in serial
two-fold dilutions beginning at this dilution, not to exceed the MVD, for use in the assay.
Based on the results illustrated in Table 4-3, the initial dilution of the test substance to be
used in the in vitro pyrogen test would be 1:4 (i.e., the lowest dilution between 50% and200% of the 0.25 EU/mL EC).
4.2.2
Interference at the MVD
If the data obtained from the experiment in Section 4.2.1 suggests the presence ofinterference at the MVD, then consideration should be given for using another validated
pyrogen test method.
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ICCVAM Test Method Evaluation Report: Appendix C4

5.0

CONTROLS

5.1

Benchmark Controls

May 2008

Benchmark controls may be used to demonstrate that the test method is functioning properly,
or to evaluate the relative pyrogenic potential of chemicals (e.g., parenteral pharmaceuticals,
medical device eluates) of a specific class or a specific range of responses, or for evaluating
the relative pyrogenic potential of a test substance. Appropriate benchmark controls shouldhave the following properties:

5.2•

consistent and reliable source(s) for the chemicals (e.g., parenteralpharmaceuticals, medical device eluates)•

structural and functional similarities to the class of substance being tested•

known physical/chemical characteristics•

supporting data on known effects in animal models•

known potency in the range of response

Endotoxin Control

The EC (i.e., PBMCs incubated with an internationally harmonized RSE) serves as the
positive control in each experiment. The results should be compared to historical values toinsure that it provides a known level of cytokine release relative to the NSC.
5.3

Negative Saline Control

The NSC (i.e., PBMCs incubated with PFS instead of the test substance) is included in eachexperiment in order to detect nonspecific changes in the test system, as well as to provide abaseline for the assay endpoints.
5.4

Solvent Control

Solvent controls are recommended to demonstrate that the solvent is not interfering with thetest system when solvents other than PFS are used to dissolve test substances.
6.0

EXPERIMENTAL DESIGN

6.1

Incubation with Test Samples and Measurement of IL-6 Release

6.1.1
Collection of Human Blood
Human volunteers that have met the donor eligibility criteria described in Section 3.1 areused as the source of WB. All components of the blood collection system (e.g., syringes,
tubes, connecting lines) must be sterile and pyrogen-free. WB is drawn by venipuncture7
from the medial cubital or cephalic vein of either the right or left arm and collected in asterile container that contains anticoagulant solution (e.g., heparin). The total volume of7

WB is obtained using Universal Precautions (e.g., latex gloves, labcoats, safety glasses) and sterile equipment(e.g., syringes, needles, collection tubes) within a hospital or clinical setting by qualified and adequately trained
personnel (i.e., registered nurse, licensed phlebotomist, or medical doctor).
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May 2008

blood collected per donor (i.e., up to 500 mL) will be dictated by experimental design anddetermined by the test method user. All subsequent handling of WB should be performed in alaminar flow hood using sterile technique to prevent contamination.
6.1.1.1 Isolation of PBMCs from WB
PBMCs are isolated from WB using density gradient centrifugation. The PBMC suspension
must be isolated within 2 hr of WB collection using Lymphoprep™. The isolated PBMC
suspension may be used immediately (Section 6.1.2) or frozen for later use (Section 6.1.1.3).
The isolation procedure described below is a modification of the manufacturer's instructions
as outlined in the ECVAM SOP for the PBMC/IL-6 pyrogen test.
To form a lower, denser layer, 15 mL of PBS and 20 mL of Lymphoprep™ should be added
to each tube containing 15 mL of WB. The tubes are then centrifuged at 340 x g for 45 min at
RT. After centrifugation, a white band of PBMCs should be visible at approximately the 25mL graduation mark on the tube. If cryopreservation of PBMCs is to be performed (seeSection 6.1.1.3), carefully remove 18 mL of supernatant above the PBMC band and transfer
it to a new tube for preparing a cryoprotective solution. The remaining supernatant above the
PBMC band should be aspirated and discarded. Using a 10 mL pipet, transfer the PBMC
layer to a new centrifuge tube.
6.1.1.2 Washing PBMCs
The PBMCs are resuspended in a total volume of 50 mL of PBS and centrifuged at 340 x g
for 15 min. The supernatant is poured off and the cellular sediment resuspended in 10 mL of
PBS by pipetting up and down several times with a serological pipet. The total volume ineach tube is adjusted to 50 mL with PBS and centrifuged at 340 x g for 10 min. Aftercentrifugation, the PBMCs should be resuspended in RPMI-C and an equal volume of cellsuspension from multiple individual donors should be pooled8.
Prior to use in the assay, the pooled PBMCs should be examined under a microscope todetermine that the morphology of the cells is consistent with the appearance of cells that
previously yielded acceptable results. It is advisable that cell number and cell viability be
determined using appropriate methods (e.g., hemocytometer and vital dye or flow cytometerand fluorescent marker). The cell count of the PBMC suspension should be adjusted to 1 x
106 cells/mL in RPMI-C. The percentage of viable PBMCs should exceed 80% for their
inclusion in the test. The results of these examinations should be included in the study report.
If PBMCs are prepared from fresh WB, then the cell suspension must be used in the assay
within 4 hr from the time of WB collection.
6.1.1.3
Procedure for Cryopreservation and Thawing of PBMCs
To freeze the PBMCs, prepare a cryoprotective solution by adding 2 mL of pyrogen-free
DMSO to the supernatant (18 mL) collected in the centrifugation procedure outlined in
Section 6.1.1.1. Cool the cryoprotective solution to between 2 and 8°C. Centrifuge theisolated PBMCs as instructed in Section 6.1.1.2 and then add 6 mL of the chilledcryoprotective solution to the cell sediment and prepare aliquots in cryotubes. The cryotubes

8

Multiple donors (i.e., a minimum of three) should meet the acceptability criteria as outlined in Section 8.0
either as a pool of multiple individual donors or as multiple individual donors tested independently.
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May 2008

are placed in a Styrofoam box for thermal insulation and slowly frozen in a -80°C freezer.
After 72 hr, the tubes can be transferred to liquid nitrogen for prolonged storage.
To thaw the cryopreserved PBMCs, submerge the tubes in a water bath at 37±1°C. After
thawing, the cell suspensions are pooled in a single 50 mL centrifuge tube and RPMI-C isadded to give a total volume of 40 mL. The PBMCs are centrifuged at 340 x g for 10 min, thesupernatant removed, and the cells resuspended in 10 mL of RPMI-C.
Prior to use in the assay, it is advisable that cell number and cell viability be examined as
described in Section 6.1.1.2. The cell count of the PBMC suspension should be adjusted to 1
x 106 cells/mL in RPMI-C. The percentage of viable PBMCs should exceed 80% for their
inclusion in the test. The results of this examination should be included in the study report.
6.1.2 Incubation Plate
Test substances are prepared at a level of dilution that did not show interference with the test
system, provided that this dilution does not exceed the MVD. Each incubation plate canaccommodate an endotoxin standard curve, a NSC, and 14 test samples (see Table 6-1).
Table 6-1

Numberof Wells

Overview of Incubation Plate Preparation in the PBMC/IL-6 Pyrogen
Test

Sample

RPMI-CEC

Test
Sample

PBMCs

µL
20

1

4
56

3

EC

100

50

0

100

NSC

100

0

02

100

Test
samples

100

0

50

100

Mix thesamples;incubate for
16 to 24 hr at
37±1°C in ahumidified
atmospherewith 5%CO2.

Mix thesamples;
immediatelytransfer to an
ELISA plate4
and runELISA.

(1-14)
Abbreviations: EC = Endotoxin control; IL-6 = Interleukin-6; NSC = Negative saline control; PBMC =Peripheral blood mononuclear cell
1
Five EC concentrations (0.063, 0.125, 0.25, 0.50, and 1.0 EU/mL) in quadruplicate
250 µL of PFS is added instead of the test sample.
3
14 test samples (n=4 each) per plate
4
An IL-6 standard curve is prepared in Columns 11 and 12 on the ELISA plate (see Table 6-3). Therefore, 80
wells are available for test samples and controls on the incubation plate.

6.1.3
Incubation Assay for IL-6 Release
Test substances should be vortexed vigorously for at least 30 min or sonicated in a bathsonicator for at least 5 min prior to use in the assay. Test substances should be prepared inserial two-fold dilutions beginning at a level of dilution that did not show interference withthe test system (see Section 4.2) in as many subsequent dilutions that are necessary to bewithin the linear range of the endotoxin standard curve, not to exceed the MVD. PBMC
samples are prepared in a microtiter plate using a laminar flow hood (refer to Section 6.1.1).
All consumables and solutions must be sterile and pyrogen-free. Each plate should be labeled
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ICCVAM Test Method Evaluation Report: Appendix C4

May 2008

appropriately with a permanent marker. An overview of the incubation plate preparation isshown in Table 6-1. The incubation procedure is outlined below:
Step 1. Refer to the suggested incubation plate template presented in Table 6-2.
Step 2. Using a pipetter, transfer 100 µL of RPMI-C into each well.
Step 3. Transfer 50 µL of test sample or 50 µL of PFS for the NSC into theappropriate wells as indicated in the template.
Step 4. Transfer 50 µL of the EC (standard curve) in quadruplicate into theappropriate wells according to the template.
Step 5. Transfer 100 µL of a well-mixed PBMC suspension into each well and mix
by gently swirling the plate.
Step 6. Mix the contents of the wells thoroughly by pipetting up and down several
times using a multichannel pipetter, changing the tips between each row in order toavoid cross-contamination.
Step 7. Place the covered plate in a tissue culture incubator for 16 to 24 hr at
37±1°C in a humidified atmosphere containing 5% CO2.
Step 8. Prior to transferring the test samples to the ELISA plate, mix the contentsof the wells by pipetting up and down using a multichannel pipetter, changing thetips between each row in order to avoid cross-contamination.
Table 6-2

Incubation Plate - Sample and Control Template

1

2

3

4

5

6

7

8

9

10

11

12

A

EC1
1.0

EC
1.0

EC
1.0

EC
1.0

TS3

TS3

TS3

TS3

TS11

TS11

Void3

Void

B

EC
0.50

EC
0.50

EC
0.50

EC
0.50

TS4

TS4

TS4

TS4

TS11

TS11

Void

Void

C

EC
0.25

EC
0.25

EC
0.25

EC
0.25

TS5

TS5

TS5

TS5

TS12

TS12

Void

Void

D

EC
0.125

EC
0.125

EC
0.125

EC
0.125

TS6

TS6

TS6

TS6

TS12

TS12

Void

Void

E

EC
0.063

EC
0.063

EC
0.063

EC
0.063

TS7

TS7

TS7

TS7

TS13

TS13

Void

Void

F

NSC

NSC

NSC

NSC

TS8

TS8

TS8

TS8

TS13

TS13

Void

Void

G

TS12

TS1

TS1

TS1

TS9

TS9

TS9

TS9

TS14

TS14

Void

Void

H

TS2

TS2

TS2

TS2

TS10

TS10

TS10

TS10

TS14

TS14

Void

Void

Abbreviations: EC = Endotoxin control; NSC = Negative saline control; TS = Test substance1
EC value (e.g., EC 1.0) represents the endotoxin concentration in EU/mL.
2
TS number (e.g., TS1) represents an arbitrary sequence for individual test substances.
3
Columns 11 and 12 are reserved for the IL-6 standard curve on the ELISA plate (see Table 6-3).

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ICCVAM Test Method Evaluation Report: Appendix C4

6.2

May 2008

ELISA to Measure IL-6 Release

6.2.1
IL-6 Standard Curve
An IL-6 standard, supplied with the ELISA kit, is used. IL-6 standards are typically suppliedin lyophilized form and should be reconstituted according to the manufacturer's instructions.
The stock solution should be diluted in RPMI-C to the following concentrations: 0, 62.5, 125,
250, 500, 1000, 2000, and 4000 pg/mL in volumes of at least 500 µL. Each well on the
ELISA plate will receive 50 µL of an IL-6 blank or standard.
6.2.2
ELISA
The manufacturer's instructions provided with the ELISA kit should be followed and atypical experimental design is outlined below. The ELISA should be carried out at RT and
therefore all components must be at RT prior to use. Frozen specimens should not be thawedby heating them in a water bath. A suggested ELISA plate template is shown in Table 6-3,
which includes a five-point EC standard curve, an eight-point IL-6 standard curve (0 to 4000
pg/mL), and available wells for up to 14 test substances and a NSC each in quadruplicate.
The EC standard curve, the NSC, and the test sample supernatants are transferred directlyfrom the incubation plate. The IL-6 standard curve is prepared as described in Section 6.2.1.
An overview of the ELISA plate preparation is shown in Table 6-4.
Step 1. After pipetting up and down very carefully three times (avoid detachmentof the adherent PBMCs) to mix the supernatant, transfer 50 µL from each well of
the Incubation Plate (A1-10; H1-10) to the ELISA plate.
Step 2. Add 50 µL of each IL-6 standard (0 to 4000 pg/mL) into the respective
wells on the ELISA plate.
Step 3. Add 200 µL of the enzyme-labeled detection antibody (neat as supplied, or
diluted, if necessary) to each of the wells.
Step 4. Cover the microtiter plate(s) with adhesive film and incubate for 2 to 3 hr
at RT.
Step 5. Decant and wash each well three times with 300 µL Buffered Wash
Solution and then rinse three times with deionized water. Place the plates upside
down and tap to remove water.
Step 6. Add 200 µL of TMB/Substrate Solution to each well and incubate at RT in
the dark for 15 min. If necessary, decrease the incubation time.
Step 7. Add 50 µL of Stop Solution to each well.
Step 8. Tap the plate gently after the addition of Stop Solution to aid in mixing.
Step 9. Read the OD450 within 15 min of adding the Stop Solution. Measurementwith a reference wavelength of 540 to 590 nm is recommended.99

The TMB chromagen is measured at OD450. However, the use of an IL-1β ELISA kit with a chromagen otherthan TMB is acceptable. The ELISA should be measured at a wavelength appropriate for the specificchromagen used.
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ICCVAM Test Method Evaluation Report: Appendix C4Table 6-3

May 2008

ELISA Plate - Sample and Control Template

1

2

3

4

5

6

7

8

9

10

11

12

A

EC1
1.0

EC
1.0

EC
1.0

EC
1.0

TS3

TS3

TS3

TS3

TS11

TS11

IL-63
0

IL-6
0

B

EC
0.50

EC
0.50

EC
0.50

EC
0.50

TS4

TS4

TS4

TS4

TS11

TS11

IL-6
62.5

IL-6
62.5

C

EC
0.25

EC
0.25

EC
0.25

EC
0.25

TS5

TS5

TS5

TS5

TS12

TS12

IL-6
125

IL-6
125

D

EC
0.125

EC
0.125

EC
0.125

EC
0.125

TS6

TS6

TS6

TS6

TS12

TS12

IL-6
250

IL-6
250

E

EC
0.063

EC
0.063

EC
0.063

EC
0.063

TS7

TS7

TS7

TS7

TS13

TS13

IL-6
500

IL-6
500

F

NSC

NSC

NSC

NSC

TS8

TS8

TS8

TS8

TS13

TS13

IL-6
1000

IL-6
1000

G

TS12

TS1

TS1

TS1

TS9

TS9

TS9

TS9

TS14

TS14

IL-6
2000

IL-6
2000

H

TS2

TS2

TS2

TS2

TS10

TS10

TS10

TS10

TS14

TS14

IL-6
4000

IL-6
4000

Abbreviations: EC = Endotoxin control; NSC = Negative saline control; TS = Test substance1
EC value (e.g., EC 1.0) represents the endotoxin concentration in EU/mL.
2
TS number (e.g., TS1) represents an arbitrary sequence for individual test substances.
3
IL-6 values in columns 11 and 12 are in pg/mL.

Table 6-4
MaterialtransferfromIncubation
Plate

Overview of ELISA Procedure

(µL)

IL-6
standard(0 to
4000
pg/mL)
(µL)

50

50

Enzymelabeled
Antibody(µL)

Cover theIncubation
Plate andincubatefor 2 to 3
hr at RT.

200

Decant andwash eachwell threetimes with
300 µL
Buffered
Wash
Solutionand threetimes with
deionized
water.

TMB/Substrate
Solution(µL)

200

Incubatefor lessthan 15
min at
RT in
dark.

Stop
Solution(µL)

50

Read eachwell at
OD450
with a 540
to 590 nm
reference
filter.

Abbreviations: OD450 = Optical density at 450 nm; RT = Room temperature

7.0

EVALUATION OF TEST METHODS

7.1

OD Measurements

The OD of each well is obtained by reading the samples in a standard microplate
spectrophotometer (i.e., plate reader) using a visible light wavelength of 450 nm (OD450) with
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ICCVAM Test Method Evaluation Report: Appendix C4

May 2008

a 540 to 590 nm reference filter (recommended)10. OD values are used to determine assay
acceptability and in the decision criteria for pyrogen detection (see Sections 8.0 and 9.0).
8.0

CRITERIA FOR AN ACCEPTABLE TESTAn EC (five-point standard curve) and a NSC should be included in each experiment. An
IL-6 standard curve should be included in each ELISA as shown in the template presented inTable 6-3. An assay is considered acceptable only if the following minimum criteria are met:•

The quadratic function of the IL-6 standard curve produces an r ≥0.9511 andthe OD of the blank control is below 0.15.

•

The endotoxin standard curve produces OD values that ascend in a sigmoidal
concentration response.

Blood donors (or a pool of blood donors) are considered to be low responders if their OD450
value obtained for 1.0 EU/mL EC is below the OD450 value obtained for 1000 pg/mL IL-6.
Blood donors (or a pool of blood donors) who produce an OD450 value for the NSC that is
above the OD450 value at 500 pg/mL IL-6 are considered to be high responders. Low andhigh responders should be excluded from analysis. The preparation being examined isrequired to pass the test with blood donations from at least three different donors (i.e., either
as a pool of three individual donors or as three individual donors tested independently).
An outlying observation that represents either a pool of multiple independent donors or asingle individual donor may be excluded if there is confirmation that the accuracy of the
medical information provided by an individual donor is suspect, or if the aberrant response isidentified using acceptable statistical methodology (e.g., Dixon's test [Dixon 1950; Barnett
and Lewis 1994], Grubbs' test [Barnett and Lewis 1994; Grubbs 1969; Iglewicz andHoughlin 1993]).
9.0

DATA INTERPRETATION/DECISION CRITERIA

9.1

Decision Criteria for Pyrogen Detection12

A test substance is considered pyrogenic when the endotoxin concentration of the test
substance exceeds the ELC for the test sample. The ELC can be calculated as shown in
Section 12.2.
10.0

STUDY REPORTThe test report should include the following information:
Test Substances and Control Substances•

Name of test substance10

The TMB chromagen is measured at OD450. However, the use of an IL-1β ELISA kit with a chromagen otherthan TMB is acceptable. The ELISA should be measured at a wavelength appropriate for the specificchromagen used.
11
Correlation coefficient (r), an estimate of the correlation of x and y values in a series of n measurements.
12

Decision criteria for individual donors were defined in the ECVAM SOP for the PBMC/IL-6 test method. Test
method users should refer to these criteria if multiple donors are tested independently.
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Purity and composition of the substance or preparation•

Physicochemical properties (e.g., physical state, water solubility)•

Quality assurance data•

Treatment of the test/control substances prior to testing (e.g., vortexing,
sonication, warming, resuspension solvent)Justification of the In Vitro Test Method and Protocol Used
Test Method Integrity•

The procedure used to ensure the integrity (i.e., accuracy and reliability) of thetest method over time•

If the test method employs proprietary components, documentation on theprocedure used to ensure their integrity from “lot-to-lot” and over time•

The procedures that the user may employ to verify the integrity of the
proprietary components

Criteria for an Acceptable Test•

Acceptable concurrent positive control ranges based on historical data•

Acceptable negative control data

Test Conditions•

Cell system used•

Calibration information for the spectrophotometer used to read the ELISA•

Details of test procedure used•

Description of any modifications of the test procedure•

Reference to historical data of the model•

Description of evaluation criteria used•

Tabulation of data from individual test samples

Results
Description of Other Effects Observed
Discussion of the Results
Conclusion
A Quality Assurance Statement for Good Laboratory Practice (GLP)-Compliant Studies•

This statement should indicate all inspections made during the study and thedates any results were reported to the Study Director. This statement should
also confirm that the final report reflects the raw data.

If GLP-compliant studies are performed, then additional reporting requirements provided in
the relevant guidelines (e.g., OECD 1998; EPA 2003a, 2003b; FDA 2003) should be
followed.
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11.0

May 2008

REFERENCES

Barnett V, Lewis T. 1994. Outliers in Statistical Data. In: Wiley Series in Probability andMathematical Statistics. Applied Probability and Statistics. 3rd Ed. New York: John Wiley &Sons.
Bleeker WK, de Groot EM, den Boer PJ, Biessels PT, Aarden LA, Bakker JC. 1994.
Measurement of interleukin-6 production by monocytes for in vitro safety testing ofhemoglobin solutions. Artif Cells Blood Substit Immob Biotechnol 22:835-840.
Dinarello CA, O'Connor JV, LoPreste G, Swift RL. 1984. Human leukocyte pyrogen test for
detection of pyrogenic material in growth hormone produced by recombinant Escherichia
coli. J Clin Microbiol 20:323-329.
Dixon WJ. 1950. Analysis of extreme values. Annals of Mathematical Statistics 21:488-506.
EPA. 2003a. Good Laboratory Practice Standards. Toxic Substances Control Act. 40 CFR
792.
EPA. 2003b. Good Laboratory Practice Standards. Federal Insecticide, Fungicide, andRodenticide Act. 40 CFR 160.
FDA. 1987. Guideline on Validation of the Limulus Amebocyte Lysate Test as an
End-product Endotoxin Test for Human and Animal Parenteral Drugs, Biological Products,
and Medical Devices. Rockville, MD:U.S. Department of Health and Human Services(DHHS), Food and Drug Administration (FDA).
FDA. 2003. Good Laboratory Practices for Nonclinical Laboratory Studies. 21 CFR 58.
Grubbs FE. 1969. Procedures for detecting outlying observations in samples. Technometrics
11(1):1-21.
Iglewicz B, Houghlin DC. 1993. How to detect and handle outliers. In ASQC Basic
Reference in Quality Control. Vol. 14. Milwaukee, WI: ASQ Quality Press, 87.
Organization for Economic Co-operation and Development (OECD). 1998. OECD Series on
Principle of Good Laboratory Practice and Compliance Monitoring. No. 1. OECD Principles
of Good Laboratory Practice (as revised in 1997). Organisation for Economic Co-operation
and Development, ENV/MC/CHEM (98)17. Paris: OECD.
Poole S, Mistry Y, Ball C, Gaines Das RE, Opie LP, Tucker G, Patel M. 2003. A rapid'one-plate' in vitro test for pyrogens. J Immunol Methods 274:209-220.
USP. 2007. The U.S. Pharmacopeia. USP30 NF25<85>. Ed. The U.S. Pharmacopeial
Convention. Rockville, MD:The U.S. Pharmacopeial Convention.

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12.0
12.1

May 2008

TERMINOLOGY AND FORMULA
Assay Sensitivity (λ)1

The variable λ is defined as the labeled sensitivity (in EU/mL) of the LAL Reagent in
endpoint assays (e.g., the BET gel-clot technique). For kinetic BET assays, λ is the lowest
point used in the endotoxin standard curve.
12.2

Endotoxin Limit Concentration (ELC)1,2

The ELC for parenteral drugs is expressed in Endotoxin Units (EU) per volume (mL) or
weight (mg). The ELC is equal to K/M, where:
K is the threshold human pyrogenic dose of endotoxin (EU) per body weight (kg). K is equal
to 5.0 EU/kg for intravenous administration. For intrathecal administration, K is equal to 0.2
EU/kg (see also Section 12.5).
M is the rabbit test dose or the maximum recommended human dose of product (mL or mg)
per body weight (kg) in a single hour period (see also Section 12.8).
For example, if a non-intrathecal product is used at an hourly dose of 10 mL per patient, thenthe ELC would be 0.50 EU/mL.
12.3

Maximum Valid Dilution (MVD)1,2

The MVD is the maximum allowable dilution of a test substance at which the endotoxin limit
can be determined. The calculation of the MVD is dependent on the ELC for a test substance.
When the ELC is known, the MVD is1:
MVD = (ELC x Product Potency [PP])/λAs an example, for Cyclophosphamide Injection, the ELC is 0.17 EU/mg, PP is 20
mg/mL, and the assay sensitivity is 0.065 EU/mL. The calculated MVD would be 1:52.3 or
1:52. The test substance can be diluted no more than 1:52 prior to testing.
If the ELC is not known, the MVD is1:
MVD = PP/Minimum Valid Concentration (MVC)
where, MVC = (λ x M)/K
where, M is the maximum human dose
As an example, for Cyclophosphamide Injection, the PP is 20 mg/mL, M is 30 mg/kg,
and assay sensitivity is 0.065 EU/mL. The calculated MVC is 0.390 mg/mL and the MVD is
1:51.2 or 1:51. The test substance can be diluted no more than 1:51 in the assay prior to
testing.
12.4

Negative Product Control (NPC)

For interference testing, the NPC is a test sample to which pyrogen-free saline (PFS) is
added. The NPC is the baseline for determination of cytokine release relative to theendotoxin-spiked PPC.
1
2

From FDA (1987)
From USP (2007)
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12.5

May 2008

Parenteral Threshold Pyrogen Dose (K)1,2

The value K is defined as the threshold human pyrogenic dose of endotoxin (EU) per bodyweight (kg). K is equal to 5.0 EU/kg for parenteral drugs except those administeredintrathecally; 0.2 EU/kg for intrathecal drugs.
12.6

Positive Product Control (PPC)

For interference testing, the PPC is a test substance spiked with the control standard
endotoxin (i.e., 0.5 EU/mL or an amount of endotoxin equal to that which produces ½ themaximal increase in optical density (OD) from the endotoxin standard curve) to insure thatthe test system is capable of endotoxin detection in the product as diluted in the assay.
12.7

Product Potency (PP)1,2

The test sample concentration expressed as mg/mL or mL/mL.
12.8

Rabbit Pyrogen Test (RPT) Dose or Maximum Human Dose (M)1,2

The variable M is equal to the rabbit test dose or the maximum recommended human dose of
product per kg of body weight in a single hour period. M is expressed in mg/kg or mL/kg andvaries with the test substance. For radiopharmaceuticals, M equals the rabbit dose or
maximum human dose/kg at the product expiration date or time. Use 70 kg as the weight of
the average human when calculating the maximum human dose per kg. If the pediatricdose/kg is higher than the adult dose, then it shall be the dose used in the formula.

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May 2008

Appendix C5
The Monocytoid Cell Line Mono Mac 6 (MM6)/IL-6 In Vitro Pyrogen Test

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ICCVAM Test Method Evaluation Report: Appendix C5

May 2008

ICCVAM Final Recommended Protocol for Future Studies Using the Monocytoid Cell
Line Mono Mac 6 (MM6)/Interleukin (IL)-6 In Vitro Pyrogen Test
PREFACEThis protocol is for the detection of Gram-negative endotoxin, a pyrogen, in parenteral drugs,
as indicated by the release of IL-6 from the monocytoid cell line Mono Mac 6 (MM6). This
protocol is based on information obtained from 1) the European Centre for the Validation of
Alternative Methods (ECVAM)1, MM6/IL-6 Background Review Document (BRD)
presented in Appendix A of the Interagency Coordinating Committee on the Validation of
Alternative Methods (ICCVAM) BRD (available at
http://iccvam.niehs.nih.gov/methods/pyrogen/pyr_brd.htm), and 2) information provided to
the National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative
Toxicological Methods (NICEATM) by Dr. Thomas Hartung, Head of ECVAM. The
ICCVAM BRD includes the ECVAM Standard Operating Procedures (SOPs) for the
MM6/IL-6 test (could be referred to as Monocyte Activation Test), which are based on themethodology published by Taktak et al. (1991). A table of comparison between the
ICCVAM recommended protocol and the ECVAM SOPs is provided in Table 1.
Users should contact the relevant regulatory authority for guidance when using thisICCVAM recommended protocol to demonstrate product specific validation, and any
deviations from this protocol should be accompanied by scientifically justified rationale.
Future studies using the MM6/IL-6 pyrogen test may include further characterization of theusefulness or limitations of the assay for regulatory decision-making. Users should be awarethat this protocol might be revised based on additional optimization and/or validation studies.
ICCVAM recommends that test method users routinely consult the ICCVAM/NICEATM
website (http://iccvam.niehs.nih.gov) to ensure that the most current protocol is used.

1

ECVAM is a unit of the Institute for Health and Consumer Protection at the European Commission's Joint
Research Centre.
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May 2008

Table 1

Comparison of ICCVAM Recommended Protocol with the ECVAM SOPs for the
MM6/IL-6 Pyrogen Test

Protocol Component
Test Substance
Decision Criteria forInterference

Incubation Plate for ELISA(The number of samples orcontrols in quadruplicate)

ELISA PlateICCVAM Protocol
Test neat or in serial dilutions
that produce no interference,
not to exceed the MVD
Mean OD2 of PPC is 50% to200% of 1.0 EU/mL EC
NSC (1)EC (5)

ECVAM SOP1
Test neat or at minimal
dilution that produces nointerference
Mean OD of PPC is 50% to200% of 1.0 EU/mL EC
NSC (1)EC (5)

TS (14)

TS (14)3

PPC (0)NPC (0)PC (0)NC (0)
Includes seven point IL-6
SC and blank in duplicate

PPC (0)
NPC3 (0)PC4 (0)NC4 (0)Includes seven point IL-6 SC
and blank in duplicate
Quadratic function of IL-6 SC r
≥0.955
Mean OD of NSC ≤0.15

Not included
Not included

ECVAM Validation SOP1
Test at MVD
Mean OD of PPC is 50% to200% of 1.0 EU/mL EC
NSC (1)EC (5)TS (2) x EC (5) spikes = 10TS
PPC (2) = 2 TS
NPC (2) = 2 TS
PC (1) = 1 TS
NC (1) = 1 TS
Not included
Not included

Not included
Mean OD of each EC >Endotoxin concentration
EC SC produces OD values
Mean OD of next lower EC(0.5 IU/mL) > backgroundthat ascend in a sigmoidal
concentration (minimum of(defined as the mean +2SD
Assay Acceptability
concentration response
4 data points needed for(n-1)
Criteria
valid SC)
PC = ±20% of the
Not included
Not included
theoretical value
Not included
Not included
OD NC < 0.200
Not included
Not included
OD PC > LOQ6
Outliers rejected using Dixon's
Outliers rejected using
Outliers rejected using
test
Dixon's test
Dixon's test
Decision Criteria for
Endotoxin concentration
Endotoxin concentration
OD TS > OD 0.5 EU/mL
Pyrogenicity
TS > ELC7 TS
TS .> ELC TS
EC
Abbreviations: EC = Endotoxin control; ELC = Endotoxin limit concentration; ELISA = Enzyme-linked immunosorbent assay;
EU = Endotoxin units; IL-6 = Interleukin-6; IU = International units; LOQ = Limit of quantification; MM6 = Mono Mac 6;
MVD = Maximum valid dilution;
NC = Negative control; NPC = Negative product control; NSC = Negative saline control; OD = Optical density; PC = Positive control;

PPC = Positive product control; SC = Standard curve; SD = Standard deviation; SOP = Standard operating procedure; TS = Test
substance
1
ECVAM MM6/IL-6 SOP and ECVAM MM6/IL-6 Validation SOP are presented in Appendix A of the ICCVAM BRD (available at
http://iccvam.niehs.nih.gov/methods/pyrogen/pyr_brd.htm).
2
Mean OD values are corrected (i.e., reference filter reading, if applicable, and NSC are subtracted).
3
In the ICCVAM MM6/IL-6 protocol, PPC and NPC are assessed in the interference test described in Section 4.3, which is performed prior
to the ELISA. In the ECVAM SOPs, PPC and NPC were only included in the ECVAM validation study.
4
PC and NC were only included in the ECVAM validation study. PC is 50 pg/mL endotoxin in saline. NC is 0.9% saline.
5
Correlation coefficient (r), an estimate of the correlation of x and y values in a series of n measurements.
6
LOQ is the mean OD of the NSC + 10x the SD of the mean OD for the NSC.
7
Where unknown, the ELC is calculated (See Section 12.2).

ICCVAM Test Method Evaluation Report: Appendix C5

1.0

May 2008

PURPOSE AND APPLICABILITY

The purpose of this protocol is to describe the procedures used to evaluate the presence ofGram-negative endotoxin, a pyrogen, in parenteral drugs. The presence of Gram-negative
endotoxin is detected by its ability to induce the release of IL-6 from Mono Mac 6 (MM6)cells, a human cell line derived from a patient with acute monocytic leukemia(Zeigler-Heitbrock et al. 1988). The concentration of IL-6 released by incubation of MM6
cells with a test substance or controls (i.e., positive and negative) is quantified using an
enzyme-linked immunosorbent assay (ELISA) that includes monoclonal or polyclonal
antibodies specific for IL-6. The amount of pyrogen present is determined by comparing thevalues of endotoxin equivalents produced by MM6 cells exposed to the test substance tothose exposed to an internationally harmonized Reference Standard Endotoxin (RSE)1 or anequivalent standard expressed in Endotoxin Units (EU)/mL. A test substance is consideredpyrogenic if the endotoxin concentration of the test substance exceeds the Endotoxin Limit
Concentration (ELC) for the test substance.
The relevance and reliability of this test method to detect non-endotoxin pyrogens have notbeen demonstrated in a formal validation study, although data are available in the literature tosuggest that this assay has the potential to serve this purpose.
2.0

SAFETY AND OPERATING PROCEDURES

All procedures should be performed following standard laboratory precautions, including theuse of laboratory coats, eye protection, and gloves. If necessary, additional precautions
required for specific chemicals will be identified in the Material Safety Data Sheet (MSDS).
The stop solution used in the ELISA kit is acidic and corrosive and should be handled withthe proper personal protective devices. If this reagent comes into contact with skin or eyes,
wash thoroughly with water. Seek medical attention, if necessary.
Tetramethylbenzidine (TMB) solution contains a hydrogen peroxide substrate and 3, 3’, 5,5’-TMB. This reagent is a strong oxidizing agent and a suspected mutagen. Appropriate
personal protection should be used to prevent bodily contact.
Bacterial endotoxin is a toxic agent (i.e., can induce sepsis, shock, vascular damage,antigenic response) and should be handled with care. Skin cuts should be covered andappropriate personal protective devices should be worn. In case of contact with endotoxin,
immediately flush eyes or skin with water for at least 15 minutes (min). If inhaled, remove
the affected individual from the area and provide oxygen and/or artificial respiration asneeded. Skin absorption, ingestion, or inhalation may produce fever, headache, andhypotension.

1

RSEs are internationally harmonized reference standards (e.g., WHO-lipopolysaccharide [LPS] 94/580
Escherichia coli [E. coli] O113:H10:K-; United States Pharmacopeia [USP] RSE E. coli LPS Lot G3E069; USP
RSE E. coli Lot G; FDA E. coli Lot EC6). Equivalent endotoxins include commercially available E.
coli-derived LPS Control Standard Endotoxin (CSE) or other E. coli LPS preparations that have been calibrated
with an appropriate RSE.
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3.0

MATERIALS, EQUIPMENT AND SUPPLIES

3.1

Source of Cells

May 2008

The MM6 cell line is a human monocytic cell line originally described by Professor H.W.L.
Ziegler-Heitbrock at the Institute for Immunology, University of Munich, Germany(Ziegler-Heitbrock et al. 1988). The MM6 cell line may be purchased from the German
Collection of Microorganisms and Cell Cultures (DSMZ, http://www.dsmz.de) byindividuals working at non-profit organizations. Prior to transaction, a legal agreement must
be reached with Professor H.W.L. Ziegler-Heitbrock stating that the cells will be used forresearch purposes only. Any contract research organization or pharmaceutical company
wanting to obtain the MM6 cell line must contact Professor H.W.L. Ziegler-Heitbrock tonegotiate a fee for provision and a royalty payment per batch of product tested. Contact
information for Professor H.W.L. Ziegler-Heitbrock is as follows: Professor Dr. H.W.L.
Ziegler-Heitbrock, University of Leicester, Dept. of Microbiology, University Road,
Leicester LE1 9HN, United Kingdom, e-mail: ziehei@gmx.de.
MM6 cells should be maintained according to the instructions provided by the DSMZ andProfessor Dr. H.W.L. Ziegler-Heitbrock, which should stipulate the permissible limit to thepassage number for these cells.
3.2

Equipment and Supplies

For all steps in the protocol, excluding the ELISA procedure, the materials that will be inclose contact with samples (e.g., pipet tips, containers, solutions) should be sterile andpyrogen-free.
3.2.1

Utilization of MM6 cells

3.2.1.1

Equipment• Centrifuge

3.2.1.2•

Hood; Bio-safety, laminar flow (recommended)•

Incubator; cell culture (37±1°C + 5% CO2)•

Inverted Microscope•

pH meter•

Pipetter; multichannel (8- or 12-channel)•

Pipetters; single-channel adjustable (20, 200, and 1000 µL)•

Repeating pipetter•

Vortex mixer•

Water bath

Consumables• Centrifuge tubes; polystyrene (15 and 50 mL)•

Combitips; repeating pipetter (1.0 and 2.5 mL)
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ICCVAM Test Method Evaluation Report: Appendix C5•

Cryotubes; screw-cap (2 mL)•

Filters; sterile, 0.22 µm•

Flasks; tissue culture•

Phosphate buffered saline (PBS); sterile•

Pipettes; sterile•

Plates; microtiter, 96-well, polystyrene, tissue culture•

Pyrogen-free saline (PFS)•

Reaction tubes; polystyrene (1.5 mL)•

RPMI-1640 cell culture medium supplemented as described in Section 4.3 toyield either RPMI-C or RPMI-M•

Tips; pipetter, sterile, pyrogen-free (20 and 200 µL)•

Tubes; polystyrene

3.2.2

ELISA

3.2.2.1

Equipment• Microplate mixer

3.2.2.2

May 2008•

Microplate reader (450 nm with an optional reference filter in the range of
540-590 nm)2•

Microplate washer (optional)•

Multichannel pipetter

Consumables• Container; storage, plastic•

Deionized water; nonsterile•

Plates; microtiter, 96-well, polystyrene•

Pyrogen-free water (PFW)•Reservoirs; fluid•

Tips; pipetter, sterile and nonsterile•

Tubes; polystyrene (12 mL)

3.2.2.3 ELISA Kit
An ELISA that measures IL-6 release is used. A variety of IL-6 ELISA kits are commerciallyavailable and the IL-6 ELISA procedure outlined in this protocol is intended to serve as an
example for using an ELISA kit. The IL-6 ELISA should be calibrated using an IL-6
2

The TMB chromagen is measured at OD450. However, the use of an IL-1β ELISA kit with a chromagen otherthan TMB is acceptable. The ELISA should be measured at a wavelength appropriate for the specificchromagen used.
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international reference standard (e.g., World Health Organization [WHO] 89/548) prior touse. The IL-6 cytokine assay kits do not provide the RSE or endotoxin equivalent; therefore,
this reagent must be purchased separately. Results obtained using these products are subject
to the assay acceptability and decision criteria described in Sections 8.0 and 9.0. IL-6 ELISA
kit components may include the following:

3.3•

ELISA plates coated with anti-human IL-6 capture antibody; monoclonal or
polyclonal•

Buffered wash solution•

Dilution buffer•

Enzyme-labeled detection antibody•

Human IL-6 reference standard

•

PFS

•

Stop solution•

TMB3/substrate solution

Chemicals•

3.4

Solutions•

4.0

Endotoxin (e.g., WHO-lipopolysaccharide [LPS] 94/580 Escherichia coli [E.
coli] O113:H10:K-; United States Pharmacopeia [USP] RSE E. coli LPS Lot
G3E069; USP RSE E. coli Lot G; U.S. Food and Drug Administration [FDA]
E. coli Lot EC6)

RPMI-1640 cell culture medium; supplemented as described in Section 4.3

ASSAY PREPARATION

All test substances, endotoxin, and endotoxin-spiked solutions should be stored as specified
in the manufacturer's instructions. The preparation of MM6 cells for use in the assay isoutlined in Section 6.1.
4.1

Endotoxin Standard Curve

An internationally harmonized RSE or equivalent is used to generate the endotoxin standard
curve. The use of any other E. coli LPS requires calibration against a RSE using theMM6/IL-6 pyrogen test. A standard endotoxin curve consisting of a Negative Saline Control(NSC) and five RSE concentrations (0.125, 0.25, 0.50, 1.0, and 2.0 EU/mL) are included in
the incubation step (refer to Table 4-1) and then transferred to the ELISA plate. To prepare
the endotoxin standard curve, first obtain a 2000 EU/mL stock solution by addition of PFW
to the lyophilized content of the stock vial by following the instructions provided by the
manufacturer (e.g., 5 mL of PFW is added to a vial containing 10,000 EU). To reconstitutethe endotoxin, the stock vial should be vortexed vigorously for at least 30 min or sonicated in

3

The use of an IL-6 ELISA kit with a chromagen other than TMB is acceptable.
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May 2008

a bath sonicator for at least 5 min. Subsequent dilutions should be vortexed vigorously
immediately prior to use. The stock solution is stable for not more 14 days when stored at 2
to 8°C or for up to 6 months when kept in a -20°C freezer. An endotoxin standard curve isprepared as described in Table 4-1 by making serial dilutions of the stock solution in PFS
with vigorous vortexing at each dilution step. Dilutions should not be stored, because diluteendotoxin solutions are not as stable as concentrated solutions due to loss of activity byadsorption, in the absence of supporting data to the contrary.
Table 4-1

Preparation of Endotoxin Standard Curve

Stock Endotoxin
EU/mL1µL of Stock
EndotoxinµL of PFS

Endotoxin
Concentration
EU/mL
204
2.0
1.0
0.50
0.25
0.125
0

20002,3
40
3960
20
100
900
2.0
500
500
1.0
500
500
0.50
500
500
0.25
500
500
0
0
1000
Abbreviations: EU = Endotoxin units; PFS = Pyrogen-free saline
Each stock tube should be resonicated and vortexed vigorously before the subsequent dilution.
1
To reconstitute the endotoxin, the stock vial should be vortexed vigorously for at least 30 min or sonicated in abath sonicator for at least 5 min. Subsequent dilutions should be vortexed vigorously immediately prior to use.
2
A 2000 EU/mL stock solution of endotoxin is prepared according to the manufacturer's instructions.
3
The stock solution is stable for not more 14 days when stored at 2 to 8°C or for up to 6 months when kept in a 20°C freezer.
4This concentration is not used in the assay.

4.2

Cell Culture Medium

MM6 cells are maintained in RPMI containing 10% FBS, denoted as RPMI-M. For use in the
ELISA procedure, the concentration of FBS is reduced to 2% and referred to as RPMI-C.
Each medium is prepared and stored as described by the manufacturer.
4.2.1

RPMI-M• Bovine insulin; 0.23 IU/mL•

FBS; heat-inactivated at 55±1°C (50 mL or a 10% final concentration)•

HEPES buffer; 20 mM•

L–Glutamine; 2 mM•

MEM non-essential amino acids; 0.1 mM•

Oxaloacetic acid; 1 mM•

Penicillin/streptomycin (10,000 IU/mL penicillin, 10 mg/mL streptomycin)•

RPMI-1640 medium (500 mL)•

Sodium pyruvate; 1 mM
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ICCVAM Test Method Evaluation Report: Appendix C5

May 2008

4.2.2
Starting a Culture of MM6 Cells
To initiate a culture of MM6 cells, remove a vial of the primary stock from liquid nitrogen.
Thaw the vial on ice. Gently mix and transfer the cells to a 50 mL centrifuge tube and add 10
mL of RPMI-M. Centrifuge at 100 x g for 5 min at room temperature (RT). Remove thesupernatant and resuspend the cells in ice-cold RPMI-M. Centrifuge at 100 x g for 5 min at
RT. Remove the supernatant and resuspend the MM6 cells in 2 mL of RPMI-M. Add 8 mL
of RPMI-M to a tissue culture flask and transfer the cell suspension to the flask. Cells should
be examined microscopically to ensure that the cells are not clumped together. Place theflasks in a cell culture incubator and maintain the cells at 37±1°C + 5% CO2.
4.2.3
Propagation of MM6 Cells
Remove the cell culture flask from the incubator and examine the cells under a microscope toto determine that the morphology of the cells is consistent with the appearance of MM6 cells
that previously yielded acceptable results. Centrifuge at 100 x g for 8 min at RT. Remove thesupernatant, resuspend the cell pellet in 4 mL of RPMI-M, and gently pipet up and down tomix. It is advisable that cell number and cell viability be determined using appropriate
methods (e.g., hemocytometer and vital dye or flow cytometer and fluorescent marker). The
percentage of cell viability should exceed 80% for further propagation. The results of these
examinations should be included in the study report. Transfer the cells (2 x 105 cells/mL) to
new tissue culture flasks and add RPMI-M. Place the flasks in a cell culture incubator andmaintain the cells at 37±1°C + 5% CO2.
4.2.4
Preparation of a MM6 Cell Bank
To initiate a bank of MM6 cells, centrifuge the cell culture(s) at 100 x g for 8 min at 2 to
8°C. Remove the supernatant and resuspend the cells in FBS at 2 to 8°C. It is advisable todetermine cell number and cell viability as outlined in Section 4.2.3 and adjust the cellconcentration to 4 x 106 cells/mL and store on ice for 10 min. Add an equal volume of icecold FBS containing 10% dimethyl sulfoxide (DMSO) drop-wise to the cell suspension (final
concentration is 2 x 106 cells/mL with 5% DMSO). Transfer the cell suspension to sterile,
pyrogen-free cryotubes (1 mL/tube). Place the tubes in a well-insulated polystyrene box andstore in a -80°C freezer for greater than 48 hours (hr) and then transfer to a liquid nitrogencontainer.
4.3

Interference Test

For every test substance lot, interference testing must be performed to check for interference
between the test substance and the cell system and/or ELISA. The purpose of the interference
test is to determine whether the test substance (or specific lot of test substance) has an effecton cytokine release.
4.3.1
Interference with the Cell System
All test substances must be labeled as pyrogen-free (i.e., endotoxin levels at an acceptablelevel prior to release by the manufacturer) to ensure that exogenous levels of endotoxin do
not affect the experimental outcome. Liquid test substances should be diluted in PFS. Solid
test substances should be prepared as solutions in PFS or, if insoluble in saline, dissolved in
DMSO and then diluted up to 0.5% (v/v) with PFS, provided that this concentration of
DMSO does not interfere with the assay. To ensure a valid test, a test substance cannot be
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ICCVAM Test Method Evaluation Report: Appendix C5

May 2008

diluted beyond its Maximum Valid Dilution (MVD) (refer to Section 12.3). The calculationof the MVD is dependent on the ELC for a test substance. The ELC can be calculated by
dividing the threshold human pyrogenic dose by the maximum recommended human dose ina single hour period (see Section 12.2) (USP 2007; FDA 1987). Furthermore, test substances
should not be tested at concentrations that are cytotoxic to MM6 cells.
4.3.1.1 Reference Endotoxin for Spiking Test Substances
The WHO-LPS 94/580 [E. coli O113:H10:K-] or equivalent internationally harmonized RSE
is recommended for preparation of the endotoxin-spike solution and the endotoxin standard
curve (see Section 4.1).
4.3.1.2 Spiking Test Substances with Endotoxin
Non-spiked and endotoxin-spiked test substances are prepared in quadruplicate and an invitro pyrogen test is performed. A fixed concentration of the RSE (i.e., 1.0 EU/mL or aconcentration equal to or near the middle of the endotoxin standard curve) is added to theundiluted test substance (or in serial two-fold dilutions, not to exceed the MVD). An
illustrative example of endotoxin spiking solutions is shown in Table 4-2. For non-spiked
solutions, 150 µL of RPMI-C and 50 µL of the test substance (i.e., equivalent to the negative
product control [NPC]) are added to a well. Endotoxin-spiked solutions are prepared byadding 100 µL of RPMI-C, 50 µL of the test substance, and 50 µL of an endotoxin-spikesolution (1.0 EU/mL) (i.e., equivalent to the positive product control [PPC]). Finally, MM6
cells (50 µL) are added to each well and the wells are mixed and incubated as outlined in
Section 6.1.3, Steps 6-7. An ELISA is then performed as outlined in Section 6.2, without theIL-6 standard curve.
Table 4-2

Preparation of Endotoxin-Spiked and Non-Spiked Solutions for
Determination of Test Substance Interference
SpikedSample Addition
RPMI-C
Endotoxin-spike solution2
Test substance (neat and each serial dilution)
MM6 cells3
Total4Abbreviations: MM6 cells = Mono Mac 61
n=4 replicates each2
Endotoxin concentration is 1.0 EU/mL in RPMI-C.
3
MM6 cells are resuspended in RPMI-C (2.5 x 106 cells/mL).
4
A total volume of 250 µL per well is used for the incubation.

100
50
50
50
250

µL/well1

Non-spiked
150
0
50
50
250

The optical density (OD) values of the endotoxin-spiked and non-spiked test substances arecalibrated against the endotoxin calibration curve. The resulting EU value of the non-spiked
test substance is subtracted from the corresponding EU value of the endotoxin-spiked test
substance at each dilution. The spike recovery for each sample dilution is calculated as apercentage by setting the theoretical value (i.e., endotoxin-spike concentration of 1.0 EU/mL)
at 100%. For example, consider the following interference test results in Table 4-3:

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ICCVAM Test Method Evaluation Report: Appendix C5Table 4-3

May 2008

Example of Interference Data Used to Determine Sample Dilution% Recovery of Endotoxin
Control
25
49
90
110

Sample Dilution
None
1:2
1:4
1:8

If a spike recovery between 50% and 200% is obtained, then no interference of the test
substance with either the cell system or the ELISA is demonstrated (i.e., the test substancedoes not increase or decrease the concentration of IL-6 relative to the endotoxin spike). The
lowest dilution (i.e., highest concentration) of a test substance that yields an endotoxin-spikerecovery between 50% and 200% is determined. The test substance is then diluted in serial
two-fold dilutions beginning at this dilution, not to exceed the MVD, for use in the assay.
Based on the results illustrated in Table 4-3, the initial dilution of the test substance to be
used in the in vitro pyrogen test would be 1:4 (i.e., the lowest dilution between 50% and200% of the 1.0 EU/mL EC).
4.3.2
Interference at the MVD
If the data obtained from the experiment in Section 4.2.1 suggests the presence ofinterference at the MVD, then consideration should be given for using another validated
pyrogen test method.
5.0

CONTROLS

5.1

Benchmark Controls

Benchmark controls may be used to demonstrate that the test method is functioning properly,
or to evaluate the relative pyrogenic potential of chemicals (e.g., parenteral pharmaceuticals,
medical device eluates) of a specific class or a specific range of responses, or for evaluating
the relative pyrogenic potential of a test substance. Appropriate benchmark controls shouldhave the following properties:

5.2•

consistent and reliable source(s) for the chemicals (e.g., parenteralpharmaceuticals, medical device eluates)•

structural and functional similarities to the class of substance being tested•

known physical/chemical characteristics•

supporting data on known effects in animal models•

known potency in the range of response

Endotoxin Control

The EC (i.e., MM6 cells incubated with an internationally harmonized RSE) serves as the
positive control in each experiment. The results should be compared to historical values toinsure that it provides a known level of cytokine release relative to the NSC.

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ICCVAM Test Method Evaluation Report: Appendix C5

5.3

May 2008

Negative Saline Control

The NSC (i.e., MM6 cells incubated with PFS instead of the test substance) is included ineach experiment in order to detect nonspecific changes in the test system, as well as toprovide a baseline for the assay endpoints.
5.4

Solvent Control

Solvent controls are recommended to demonstrate that the solvent is not interfering with thetest system when solvents other than PFS are used to dissolve test substances.
6.0

EXPERIMENTAL DESIGN

6.1

Incubation with Test Samples and Measurement of IL-6 Release

6.1.1
Preincubation of MM6 Cells
To perform an ELISA on the following day, obtain an MM6 cell suspension (30 to 50 mL)
from propagation flasks and centrifuge at 100 x g for 8 min at RT. Remove the supernatant,resuspend the cell pellet in 2 mL of RPMI-C and gently pipet up and down to mix. It isadvisable to determine cell number and cell viability as outlined in Section 4.2.3. The
percentage of viable MM6 cells should exceed 80% to be suitable for use in the test. The
results of these examinations should be included in the study report. Transfer the cells (4 x
105 cells/mL) to new tissue culture flasks and add RPMI-C. Place the flasks in a cell culture
incubator and maintain the cells at 37±1°C + 5% CO2 for 16 to 24 hr. In general, thepreincubation of 2.0 x 107 cells in 50 mL RPMI-C will provide enough cells for one 96-wellassay plate
6.1.2
Preparation of MM6 Cells for the Incubation Assay
Prepare the MM6 cells just prior to addition to the incubation plate (Section 6.1.3, Step 5).
Centrifuge 30 to 50 ml of cell suspension at 100 x g for 8 min at RT. Pour off the supernatantand resuspend the cells in approximately 2 ml of RPMI-C. It is advisable that cell number
and cell viability be determined as outlined in Section 4.2.3. The percentage of viable MM6
cells should exceed 80% to be suitable for use in the test. The results of these examinations
should be included in the study report. Dilute the cells with RPMI–C to a volume that gives aconcentration of 2.5 x 106 cells/ml.
6.1.3
Incubation Plate
Test substances should be vortexed vigorously for at least 30 min or sonicated in a bathsonicator for at least 5 min prior to use in the assay. Test substances should be prepared inserial two-fold dilutions beginning at a level of dilution that did not show interference withthe test system (see Section 4.2) in as many subsequent dilutions that are necessary to bewithin the linear range of the endotoxin standard curve, not to exceed the MVD. Each
incubation plate can accommodate an endotoxin standard curve, a NSC, and 14 test
substances (see Table 6-1).

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ICCVAM Test Method Evaluation Report: Appendix C5Table 6-1
Numberof Wells

May 2008

Overview of Incubation Plate Preparation in the MM6/IL-6 Pyrogen Test

Sample

RPMI-C

Test
Sample

MM61

50

0

100

3

100
100

EC
µL

20

2

EC

100

4

NSC

100

0

0

564

Test
samples

100

0

50

Mix thesamples;incubate for
16 to 24 hr at
37±1°C in ahumidified
atmospherewith 5%CO2.

Mix thesamples;
immediatelytransfer to an
ELISA plate5
and runELISA.

(1-14)
Abbreviations: EC = Endotoxin control; IL-6 = Interleukin-6; NSC = Negative saline control; MM6 = Mono
Mac 61
MM6 cell concentration is 2.5 x 106 cells/mL.
2
Five EC concentrations (0.125, 0.25, 0.50, 1.0, and 2.0 EU/mL) in quadruplicate
3
50 µl of PFS is added instead of the test sample.
4
14 test samples (n=4 each) per plate
5
An IL-6 standard curve is prepared in Columns 11 and 12 on the ELISA plate (see Table 6-3). Therefore, 80
wells are available for test samples and controls on the incubation plate.

6.1.4
Incubation Assay for IL-6 ReleaseMM6 cells are prepared in a microtiter plate using a laminar flow hood (refer to Section
6.1.2). All consumables and solutions must be sterile and pyrogen-free. Each plate should belabeled appropriately with a permanent marker. An overview of the incubation plate
preparation is shown in Table 6-1. The incubation procedure is outlined below:
Step 1. Refer to the suggested incubation plate template presented in Table 6-2.
Step 2. Using a pipetter, transfer 100 µL of RPMI-C into each well.
Step 3. Transfer 50 µL of test sample or 50 µL of PFS for the NSC into theappropriate wells as indicated in the template.
Step 4. Transfer 50 µL of the EC (standard curve) in quadruplicate into theappropriate wells according to the template.
Step 5. Transfer 100 µL of a well-mixed MM6 cell suspension into each well.
Step 6. Place the covered plate in a tissue culture incubator for 16 to 24 hr at
37±1°C in a humidified atmosphere containing 5% CO2.
Step 7. Remove 150 µL of the supernatant from each well, without disrupting thecells, and transfer to the IL-6 ELISA plate.

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ICCVAM Test Method Evaluation Report: Appendix C5Table 6-2

May 2008

Incubation Plate - Sample and Control Template

1

2

3

4

5

6

7

8

9

10

11

12

A

EC1
2.0

EC
2.0

EC
2.0

EC
2.0

TS3

TS3

TS3

TS3

TS11

TS11

Void3

Void

B

EC
1.0

EC
1.0

EC
1.0

EC
1.0

TS4

TS4

TS4

TS4

TS11

TS11

Void

Void

C

EC
0.50

EC
0.50

EC
0.50

EC
0.50

TS5

TS5

TS5

TS5

TS12

TS12

Void

Void

D

EC
0.25

EC
0.25

EC
0.25

EC
0.25

TS6

TS6

TS6

TS6

TS12

TS12

Void

Void

E

EC
0.125

EC
0.125

EC
0.125

EC
0.125

TS7

TS7

TS7

TS7

TS13

TS13

Void

Void

F

NSC

NSC

NSC

NSC

TS8

TS8

TS8

TS8

TS13

TS13

Void

Void

G

TS12

TS1

TS1

TS1

TS9

TS9

TS9

TS9

TS14

TS14

Void

Void

H

TS2

TS2

TS2

TS2

TS10

TS10

TS10

TS10

TS14

TS14

Void

Void

Abbreviations: EC = Endotoxin control; NSC = Negative saline control; TS = Test substance1
EC value (e.g., EC 2.0) represents the endotoxin concentration in EU/mL.
2
TS number (e.g., TS 1) represents an arbitrary sequence for individual test substances.
3
Columns 11 and 12 are reserved for the IL-6 standard curve on the ELISA plate (see Table 6-3).

6.2

ELISA to Measure IL-6 Release

6.2.1

IL-6 Standard Curve

An IL-6 standard supplied with the ELISA kit is used. IL-6 standards are typically suppliedin lyophilized form and should be reconstituted according to the manufacturer's instructions.
The stock solution should be diluted in RPMI-C to the following concentrations: 0, 62.5, 125,
250, 500, 1000, 2000, and 4000 pg/mL in volumes of at least 500 µL. Each well on the
ELISA plate will receive 50 µL of an IL-6 blank or standard.
6.2.2
ELISA
The manufacturer's instructions provided with the ELISA kit should be followed and atypical experimental design is outlined below. The ELISA should be carried out at RT and
therefore all components must be at RT prior to use. Frozen specimens should not be thawedby heating them in a water bath. A suggested ELISA plate template is shown in Table 6-3,
which includes a five-point EC standard curve, an eight-point IL-6 standard curve (0 to 4000
pg/mL), and available wells for up to 14 test substances and a NSC each in quadruplicate.
The EC standard curve, the NSC, and the test sample supernatants are transferred directlyfrom the incubation plate. The IL-6 standard curve is prepared as described in Section 6.2.1.
An overview of the ELISA plate preparation is shown in Table 6-4.

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ICCVAM Test Method Evaluation Report: Appendix C5

May 2008

Step 1. After pipetting up and down very carefully three times (avoid detachmentof the adherent MM6 cells) to mix the supernatant, transfer 50 µL from each wellof the Incubation Plate (A1-10; H1-10) to the ELISA plate.
Step 2. Add 50 µL of each IL-6 standard (0 to 4000 pg/mL) into the respective
wells on the ELISA plate.
Step 3. Add 200 µL of the enzyme-labeled detection antibody (neat as supplied, or
diluted, if necessary) to each of the wells.
Step 4. Cover the microtiter plate(s) with adhesive film and incubate for 2 to 3 hr
at RT.
Step 5. Decant and wash each well three times with 300 µL Buffered Wash
Solution and then rinse three times with deionized water. Place the plates upside
down and tap to remove water.
Step 6. Add 200 µL of TMB/Substrate Solution to each well and incubate at RT in
the dark for 15 min. If necessary, decrease the incubation time.
Step 7. Add 50 µL of Stop Solution to each well.
Step 8. Tap the plate gently after the addition of Stop Solution to aid in mixing.
Step 9. Read the OD450 within 15 min of adding the Stop Solution. Measurementwith a reference wavelength of 540 to 590 nm is recommended4.

4

The TMB chromagen is measured at OD450. However, the use of an IL-1β ELISA kit with a chromagen otherthan TMB is acceptable. The ELISA should be measured at a wavelength appropriate for the specificchromagen used.
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ICCVAM Test Method Evaluation Report: Appendix C5Table 6-3

May 2008

ELISA Plate - Sample and Control Template

1

2

3

4

5

6

7

8

9

10

11

12

A

EC1
2.0

EC
2.0

EC
2.0

EC
2.0

TS3

TS3

TS3

TS3

TS11

TS11

IL-63
0

IL-6
0

B

EC
1.0

EC
1.0

EC
1.0

EC
1.0

TS4

TS4

TS4

TS4

TS11

TS11

IL-6
62.5

IL-6
62.5

C

EC
0.50

EC
0.50

EC
0.50

EC
0.50

TS5

TS5

TS5

TS5

TS12

TS12

IL-6
125

IL-6
125

D

EC
0.25

EC
0.25

EC
0.25

EC
0.25

TS6

TS6

TS6

TS6

TS12

TS12

IL-6
250

IL-6
250

E

EC
0.125

EC
0.125

EC
0.125

EC
0.125

TS7

TS7

TS7

TS7

TS13

TS13

IL-6
500

IL-6
500

F

NSC

NSC

NSC

NSC

TS8

TS8

TS8

TS8

TS13

TS13

IL-6
1000

IL-6
1000

G

TS12

TS1

TS1

TS1

TS9

TS9

TS9

TS9

TS14

TS14

IL-6
2000

IL-6
2000

H

TS2

TS2

TS2

TS2

TS10

TS10

TS10

TS10

TS14

TS14

IL-6
4000

IL-6
4000

Abbreviations: EC = Endotoxin control; NSC = Negative saline control; TS = Test substance1
EC value (e.g., EC 2.0) represents the endotoxin concentration in EU/mL.
2
TS number (e.g., TS1) represents an arbitrary sequence for individual test substances.
3
IL-6 values in columns 11 and 12 are in pg/mL.

Table 6-4

Overview of ELISA Procedure

MaterialtransferfromIncubation
Plate (µ L)IL-6standard(0 to
4000
pg/mL)(µL)

Enzymelabeled
Antibody
(µL)

50

50

200

Cover theIncubation
Plate andincubatefor 2 to 3
hr at RT.

Decantand washeach wellthree
times with
300 µL
Buffered
Wash
Solutionand threetimes with
deionized
water.

TMB/Substrate
Solution(µL)

200

Abbreviations: OD450 = Optical density at 450 nm; RT = Room temperature

C-113

Incubatefor lessthan15min at
RT in
dark.

Stop
Solution(µL)

50

Read eachwell at
OD450
with a 540
to 590 nm
reference
filter.

ICCVAM Test Method Evaluation Report: Appendix C5

7.0

EVALUATION OF TEST RESULTS

7.1

OD Measurements

May 2008

The OD of each well is obtained by reading the samples in a standard microplate
spectrophotometer (i.e., plate reader) using a visible light wavelength of 450 nm (OD450) witha reference filter of 540 to 590 nm (recommended)5. OD values are used to determine assay
acceptability and in the decision criteria for pyrogen detection (see Sections 8.0 and 9.0).
8.0

CRITERIA FOR AN ACCEPTABLE TESTAn EC (five-point standard curve) and a NSC should be included in each experiment. An
IL-6 standard curve should be included in each ELISA as shown in the template presented inTable 6-3. An assay is considered acceptable only if the following minimum criteria are met:•

The quadratic function of the IL-6 standard curve produces an r ≥0.956 and the
OD of the blank control is below 0.15.

•

The endotoxin standard curve produces OD values that ascend in a sigmoidal
concentration response.

An outlying observation may be excluded if the aberrant response is identified using
acceptable statistical methodology (e.g., Dixon's test [Dixon 1950; Barnett and Lewis 1994]
or Grubbs' test [Barnett and Lewis 1994; Grubbs 1969; Iglewicz and Houghlin 1993]).
9.0

DATA INTERPRETATION/DECISION CRITERIA

9.1

Decision Criteria for Pyrogen Detection

A test substance is considered pyrogenic when the endotoxin concentration of the test
substance exceeds the ELC for the test sample. The ELC can be calculated as shown in
Section 12.2.
10.0

STUDY REPORTThe test report should include the following information:
Test Substances and Control Substances

5•

Name of test substance•Purity and composition of the substance or preparation•

Physicochemical properties (e.g., physical state, water solubility)•

Quality assurance data•

Treatment of the test/control substances prior to testing (e.g., vortexing,
sonication, warming, resuspension solvent)

The TMB chromagen is measured at OD450. However, the use of an IL-1β ELISA kit with a chromagen otherthan TMB is acceptable. The ELISA should be measured at a wavelength appropriate for the specificchromagen used.
6
Correlation coefficient (r), an estimate of the correlation of x and y values in a series of n measurements.
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May 2008

Justification of the In Vitro Test Method and Protocol Used
Test Method Integrity•

The procedure used to ensure the integrity (i.e., accuracy and reliability) of thetest method over time•

If the test method employs proprietary components, documentation on theprocedure used to ensure their integrity from “lot-to-lot” and over time•

The procedures that the user may employ to verify the integrity of the
proprietary components

Criteria for an Acceptable Test•

Acceptable concurrent positive control ranges based on historical data•

Acceptable negative control data

Test Conditions•

Cell system used•

Calibration information for the spectrophotometer used to read the ELISA•

Details of test procedure used•

Description of any modifications of the test procedure•

Reference to historical data of the model•

Description of evaluation criteria used•

Tabulation of data from individual test samples

Results
Description of Other Effects Observed
Discussion of the Results
Conclusion
A Quality Assurance Statement for Good Laboratory Practice (GLP)-Compliant Studies•

This statement should indicate all inspections made during the study and thedates any results were reported to the Study Director. This statement should
also confirm that the final report reflects the raw data.

If GLP-compliant studies are performed, then additional reporting requirements provided in
the relevant guidelines (e.g., OECD 1998; EPA 2003a, 2003b; FDA 2003) should be
followed.
11.0

REFERENCES

Barnett V, Lewis T. 1994. Outliers in Statistical Data. In: Wiley Series in Probability andMathematical Statistics. Applied Probability and Statistics. 3rd Ed. New York: John Wiley &Sons.
Dixon WJ. 1950. Analysis of extreme values. Annals of Mathematical Statistics 21:488-506.
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ICCVAM Test Method Evaluation Report: Appendix C5

May 2008

EPA. 2003a. Good Laboratory Practice Standards. Toxic Substances Control Act. 40 CFR
792.
EPA. 2003b. Good Laboratory Practice Standards. Federal Insecticide, Fungicide, andRodenticide Act. 40 CFR 160.
FDA. 1987. Guideline on Validation of the Limulus Amebocyte Lysate Test as an
End-product Endotoxin Test for Human and Animal Parenteral Drugs, Biological Products,
and Medical Devices. Rockville, MD:U.S. Department of Health and Human Services(DHHS), Food and Drug Administration (FDA).
FDA. 2003. Good Laboratory Practices for Nonclinical Laboratory Studies. 21 CFR 58.
Grubbs FE. 1969. Procedures for detecting outlying observations in samples. Technometrics
11(1):1-21.
Iglewicz B, Houghlin DC. 1993. How to detect and handle outliers. In: ASQC Basic
Reference in Quality Control. Vol. 14. Milwaukee, WI: ASQ Quality Press.
Organization for Economic Cooperation and Development (OECD). 1998. OECD Series on
Principle of Good Laboratory Practice and Compliance Monitoring. No. 1. OECD Principles
of Good Laboratory Practice (as revised in 1997). Organisation for Economic Co-operation
and Development (OECD), ENV/MC/CHEM (98)17. Paris: OECD.
Taktak YS, Selkirk S, Bristow AF, Carpenter A, Ball C, Rafferty B, Poole S. 1991. Assay ofpyrogens by interleukin-6 release from monocytic cell lines. J Pharm Pharmacol 43:578-582.
USP. 2007. The U.S. Pharmacopeia. USP30 NF25<85>. Ed. The U.S. Pharmacopeial
Convention. Rockville, MD:The U.S. Pharmacopeial Convention.

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ICCVAM Test Method Evaluation Report: Appendix C5

12.0
12.1

May 2008

TERMINOLOGY AND FORMULA
Assay Sensitivity (λ)1

The variable λ is defined as the labeled sensitivity (in EU/mL) of the LAL Reagent in
endpoint assays (e.g., the BET gel-clot technique). For kinetic BET assays, λ is the lowest
point used in the endotoxin standard curve.
12.2

Endotoxin Limit Concentration (ELC)1,2

The ELC for parenteral drugs is expressed in Endotoxin Units (EU) per volume (mL) or
weight (mg). The ELC is equal to K/M, where:
K is the threshold human pyrogenic dose of endotoxin (EU) per body weight (kg). K is equal
to 5.0 EU/kg for intravenous administration. For intrathecal administration, K is equal to 0.2
EU/kg (see also Section 12.5).
M is the rabbit test dose or the maximum recommended human dose of product (mL or mg)
per body weight (kg) in a single hour period (see also Section 12.8).
For example, if a non-intrathecal product were used at an hourly dose of 10 mL per patient,then the ELC would be 0.50 EU/mL.
12.3

Maximum Valid Dilution (MVD)1,2

The MVD is the maximum allowable dilution of a test substance at which the endotoxin limit
can be determined. The calculation of the MVD is dependent on the ELC for a test substance.
When the ELC is known, the MVD is1:
MVD = (ELC x Product Potency [PP])/λAs an example, for Cyclophosphamide Injection, the ELC is 0.17 EU/mg, PP is 20
mg/mL, and the assay sensitivity is 0.065 EU/mL. The calculated MVD would be 1:52.3 or
1:52. The test substance can be diluted no more than 1:52 prior to testing.
If the ELC is not known, the MVD is1:
MVD = PP/Minimum Valid Concentration (MVC)
where, MVC = (λ x M)/K
where, M is the maximum human dose
As an example, for Cyclophosphamide Injection, the PP is 20 mg/mL, M is 30 mg/kg,
and assay sensitivity is 0.065 EU/mL. The calculated MVC is 0.390 mg/mL and the MVD is
1:51.2 or 1:51. The test substance can be diluted no more than 1:51 in the assay prior to
testing.
12.4

Negative Product Control (NPC)

For interference testing, the NPC is a test sample to which pyrogen-free saline (PFS) is
added. The NPC is the baseline for determination of cytokine release relative to theendotoxin-spiked PPC.
1
2

From FDA (1987)
From USP (2007)
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ICCVAM Test Method Evaluation Report: Appendix C5

12.5

May 2008

Parenteral Threshold Pyrogen Dose (K)1,2

The value K is defined as the threshold human pyrogenic dose of endotoxin (EU) per bodyweight (kg). K is equal to 5.0 EU/kg for parenteral drugs except those administeredintrathecally; 0.2 EU/kg for intrathecal drugs.
12.6

Positive Product Control (PPC)

For interference testing, the PPC is a test substance spiked with the control standard
endotoxin (i.e., 0.5 EU/mL or an amount of endotoxin equal to that which produces ½ themaximal increase in optical density (OD) from the endotoxin standard curve) to insure thatthe test system is capable of endotoxin detection in the product as diluted in the assay.
12.7

Product Potency (PP)1,2

The test sample concentration expressed as mg/mL or mL/mL.
12.8

Rabbit Pyrogen Test (RPT) Dose or Maximum Human Dose (M)1,2

The variable M is equal to the rabbit test dose or the maximum recommended human dose of
product per kg of body weight in a single hour period. M is expressed in mg/kg or mL/kg andvaries with the test substance. For radiopharmaceuticals, M equals the rabbit dose or
maximum human dose/kg at the product expiration date or time. Use 70 kg as the weight of
the average human when calculating the maximum human dose per kg. If the pediatricdose/kg is higher than the adult dose, then it shall be the dose used in the formula.

C-118

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May 2008

Appendix D
D1

D2
D3

Federal Register Notices, Public Comments, and Relevant
SACATM Meeting Minutes
Federal Register Notices.......................................................................................D-3
D1-1 Vol. 70, No. 241, pp. 74833-74834, December 16, 2005: Peer Panel
Evaluation of In Vitro Pyrogenicity Testing Methods: Request forComments, Nominations of Experts, and Submission of In Vivo and In
Vitro Data.............................................................................................................D-5
D1-2 Vol. 71, No. 238, pp. 74533-74534, December 12, 2006:
Announcement of an Independent Scientific Peer Review Meeting on the
Use of In Vitro Pyrogenicity Testing Methods; Request for Comments............D-7
D1-3 Vol. 72, No. 89, pp. 26395-26396, May 9, 2007: Peer Review Panel
Report on Five In Vitro Pyrogen Test Methods: Availability and Request
for Public Comments...........................................................................................D-9
Public Comments Received in Response toFederal Register Notices..................................................................................... D-11
ICCVAM Evaluation of In Vitro Pyrogen Test Methods:
Excerpt from SACATM Draft Meeting Minutes ............................................. D-43

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D-2

May 2008

ICCVAM Test Method Evaluation Report: Appendix D1

May 2008

Appendix D1Federal Register Notices
D1-1 Federal Register, Vol. 70, No. 241, pp. 74833-74834, December 16, 2005: Peer
Panel Evaluation of In Vitro Pyrogenicity Testing Methods: Request for Comments,
Nominations of Experts, and Submission of In Vivo and In Vitro Data ........................D-5
D1-2 Federal Register, Vol. 71, No. 238, pp. 74533-74534, December 12, 2006:
Announcement of an Independent Scientific Peer Review Meeting on the Use of In Vitro
Pyrogenicity Testing Methods; Request for Comments ................................................D-7
D1-3 Federal Register, Vol. 72, No. 89, pp. 26395-26396, May 9, 2007: Peer Review
Panel Report on Five In Vitro Pyrogen Test Methods: Availability and Request for
Public Comments ............................................................................................................D-9

D-3

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D-4

May 2008

ICCVAM Test Method Evaluation Report: Appendix D1

May 2008

Federal Register / Vol. 70, No. 241 / Friday, December 16, 2005 / Notices
DEPARTMENT OF HEALTH AND
HUMAN SERVICES
National Toxicology Program (NTP),NTP Interagency Center for the
Evaluation of Alternative Toxicological
Methods (NICEATM); Peer Panel
Evaluation of In Vitro Pyrogenicity
Testing Methods: Request forComments, Nominations of Experts,
and Submission of In Vivo and In Vitro
Data
AGENCY: National Institute ofEnvironmental Health Sciences(NIEHS), National Institutes Of Health(NIH).
ACTION: Request for comments,
nominations of scientific experts, andsubmission of data.
SUMMARY: NICEATM, in collaboration

with the Interagency Coordinating
Committee on the Validation of
Alternative Methods (ICCVAM), is
considering convening an independent
peer review panel (hereafter, ‘‘Panel’’) toevaluate the validation status of five in
vitro pyrogenicity test methods: (1)Human PBMC/IL–6 in vitro pyrogen test
(PBMC/IL–6), (2) human whole blood/
IL–1 in vitro pyrogen test (WB/IL–1), (3)human whole blood/IL–1 in vitro
pyrogen test: application of
cryopreserved human whole blood cryo
(WB/IL–1), (4) the human whole blood/
IL–6 in vitro pyrogen test (WB/IL–6),
and (5) an alternative in vitro pyrogentest using the human monocytoid cellline MONO MAC–6 (MM6/IL6).
NICEATM requests public comments as
to the appropriateness and relative
priority of this activity. In addition,
NICEAM requests the nomination ofexpert scientists for consideration as
potential Panel members in the event a
Panel meeting occurs. Finally,
NICEATM requests the submission ofdata from the rabbit pyrogenicity test,
the bacterial endotoxin test (BET), andin vitro pyrogenicity testing with themethods listed above.
DATES: Comments, nominations ofexpert scientist, and data submissions
should be received by January 17, 2006.
ADDRESSES: Correspondence should besent by mail, fax, or email to Dr.
William S. Stokes, NICEATM, NIEHS, P.
O. Box 12233, MD EC–17, Research
Triangle Park, NC, 27709, (phone) 919–
541–2384, (fax) 919–541–0947, (e-mail)niceatm@niehs.nih.gov.
SUPPLEMENTARY INFORMATION:
Background
The European Committee on the
Validation of Alternative Methods(ECVAM) conducted a validation study
to independently evaluate theusefulness of five in vitro pyrogenicity
assays (PBMC/IL–6, WB/IL–1, cryo WB/
IL–1, WB/IL–6, and MM6/IL6). In June
2005, ECVAM submitted backgroundreview documents (BRDs) for these fivemethods to NICEATM for consideration
as replacements for the currently
required tests (i.e., rabbit pyrogen tests
and the BET). ICCVAM and NICEATM
reviewed the BRDs for completenessand concluded that these five in vitro
test methods appear to haveconsiderable potential for pyrogenicity
testing, but the sponsors needed toprovide additional information prior toa formal review by a Panel. Pending
receipt and review of the requested
information, ICCVAM and NICEATM
will determine the priority of anevaluation of these test methods. If
convened, the Panel would (1) peer
review the BRDs for the test methods,
and (2) determine whether the data
cited in the BRDs support draft ICCVAM
Test Method Recommendations
regarding the proposed usefulness,
limitations, and validation status of thetest methods. If appropriate, the Panelmight also formulate conclusions on theadequacy of any draft recommended
performance standards, any proposed
future validation studies, draft
standardized test method protocols,
and/or reference substances. In making
their conclusions andrecommendations, the Panel considers
all available information including thescientific studies cited in the draft BRD,
public comments, and any new
information identified during the peer
review.
Request for Public Comments andNominations of Scientific Experts
NICEATM requests public commentson the appropriateness and relative
priority of the proposed Panel review
activity. In addition, NICEAM requests
the nomination of scientists with
relevant knowledge and experience topotentially serve on the Panel should it
be convened. Areas of relevant expertise
include, but are not limited to:physiology, pharmacology,
immunology, pyrogenicity testing inanimals, development and use of invitro methodologies, biostatistical data
analysis, knowledge of chemical data
sets useful for validation of toxicity
studies, and hazard classification ofchemicals and products. Eachnomination should include the person’s
name, affiliation, contact information(i.e., mailing address, e-mail address,
telephone and fax numbers), and a briefsummary of relevant experience andqualifications.

Request for Data
NICEATM invites the submission ofdata from standard in vivo rabbit
pyrogen testing, the BET, and in vitro
pyrogenicity testing using the methods
detailed above. Although data can be
accepted at any time, data submitted by
the deadline listed in this notice would
be considered during an evaluation of
the validation status of the fivepyrogenicity testing methods should
this activity occur. Submitted data will
be used to further evaluate theusefulness and limitations of in vitro
pyrogenicity test methods and may be
included in future NICEATM andICCVAM reports and publications as
appropriate. The data will also be
included in a NICEATM database tosupport the investigation of other test
methods for assessing pyrogenicity.
When submitting chemical andprotocol information/test data, pleasereference this Federal Register noticeand provide appropriate contact
information (name, affiliation, mailingaddress, phone, fax, e-mail, andsponsoring organization, as applicable).
NICEATM prefers data to besubmitted as copies of pages from study
notebooks and/or study reports, if
available. Raw data and analyses
available in electronic format may also
be submitted. Each submission for achemical should preferably include thefollowing information, as appropriate:• Common and trade name• Chemical Abstracts Service Registry
Number (CASRN)• Chemical class• Product class• Commercial source• In vitro pyrogenicity test protocol
used• In vitro pyrogenicity test results• BET test protocol used
• BET test results• In vivo rabbit pyrogen test protocol
used• Individual animal responses• The extent to which the study
complied with national orinternational Good Laboratory
Practice (GLP) guidelines• Date and testing organization
Background Information on ICCVAM
and NICEATMICCVAM is an interagency committee
composed of representatives from 15
Federal regulatory and research agencies
that use or generate toxicologicalinformation. ICCVAM conducts
technical evaluations of new, revised,and alternative methods with regulatory
applicability and promotes the scientificvalidation and regulatory acceptance of
toxicological test methods that more

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Federal Register / Vol. 70, No. 241 / Friday, December 16, 2005 / Notices

accurately assess the safety and hazardsof chemicals and products and thatrefine, reduce, or replace animal use.
The ICCVAM Authorization Act of 2000
(Pub. L. 106–545, available at http://
iccvam.niehs.nih.gov/about/
PL106545.htm) establishes ICCVAM as apermanent interagency committee of the
NIEHS under the NICEATM. NICEATM
administers the ICCVAM and provides
scientific and operational support for
ICCVAM-related activities. NICEATM
and ICCVAM work collaboratively toevaluate new and improved test
methods applicable to the needs ofFederal agencies. Additional
information about ICCVAM and
NICEATM can be found at the following
Web site: http://
www.iccvam.niehs.nih.gov.
Dated: Decmeber 5, 2005.
Samuel H. Wilson,
Deputy Director, National Institute ofEnvironmental Health Sciences.
[FR Doc. E5–7410 Filed 12–15–05; 8:45 am]
BILLING CODE 4140–01–P

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16DEN1ICCVAM Test Method Evaluation Report: Appendix D1Federal Register / Vol. 71, No. 238 / Tuesday, December 12, 2006 / Notices

DEPARTMENT OF HEALTH AND
HUMAN SERVICES
National Toxicology Program (NTP),NTP Interagency Center for the
Evaluation of Alternative Toxicological
Methods (NICEATM); Announcementof an Independent Scientific Peer
Review Meeting on the Use of In Vitro
Pyrogenicity Testing Methods;
Request for Comments
AGENCY: National Institute ofEnvironmental Health Sciences(NIEHS), National Institutes of Health(NIH).
ACTION: Meeting announcement andrequest for comments.

Backgroundjlentini on PROD1PC65 with NOTICESSUMMARY: NICEATM in collaboration

with the Interagency Coordinating
Committee on the Validation of
Alternative Methods (ICCVAM)
announces an independent scientificpeer review meeting to evaluate thevalidation status of five in vitro
pyrogenicity test methods: (1) Human
PBMC/IL–6 in vitro pyrogen test
(PBMC/IL–6), (2) human whole blood/
IL–1 in vitro pyrogen test (WB/IL–1), (3)human whole blood/IL–1 in vitro
pyrogen test: application of
cryopreserved human whole blood (cryo
WB/IL–1), (4) the human whole blood/
IL–6 in vitro pyrogen test (WB/IL–6),
and (5) an alternative in vitro pyrogentest using the human monocytoid cellline MONO MAC–6 (MM6/IL6). These
five in vitro test methods are proposed
as replacements for the in vivo rabbit
pyrogen test (RPT). At this meeting, ascientific panel will peer review thedraft background review document(BRD) on each test method, evaluate theextent that the BRD addresses
established validation and acceptancecriteria for each test method, andprovide comment on draft ICCVAM
recommendations on the proposed useof these test methods, draft test method
protocols, and draft performancestandards. NICEATM invites publiccomments on the draft BRDs, draft
ICCVAM test method recommendations,

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draft test method protocols, and draft
performance standards.
DATES: The meeting will be held on
February 6, 2007, from 8:30 a.m. to 5
p.m. The meeting is open to the publicwith attendance limited only by the
space available. In order to facilitate
planning for this meeting, persons
wishing to attend are asked to registerby January 23, 2007, via the
ICCVAM/NICEATM Web site(http://iccvam.niehs.nih.gov).
Comments should be sent by mail, fax,
or email to the address given below by
January 26, 2007.
ADDRESSES: The meeting will be held at
the National Institutes of Health (NIH),
Natcher Conference Center, 45 Center
Drive, Bethesda, MD 20892.
FOR FURTHER INFORMATION CONTACT: Dr.
William S. Stokes, Director of
NICEATM, NIEHS, P.O. Box 12233, MD
EC–17, Research Triangle Park, NC,
27709, (phone) 919–541–2384, (fax)919–541–0947, (e-mail)niceatm@niehs.nih.gov. Courier address:
NICEATM, 79 T.W. Alexander Drive,
Building 4401, Room 3128, Research
Triangle Park, NC 27709.
SUPPLEMENTARY INFORMATION:
The European Centre for the
Validation of Alternative Methods(ECVAM) conducted a validation study
to independently evaluate theusefulness and limitations of five in
vitro pyrogenicity test methods (PBMC/
IL–6, WB/IL–1, cryo WB/IL–1, WB/IL–6,
and MM6/IL6). In June 2005, ECVAM
submitted BRDs for these five methods
to NICEATM for consideration as
replacements for the currently required
test, the RPT. ICCVAM and NICEATM
reviewed the BRDs for completenessand concluded that these five in vitro
test methods appear to haveconsiderable potential for pyrogenicity
testing, but that the sponsor needed toprovide additional information prior toa formal scientific review by an expert
panel. In anticipation of proceedingwith an evaluation of these test
methods, ICCVAM and NICEATM
requested public comments as to theappropriateness and relative priority ofa panel review activity and thenomination of scientists with relevant
knowledge and experience topotentially serve on the panel (Federal
Register Vol. 70, No. 241, pp. 74833–4,
December 16, 2005). NICEATM also
requested submission of data using thestandard in vivo rabbit pyrogen test, thebacterial endotoxin test (BET), and invitro pyrogenicity tests. These requests
were sent directly to over 100 interested

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74533

stakeholders; no additional data werereceived.
In March 2006, ECVAM responded to
the ICCVAM/NICEATM request for
information by providing a revised BRD
for each test method. ICCVAM and
NICEATM drafted a BRD that combines
all of the available information on thefive in vitro pyrogenicity test methods
into a single document and includes
each of the ECVAM BRDs as anappendix. Based on this information,
ICCVAM developed draft test method
recommendations regarding the
proposed usefulness, limitations, andvalidation status of these test methods.
ICCVAM subsequently recommended
that an independent scientific panel beconvened to (1) peer review the draft
BRD for the test methods and (2)determine whether the data andanalyses in the draft BRDs support thedraft ICCVAM test method
recommendations. The panel will also
be asked to comment on the adequacyof the draft recommended performancestandards, proposed future validation
studies, draft standardized test method
protocols, and recommended reference
substances. In making their conclusions
and recommendations, NICEATM will
ask the panel to consider all available
information including the scientificstudies cited in the draft BRD, publiccomments, and any new information
identified during the peer review.
Peer Review Panel Meeting
The purpose of this meeting is thescientific peer review evaluation of thevalidation status of five in vitro
pyrogenicity test methods as
replacements for the RPT. First, thepanel will review the draft BRD on thecurrent status of five in vitro test
methods for the detection ofpyrogenicity and evaluate the extent
that established validation andacceptance criteria are addressed foreach test method (Validation andRegulatory Acceptance of Toxicological
Test Methods: A Report of the ad hoc
Interagency Coordinating Committee on
the Validation of Alternative Methods,
NIH Publication No. 97–981, http://
iccvam.niehs.nih.gov). Next, the panel
will comment on the extent to which
the ICCVAM recommendations aresupported by the information provided
in the BRD and on the proposed use of
these test methods, draft test method
protocols, draft performance standards,
and any proposed validation studies.
Information about the panel meeting,including a roster of the panel members
and the draft agenda, will be madeavailable two weeks prior to the meetingon the ICCVAM/NICEATM Web site(http://iccvam.niehs.nih.gov) or can be

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May 2008

74534

obtained after that date by contacting
NICEATM (see FOR FURTHERINFORMATION CONTACT above).
Attendance and Registration
This public meeting will take place
February 6, 2007, at the NIH Campus,
Natcher Conference Center, Bethesda,
MD (a map of the NIH campus and othervisitor information are available at
http://www.nih.gov/about/visitor/
index.htm). The meeting begins at 8:30
a.m. and will conclude at approximately
5 p.m. Persons needing special
assistance, such as sign language
interpretation or other reasonableaccommodation in order to attend,
should contact 919–541–2475 (voice),
919–541–4644 TTY (text telephone),through the Federal TTY Relay System
at 800–877–8339, or by e-mail toniehsoeeo@niehs.nih.gov. Requests
should be made at least seven businessdays in advance of the event.

jlentini on PROD1PC65 with NOTICES

Availability of the BRD and Draft
ICCVAM Recommendations
NICEATM prepared a BRD on five in
vitro pyrogenicity test methods that
describes the current validation status of
the in vitro test methods and contains
all of the data and analyses supporting
this validation status. The draft BRDs,
draft ICCVAM test method
recommendations, draft test method
protocols, and draft test method
performance standards are available
from the ICCVAM/NICETAM Web site(http://iccvam.niehs.nih.gov) or bycontacting NICEATM (see FOR FURTHERINFORMATION CONTACT above).
Request for Comments
NICEATM invites the submission of
written comments on the BRDs, draft
ICCVAM test method recommendations,draft test method protocols, and draft
test method performance standards.
When submitting written comments, it
is important to refer to this Federal
Register notice and include appropriate
contact information (name, affiliation,
mailing address, phone, fax, e-mail, andsponsoring organization, if applicable).
Written comments should be sent by
mail, fax, or e-mail to Dr. William
Stokes, Director of NICEATM, at theaddress listed above, not later thanJanuary 26, 2007. All comments
received will be placed on the ICCVAM/NICEATM Web site (http://
iccvam.niehs.nih.gov), sent to the panel
and ICCVAM agency representatives,and made available at the meeting.
This meeting is open to the public
and time will be provided for thepresentation of public oral comments at
designated times during the peer
review. Members of the public who

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wish to present oral statements at themeeting (one speaker per organization)
should contact NICEATM (see FOR
FURTHER INFORMATION CONTACT above) nolater than January 26, 2007. Speakers
will be assigned on a consecutive basis
and up to seven minutes will be allotted
per speaker. Persons registering to makecomments are asked to provide
NICEATM a written copy of theirstatement by January 26, 2007, so that
copies can be distributed to the panel
prior to the meeting or if this is notpossible to bring 40 copies to themeeting. Written statements can
supplement and expand the oralpresentation. Each speaker is asked toprovide contact information (name,affiliation, mailing address, phone, fax,
e-mail, and sponsoring organization, if
applicable) when registering to makeoral comments.
Summary minutes and the panel’s
final report will be available following
the meeting on the ICCVAM/NICEATM
Web site (http://iccvam.niehs.nih.gov).
ICCVAM will consider the panel’s
conclusions and recommendations andany public comments received in
finalizing their test method
recommendations and performancestandards for these methods.

Dated: November 27, 2006.
Samuel H. Wilson,
Deputy Director, National Institute ofEnvironmental Health Sciences and National
Toxicology Program.
[FR Doc. E6–21038 Filed 12–11–06; 8:45 am]
BILLING CODE 4140–01–P

Background Information on ICCVAM
and NICEATMICCVAM is an interagency committee
composed of representatives from 15
Federal regulatory and research agencies
that use or generate toxicologicalinformation. ICCVAM conducts
technical evaluations of new, revised,and alternative methods with regulatory
applicability and promotes the scientificvalidation and regulatory acceptance of
toxicological test methods that moreaccurately assess the safety and hazardsof chemicals and products and thatrefine, reduce, and replace animal use.
The ICCVAM Authorization Act of 2000
(42 U.S.C. 285l–3, available at
http://iccvam.niehs.nih.gov/about/
PL106545.htm) establishes ICCVAM as apermanent interagency committee of the
NIEHS under the NICEATM. NICEATM
administers ICCVAM and provides
scientific and operational support for
ICCVAM-related activities. NICEATM
and ICCVAM work collaboratively toevaluate new and improved test
methods applicable to the needs offederal agencies. Additional information
about ICCVAM and NICEATM can befound at the following Web site:
http://iccvam.niehs.nih.gov.

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May 2008

Federal Register / Vol. 72, No. 89 / Wednesday, May 9, 2007 / Notices
SUPPLEMENTARY INFORMATIONDEPARTMENT OF HEALTH AND
HUMAN SERVICES
National Toxicology Program (NTP),NTP Interagency Center for the
Evaluation of Alternative Toxicological
Methods (NICEATM); Peer Review
Panel Report on Five In Vitro Pyrogen
Test Methods: Availability and Request
for Public Comments
AGENCY: National Institute ofEnvironmental Health Sciences(NIEHS), National Institutes of Health(NIH).
ACTION: Request for comments.
SUMMARY: NICEATM in collaboration

with the Interagency Coordinating
Committee on the Validation of
Alternative Methods (ICCVAM)
convened an independent scientificpeer review panel meeting on February
6, 2007, to evaluate the validation statusof five in vitro pyrogen test methods
proposed as replacements for the Rabbit
Pyrogen Test (RPT). The peer review
panel (‘‘the Panel’’) report from thismeeting is now available. The reportcontains (1) the Panel’s evaluation of thevalidation status of the methods and (2)the Panel’s comments and conclusions
on draft ICCVAM test method
recommendations. NICEATM invites
public comment on the Panel’s report.
The report is available on the
NICEATM/ICCVAM Web site at (http://
iccvam.niehs.nih.gov/methods/pyrogen/pyrogen.htm) or by contacting
NICEATM (see FOR FURTHERINFORMATION CONTACT below).
DATES: Written comments on the Panelreport should be received by June 25,
2007.

sroberts on PROD1PC70 with NOTICESADDRESSES: Comments should

preferably be submitted electronically
via the NICEATM/ICCVAM Web site:
http://iccvam.niehs.nih.gov/contact/
FR_pubcomment.htm. Comments canalso be submitted by e-mail toniceatm@niehs.nih.gov. Writtencomments can be sent by mail or fax toDr. William S. Stokes, NICEATM
Director, NIH/NIEHS, P.O. Box 12233,
MD EC–17, Research Triangle Park, NC
27709, (fax) 919–541–0947, (e-mail)niceatm@niehs.nih.gov. Courier address:
NICEATM, 79 T.W. Alexander Drive,
Building 4401, Room 3128, Research
Triangle Park, NC 27709.
FOR FURTHER INFORMATION CONTACT:Other correspondence should bedirected to Dr. William S. Stokes,
NICEATM Director (919–541–2384 or
niceatm@niehs.nih.gov).

Background
The European Centre for the
Validation of Alternative Methods(ECVAM) submitted five in vitro
pyrogen test methods to ICCVAM forevaluation in 2006. The proposed test
methods include:
1. The Human Whole Blood(WB)/IL–1 In Vitro Pyrogen Test:
Application of Cryopreserved Human
WB
2. The Monocytoid Cell Line Mono
Mac 6 (MM6)/IL–6 In Vitro Pyrogen Test
3. The Human PBMC/IL–6 In Vitro
Pyrogen Test
4. The Human WB/IL–1 In Vitro
Pyrogen Test
5. The Human WB/IL–6 In Vitro
Pyrogen Test
These test methods are based on themeasurement of proinflammatory
cytokines released from either fresh or
cryopreserved human blood cells or ahuman monocytoid line in response to
the presence of Gram-negative
endotoxin in parenteralpharmaceuticals. NICEATM andICCVAM prepared a comprehensive
background review document (BRD)
that included the available data for thefive test methods and a separate
document containing ICCVAM test
method recommendations. At the peer
review meeting, the Panel reviewed the
BRD and evaluated the extent to which
the ICCVAM criteria for validation andacceptance had been adequately
addressed for the intended purpose of
these test methods. The Panel also
provided comments on the ICCVAM
draft test method recommendations
regarding the proposed usefulness andlimitations, standardized protocols,
performance standards, and futurestudies. The Panel’s conclusions andrecommendations on the five in vitro
pyrogen test methods are described in
the Peer Review Panel Final Report: Five
In Vitro Pyrogen Test Methods (available
at: http://iccvam.niehs.nih.gov/
methods/pyrogen/pyrogen.htm). The
draft BRD and the draft test method
recommendations are available at http://
iccvam.niehs.nih.gov/methods/pyrogen/pyrogen.htm.
Request for Comments
NICEATM invites the submission of
written comments on the Panel’s report.
When submitting written comments
please refer to this Federal Register
notice and include appropriate contact
information (name, affiliation, mailingaddress, phone, fax, email, andsponsoring organization, if applicable).
All comments received by the deadline
listed above will be placed on the

NICEATM/ICCVAM Web site (http://
ntp-apps.niehs.nih.gov/iccvampb/
searchPubCom.cfm) and made available
to ICCVAM. In addition, there will be an
opportunity for oral public commentson the Panel’s report during a meetingof the Scientific Advisory Committee on
Alternative Toxicological Methods(SACATM) scheduled for June 12, 2007.
Information concerning the SACATM
meeting will be published in a separate
Federal Register notice and available on
the SACATM website: (http://
ntp.niehs.nih.gov/go/7441). Any writtencomments on the Panel report received
prior to June 7, 2007, will be distributed
to SACATM.
ICCVAM will consider the Panelreport along with the SACATM andpublic comments as it finalizes
recommendations for the five in vitro
pyrogen test methods. An ICCVAM test
method evaluation report, which
includes the ICCVAM final
recommendations, will be forwarded toappropriate federal agencies for theirconsideration. This report will also be
available to the public on the
NICEATM/ICCVAM Web site and by
request from NICEATM.
Background Information on ICCVAM,NICEATM, and SACATMICCVAM is an interagency committee
composed of representatives from 15
federal regulatory and research agencies
that use, generate, or disseminate
toxicological information. ICCVAM
conducts technical evaluations of new,revised, and alternative methods withregulatory applicability and promotes
scientific validation and regulatory
acceptance of toxicological test methods
that more accurately assess safety andhazards of chemicals and products andthat refine, reduce, and replace animal
use. The ICCVAM Authorization Act of2000 (42 U.S.C. 285l–3, available at
http://iccvam.niehs.nih.gov/docs/
about_docs/PL106545.pdf) establishes
ICCVAM as a permanent interagency
committee of the NIEHS under
NICEATM. NICEATM administers
ICCVAM and provides scientific andoperational support for ICCVAM-related
activities. NICEATM and ICCVAM work
collaboratively to evaluate new andimproved test methods applicable to theneeds of Federal agencies. Additional
information about ICCVAM and
NICEATM can be found at the ICCVAM/NICEATM Web site (http://
iccvam.niehs.nih.gov).
Additional information about
SACATM, including the charter, roster,and records of past meetings, can befound at http://ntp.niehs.nih.gov/go/
167.

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Federal Register / Vol. 72, No. 89 / Wednesday, May 9, 2007 / Notices

Dated: April 30, 2007.
Samuel H. Wilson,
Deputy Director, National Institute ofEnvironmental Health Sciences and National
Toxicology Program.
[FR Doc. E7–8896 Filed 5–8–07; 8:45 am]

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May 2008

Appendix D2
Public Comments Received in Response to Federal Register Notices
Comments in Response to Federal Register, Vol. 70, No. 241, pp. 74833-74834,
December 16, 2005: Peer Panel Evaluation of In Vitro Pyrogenicity Testing
Methods: Request for Comments, Nominations of Experts, and Submission of
In Vivo and In Vitro Data
1. Dr. Pilar Vindarell (Facultat de Farmacia, Barcelona, Spain) ....................D-13
2. Sadhana Dhruvakumar (People for the Ethical Treatment of Animals[PETA]), Dr. Martin Stephens (Humane Society of the United States[HSUS]), Dr. Chad Sandusky (Physician’s Committee for Responsible
Medicine [PCRM]), Sue Leary (Alternatives Research andDevelopment Foundation [ARDF])...............................................................D-14
Comments in Response to Federal Register, Vol. 71, No. 238, pp. 74533-74534,
December 12, 2006: Announcement of an Independent Scientific Peer Review
Meeting on the Use of In Vitro Pyrogenicity Testing Methods; Request forComments
1. Kristie Stoick and Dr. Chad Sandusky (PCRM), Dr. Martin Stephens(HSUS), Dr. Catherine Willett (PETA), Sue Leary (ARDF),
Tracie Letterman (American Anti-Vivisection Society),
Sara Amundson (Doris Day Animal League)...............................................D-19
2. Dr. Mary Lou Chapek (MVP Laboratories Inc.) .........................................D-24
3. Steven Myers and Anita Sawyer (Becton, Dickinson and Co.) ....................D-27
4. Dr. Erik Wind Hansen and Michael Timm (University of Copenhagen) ....D-29
Comments in Response to Federal Register, Vol. 72, No. 89, pp. 26395-26396,
May 9, 2007: Peer Review Panel Report on Five In Vitro Pyrogen Test Methods:
Availability and Request for Public Comments
1. Kristie Stoick (PCRM) ..................................................................................D-34
2. Dr. Thomas Montag-Lessing and Dr. Ingo Spreitzer(Paul Ehrlich Institute) .................................................................................D-37
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May 2008

ICCVAM Test Method Evaluation Report: Appendix D2
From :
Sent :
To:
Subjec t :May 2008

MA. PILAR VINARDELL MARTINEZ-HIDALGO
Tuesday, December 20, 2005 8:08 AM
NIEHS NICEATM
peer panel pyrogens

Dear Dr Stockes
I am pleased to send you our paper* related to studies of pyrogens invitro. I hope it will be of interest for your work. I have collaborated
before with the het-cam test.
Sincerely yours
Dr. Pilar Vinardell
Dept Fisiologia-Divisio IV
Facultat de Farmacia
Av. Joan XXIII s/n
08028 Barcelona (Spain)*Martinez V, Mitjans M, Vinardell MP. 2004. TNFα Measurement in Rat and Human
Whole Blood as an In Vitro Method to Assay Pyrogens and its Inhibition by
Dexamethasone and Erythromycin. J. Pharm. Sci. 93:2718-2723.

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ICCVAM Test Method Evaluation Report: Appendix D2

May 2008

January 17, 2006
Dr. William Stokes
Director, NICEATM
National Institute of Environmental Health Sciences
P.O. Box 12233, MD EC-17
Research Triangle Park, NC 27709
Via electronic transmission to: niceatm@niehs.nih.gov
Dear Dr. Stokes:
These comments are submitted on behalf of People for the Ethical Treatment of Animals,
Humane Society of the United States, Physicians Committee for Responsible Medicine, and theAlternatives Research & Development Foundation, a coalition of animal protection, alternatives
development, and health advocacy organizations representing more than 10 million Americans in
response to a December 16, 2005 notice in the Federal Register inviting public comment on a
proposed peer review panel evaluation of five human biology-based in vitro pyrogenicity test
methods. We consider these methods to have great potential to replace the existing animal-based
methods and we appreciate the work that has gone into the development of the backgroundreview documents (BRDs) by the European Center for the Validation of Alternative Methods(ECVAM) and into their preliminary review by Interagency Coordinating Committee on the
Validation of Alternative Methods (ICCVAM).
We believe that an international peer review of these novel pyrogenicity test methods isappropriate, necessary, and should be given extremely high priority. A thorough yet expeditious
review of these tests by an expert panel resulting in the endorsement of at least one proposed test
method should be viewed as a potential quick win in the efforts of ICCVAM to meet its statutory
mandate to promote the replacement, reduction, or refinement of animal-based testing (42 U.S.C.
Sec. 2851-3(b)).
Need for Speedy Review of Novel Pyrogenicity Tests
Pyrogenicity testing is most commonly used to ensure that medical treatments (particularly
injectable medicines or implanted devices) are free of fever-inducing contaminants. Currently
used methods of animal-based pyrogenicity testing have significant scientific and practicallimitations (described below). Human-biology based pyrogenicity tests are more sensitive, moreconsistent, and more versatile, but most importantly, more accurate. Since they are based onhuman immune system responses, they represent the most relevant and best possible means ofpredicting human pyrogenic potential. Swift validation of the proposed in vitro tests andreplacement of animal-based pyrogenicity tests is necessary to best safeguard consumer safety.
Use of the five novel test methods will also better protect the public because they enable testingthat was not previously possible due to the limitations of the animal tests. For example, they
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May 2008

Dr. William Stokes
January 17, 2005
Page 2enable direct testing of air filters in buildings so that airborne pyrogens can be detected andeliminated; they enable direct testing for pyrogens bound to the surfaces of medical devices,previously not possible; and they enable the testing of cell culture media in order to guarantee its
pyrogen-free status. The existing animal-based testing methods are inadequate for testing many
important upcoming areas of therapeutics (especially cellular products) which can be tested
using the novel human biology-based methods (Hartung et al. ATLA 29, 99-123; 2001).
The proposed methods are already in use by over 200 laboratories around the world (EU press
release 12/5/03 Reference: IP/03/662) and interest from industry is quite high, thus it isimperative that US federal agencies issue a stance on the validity of these methods. Of themethods under consideration, those utilizing human whole blood (fresh or cryopreserved) andmeasuring the production of Interleukin-1 are particularly advanced (Methods #2 & 3 in Federal
Register notice). These methods have been commercialized as test kits that produce results
within a day by the European company Milenia as “PyroCheck” and in the US by Charles River
Labs as “Endosafe-IPT.” Customers are already using these test kits but cannot stop using theanimal-based tests until they know that Agencies will accept their results.
The EU, primarily through ECVAM, has invested considerable resources into the development
and international validation of the five submitted in vitro test methods with the involvement ofover 60 groups from academia, industry, and regulatory bodies. Descriptions of this work havebeen published in numerous scientific journals. The European Pharmacopeia has installed aninternational expert group to draft a General Method for these tests and we understand that the
ECVAM Scientific Advisory Committee (ESAC) will shortly review the methods and make astatement on their validity. Whereas the vast majority of novel non-animal test methods arevalidated in the EU years prior to consideration in the US, this submission represents an exciting
first opportunity for the US to concurrently evaluate a test method in parallel with the EU. This
adds to the imperative that a panel is convened and a review is conducted in a timely manner.
Limitations of Currently Used Pyrogenicity Tests
The considerable limitations of the existing animal-based pyrogenicity tests create anotherimportant imperative. The rabbit pyrogenicity test, developed in the 1940s, still consumes anestimated 400,000 rabbits per year (Hartung et al. ATLA 30, 49-51; 2002). Animals are locked infull-body restraints while their temperature is monitored through rectal probes and suffer effects
which can include fever, breathing problems, organ failure, and fatal shock. Like all animalbased tests, the rabbit pyrogenicity test is time-consuming, costly, and gives results that are
species-specific: The potency of pyrogens varies by up to 10,000 in different mammals((Hartung et al. ALTEX 15, 17-18; 1998). However, the rabbit test is scientifically problematic inmany additional ways. Even at the highest injected volumes, the detection limit of the rabbit test
is above the human fever threshold: humans show a fever response at concentrations as low as
30pg LPS/ml while rabbits’ sensitivity varies between 50 and 350 pg LPS/ml. In contrast, thehuman whole blood IL-1 test has a sensitivity of 10pg LPS/ml (Hartung et al. ATLA 29, 99-123;
2001). In addition, the sensitivity of the rabbit test varies depending on the strain, age and genderof rabbit used. Other important problems include the fact that the rabbit test often only gives a
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Dr. William Stokes
January 17, 2005
Page 3

pass/fail, rather than a quantitative, answer; that results are influenced by animal distress as well
as seasonal variation; and that inconclusive results necessitating test repetition are common.
Lastly, the rabbit pyrogenicity test does not work for many classes of substances including
important new therapies such as cellular products or species-specific agents, as well as
chemotherapeutics, radiopharmaceuticals, certain biologicals and antibiotics, drugs that cause
immune reactions, drugs that influence body temperature such as
sedatives/analgesics/anesthetics, and vitamins.
The in vitro Limulus Amoebocyte Lysate (LAL) assay, also known as the bacterial endotoxin
test or BET, was developed in the 1970s and has largely replaced the rabbit test where possible,
but it has severe limitations as well. The most important limitation of the LAL assay is that it
only detects endotoxins (components of gram-negative bacteria) but not other pyrogens
including gram-positive bacteria, fungi, and viruses. Thus, the LAL assay is used extensively forpharmaceutical testing and for in-process monitoring in biological production but is not suitable
as a final release test for complex biologically-derived products that may contain non-endotoxin
pyrogens, for material-mediated pyrogenicity, or for substances that chemically or physicallyinterfere with the clotting reaction in the LAL test such as proteins or lipids. It cannot be used forthe testing of biological products such as vaccines, immunoglobulins, and clotting factors. In
addition, the accuracy of the LAL test for predicting human pyrogens and their potencies isquestionable since it is based on the defense system of an arthropod (the coagulation ofhorseshoe crabs’ blood) which is not mechanistically relevant to the human response (Hartung etal. ALTEX 15, 9-10; 1998). It is also important to note that the blood used in the LAL assay is
obtained by harvesting crabs from the ocean floor and draining ~30% of their blood, which cancause them injury, disrupts their natural life cycles, and depletes their populations, which may
make availability of their blood more limited in the future. For ethical and welfare reasons, thistest should be replaced as soon as possible.
The rabbit assay is a poor and inadequate test in numerous ways, but the limitations of the LAL
assay have led to its continued use. For decades, these tests have been used complementary, but
in fact, they are simply limited in different ways and their combined use leaves many gaps inconsumer protection and much to be desired. In addition, the two animal-based tests are difficult
to correlate with each other. Since the proposed human biology-based tests can detect nonendotoxin pyrogens, they should at the very least completely replace the outdated rabbit
pyrogenicity test in final release testing. However, the human biology-based test should also
replace the LAL test which should not be conducted if a more humane and relevant humanbiology-based in vitro test is available, which will clearly better safeguard human health.
Human Biology-Based Pyrogenicity Tests
Our understanding of human immunology has advanced rapidly over the last 20 years, and thisrepresents the first opportunity to reflect this in our methods of testing for pyrogenicity. The firstinterleukins were cloned in 1984, leading to an understanding of the mechanism of pyrogenicity:
When an “exogenous pyrogen” enters the bloodstream, cells of the immune system produce“endogenous pyrogens” (interleukins) that signal the brain to generate a fever. The first human
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Dr. William Stokes
January 17, 2005
Page 4

blood-based in vitro pyrogenicity tests were developed over a decade ago (Hartung & Wendel
ALTEX 12, 70-75; 1995), based on measuring the production of interleukins in response to thetest substance. Such methods are physiologically and mechanistically relevant and thus arecapable of detecting all classes of human pyrogens (though there are a few limitations, such as
testing for contamination of drugs that interact with immune cells, however this limitation also
applies to the rabbit test).
Human biology-based pyrogenicity tests have since undergone extensive development andevaluation. The five tests proposed for consideration vary in their use of human whole blood(fresh or cryopreserved), cells isolated from blood, or immune cell lines, and in the interleukin
response they measure, but otherwise work on the same principle. It will be up to the panel todecide whether all of these test methods accurately model the pyrogenic response with thenecessary accuracy and sensitivity, and whether it varies by application.
The proposed human biology-based tests have almost every advantage over the existing animalbased tests: They are more biologically relevant, more reproducible, and more broadly applicablethan the animal-based alternatives. They are speedier, more cost-effective, less laborious, andmore humane. They are very sensitive; as mentioned above, the whole blood IL-1 test has beenshown to have a sensitivity of 10pg LPS/ml, far below the human fever threshold. (As previously
discussed, the rabbit test is far less sensitive and consistent, and neither the rabbit or LAL tests
have ever been formally validated to demonstrate either intra- and inter-laboratory
reproducibility, much less their relevance to human beings. Thus, when the expert panel
considers the proposed novel methods, it is especially important to avoid the common pitfall ofusing the animal data as the “gold standard” in assessing false positive and negative rates.)
In conclusion, the submitted BRDs represent an ideal opportunity to conduct an expeditious
review of well-validated non-animal methods and fully replace outdated animal tests withmodern, improved alternatives as per ICCVAM’s mandate. ICCVAM’s endorsement will be key
in encouraging US government agencies and industry to develop the necessary confidence inthese innovative methods. Led by the FDA, Agencies should require these tests as the newstandards in place of the animal tests, for which there will be no adequate rationale for continued
use. The rabbit test in particular should be deleted from pharmacopeias and regulatory guidance
and not accepted by Agencies once the new tests are validated.
With all this in mind, we strongly urge ICCVAM to move ahead quickly to convene a panel ofexperts who can make the necessary scientific judgments regarding the proposed tests with aview towards a speedy affirmation of their respective values in assessing pyrogenic potential.
Consumer safety, scientific rigor, and animal welfare concerns will all be best served bypromoting the use of these accurate, sensitive, and humane tests.

Thank you for your attention and responsiveness to these comments.
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ICCVAM Test Method Evaluation Report: Appendix D2

May 2008

Dr. William Stokes
January 17, 2005
Page 5Sincerely,

Sadhana Dhruvakumar
Director, Medical Testing Issues
People for the Ethical Treatment of Animals

Martin L. Stephens, Ph.D.
Vice President, Animal Research Issues
Humane Society of the United States

Chad B. Sandusky, Ph.D.
Director of Toxicology and ResearchPhysicians Committee for Responsible Medicine

Sue Leary
President
Alternatives Research & Development Foundation
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ICCVAM Test Method Evaluation Report: Appendix D2

May 2008

January 26, 2007
Via email to: niceatm@niehs.nih.gov
Dr. William Stokes
Director, NICEATM
National Institute of Environmental Health Sciences
P.O. Box 12233, MD EC-17
Research Triangle Park, NC 27709
Re:

Federal Register Vol. 70, No. 238, pp 74533-4, December 12, 2006: NTP Interagency Center for
the Evaluation of Alternative Toxicological Methods; Announcement of an Independent ScientificPeer Review Meeting on the Use of In Vitro Pyrogenicity Testing Methods; Request for Comments

Dear Dr. Stokes:
These comments are submitted on behalf of the more than 10 million U.S. members of the PhysiciansCommittee for Responsible Medicine, People for the Ethical Treatment of Animals, the Humane Society of
the United States, the Alternatives Research & Development Foundation, the American Anti-Vivisection
Society, and the Doris Day Animal League. We appreciate the opportunity to review ICCVAM’s
recommendations for five in vitro pyrogenicity tests (IVPTs) conducted using either human whole bloodor human monocytic cell lines, and to provide comments regarding ICCVAM’s “Draft Test Method
Recommendations” (Recommendations) and “Draft Background Review Document” (BRD) on thesemethods. These comments incorporate by reference an earlier submission dated January 17, 2006.
At the outset, it should be stated that the parties to this submission have always endeavored to regard
ICCVAM and its member agencies as federal partners who share our commitment to reducing, refining,
and ultimately replacing the use of animals in regulatory toxicology. However, the abbreviated number ofmethods reviewed by ICCVAM and accepted by federal agencies in recent years raises concern over the
genuine commitment to progress in the 3Rs by some federal agencies and/or their representatives on
ICCVAM. The pyrogenicity BRD and Recommendations currently under discussion represent a glaring
case in point.
ICCVAM’s Recommendations accept the use of IVPTs only for the detection of lipopolysaccharidemediated (LPS) pyrogenicity induced by gram-negative bacterial endotoxins “in materials currently tested
in the RPT” (rabbit pyrogen test). Thus, for practical purposes, ICCVAM’s Recommendations do notsupport the use or regulatory acceptance of these methods for the detection of gram-positive bacterial,
fungal, or viral pyrogens. Moreover, ICCVAM specifically states that it does not regard the IVPTs as full
replacements for the Limulus amebocyte lysate (LAL). Its Recommendations further state that in order tobe considered as potential replacements for the RPT for the detection of non-LPS-mediated pyrogenicity,“additional studies that include a broader range of pyrogenic materials are recommended…such studies
should include parallel RPT testing.” More specifically, “when a positive non-endotoxin-mediated RPT
result is encountered, this same sample should be subsequently tested in vitro.”1
Despite the extensive discussion of the 3Rs throughout the BRD and Recommendations, it is not clear if
or how ICCVAM’s Recommendations could contribute to a meaningful reduction in animal use in

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pyrogenicity testing if in fact we are not looking to replace the BET and continued comparisons to—and
confirmatory testing in—the RPT are required for these methods.
We therefore strongly urge ICCVAM to significantly revise its Recommendations and BRD tomore accurately reflect the potential use of these methods as full replacements for both the
LAL and RPT. The available evidence shows that the IVPTs are fully valid for the detection of
all pyrogens. We also strongly encourage ICCVAM to delete the recommendation regarding
the conduct of de novo RPTs to further demonstrate in vivo/in vitro concordance.
General Comments
There are a number of disadvantages to current pyrogen-detection methods. These have been discussed
previously, but necessitate a brief mention. The RPT exposes live rabbits to painful or distressing
experiences; requires trans-species extrapolation; is less sensitive than the human fever threshold;2 andis ill equipped to handle substances such as cellular products, radiopharmaceuticals, certain biologicals,
and medical devices. The LAL also requires species extrapolation, can only detect LPS, and cannot be
used for substances that interfere with the clotting process, biologicals ,or the direct assessment of
medical devices.
Despite references to the 3Rs, the RPT is still used extensively, especially for complex biologically derived
products and end-product release testing. Indeed, it is estimated that up to 400,000 rabbits per year areused,3 and the LAL, despite catch-and-release procedures, results in an approximate 15% mortality rate.4
It is therefore imperative, for both ethical and scientific reasons, that both of these tests are replaced by
the alternatives presented here for endorsement.
In addition to the obvious ethical advantages of human whole and/or cellular blood pyrogenicity tests,
the IVPTs have numerous scientific advantages. The first is the elimination of species extrapolation
issues, since the proposed test methods are direct in vitro models of the human fever response.
Additionally, because the pyrogenic response is a blood-mediated reaction, IVPTs are not limited bypotential in vivo/in vitro extrapolation considerations, as some in vitro tests might be. The IVPTs are
sensitive and can detect all potential pyrogens, not only LPS. They can be used to evaluate traditional
pharmaceuticals as well as medical devices, species-specific cellular/biological therapies, cell culture
media, air quality assessments, and human serum albumin, among other materials. The IVPTs could also
be easily adapted into species-specific pyrogenicity tests for veterinary products.
The methods presented to the panel have undergone a full quantitative validation study. The validation
studies were conducted in order to certify the IVPTs as appropriate for replacement of both the RPT and
the LAL. The concordances and sensitivities for all five human blood-based methods are over 90%;
specificities are above 80%; and all methods demonstrate low false-positive and -negative rates.5 In
comparison, historical data from 171 rabbits were used to calculate a theoretical sensitivity of 57.9% anda theoretical specificity of 88.3% for the RPT.4
Clearly, the IVPT methods, after 20 years of research and refinement, are a wholly superior way to detect
pyrogens in medicinal products. However, the animal protection community has serious concerns related
to the duplication of review efforts, as evidenced by the time ICCVAM has taken to arrive at this pointwith the IVPTs. As discussed in another recent set of public comments, ICCVAM continues to invest
substantial time and resources in what are regarded by many as redundant and unnecessarily duplicativeevaluations of 3Rs methods that have already undergone successful validation, independent peer review,
and/or international acceptance in other jurisdictions. We therefore question the value of subjecting the
IVPTs to multiple peer reviews––particularly when the animal-based RPT and LAL have not been subject
to a level of scrutiny even closely resembling that of an ECVAM or ICCVAM validation study.
Specific Recommendations

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ICCVAM Test Method Evaluation Report: Appendix D2

May 2008

Accept IVPTs as full replacements for the LAL
It is unclear why ICCVAM has chosen not to consider the IVPTs as appropriate for replacement of both
the RPT and the LAL. With the validation of the IVPTs using an endotoxin standard, the LAL has become
redundant. If there are specific cases of which we are not aware that require the LAL, exceptions can bemade, but surely for ethical and scientific reasons the IVPTs should in general replace the LAL.

Certify the IVPTs valid for the detection of all pyrogens; conduct a “retrospective validation,” if needed.
The mechanism of action behind the detection of LPS in the LAL, and hence the reason for its pyrogenspecificity, is unique to arthropods. The mechanism of action, if not the magnitude of response, behindthe detection of pyrogens in the RPT and the IVPTs is the same. Since the RPT is currently used to detect
all pyrogens, there is no biologically sound rationale to conclude that the IVPTs cannot also detect all
pyrogens—at a level at least equivalent to the RPT. ICCVAM documents drafted for review today state asmuch.
Indeed, BRDs submitted by ECVAM, draft BRDs posted by ICCVAM, and other materials list between 15
and 30 published studies discussing the detection of pyrogens, including non-LPS pyrogens, in humanserum albumin, pharmaceuticals, and other materials. Some studies used clinically positive materials, andsome used comparisons to the traditional in vivo or an in vitro version of the RPT.6-8 One of thesestudies6 compared the WB/IL-1 IVPT and the RPT using 96 batches of parenteral pharmaceuticals. Of all
test substances, only one tested positive in all three (RPT, LAL, and WB/IL-1) test systems. The
remaining 95 were negative in all test systems. ECVAM has also provided detailed testing results ofmaterials with the IVPT methods that were determined to be positive for pyrogenic activity during clinicalexperience. Results were favorable in all assessments.4
It is at best perplexing to see peer review reports and testing recommendations stop short of giving the
IVPT methods full validated certification, and only recommend the use of these methods for the detectionof LPS-mediated pyrogenicity. While most pyrogenicity is indeed related to LPS, the ICCVAM draft
recommended test method uses and future studies virtually guarantee that the RPT will not be replacedin the foreseeable future, as it will be needed to certify regulated end products completely “pyrogenfree.”
Given the results of Jahnke6 above, it is further difficult to envision the concurrent in vivo/in vitro study
recommended by ICCVAM. Hundreds of rabbits could be used in an unnecessary quest to get enough
non-LPS-mediated pyrogenicity reactions in rabbits to subsequently confirm using the IVPT methods.
For ethical reasons, the ECVAM validation did not include such concurrent testing. Instead, the study
chose LPS, a model pyrogen, to represent the pyrogen reaction and validate the in vitro test systems.
There is no scientific reason to suspect that the IVPTs will not detect the full range of pyrogens.
Published evidence supports this hypothesis,7-10 as does supporting evidence submitted by ECVAM in
early 2006.4 If necessary, a coordinated assessment of such evidence—a retrospective validation ofsorts—should more than allay any concerns about the applicability of the IVPTs to all varieties ofpyrogen.

Articulate more clearly a path to full replacement
Investments in IVPTs by industry and the public sector are increasing. At least one American company,
Charles River Laboratories, has for some time offered an IVPT assay for use in the detection of the rangeof pyrogens for research use. At least 200 laboratories worldwide have worked with or offer similar
assays. Faith in the continued growth of these methods is clearly held by industry, academia, andgovernment alike. With approval and continued use, we are confident that the IVPT methods will become

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May 2008

the “Gold Standard” for human pyrogen detection. The ICCVAM recommendations as currently written
will limit the usefulness of these assays, and fail to achieve real reductions in animal use in a timely
manner. We urge ICCVAM to revise its Recommendations as outlined above—and offer detailed guidanceon how prospective end-users can adopt the IVPTs and put them into immediate practice.
Thank you for your attention to these comments.
Kristie Stoick, MPH
Chad Sandusky, PhD
Physicians Committee for Responsible Medicine
Martin Stephens, PhD
The Humane Society of the United States
Catherine Willett, PhD
People for the Ethical Treatment of Animals
Sue Leary
Alternatives Research & Development Foundation
Tracie Letterman, Esq
American Anti-Vivisection Society
Sara Amundson
Doris Day Animal League
References
1. National Toxicology Program NECEATM. 1 Dec 2006. Draft ICCVAM TEST METHODSRECOMMENDATIONS: in vitro pyrogenicity test methods. Accessed at
http://iccvam.niehs.nih.gov/methods/pyrodocs/pyroBRDU.htm on January 23, 2007.
2. Hartung T, Aaberge I, Berthold S, Carlin G, Charton E, Coecke S, Fennrich S, Fischer M, Grommer
M, Halder M, Haslov K, Jahnke M, Montag-Lessing T, Poole St, Schectman L, Wendel A, WernerFelmayer G. 2001. Novel pyrogen tests based on the human fever reaction. ATLA 29:99-123.
3. Hartung T and the Human(e) Pyrogen Test Study Group. 2002. Comparison and validation ofnovel pyrogen tests based on the human fever reaction. Altern Lab Anim. 30 Suppl 2:49-51.
4. European Commission: ECVAM. 24 Mar 2006. Response to the ICCVAM-PWG Questions for
ECVAM regarding the five in vitro pyrogenicity test method submissions. Accessed at
http://iccvam.niehs.nih.gov/methods/pyrodocs/pyroBRDU.htm on January 23, 2007.
5. National Toxicology Program NICEATM. 1 Dec 2006. Draft BACKGROUND REVIEW DOCUMENT:
Current status of five in vitro pyrogenicity test methods. Accessed at
http://iccvam.niehs.nih.gov/methods/pyrodocs/pyroBRDU.htm on January 23, 2007.
6. Jahnke M, Weignad, Sonntag H-G. 2000. Comparative testing for pyrogens in parenteral drugs
using the human whole blood pyrogen test, the rabbit in vivo pyrogen test and the LAL test.
European J Parent Sci 5(2):39-44.

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7. Nakagawa Y, Maeda H, Murai T. 2002. Evaluation of the in vitro pyrogen test system based on
proinflammatory cytokine release from human monocytes: Comparison with a human whole
blood culture test system and with the rabbit pyrogen test. Clinical and Diagnostic Laboratory
Immunology 9(3):588-597.
8. Schindler S, Bristow A, Cartmell T, Hartung T, Fennrich S. 2003. Comparison of the reactivity ofhuman and rabbit blood towards pyrogenic stimuli. ALTEX 20:59-63.
9. Schindler S, Spreitzer I, Löschner B, Hoffmann S, Hennes K, Halder M, Brügger P, Frey E,
Hartung T, Montag T. 2006. International validation of pyrogen tests based on cryopreserved
human primary blood cells. J Immuno Methods 316:42-51.
10. Pool E, Johaar G, James S, Peterson I, Bouic P. 1999. Differentiation between endotoxin and nonendotoxin pyrogens in human albumin solutions using an ex vivo whole blood culture assay. J
Immunoassay 20(1&2):79-89.

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February 5, 2007
Via email to: niceatm@niehs.nih.gov

Dr. William Stokes
Director, NICEATM
National Institute of Environmental Health Sciences
P.O. Box 12233, MD EC-17
Research Triangle Park, NC 27709
Re:

Federal Register Vol. 70, No. 238, pp 74533-4, December 12, 2006: NTP
Interagency Center for the Evaluation of Alternative Toxicological Methods;
Announcement of an Independent Scientific Peer Review Meeting on the Use of
In Vitro Pyrogenicity Testing Methods; Request for Comments

Dear Dr. Stokes:
I have taken the opportunity to review ICCVAM’s recommendations for five in vitro
pyrogenicity tests (IVPTs) and to provide comments regarding ICCVAM’s “Draft Test
Method Recommendations” (Recommendations) and “Draft Background Review
Document” (BRD) on these methods.
I have always regarded ICCVAM and its member agencies as federal partners who share
my commitment to the 3 R’s, reducing, refining, and ultimately replacing the use ofanimals in regulatory testing. I have been greatly disappointed at the minimal number ofmethods reviewed by ICCVAM and accepted by federal agencies over the past 15 years
and would like to see progress in this area, not just stagnation. The pyrogenicity BRD
and Recommendations currently under discussion indicate to me that there is a lack oflogical focus. I propose a two phase approach whereby ICCVAM can demonstrate
success.
The summaries and data provided in the BRD indicate that the five proposed in vitro
pyrogenicity tests are only being evaluated and validated for their ability to measure the

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pyrogenic response produced by endotoxin. Even then, only a few pharmaceuticalproducts were tested by spiking with known amounts of endotoxin. Replacing the RPT
fully with the in vitro pyrogenicity tests is a noble and worthwhile project. I support it
fully. However, the testing still to be conducted is extraordinary. Numerous types of
products need to be evaluated (some of which have been reported by ECVAM) and nonendotoxin pyrogens must be tested. I would strongly suggest that the ICCVAM proceedwith a phased project in order to demonstrate that something can be accomplished rather
quickly and animals’ lives can be saved.
I propose that Phase I would concentrate on replacing the BET with one or more of the invitro pyrogenicity tests, a task that appears less daunting than replacement of the RPT.
The Phase I testing is important because use of the in vitro pyrogenicity tests instead of
the BET would eliminate the need for horseshoe crabs to die during or after the processof removing the hemolymph. Additionally, the in vitro pyrogenicity tests use human
components instead of non-human horseshoe crab hemolymph that could be argued to beless relevant to the human fever response.
There is already a significant amount of work reported in the BRD indicating that the fivein vitro pyrogenicity tests can detect endotoxin pyrogens with accuracy and sensitivity.
Thus, Phase I would only require validation against the BET for those products that cancurrently be tested in the BET.
It appears from the ECVAM information that the in vitro pyrogenicity tests can actually
test more varied products since there is no interference with these test systems. Such lackof interference could also be demonstrated during Phase I by spiking an array of test
products with known endotoxin levels and demonstrating accuracy, specificity and lackof interference.
As for Phase II, I would strongly suggest that the ICCVAM select one or two of the invitro tests based on the results obtained so far, and use them in validation studies against
the RPT in order to replace that test completely. The reason for selecting only one or twoof the in vitro tests is based on the fact that three of the five proposed in vitro
pyrogenicity tests require fresh human blood that must be collected within 4 hours of
running the test. In today’s world, such a task is difficult to say the least. The cell
culture assay appears much more adaptable to ease of use. That would certainly be oneof my choices.
Phase II would still be complex, as now the focus would be on total replacement of the
RPT with one or two of the in vitro pyrogenicity tests. However, evaluation and initial
validation of one or two tests is less of a challenge than trying to evaluate and validate
five tests.
Phase II evaluation would require evaluation and validation of all materials currently
tested in the RTP as well as all of the types of pyrogens currently quantified in the RPT.

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Because standards are not available for all of the types of pyrogens, such standards would
have to be developed. Another possibility would be to find products that failed the RPT
and use those for validation purposes (less difficult but less scientific). As you alreadyknow, this could require years. At least, if Phase I was complete, there could be ademonstration that ICCVAM had accomplished some of its goal of replacement ofanimal tests with in vitro tests.
I hope that ICCVAM will consider my recommendations.
Best Regards,

Mary Lou Chapek, President and CEO
MVP Laboratories, Inc.

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March 12, 2007
Via e-mail to: niceatm@niehs.nih.gov
Dr. William Stokes
Director, NICETAM
National Institute of Environmental Health Sciences
PO Box 12233, MD ED-17
Research Triangle Park, NC 27709
Dear Dr. Stokes:
These comments are intended to be a follow-up to the recent NICETAM-sponsored Independent ScientificPeer Review: Five In Vitro Test Methods Proposed for Assessing Potential Pyrogenicity of Pharmaceuticals
and Other Products (February 6, 2007). They are supported by the larger Animal protection community,
including the more than 10 million members of the Physicians Committee for Responsible Medicine,
People for the Ethical Treatment of Animals, the Humane Society of the United States, and the Doris Day
Animal League.
Based on information communicated to me at this meeting, I understand that these additional comments
are accepted because of limited time available during my oral public comment. I appreciate theopportunity to submit these additional comments and I urge NICETAM to take them into account when
considering the Peer Review Panel’s (PRP) recommendations and conclusions.
Panel Recommendations
As you know, the ICCVAM recommendations were written with the intention that the in vitro pyrogenicity
tests (IVPTs) would replace a small subset of rabbits used in the rabbit pyrogen test (RPT), but not theLimulus amebocyte lysate (LAL), nor rabbits used for non-endotoxin-mediated pyrogenicity testing. As
you know, the PRP did not even agree with these limited recommendations. The PRP did, however, makeseveral of their own recommendations. I was pleased to hear of some of them and hope that ICCVAM willconsider putting them into place as quickly as possible (abridged below):
1. Human data on pyrogens is extensive and should be analyzed, presented, and consulted.
2. More discussions on the financial and ethical costs (including monetary values and animal
numbers) associated with the RPT are needed.
3. Individual product-specific validation studies are required and may negate the need for a large
validation study.
However, several of the panel’s observations and recommendations seemed nonsensical, irrelevant, or
inappropriate (abridged below):
1. The methods should not be called “in vitro pyrogen tests” because only bacterial endotoxin was
evaluated.
2. The in vivo reference data is not adequate and/or of unknown quality.
3. The calculated “theoretical sensitivity” of the RPT data used in the validation study does notreflect current practice and regulatory use.
4. The IVPT validation data should be quantitative

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5. Concordance between the IVPTs and the RPT is not demonstrated.
Considerations as to the realities of a validation study are clearly not recognized by the PRP, as reflected
in its deliberations and recommendations. The validation study conducted by ECVAM used scientificallyjustified in vivo reference data, a scientifically-justified method to calculate the theoretical sensitivity of
the RPT data (as admitted by the panel), and generated data with theoretical concordance values
presented. Indeed, the RPT itself does not give quantitative data, but a decision of “pyrogenic” or “notpyrogenic,” while the in vitro methods do have this potential.
What went wrong?
Panel Selection: This meeting reflects a growing concern of the animal protection community thatICCVAM is more interested in picking new non-animal methods apart than in seriously considering themfor adoption. Accepted peer review process guidelines state: “Peer reviewers should include individuals
who will not be affected by the outcome of the results, but who are well-versed in the relevant
experimental techniques and the specific method under review.”1 However, many of the panel members
were either demonstrably biased against the IVPTs, silent, or ignorant of the validation and acceptanceprocedures, the PRP’s role, or the ICCVAM process. Too often it seems that panelists have unreasonable
expectations regarding every minute detail of the alternative methods, without a clear understanding of
the limitations of the current animal-based tests. This was especially true in this meeting. Random
selection of panel members from the scientific topic of interest biases every single panel towards the null
hypothesis, leaving an unreasonably high barrier over which the new alternative methods cannot cross.
Charge/Question Wording: It was clear from the deliberation among the PRP that the panel members had
no clear idea of their task, and were unnecessarily confused by the questions posed to them by ICCVAM.
One question elicited an hour’s debate over what the question actually meant. Simplification of thequestions posed to the panel, as well as a pre-meeting orientation, is in order. For example, there wasclearly little or no background information provided on the limitations of the animal tests. An orientation
process could also help the panelists stay focused. The panel deviated too often from the task at hand
into both broad and detailed scientific questions that had no bearing on the validation of the IVPTs. For
example, one panel recommendation stated that an explanation should be given as to why in vitro
responses are a better reflection of in vivo human responses than in vivo rabbit responses. Whilebiological relevance is important, it has already been demonstrated; this recommendation has no bearing
on the validation status of the assays as presented, whether it is true or not.
Validation Study Considerations
Despite public testimony given at the time of the meeting, the PRP did not take the realities of validation
studies, nor this particular one, into account. First, the validation study selected a small set ofpharmaceuticals and spiked them with endotoxin, because endotoxin standard is the only standard
available, and the majority of febrile reactions are due to endotoxin. As is often done with animal tests,
practical experience over the past couple of decades led the validation study directors to surmise that themethods would also work with non-endotoxin pyrogens, and with medical devices and blood products.
Pages of data were provided to the PRP to support this conclusion. Given that the methods would require
product-specific validation in the future, and limited resources for the validation study, a large,
complicated validation study was not called for. Further, parallel rabbit testing, for animal welfare
reasons, could not be conducted. So, the study directors designed an approach that would allow the useof historical RPT data of a comparable nature. It was determined that in order to “pass,” the IVPTs would
1

NIEHS (National Institute of Environmental Health Sciences), Validation and Regulatory Acceptance of
Toxicological Test Methods: A Report of the ad hoc Interagency Coordinating Committee on the
Validation of Alternative Methods, NIH Publication No. 97-3981, NIEHS, Research Triangle Park , North Carolina,
U.S.A., 1997, section 2.4.2.6.

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be validated with spiked products at a level of detection comparable with the most sensitive rabbit
species. Even with these “unacceptable” sensitivity and specificity values, the IVPTs still surpassed theperformance of the RPT. Comments to this effect from the study directors themselves were ignored by
the PRP.
The PRP showed no tolerance for adopted tenets of the validation process that call for flexibility: “…thetest validation process should be highly flexible and adapted to the specific test and its proposed use.”2
These procedures do not require direct comparison of in vivo/in vitro methods, and indeed, the BRD andother documentation submitted to the PRP contain all of the Validation Criteria listed in the abovereferenced ICCVAM document.
The Way Forward
I would like to reiterate the animal protection community’s initial comments, sent before the meeting:“We therefore strongly urge ICCVAM to significantly revise its Recommendations andB[ackground] R[eview] D[ocument] to more accurately reflect the potential use of
these methods as full replacements for both the LAL and the RPT. The available
evidence shows that the IVPTs are fully valid for the detection of all pyrogens. We
also strongly encourage ICCVAM to delete the recommendation regarding the
conduct of de novo RPTs to further demonstrate in vivo/in vitro concordance.”3
Our organizations stand by these initial recommendations. However, given the PRP’s final
recommendations, we request that ICCVAM coordinate with the pharmaceutical and medical
devices industry to conduct product-specific validation on a set of pre-selected products anddevices to serve as further validation work. Since this work will need to be conducted anyway, andwould be acceptable to the Food and Drug Administration, this would be an appropriate way forward.
Further delays or de novo validation work would result in the deaths of thousands of additional animals is
not recommended.
Thank you for your attention to these comments. I can be reached at kstoick@pcrm.org or 510.834.8320
with any questions.
Sincerely,

Kristie Stoick, MPH
Physicians Committee for Responsible Medicine

2

NIEHS (National Institute of Environmental Health Sciences), Validation and Regulatory Acceptance of
Toxicological Test Methods: A Report of the ad hoc Interagency Coordinating Committee on the
Validation of Alternative Methods, NIH Publication No. 97-3981, NIEHS, Research Triangle Park , North Carolina,
U.S.A., 1997, sections 2.4.7 and 2.5.
3Letter submitted to NICEATM January 26, 2007.

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Dr. Thomas Montag-Lessing
Dr. Ingo Spreitzer
Paul Ehrlich InstituteLangen, Germany(www.pei.de)Via e-mail to: niceatm@niehs.nih.gov
Dr. William Stokes
Director, NICETAM
National Institute of Environmental Health Sciences
PO Box 12233, MD ED-17
Research Triangle Park, NC 27709

Dear Dr. Stokes:
Please, find below our comments to the “Independent Peer Review Panel Report:
Five In vitro Test Methods Proposal for Assessing Potential Pyrogenicity of
Pharmaceuticals and Other Products”.

Comment to A 1.2.1 Criterion 4 (page 3), also comment to A 4.6 (page 12)
The PRP discussed critically whether the right end-points had been set in thevalidation study regarding sensitivity (respectively detection limits) of the tests. We
are wondering why the PRP did not consider the internationally accepted endotoxin
limits. They are regulated in the respective monographs for endotoxin testing(Bacterial Endotoxin Test, BET) since decades in the international pharmacopoeias.
The endotoxin limit for parenteral drugs intended for intravenous administration is 5
International Units endotoxin (E.U., to calibrate using the WHO Endotoxin Standard
which is identical with the US Endotoxin Standard) per kilogram body weight of thepatient (in the past: administration during one hour period; following the current ICH
document: as a bolus injection). Exactly this endotoxin limit had been used for
calculation of the detection limits in examining the involved drugs. As usual inpyrogen testing, a patient having a body weight of 70 kg (corresponding to a
maximal endotoxin content of 350 E.U. of the whole volume of the given drug) had
been considered for calculation. Furthermore, the WHO Endotoxin Standard had
been used in the study.
Additionally, the sensitivity respectively the detection limit of Rabbit Pyrogen Test
(RPT) had been considered in the study design. The sensitivity of RPT can becalculated considering the fever threshold respectively the threshold of significant
temperature increase of rabbits. The most sensitive rabbit strains show a fever
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threshold of 5 E.U. per kilogram body weight (see papers Hoffmann et al. 2005,
Journal of Immunological Methods, Vol. 298, pp. 161-173, and Hoffmann et al. 2005,
Journal of Endotoxin Research, Vol. 11, pp. 1-7). This endotoxin concentration may
be contained in maximally 10 milliliter which represents the highest allowed burden
for the rabbits following the animal protection lows. In consequence, the sensitivityof RPT is represented by 0.5 E.U. per milliliter (5 E.U. in 10 ml = 0.5 E.U. per ml)
corresponding to 50 pg/ml. This endotoxin concentration had been used for setting
the detection limits of the five In vitro Test methods and it is (at least) fulfilled by all
tests. So the five alternative pyrogen tests meet worst case conditions of RPT andguarantee, therefore, a high safety level for the patients.
It has to be mentioned here that the endotoxin limit regulation mentioned above (5E.U. per kg body weight of the recipient) comes directly from rabbit’s sensitivity.
Preparing the implementation of BET into the pharmacopoeias decades ago, thesafety level of the drugs was the most important criterion. In this time, only datafrom the rabbit were available and, consequently, they were used for definition ofendotoxin limits. This was a wise decision since the fever threshold of human beings
lies in a range of 10 - 20 E.U. per kg body weight and, therefore, the safety of drugs
regarding potential pyrogenicity is guaranteed. Taking into account the backgroundof endotoxin limits, it is surprising when the expert panel used the phrasing“theoretical sensitivity” of the RPT. As demonstrated above, the calculations for thevalidation study reflects exactly the current practice and the regulatory use.
Comment to A 1.2.1 Criterion 5 (page 3)
The PRP stated: “The new test methods clearly take longer to produce definitive
results”. This statement does not consider the mandatory pre-test for RPT which has
to be performed two days prior to the main test employing the same animals (i.e.
RPT lasts all together not less than 48 hours). The in vitro tests are usually
performed within less than 20 hours (i.e. incubation of the cells overnight andmeasuring the cytokine content in ELISA next morning). If necessary, the tests canbe performed within 10 hours by shortening the cell culture to 6 hours.
Comment to A 1.2.2 (page 3)
There is a contradiction in this passage. On the one, hand it is stated: “The RPT(Rabbit Pyrogen Test) detects both endotoxin and non-endotoxin pyrogens, but thein vitro pyrogen tests have not been validated for non-endotoxin pyrogens.
Therefore, they cannot be considered complete replacements for the RPT.” On theother hand, it is stated: “The BET (Bacterial Endotoxin Test) detects endotoxin inmost cases and is used instead of the RPT for this purpose.” It is not understandable
why the in vitro tests, able to detect endotoxin, cannot replace the RPT but BET, able
to detect only endotoxin, can.
There is a clear need for tests able to detect non-endotoxin pyrogens (for examples
regarding adverse reactions caused by non-endotoxin pyrogens, see comment to A
4.0 below, please). The PRP mentioned several times in the report that nonendotoxin pyrogens were not included in the validation study. This holds true but
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there is a broad specter of publications demonstrating that in vitro pyrogen test
methods are able to detect non-endotoxin pyrogens. This includes papers whichapplied the same procedure for pyrogen testing using human whole blood as used in
the validation study (e.g. Hermann et al. European Journal of Immunology, 2002,
Vol. 32, pp. 541-551, and Morath et al., Infection and Immunity, 2002, Vol. 70, pp.
938-944). One would appreciate if the PRP (at least) had mentioned thosepublications.
Comment to A 1.3.2 (page 4)
The PRP stated: “A major concern is the lack of validation of these new assays
directly compared to the RPT.” There were data available on several studies
regarding comparison of RPT and in vitro pyrogen tests as used in the validation
study. The first study (Spreitzer at al., Altex, 2002, Vol. 19, pp. 73-75) concerns acomparative study of Rabbit Pyrogen Test and Human Whole Blood Assay
implementing 29 batches of 10 different Human Serum Albumins from 5
manufacturers. All together, 261 rabbits were included in the study. Two endotoxin
spike concentrations in the range of RPT detection limit were used. There was no
failure in the in vitro pyrogen test. Actually, the in vitro test appeared more sensitive
than the RPT. In the second study (Andrale et al. International Journal of
Pharmaceutics, 2003, Vol. 265, pp. 115-124) a broad range of parenterals (15different drugs) were tested comparing RPT and BET with Human Whole Blood Assay
and, additionally, with Human Peripheral Blood Mononuclear Cell (PBMC) Test. The
two in vitro tests showed good agreement overall, both with each other and with
BET and the RPT. The third study concerns a comparison of six different Coagulation
Factor VIII Concentrates (3 lots each) in RPT and in Human Whole Blood Assay. 162
rabbits were included in the study; two different endotoxin spikes in the range of
RPT detection limit were used. As in the above mentioned albumin study, no failure
was seen in the in vitro assay. Again, the in vitro test appeared more sensitive as the
RPT. The latter study is not published yet but, due to our knowledge, the data had
been provided to the PRP.
Comment to A 3.1 (page 9)
The PRP stated: “No ‘classical’ examples of biological products or medical devices
were included; thus, the validation of either of these categories has not been
provided.”
It should be mentioned that Coagulation Factor VIII concentrate had been includedin the pre-validation study where it was successfully tested. This preparation could
not be considered in main study because of its high price. Additionally, see the above
comment to A 1.3.2, please. Human Serum Albumin and Coagulation Factor VIII
concentrate belong to the ‘classical’ biological products.
Comment to A 3.4 (page 10)
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The PRP stated: “The coding procedures were adequate for the assessment ofrelevance during the validation studies. However, the identity of substances used in
the reproducibility analyses was not blinded (although the spike concentrations
were). A reason was not given.”
It is commonly known that a pharmaceutical company has to perform for any test
the so called product validation in order to exclude potential interferences of the
preparation with the test system (e.g. inhibition of the test by the drug). Of course,
the best approach is to perform the product validation using a clean batch of the
product. The latter procedure had been chosen for the validation study considering
the practice in pharmaceutical industry.
Comment to A 4.0 (page 10)
The PRP stated: “… a summary of reference data demonstrating whether substancesthat were shown to be pyrogenic in humans either passed or failed the RPT, BET, orin vitro tests would have been useful.” There are data published regarding adverse
reactions (fever respectively pro-inflammatory reactions) in patients caused by drugs
which were negative in RPT as well as in BET, but could be tested positive using invitro pyrogen tests. The first event happened with a Tick Borne Encephalitis Vaccine
which induced fever up to cramps and hospitalization in around 50 percent of therecipients. As mentioned above, both RPT and BET remained negative in testing the
product. In contrast, this product produced positive results with blood samples of 50
percent of the donors applying the in vitro pyrogen test (Whole Blood Test, Fischer
et al., Altex, 2001, Vol. 18, pp. 47-49). Another example concerns a dialysis solution
which caused aseptic peritonitis in the patients (Martis et al. Lancet, 2005, Vol. 365,
pp. 588-594). Again, both RPT and BET were negative whereas the in vitro pyrogentest (PBMC Test) could identify the incriminated batches. These two examples
demonstrate that the in vitro pyrogen tests are in certain cases superior to the RPT
since they are working in the ‘homologous system’ (i.e. human indicator cells andfever/pro-inflammatory reactions in humans).
It should be pointed out that the PRP should know the above cited cases as one ofits members was in touch with both of them.
Comment to A 4.3 (page 11)
The PRP stated: “Archived records have not been audited by ECVAM or ICCVAM.”
This statement is wrong as the archived records have been audited by ECVAM in PEI.
Comment to A 4.4 (page 11)
The PRP stated: “However, the PEI did not have formal GLP accreditation (refer to
Section 5.5, ECVAM request for additional information).”
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As the Federal Agency of Sera and Vaccines, the unit for pyrogen and endotoxin
testing of the Paul Ehrlich Institute (PEI) is accredited following ISO 17025 (for thelists of accredited methods in PEI see
http://www.pei.de/cln_049/nn_162948/EN/infos-en/pu-en/11-quality-managementen/accredited-methods-en/akkreditierungen-inhalt-en.html, please).
Despite the above cited ECVAM answer, it should be mentioned here that one of the
PRP members visited the PEI unit for pyrogen and endotoxin testing and knows its
accreditation status.
Comment to A 5.1.1 (page 12)
The PRP stated: “Quality control (QC) testing of cell viability is not performed.
Viability testing of human cells before and after incubation should be performed.”
This statement does not consider how the tests are designed. It is one of theadvantages of the in vitro pyrogen tests that additional testing of cell viability is notnecessary. The functionality of cells is controlled in every test via reaction of
monocytes to endotoxin controls which have to induce a defined minimum ofcytokine concentration. This internal quality control gives more information on thestatus of the monocytes than a viability test; viability test indicates only that the cells
are living whereas functionality test indicates that cells are living and able to react.
Comment to A 10.2 (page 21)
The PRP stated: “The discussion that reduction of the use of animals (i.e., rabbits)
will be associated with the increased use of another animal (i.e., humans) is
inadequate.”
This statement seems to be far away from practice. It is commonly known thatworldwide millions of people are donating blood (for example, more than 5 millions
blood donations per year in Germany). One whole blood donation consists of 500 ml
blood, a volume which would be theoretically sufficed for 5,000 to 50,000 whole
blood pyrogen tests. Therefore, the use of human blood for pyrogen testing would
lead to a marginal increase of blood donation. Blood donors are mainly volunteers
offering their blood for philanthropic reasons. Donating blood for safety testing ofdrugs for human use is a philanthropic attitude, too.
Comment to A 11.4 (page 22)
The PRP stated: “Furthermore, the in vitro pyrogen test methods are dependent on
the availability of donors or blood supplies, which might further restrict the frequencyof which these tests can be performed.”
This statement does not consider the cryo-preserved blood since it would beavailable at any time.
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ICCVAM Test Method Evaluation Report: Appendix D2Thank you for your attention to these comments.
Sincerely,
Dr. Thomas Montag-Lessing
Dr. Ingo Spreitzer

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Appendix D3
SACATM Comments: ICCVAM Evaluation of In Vitro Pyrogen Test Methods

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The following is excerpted from the final minutes of the SACATM meeting convened onJune 12, 2007. The full meeting minutes will be available online at http://ntpserver.niehs.nih.gov/ntpweb/index.cfm?objectid=AF6CC417-F1F6-975E75B5F3FF7DF1CDDC.
Overview of the ICCVAM Evaluation of In Vitro Pyrogen Test Methods
Dr. Richard McFarland, U.S. Food and Drug Administration (FDA), ICCVAM
Pyrogenicity Working Group (PWG) Chair, presented an update on ICCVAM’s ongoingevaluation of five in vitro human cell-based pyrogen test methods nominated for review
by the European Centre for the Validation of Alternative Methods (ECVAM).
Pyrogenicity is defined as an increase in body temperature following the release of proinflammatory cytokines [e.g., interleukin (IL)-1, IL-6, and tumor necrosis factor-α (TNFα)] by leukocytes. Pyrogens may be found in processing and packaging materials,
chemicals, or parenteral pharmaceuticals, biologicals, and medical devices. Bacterial
endotoxin, a component of the outer cell wall of Gram-negative bacteria, is one of themost potent pyrogenic materials. Pyrogen testing is important to prevent the introductionof endotoxin or non-endotoxin pyrogen-contaminated products into humans or animals.
Currently there are two accepted pyrogen tests. The Rabbit Pyrogen Test (RPT), whichmeasures a temperature rise in rabbits injected with a test substance, can detect both
endotoxin and non-endotoxin pyrogens. The Bacterial Endotoxin Test (BET), also
referred to as the Limulus Amoebocyte Lysate (LAL) Test, detects endotoxin by its
ability to activate a serine-protease catalytic cascade.
In June 2005, ECVAM submitted background review documents (BRDs) on fivemethods for consideration by NICEATM as replacements for the RPT. The methods are:• Human Whole Blood (WB)/Interleukin (IL)-1 In Vitro Pyrogen Test
• Human WB/IL-1 In Vitro Pyrogen Test: Application of Cryopreserved Human
WB• Human WB/IL-6 In Vitro Pyrogen Test
• Human Peripheral Blood Mononuclear Cell (PBMC)/IL-6 In Vitro Pyrogen Test
• In Vitro Pyrogen Test using the monocytoid cell line, Mono Mac 6 (MM6)/IL-6
Before describing the evaluation process, Dr. McFarland listed the members of the PWG,
provided a time line for the various activities connected with the evaluation process, anddescribed the ICCVAM acceptance and validation criteria for alternative test methods.
Following a prescreen evaluation, NICEATM requested additional information andclarification from ECVAM in regard to the data provided in their BRDs. ECVAM
submitted revised BRDs that addressed these requests. Subsequently, ICCVAM prepareda draft ICCVAM BRD that contained a comprehensive review of all available data andinformation regarding the usefulness and limitations of the five alternative in vitro
pyrogen test methods and described the current validation status of the test methods
including their relevance, reliability, scope of substances tested, and the availability of astandardized test method protocol for each test method.

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ICCVAM Test Method Evaluation Report: Appendix D3

May 2008

The major difference among the five test methods is the cell types used; the methodology
used for the test methods is very similar. Briefly, the test substance is applied to culturesof the specific human-derived cells, which are then incubated for 16-24 hr. The
concentration of pro-inflammatory cytokines (e.g., IL-1β, IL-6) is quantified via acytokine-specific enzyme-linked immunosorbent assay (ELISA). The endotoxin activityof a test substance is calculated by comparing the induced cytokine release with thatinduced by the endotoxin standard.
The test methods were reviewed for their ability to detect the presence of Gram-negative
endotoxin when several parenteral pharmaceuticals were spiked with the endotoxin
standard at several different concentrations. The reference pharmaceuticals wereconsidered positive for endotoxin if the endotoxin content was > 0.5 endotoxin units(EU)/mL. Differences were found in the performance of the five test methods. Based on
the information contained in the BRD, ICCVAM developed draft recommendations forthe use, formulated draft performance standards and draft test method protocols for eachtest method, and identified proposed future studies.
ICCVAM’s draft recommendations on test method uses and limitations was that, basedon the validation studies with a limited number of pharmaceuticals, there is sufficient
information to substantiate the use of these test methods for the detection of pyrogenicity
mediated by Gram-negative endotoxins in materials that are currently tested in the RPT,
subject to product-specific validation to demonstrate equivalency. Further, ICCVAM’s
draft recommendations stated that although the five in vitro test methods may be capableof detecting a wider range of pyrogens than was tested, the data in the BRDs do notsupport this broader application. One limitation of the validation study was the lack of adirect comparison of the results for the same test substances in the proposed in vitro test
methods versus the RPT.
ICCVAM also provided draft recommendations for performance standards for these fivein vitro test methods for consideration by the peer review panel and for public comment;
the purpose of performance standards are to ensure that any proposed mechanistically andfunctionally similar proposed test method meets acceptable standards. Performancestandards include essential test method components based upon common structural,functional, and procedural elements that should be included in the protocol of amechanistically and functionally similar proposed test method; recommended reference
substances for evaluating the relevance and reliability of the proposed test method andthe performance characteristics (relevance and reliability values) that should be met or
exceeded. ICCVAM also recommended draft standardized protocols that were based on
those used in the ECVAM validation study. Finally, ICCVAM recommended futurestudies that included the testing of a broader range of pyrogenic materials underconditions where the in vitro pyrogen test(s) and the RPT were run in parallel to be able
to directly compare the results.
Peer Panel Report
ICCVAM and NICEATM held a peer review panel meeting on February 6, 2007, toreview the five in vitro pyrogenicity test methods. Dr. Karen Brown, DRL Pharma and

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May 2008

Pair O’Doc’s Enterprises, chair of the peer panel, said the task was daunting because thepanel was tasked to complete the evaluation of the five in vitro test methods in one day.
She recognized the hard work and diligence of the panel.
The charge to the peer review panel was to review the draft BRDs for completeness,
assess whether each applicable criterion for validation and acceptance of the test method
had been appropriately addressed, and consider whether the information in the BRD
supported the draft ICCVAM recommendations for the draft standardized protocols, thedraft test method performance standards, and the draft proposed future studies.
The panel concluded that the explanation in the BRD of the usefulness and limitations of
the in vitro pyrogenicity test methods and of the description of the current validation
status of these methods was sufficient. However, they identified a number of deficiencies
in the BRD, which are briefly described below.
1. There were some sections where additional details would have improved the
document. For example, the panel wanted information included about (1) the
number of RPTs conducted per year to evaluate bacterial endotoxin, (2) the
number of rabbits used for pyrogenicity testing per year, and (3) the costs andlogistical considerations for either setting up the cell culture for the MM6 test or
obtaining human blood for the other tests.
2. The rationale for selecting the test substances for evaluating the five in vitro test
methods was flawed because it did not represent the range of products tested forbacterial endotoxin using the RPT and seven of the 10 substances were not tested
in the RPT but rather in the BET. For example, no biologicals or medical devices
were evaluated. The panel felt that the number of substances tested in thevalidation study was not adequate to evaluate whether a specific test method
could replace the RPT.
3. The in vivo RPT reference data were limited to one strain of rabbit tested in one
laboratory by one protocol using two sources of bacterial endotoxin.
4. The evaluation of the relevance of each test method was adequately demonstrated
and discussed in the BRDs, but was limited by the ability to judge a positive
versus a negative response based on 0.5 endotoxin units (EU)/ mL. Since samples
were only spiked with bacterial endotoxin, the relevance was only demonstrated
for the detection of this type of pyrogen, and there was no evaluation for theability to detect non-endotoxin pyrogens.
5. The discussion on concordance in the RPT is speculative because there was noparallel testing with the RPT, and the RPT performance was modeled statistically.
6. The whole blood IL-1 test is inadequate because there were too many false
positives and false negatives; however the IL-6 assay appeared to perform better.

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It would have been more appropriate to compare these in vitro tests directly with
the BET, since only bacterial endotoxin samples were used.
7. Test method reliability was acceptable in both within and between laboratory
studies; however, a quantitative assessment of intra- and inter-laboratory
variability would have been more informative. A statistical assessment providing
acceptability criteria should have been performed to test the hypothesis that there
were no differences among groups.
8. The assessment of test method reliability had the following deficiencies:a. There was a high exclusion rate for individual runs of the whole blood IL-1
assay due to excessive variability among the four replicates.
b. The agreement across three validation laboratories was only 57% for thewhole blood IL-1 assay.
c. The same subset of drugs tested for sensitivity and specificity should havebeen tested for reliability.
Most of the panel agreed that application of the validation criteria to determine theusefulness and limitations of these test methods to replace the RPT under conditions
where the test was for the presence of Gram-negative endotoxin was adequately
addressed in the BRDs.
The panel concluded that the usefulness of the test methods to detect Gram-negative
endotoxin was not assessed properly to determine their concordance with the RPT or tocompare their relevance with the BET. The assessment of the usefulness was limited
because non-endotoxin pyrogens were not included, and the pure form of the test
materials may stimulate cytokine production.
The panel agreed that the BRDs did support the proposed standardized test method
protocols if the list of its inadequacies were fully addressed. The panel noted that to
reduce variability, similar acceptance criteria must be used for multiple blood donors andsimilar exclusion rules must be used for each test method. They recommended that amore specific protocol be developed that details recruitment of human blood donors,
selection criteria for donors, as well as conditions for veinipuncture.
The panel concluded that the test method performance standards were not supported by
the BRD. Statements about the five methods’ accuracy and reliability were not supported
because two assays demonstrated false-positive results greater than 16 % and the in vitro
test methods should have been compared to both the BET and RPT. Also, the panel
thought that the small list of substances was inadequate to assess whether these test
methods could replace the RPT. Test substances need to include all classes of endotoxins
as well as non-endotoxin pyrogens.
The panel agreed that additional studies should be performed, and that ICCVAM should
consider their comments and recommendations. They suggested (1) establishment of a

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May 2008

repository of clinically identified pyrogens to use in future validation studies, (2)inclusion of both endotoxin and non-endotoxin pyrogens in future validation studies, (3)prospective comparison of any in vitro tests with the RPT and BET, and (4) evaluation of
IL-1 and IL-6 levels in the in vitro tests and their correlation with levels produced in
rabbits exposed to similar levels of endotoxin.
Overall, the peer review panel concluded that these five test methods could be applicablefor a wider range of pyrogens and test materials if they were adequately validated forsuch uses. It is important to recognize that, despite the panel’s concerns about theperformance of these five in vitro test methods, the FDA has a formal process formaterials regulated under 21CFR610.9 (e.g., parenteral drugs) that allows drug
manufacturers to qualify in vitro test methods for identifying Gram-negative endotoxin,
on a case-by-case basis.
Public comments:
Dr. Freedman identified the written comments submitted by Physicians Committee for
Responsible Medicine (PCRM).
Ms. Kristy Stoick, PCRM, said her organization submitted written comments after thepeer review panel meeting. PCRM was disappointed with the ICCVAM draft
recommendations and the peer review panel report. Since federal regulations specify thatthese methods must undergo product specific validation for pyrogenicity, she encouraged
SACATM to recommend that ICCVAM help facilitate further development of thesemethods by companies so the regulatory community can begin to use them as soon as
possible. She did not support additional in vivo validation studies.
Dr. Thomas Hartung, ECVAM, joined the public for this specific agenda item because of
a conflict of interest as a patent holder for the methods. Three of the in vitro test methods
were based on his research and he had coordinated the validation study prior to joining
ECVAM. He was pleased that the European Pharmacoepia will hold a peer review panel
to review and accept these methods. He was disappointed with the outcome of the peer
review panel meeting. He noted that pyrogenicity tests are very expensive and theapproval and release of a single product can cost several hundred thousand dollars. The
validation studies were set up to assess whether the new tests would outperform the oldtests within a set threshold. Only 50% of the samples would be positive in the mostsensitive rabbit strain. All of the in vitro assays have an accuracy of around 90%. He
outlined six points where the BRD had been criticized.
1. ICCVAM said the BRD is deficient due to the limited data for only 10
pharmaceutical substances from the validation studies, which alone cost $6M.
The recommendations for additional studies from the peer review panel would
cost between $20-40M and they would be a waste of resources because a productspecific validation process would be required for each application. To help
contain cost, the tests described in the BRD were designed to emphasize theaccuracy of the method to detect pyrogens near the threshold.

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ICCVAM Test Method Evaluation Report: Appendix D3

May 2008

2. The peer review panel did not acknowledge the difference in status of the fivemethodologies. Some methods are used in more than 80 laboratories while others
are used infrequently; however, the same criticisms were applied to all of themethods.
3. The BRD recommended that parallel testing be conducted with the RPT.
However, parallel testing in rabbits is unnecessary because these studies havebeen performed for 65 years using a WHO standard as a reference material. The
outcome from rabbit testing is so predictable that ethically it is not warranted.
Also, in the European Union, it will be impossible for ECVAM to carry out thesein vivo tests especially as the new methodologies have shown partial concordance.
4. Endotoxins are only tested in the BET assay, and this assay has replaced the RPT
for about 90% of substances; the remaining 10% of substances consist of nonendotoxin pyrogen products that interfere with the BET. He asked why the newtests have to meet higher standards than the BET, which has been endorsed for thetesting of many pyrogenic products. He noted that no reference non-endotoxin
pyrogens are suitable for validation purposes in rabbits and humans; therefore,
inclusion of such controls is scientifically impossible.
5. High endotoxin concentrations will be detected accurately in the RPT, BET, or
any of the new in vitro pyrogenicity assays. Hence, a concentration near 50 pg ofendotoxin, which is equivalent to 0.5 EU and is the threshold for rabbits, waschosen for the assays. Additional concentrations of 100 pg and 25 pg were also
selected. Even though the assays were challenged at these low concentrations,they were 90% accurate. False positives were due to spikes at half the thresholdindicating that the assays are too sensitive.
6. The new assays were evaluated fairly in comparison to the limitations of theexisting tests. The rabbit test, which has a number of limitations, has never been
properly validated for non-endotoxin pyrogens. The BET does not detect all
Gram-positive endotoxins although the new assays have shown some capability
for doing so.
In conclusion, the proposed test methods for which data sets have been provided
perform better than the BET and RPT. Dr. Hartung proposed that the rabbit assay bereplaced with the in vitro assays because the RPT cannot match their performance, asreported in the BRD.
SACATM Discussion.
SACATM was asked to address questions regarding the peer review panel’s conclusion
and recommendations of the draft ICCVAM BRD with regard to its completeness; thepanel’s identification of errors or omissions; whether ICCVAM’s applicable criteria forvalidation and acceptance of toxicological test methods were addressed; and to provide
comments on the draft ICCVAM test methods recommendations, usefulness of the test

D-50

ICCVAM Test Method Evaluation Report: Appendix D3

May 2008

methods, the test method protocols, proposed performance standards, as well as proposed
additional studies.
Dr. Barile, a lead discussant, said there was no question about the usefulness ofpyrogenicity testing and the urgency and importance of validating these tests. In
combination, some of these tests will contribute to the reduction of animal usage. One
major deficiency of present pyrogenicity testing is that the RPT only detects about 50%of the endotoxins. Some of the proposed in vitro tests had false negative responses in therange of 10% while the IL-1 assay had a false negative response of 27%. These false
negative responses could be due to consistently higher variability among some donors,
which would be a limitation relative to a whole blood human assay. He expressed
concern that the IL6 ELISA test, marketed by Novartis, is a proprietary test and he would
not recommend approving a method without knowing the experimental details. He
agreed with Dr. Hartung that parallel testing in rabbits was unnecessary during
development of the testing methodologies. However, a comparison to RPT data isnecessary so that a valid concordance or regression analysis between the in vivo and invitro methods can be undertaken. He said samples spiked with endotoxin are notrepresentative of real world samples such as a biological vaccine or a solubilized
pharmaceutical product. There is no solubility problem associated with the testing of
biological vaccines in rabbits, but insolubility is a problem in in vitro tests even if the test
article is in suspension and this technicality must be addressed. He believes that the cell
culture methods are more developed than the whole blood methods for validation
purposes. A few additional studies, which address the panel’s recommendations, would
allow the cell culture pyrogenicity tests to receive validation status.
Dr. McClellan said he was generally pleased with the draft BRD until he heard Dr.
Hartung’s statement. He did not believe that the BRD is adequate nor can he compliment
the peer review panel on its report. He wondered how this difference of opinion would
be resolved and asked Dr. Brown to comment.
Dr. Freedman said he was confident that all of SACATM’s comments would be taken
into account by ICCVAM and, if necessary, ICCVAM could reconvene the expert panel.
Dr. Brown said ECVAM produced a reasonably comprehensive BRD, but the panel was
not able to address all of the components of the individual in vitro methods because time
for discussion was limited. Some of the details were missing or difficult to understand;however, she felt that given more time to discuss these methods, the panel might havebeen able to provide a stronger recommendation for one or more of the assays.
Personally, she felt that the MM6 assay has the greatest potential and several of the otherpanel members agreed. The most bothersome aspect for the panel was trying to identify
the specifics of the validation protocols. She noted that for an in vitro assay it is critical
to identify every component and every single condition of the assay completely, but thisinformation was not provided, particularly for the MM6 test method. She was impressedwith the cell culture methodology, although specifics such as cell passage levels, or how
many cells are used in a test were lacking. She felt that the panel did not seem tounderstand cell culture methodology and its related costs. Consequently, they got side-

D-51

ICCVAM Test Method Evaluation Report: Appendix D3

May 2008

tracked in specifics, which hindered them from making progress and reachingconclusions.
Dr. Brown said she does not believe that it is necessary to run in vivo assays in parallelwith the in vitro assays. She is unsure how one can run a regression analysis with one
test that is 90% accurate and a second that is 50% accurate. She questioned whether it isnecessary to validate an in vitro test against an animal test that is not as accurate as the invitro assay itself.
Dr. McClellan said that Dr. Hartung disclosed his own potential biases, concerns, andbackground. He asked whether Dr Hartung was suggesting that two of the assays shouldhave received more attention and wondered which of the assays Dr. Hartung thought
were appropriately validated and whether he might focus the panel toward those assays.
Dr. Stokes said that in the future NICEATM would set aside at least two days for a peer
review meeting, so that a panel can fully understand the methodologies before they
deliberate on the evaluation questions.
Dr. Qu had some comments on the panel’s concern about data transformations. The
panel was not sure if the data were transformed and whether or not the use of a “t” test
for their analysis was appropriate. She said it is not necessary to use a “t” test even if thedata are normal. A non-parametric test such as the permutation test, which does not
require transformation, could be used. Dr. Qu noted also that it is important to control for
false positives when doing a multiple comparison for several tests. By doing multiple
comparisons, it is possible to obtain a statistically significant difference that is notbiologically significant. One approach to dealing with this problem is to use a more
stringent level of significance.
Dr. Becker welcomed the proposed longer time frame for a peer review meeting. He
suggested that it might be useful to convene a meeting with a core panel of validation
experts and then have subject-specific experts to address specific assays.

D-52

ICCVAM Test Method Evaluation Report: Appendix E

May 2008

Appendix E
ESAC Statement on the Validity of In Vitro Pyrogen Tests
ESAC Statement on the Validity of In Vitro Pyrogen Tests ..........................................E-3

E-1

ICCVAM Test Method Evaluation Report: Appendix E

May 2008[This Page Intentionally Left Blank]

E-2

ICCVAM Test Method Evaluation Report: Appendix E

May 2008

EUROPEAN COMMISSION
DIRECTORATE GENERAL JRC
JOINT RESEARCH CENTRE
Institute for Health and Consumer Protection
European Centre for the Validation of Alternative Methods (ECVAM)

STATEMENT ON THE VALIDITY OF IN-VITRO PYROGEN TESTSAt its 24th meeting, held on 20-21 March 2006 at the European Centre for thevalidation of alternative methods (ECVAM), Ispra, Italy, the non-Commission
members of the ECVAM Scientific Advisory Committee (ESAC)1 unanimously
endorsed the following statement:
Following a review of scientific reports and peer reviewed publications on thefollowing range of in-vitro pyrogen tests:
1.
2.
3.
4.
5.

Human Whole Blood IL-1,
Human Whole Blood IL-6,
PBMC IL-6,
MM6 IL-6, andHuman Cryopreserved Whole Blood IL-1,

it is concluded that these tests have been scientifically validated for the detection ofpyrogenicity mediated by Gram-negative endotoxins, and quantification of thispyrogen, in materials currently evaluated and characterized by rabbit pyrogen tests.
These methods have the potential to satisfy regulatory requirements for the detection
and quantification of these pyrogens in these materials subject to product-specificvalidation.
The test methods have the capacity of detecting pyrogenicity produced by a wider
range of pyrogens, but the evidence compiled for, and considered within this peer
review and validation process, is not sufficient to state that full scientific validation ofthis wider domain of applicability has been demonstrated and confirmed.
Thus, the above test methods can currently be considered as full replacements for theevaluation of materials or products where the objective is to identify and evaluate
pyrogenicity produced by Gram-negative endotoxins, but not for other pyrogens.
This endorsement takes account of the dossiers prepared for peer review; the views of
independent experts who evaluated the dossiers against defined validation criteria;
supplementary submissions made by the Management Team; and the considered viewof the Peer Review Panel appointed to oversee the process.
Thomas Hartung
Head of Unit
ECVAM
Institute for Health & Consumer Protection
Joint Research Centre
European Commission
Ispra
21 March 2006

E-3

ICCVAM Test Method Evaluation Report: Appendix E

May 2008

1. The ESAC was established by the European Commission, and is composed ofnominees from the EU Members States, industry, academia and animal
welfare, together with representatives of the relevant Commission services.
This statement was endorsed by the following Members of the ESAC:
Prof Helmut Tritthart (Austria)
Dr Dagmar Jírová (Czech Republic)
Prof Elisabeth Knudsen (Denmark)Dr Timo Ylikomi (Finland)Prof André Guillouzo (France)
Dr Manfred Liebsch (Germany)
Dr Efstathios Nikolaidis (Greece)
Dr Katalin Horvath (Hungary)Prof Michael Ryan (Ireland)
Dr Annalaura Stammati (Italy)
Dr Mykolas Maurica (Lithuania)
Prof Eric Tschirhart (Luxembourg)
Dr Jan van der Valk (The Netherlands)
Dr Dariusz Sladowski (Poland)Prof Milan Pogačnik (Slovenia)
Dr Argelia Castaño (Spain)
Dr Patric Amcoff (Sweden)
Dr Jon Richmond (UK)Dr Odile de Silva (COLIPA)
Dr Julia Fentem (ECETOC)Dr Nathalie Alépée (EFPIA)Prof Robert Combes (ESTIV)Dr Maggy Jennings (Eurogroup for Animal Welfare)
Mr Roman Kolar (Eurogroup for Animal Welfare)
The following Commission Services and Observer Organisations wereinvolved in the consultation process, but not in the endorsement process itself.
Mr Thomas Hartung (ECVAM; chairman)
Mr Jens Linge (ECVAM; ESAC secretary)
Mr Juan Riego Sintes (ECB)
Ms Beatrice Lucaroni (DG Research, Unit F.5)
Mr Sylvain Bintein (DG Environment, Unit C.3)
Mr Sigfried Breier (DG Enterprise, Unit F.3)
Prof Dr Constantin Mircioiu (Romania)
Dr William Stokes (NICEATM, USA)
Prof Dr Vera Rogiers (ECOPA)

E-4

ICCVAM Test Method Evaluation Report: Appendix E

May 2008

Annex
The novel pyrogen tests are based on the human fever reaction. Monocytoid cells,
either primary from human blood or as propagated cell lines, detect pyrogens ofdifferent chemical nature and respond by the release of inflammatory mediators suchas cytokines. Since lipopolysaccharides from Gram-negative bacteria are the only
type of proven pyrogen, for which an International reference material is available, thetests were standardised to detect the presence of significantly less than 0.5 Endotoxin
Units of this preparation, which is considered to be the threshold level for fever
induction in the most sensitive rabbit species according to pharmacopoeia test
procedures.
The five tests which were sufficiently reproducible and exceeded the rabbit test withregard to sensitivity and specificity for the detection of lipopolysaccharide spiked
samples, differ with regard to cell source and preparation, cryopreservation andcytokine measured. The tests have been described elsewhere (1-4). The concept ofthe validation study (5) and the international validation studies are available (6-7).
1. Poole, S., Thorpe, R., Meager, A., Hubbard, A.R., Gearing, A.J. (1988) Detectionof pyrogen by cytokine release. Lancet 8577, 130.
2. Taktak, Y.S., Selkirk, S., Bristow, A.F., Carpenter, A., Ball, C., Rafferty, B., Poole,
S. (1991) Assay of pyrogens by interleukin-6 release from monocytic cell lines. J.
Pharm. Pharmacol. 43, 578.
3. Hartung, T., Wendel, A. (1996) Detection of pyrogens using human whole blood.
In Vitro Toxicol. 9, 353.
4. Schindler S, Asmus S, von Aulock S, Wendel A, Hartung T and Fennrich S. (2004)
Cryopreservation of human whole blood for pyrogenicity testing. J. Immunol. Meth.
294, 89-100.
5. Hartung, T., Aaberge, I., Berthold, S., Carlin, G., Charton, E., Coecke, S., Fennrich,S., Fischer, M., Gommer, M., Halder, M., Haslov, K., Jahnke, M., Montag-Lessing, T.,
Poole, S., Schechtman, L., Wendel, A., Werner-Felmayer, G. (2001) Novel pyrogentests based on the human fever reaction. The report and recommendations of ECVAM
Workshop 43. European Centre for the Validation of Alternative Methods. Altern.
Lab. Anim. 29, 99.
6. Hoffmann S, Peterbauer A, Schindler S, Fennrich S, Poole S. Mistry Y, MontagLessing T, Spreitzer I, Loschner B, vam Aalderen M, Bos R, Gommer M, Nibbeling
R, Werner-Felmayer G, Loitzl P, Jungi T, Brcic M, Brugger P, Frey E, Bowe G,
Casado J, Coecke S, de Lange J, Mogster B, Naess LM, Aaberge IS, Wendel A and
Hartung T. (2005) International validation of novel pyrogen tests based on the humanfever reaction. J. Immunol. Meth. 298, 161-173.
7. Schindler S, Spreitzer I, Loschner, Hoffmann S, Hennes K, Halder M, Brügger P,
Frey E, Hartung T and Montag T. (2006) International validation of pyrogen tests
based on cryopreserved human primary blood cells. J. Immunol. Meth. 316, 42-51.
E-5

ICCVAM TEST METHOD EVALUATION REPORTThe Reduced Murine Local Lymph Node Assay:
An Alternative Test Method Using Fewer Animals to Assess theAllergic Contact Dermatitis Potential of Chemicals and Products

NIH Publication Number 09-6439

ICCVAM TEST METHOD EVALUATION REPORTThe Reduced Murine Local Lymph Node Assay:
An Alternative Test Method Using Fewer Animals to Assess theAllergic Contact Dermatitis Potential of Chemicals and Products

U.S. Department of Health and Human Services

NIH Publication Number 09-6439

National Toxicology Program
P.O. Box 12233
Research Triangle Park, NC 27709

Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM)
National Toxicology Program (NTP) Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM)
National Institute of Environmental Health Sciences
National Institutes of Health
U.S. Public Health Service
Department of Health and Human Services

Draft ICCVAM Recommended Test Method Protocol: Cryo WB/IL-1β Pyrogen Test

01 Dec 2006

Appendix B1

Draft ICCVAM Recommended Protocol for Future Studies Using the
Cryopreserved Whole Blood (Cryo WB)/Interleukin-1β (IL-1β)
Test Method

Draft ICCVAM Recommended Test Method Protocol: Cryo WB/IL-1β Pyrogen Test

[This Page Intentionally Left Blank]

01 Dec 2006

Draft ICCVAM Recommended Test Method Protocol: Cryo WB/IL-1β Pyrogen Test

01 Dec 2006

1

Draft ICCVAM Recommended Protocol for Future Studies Using the Cryopreserved

2

Whole Blood (Cryo WB)/Interleukin-1β (IL-1β) Test Method

3
4

PREFACE

5
6This proposed protocol for the detection of pyrogenicity is based on information obtained

7

from 1) The European Centre for the Validation of Alternative Methods (ECVAM) Cryo8

WB/IL-1β Background Review Document (BRD) presented in Appendix A of the draft

9Interagency Coordination Committee on the Validation of Alternative Methods (ICCVAM)

10

Pyrogenicity Test Method BRD, which includes ECVAM Standard Operating Procedures

11(SOPs) for the Cryo WB/IL-1β test method, and 2) Information provided to the National

12

Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative

13

Toxicological Methods (NICEATM) by Dr. Thomas Hartung, Head of ECVAM. The

14

ECVAM SOPs are based on the Cryo WB/IL-1β methodology first described by Schindler et15

al. (2004). A table of comparison between the draft ICCVAM recommended protocol and the16

ECVAM SOP is provided in Table 1. Future studies using the Cryo WB/IL-1β test method17

may include further characterization of the usefulness or limitations of the assay for18

regulatory decision-making. Users should be aware that the proposed test method protocol

19

might be revised based on additional optimization and/or validation studies. ICCVAM

20

recommends that test method users routinely consult the ICCVAM/NICEATM website

21(http://iccvam.niehs.nih.gov/) to ensure that the most current test method protocol is used.

22

B1-1

Draft ICCVAM Recommended Test Method Protocol: Cryo WB/IL-1β Pyrogen Test

22

Table 101 Dec 2006

Comparison of Draft ICCVAM Recommended Test Method Protocol

23

with the ECVAM SOP for the Cryopreserved Whole Blood (Cryo24

WB)/Interleukin-1β (IL-1β) Pyrogen Test
Protocol Component
Test Substance

Incubation Plate(number of control ortest groups at n=4 each)
ELISA Plate
Decision Criteria forInterference

Assay Acceptability Criteria

25
26
27
28
29
30
31
32
33

Draft ICCVAM Protocol
Test neat or at minimal dilutionthat produces 50% to 200% of 1
EU/mL EC
NSC (1)EC (5)
TS (14)
PPC1 (0)
NPC1 (0)Includes seven point IL-1β SC andblank in duplicate
0.5 x Median OD4502 of 1 EU/mL
EC <2x Median OD450 of 1 EU/mL
EC
Mean OD450 of 0.5 EU/mL EC ≥1.6X Mean OD450 of NSC
Mean OD450 of PPC is 50% to200% of 0.5 EU/mL EC
Mean OD450 of NSC ≤0.153
Not included

ECVAM SOP
Test at MVD
Same as ICCVAM protocol
EC (2)TS (3) x EC (5) spikes
PPC (3)
NPC (3)Same as ICCVAM protocol
Mean OD4502 of PPC ≥ 1.6X Mean
OD450 of NPC
Same as ICCVAM protocol
Same as ICCVAM protocol
Mean OD450 of NSC ≤0.10
If one OD450 of the 1.0 EU/mL
EC>Max, the ELISA may berepeated at reduced incubation time

EC SC produces OD450 values thatascend in a sigmoidal
Same as ICCVAM protocol
concentration response
Quadratic function of IL-1β SC r2
Same as ICCVAM protocol≥0.953
Decision Criteria for
OD450 Test sample ≥ OD450 0.5
Same as ICCVAM protocol
Pyrogenicity
EU/mL EC
Abbreviations: EC = Endotoxin control; MVD = Maximum Valid Dilution; NPC = Negative Product Control;
NSC = Normal saline control;
PPC = Positive Product Control; SC = Standard curve; TS = Test substance1
PPC and NPC are evaluated during the interference test.
2
Median or mean OD450 values are corrected (i.e., reference filter reading, if applicable, and NSC are
subtracted).
3
Criteria originated from PBMC SOP.

34

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34

1.0

35

The purpose of this protocol is to describe the procedures used to evaluate the presence of a36

pyrogen (i.e., Gram-negative endotoxin) in a test substance. The presence of Gram-negative

37

endotoxin is detected by its ability to induce cytokine IL-1β release from monocytoid cells in

38

human Cryo WB. The quantity of IL-1β released is obtained using an enzyme-linked

39

immunosorbent assay (ELISA) that includes monoclonal or polyclonal antibodies specific for40

IL-1β. Release of IL-1β is measured by incubation of Cryo WB with test substances or41

controls (i.e., positive, negative). The amount of pyrogen present is determined by comparing42

the values of endotoxin equivalents produced by cells exposed to the test substance to those

43

exposed to an internationally-harmonized Reference Standard Endotoxin (RSE)1 or an

44

equivalent standard expressed in Endotoxin Units (EU)/mL. Based on a rabbit threshold

45

pyrogen dose of 0.5 EU/mL, which was established in a retrospective evaluation of rabbit

46

pyrogen test (RPT) data, a test substance is considered pyrogenic if it induces a level of IL-

47

1β release equal to or greater than that induced by 0.5 EU/mL of endotoxin.

48

The focus of this protocol is on the use of the Cryo WB/IL-1β test method, specifically for49

the detection of Gram-negative endotoxin in parenteral pharmaceuticals. The relevance and50

reliability for non-endotoxin pyrogens (e.g., lipoteichoic acid) has not been demonstrated in a51

formal validation study.

52

2.0

53

All procedures for procurement of eligible blood donors and blood donations should follow

54

the regulations and procedures set forth by institutional guidelines for utilization of human55

substances, which include but are not limited to blood, tissues, and tissue fluids. Standard

56

laboratory precautions are recommended including the use of laboratory coats, eye57protection, and gloves. If necessary, additional precautions required for specific study

58

substances or hazardous chemicals will be identified in the Material Safety Data Sheet

59(MSDS).
1

PURPOSE AND APPLICABILITY

SAFETY AND OPERATING PRECAUTIONSRSEs are internationally-harmonized reference standards (e.g., WHO E. coli Lipopolysaccharide [LPS] 94/580[0113:H10:K-]; USP RSE Lot G3E069; FDA Lot EC-6). Equivalent endotoxins include commercially available
E. coli-derived LPS Control Standard Endotoxin (CSE) or other E. coli LPS preparations that have beencalibrated with an appropriate RSE.
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60

The stop solution used in the ELISA kit is acidic and corrosive and should be handled with61

the proper personal protective devices. If this reagent comes into contact with skin or eyes,

62

wash thoroughly with water. Seek medical attention, if necessary.

63

Tetramethylbenzidine (TMB) solution contains a hydrogen peroxide substrate and 3, 3', 5, 5'-64

TMB. This reagent is a strong oxidizing agent and a suspected mutagen. Appropriate

65

personal protection should be used to prevent bodily contact.

66

Bacterial endotoxin is a toxic agent (i.e., can induce sepsis, shock, vascular damage,67

antigenic response) and should be handled with care. Skin cuts should be covered and68

appropriate personal protective devices should be worn. In case of contact with endotoxin,69

immediately flush eyes or skin with water for at least 15 min. If inhaled, remove the affected70

individual from the area and provide oxygen and/or artificial respiration as needed. Skin71

absorption, ingestion, or inhalation may produce fever, headache, and hypotension.

72

3.0

MATERIALS, EQUIPMENT, AND SUPPLIES

73

3.1

Source of Cells

74

Leukocytes from WB are the source of cells for cytokine production in the Cryo WB/IL-1β

75

test method (Hartung and Wendel, 1999; Schindler et al., 2004, 2006). WB is obtained from76

healthy human volunteers who have provided their consent according to established77

institutional guidelines. Volunteers are expected not to have taken any drugs (e.g.,78

prescription drugs, recreational drugs, herbal drugs) and to have been free from illness for at

79

least two weeks prior to donation.

80

The WB is processed and cryopreserved using either the Konstanz method developed at the81

University of Konstanz (Schindler et al., 2004) or the PEI method developed at the Paul82

Ehrlich Institute (Schindler et al., 2006).

83

3.2

84

For all steps in the protocol, excluding the ELISA procedure, the materials that will be in85

close contact with samples and/or blood cells (e.g., pipet tips, containers, solutions) should be86

sterile and free from detectable pyrogens.

Equipment and Supplies

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87

3.2.1

Blood Incubation

88

3.2.1.1

Equipment

89•

Centrifuge

90•

Hood; Bio-safety, laminar flow (recommended)91•

Incubator; cell culture (37±1°C + 5% CO2)

92•

Pipetter; multichannel (8- or 12-channel)

93•

Pipetters; single-channel adjustable (20 and 200 µL)

94•

Repeating pipetter

95•

Vortex mixer

96

3.2.1.2

01 Dec 2006

Consumables

97•

Centrifuge tubes; nonpyrogenic, polystyrene (15 and 50 mL)

98•

Combitips; repeating pipetter (1.0 and 2.5 mL)

99•

Needle set; Sarstedt multifly, pyrogen-free, 19 mm, 21 gauge for S-Monovette

100•

Plates; microtiter, nonpyrogenic, 96-well, polystyrene, tissue culture

101•

Reaction tubes; polystyrene (1.5 mL)

102•

Reservoirs; fluid103•

RPMI-1640 cell culture medium

104•

Tips; pipetter, sterile, pyrogen-free (20 and 200 µL)

105•

Tubes; Sarstedt S-Monovette, 7.5 mL, heparinized for blood collection

106

3.2.2

ELISA

107

3.2.2.1

Equipment

108•

Microplate mixer

109•

Microplate reader (450 nm with an optional reference filter in the range of

110
111

600-690 nm)•

Microplate washer (optional)
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112
113

•
3.2.2.2

01 Dec 2006

Multichannel pipetter

Consumables

114•

Container; storage, plastic

115•

Deionized water; nonsterile

116•

Plates; microtiter, nonpyrogenic, 96-well, polystyrene, tissue culture

117•

Pyrogen-free water (PFW)118•

Reservoirs; fluid

119•

Tips; pipetter, nonsterile

120•

Tubes; polystyrene (12mL)

121

3.2.2.3

122

An ELISA that measures IL-1β release from Cryo WB is used. A variety of IL-1β ELISA

123

kits are commercially available and the IL-1β ELISA procedure outlined in this protocol is124

intended to serve as an example for using an ELISA kit. If the user prefers to prepare an in-

125

house ELISA, then additional reagents would be required. The IL-1β ELISA should be

126calibrated using an international reference standard (e.g., WHO 86/680) prior to use. The IL-127

1β cytokine assay kits do not provide the RSE or endotoxin equivalent; therefore, this128

reagent must be purchased separately. Results obtained using these products are subject to

129

the assay acceptability and decision criteria described in Sections 8.0 and 9.0. IL-1β ELISA

130

kit components may include the following:131

ELISA Kit•

132

ELISA plates coated with anti-human IL-1β capture antibody; monoclonal or
polyclonal

133•

Buffered wash solution

134•

Dilution buffer

135•

Enzyme-labeled detection antibody

136•

Human IL-1β reference standard

137•

Pyrogen-free saline (PFS)
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138•

Stop solution

139•

TMB/substrate solution

140

3.3

141

Chemicals
•

142
143

01 Dec 2006

Endotoxin (e.g., WHO E. coli LPS 2nd International Standard 94/580; USP
RSE E. coli LPS Lot G3E069; USP RSE E. coli Lot G; FDA E. coli Lot EC6)

3.4

144

Solutions•

RPMI-1640 cell culture medium

145

4.0

ASSAY PREPARATION146

All test substances, endotoxin, and endotoxin-spiked solutions should be stored at 4°C.

147

4.1

148

An internationally harmonized RSE or equivalent is used to generate the endotoxin standard

149

curve. The use of any other E. coli LPS requires calibration against a RSE using the Cryo150

WB/IL-1β test method.

151

A standard endotoxin curve consisting of a Normal Saline Control (NSC) and five RSE

152

concentrations (0.25, 0.50, 1.0, 2.5, and 5.0 EU/mL) are included in the incubation step (refer153

to Table 4-1) and then transferred to the ELISA plate. To prepare the endotoxin standard

154

curve, first obtain a 2000 EU/mL stock solution by addition of PFW to the lyophilized

155

content of the stock vial by following the instructions provided by the manufacturer (e.g., for156

a vial containing 10,000 EU, 5 mL of PFW is added). To reconstitute the endotoxin, the stock

157

vial should be vortexed vigorously for at least 30 min or sonicated in a bath sonicator for 5

158

min. The stock solution is stable for 14 days when stored at 2 to 8°C. An endotoxin standard

159

curve is prepared by making serial dilutions of the stock solution in PFS as described in160

Table 4-1.

Endotoxin Standard Curve

161

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161
162

Table 4-1

Preparation of Endotoxin Standard Curve

Endotoxin
Concentration
EU/mL
20001,2
50
1950
503
50
100
900
5.0
5.0
500
500
2.5
2.5
400
600
1.0
1.0
500
500
0.50
0.50
500
500
0.25
0
0
1000
0
Abbreviations: EU = Endotoxin units; PFS = Pyrogen-free saline
Each stock tube should be vortexed vigorously prior to its use to make the subsequent dilution.
1
A 2000 EU/mL stock solution of endotoxin is prepared according to the manufacturer's instructions.
2
The stock solution of USP RSE may be stored in aliquots and kept at -20°C for up to 6 months. Do not store
the endotoxin at -80°C.
3This concentration is not used in the assay.
Stock Endotoxin
EU/mL

163
164
165
166
167
168
169

µL of Stock
EndotoxinµL of PFS

170

4.2

Test Substances

171

Liquid test substances should be tested neat or, if interference is detected (see Section 4.2.1),

172

diluted in PFS. Solid test substances should be prepared as solutions in PFS or, if insoluble in173

saline, dissolved in dimethylsulfoxide (DMSO) then diluted up to 0.5% (v/v) with PFS,

174

provided that this concentration does not interfere with the assay. The test substances should

175

be vortexed vigorously for at least 30 min or sonicated in a bath sonicator for 5 min.

176

4.2.1

177

Interference testing must be carried out on any test sample for which no interference

178

information is available. The purpose of the interference test is to determine the lowest

179

dilution (i.e., highest concentration) of a test substance from which an endotoxin spike can be

180

detected (i.e., based on the decision criteria described in 4.2.1.2). However, to ensure a valid

181

test, a test substance should not be diluted beyond its Maximum Valid Dilution (MVD).

182

For many marketed products, values for the MVD and the Endotoxin Limit Concentration

183(ELC) are published in the U.S. Pharmacopeia, the European Pharmacopoeia, and/or Food

184

and Drug Administration (FDA) guidelines. However if one or both of these values are not

185

available, then calculation of the MVD is dependent on the ELC (see Section 12.3). If

186

unknown, the ELC can be approximated by dividing the maximum hourly dose of theInterference Testing

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187

product by the hourly dose received per patient. For example, if a product is used at an

188hourly dose of 100 mg per patient, then the ELC would be 350 EU/100 mg or 3.5 EU/mg.

1894.2.1.1

190

The WHO-LPS 94/580 [E. coli O113:H10:K-] or equivalent international RSE (e.g., USP

191

G3E069, FDA EC-6) is recommended for preparation of the endotoxin control (EC). If a

192

different E. coli LPS is used and the potency relative to the RSE is not provided, then each193

lot must be calibrated against the RSE in the Cryo WB/IL-1β test method. For interference194

testing, an endotoxin standard curve (see Section 4.1) should be included on each plate.

195

4.2.1.2

196

For interference testing, non-spiked and endotoxin-spiked test substances are prepared in197

microplate wells (n=4 replicates) and an in vitro pyrogen test is performed. Either RPMI or a

198

fixed concentration (a concentration selected from the middle of the EC standard curve) of

199

the RSE (i.e., 1 EU/mL) in RPMI is added to the test substance in serial two-fold dilutions.

200

An illustrative example of endotoxin spiking solutions is shown in Table 4-2. For non-spiked

201

solutions, 200 µL of RPMI is added to a well followed by 20 µL of the test substance (neat or

202

at serial dilution) and 20 µL of WB. Endotoxin-spiked solutions are prepared by adding 180

203µL of RPMI to each well followed by 20 µL of the test substance (neat or at serial dilution)204

and 20 µL of WB. Then, 20 µL of a 1 EU/mL solution of endotoxin in RPMI is added and205

the well contents are mixed (see example presented in Table 4-2).

Reference Endotoxin for Spiking Test Substances

Spiking Test Substances with Endotoxin

206

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206
207

Table 4-2

Preparation of Endotoxin-Spiked and Non-spiked Solutions for

208

Determination of Test Substance Interference in the Incubation and

209

ELISA Test Systems
SpikedSample Addition

Non-spiked1µL/well

210
211
212
213
214
215
216

RPMI
Endotoxin spike solution2
Test substance (neat and each serial dilution)
Cryo WB
Total3Abbreviations: Cryo WB = Cryopreserved whole blood1
n=4 replicates each2
Endotoxin concentration is 1.0 EU/mL in RPMI.
3
A total volume of 240 µL per well is used for the incubation.

180
20
20
20
240

200
0
20
20
240

The lowest dilution of the test substance that yields an endotoxin spike recovery of 50% to217

200% in the pyrogen test is determined. The optical density (OD) values of the endotoxin-

218

spiked and non-spiked test substances are calibrated against the endotoxin calibration curve.

219

The resulting EU value of the non-spiked test substance is subtracted from the corresponding

220

EU value of the endotoxin-spiked test substance at each dilution. The % recovery for each221

sample dilution is then determined from the endotoxin spike solution concentration set to222

100%. For example, consider the following interference test results in Table 4-3:

223

Table 4-3

224

Example of Interference Data Used to Determine Sample Dilution for
Assay

Sample Dilution
None
1:2
1:4
1:8

% Recovery of Endotoxin Control
25
49
90
110

225
226
227

Based on these results, the dilution of the test substance used in the in vitro pyrogen test

228

would be 1:4 (i.e., the lowest dilution between 50% and 200% of the 1 EU/mL EC).

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229

4.2.2

230

If the data obtained from the experiment in Section 4.2.1 suggests the presence of231interference, then a subsequent experiment similar to that described in Section 4.2.1 would

232

need to be performed to confirm that the test substance(s) does not directly interfere with the233

ELISA. For this experiment, an ELISA would be performed in the absence of Cryo WB.

234

5.0

CONTROLS235

5.1

Negative Control

236

A negative control (e.g., PFS is added instead of the test sample) is included in each237

experiment in order to detect nonspecific changes in the test system, as well as to provide a

238baseline for the assay endpoints.

239

5.2

240

Solvent controls are recommended to demonstrate that the solvent is not interfering with the241test system when solvents other than PFS are used to dissolve test substances.

242

5.3

243

An internationally standardized EC (e.g., WHO 94/580; USP G3E069; 0.5 EU/mL) is

244

included in each experiment to verify that an appropriate response is induced.

245

5.4

246

Benchmark controls may be used to demonstrate that the test method is functioning properly,

247

or to evaluate the relative pyrogenic potential of chemicals (e.g., parenteral pharmaceuticals,

248

medical device eluates) of a specific class or a specific range of responses, or for evaluating249

the relative pyrogenic potential of a test substance. Appropriate benchmark controls should250

have the following properties:

251

Interference with ELISA System

Solvent Control

Positive Control

Benchmark Controls•

252

consistent and reliable source(s) for the chemicals (e.g., parenteralpharmaceuticals, medical device eluates)

253•

structural and functional similarities to the class of substance being tested

254•known physical/chemical characteristics
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255•

supporting data on known effects in animal models

256•

known potency in the range of response

01 Dec 2006

257

5.5

Positive Product Control (PPC)258

The PPC is a test substance diluted to a level that does not interfere with the test method and259

does not exceed the MVD. The PPC is obtained by spiking a test substance with a known EC

260(e.g., 1 EU/mL) and demonstrating that 50% to 200% of the EC is recovered.

261

5.6

262

The NPC is the test substance diluted to the MVD and then spiked with PFS. It is the263

negative control for the PPC.

264

6.0

EXPERIMENTAL DESIGN

265

6.1

Incubation with Test Samples and Measurement of IL-1β Release

266

6.1.1

Collection of Human Blood267

WB is obtained from healthy human volunteers who have provided their consent according268

to established institutional guidelines. Volunteers are expected not to have taken any drugs

269

and to have been free from illness for at least two weeks prior to donation. The criteria for

270

rejection of data from donors that are low responders or that are suspect due to veracity of

271

health information is addressed in Section 8.0.

272Fresh WB is drawn by venipuncture using a multifly needle set and collected in heparinized273

tubes (15 IU/mL lithium heparin). All components of the blood collection system (i.e.,274

syringes, tubes, connecting lines) must be sterile and pyrogen-free.

275

6.1.2

276

Two methods are available for cryopreservation of blood 1) The PEI method developed at the277

Paul Ehrlich Institute and 2) The Konstanz method developed at the University of Konstanz.

Negative Product Control (NPC)

Cryopreservation Procedure

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278

6.1.3

PEI Method of Cryopreservation

279

In the PEI method (Schindler et al., 2006), heparinized WB pooled from five donors is frozen

280

at -80°C in a cryoprotective phosphate buffer (Soerensen's) containing 10% (v/v) pyrogen-281

free, clinical-grade DMSO in cryotubes.

282

6.1.3.1

283

In the Konstanz method (Schindler et al., 2004), pyrogen-free, clinical grade DMSO is added284

directly to the blood of individual donors at a final concentration of 10% (v/v). The blood is285

then pooled and 1.2 mL aliquots are placed in cryotubes. The blood is frozen in a computer-286

controlled freezer using several cycles of programmed freezing down to -120°C. Tubes of287

blood are then removed from the instrument and placed in liquid nitrogen.

288

6.1.3.2

289

Calculate the volume of Cryo WB needed to carry out the assay (20 µL/well or 1.92 mL/96-

290

well plate) and remove a sufficient number of aliquots from the freezer. Place the tubes in an

291

incubator at 37±1°C and allow them to thaw for 15 min. In a laminar flow hood, unscrew the292caps and pool the Cryo WB in a centrifuge tube. Mix the tubes by gentle inversion. Do Not

293

Vortex.

294

6.1.4

295

Test substances are prepared at a level of dilution that did not show interference with the test

296

system or for which it is known that interference does not occur. Each incubation plate can297

accommodate an endotoxin standard curve, a NSC, and 14 test samples (see Table 6-1).

Konstanz Method of Cryopreservation

Thawing Procedure

Incubation Plate

298

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298
299

Table 6-1

300

Overview of Incubation Plate Preparation in the Cryo WB/IL-1β Test
Method (PEI Method)

Mix thesamples;
RPMI
EC
Cryo WB
NumberSampleimmediatelyof Wells
transfer to an
ELISA plate4µL2
and run
20
EC
180
20
0
40
ELISA
or
4
NSC
220
0
0
20
store
plate
at
Test
-20°C or 563
samples
200
0
20
20
80°C.
(1-14)
Abbreviations: EC = Endotoxin control; NSC = Negative saline control; Cryo WB = Cryopreserved wholeblood
1
For the Konstanz method of cryopreservation, 20 µL of Cryo WB is used and the volume of RPMI is adjusted
to 200 µL.
2Five EC concentrations (0.25, 0.50, 1.0, 2.5, 5.0 EU/mL) in quadruplicate.
3
14 test samples (n=4) per plate.
4
An IL-1β standard curve is prepared in Columns 11 and 12 on the ELISA plate. Therefore, 80 wells areavailable for test samples and controls on the incubation plate.
Test
Sample

301
302
303
304
305
306
307
308
309

1

Mix thesamples;incubateovernight at
37±1°C in ahumidified
atmospherewith 5%
CO2.

310

6.1.5

Incubation Assay for IL-1β Release

311

Blood samples are prepared in a microtiter plate using a laminar flow hood. All consumables

312

and solutions must be sterile and pyrogen-free. Each plate should be labeled appropriately

313

with a permanent marker. An overview of the incubation plate preparation is shown in Table

314

6-1. The incubation procedure is outlined below:

315

Step 1. Refer to the incubation plate template presented in Table 6-2.

316

Step 2. Using a pipetter, transfer either 200 or 180 µL of RPMI into each well (for317

the Konstanz or PEI method of cryopreservation, respectively – refer to Step 5318

below).

319

Step 3. Transfer 20 µL of test sample into the appropriate wells as indicated in the320

template.

321

Step 4. Transfer 20 µL of the EC (standard curve) and the NSC controls in

322

quadruplicate into the appropriate wells according to the template.

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323

Step 5. Transfer either 20 or 40 µL of Cryo WB (for the Konstanz or PEI method324

of cryopreservation, respectively) into each well and mix by gently swirling the325

plate.

326

Step 6. Mix the contents of the wells thoroughly by gently pipetting up and down

327

five times using a multichannel pipetter, changing the tips between each row in

328

order to avoid cross-contamination.

329

Step 7. Place the covered plate in a tissue culture incubator for 10 to 24 hr at

330

37±1°C in a humidified atmosphere containing 5% CO2.

331

Step 8. If using the Konstanz method, freeze the plate at -20°C or -80°C until the332

contents of the well are completely frozen and then, thaw the plate at RT or in a333

water bath not exceeding 37°C.

334

Step 9. Prior to transferring the test samples onto the ELISA plate, mix the335

contents of the wells by pipetting up and down three times using a multichannel

336

pipetter, changing the tips between each row in order to avoid cross-contamination.

337

Note: The aliquots may be tested immediately in the ELISA or stored at -20°C or

338-80°C for testing at a later time. After transfer to the ELISA plate, freeze the339

remaining aliquots at -20°C or -80°C for subsequent experiments, if necessary (see340

Assay Acceptability and Decision Criteria in Sections 8.0 and 9.0).

341

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341
342

Table 6-2
1

Incubation Plate - Sample and Control Template
2

3

4

5

6

7

8

9

10

11

EC1
EC
EC
EC
TS3
TS3
TS3
TS3
TS11 TS11
Void3
5.0
5.0
5.0
5.0
EC
EC
EC
EC
B
TS4
TS4
TS4
TS4
TS11 TS11
Void
2.5
2.5
2.5
2.5
EC
EC
EC
EC
C
TS5
TS5
TS5
TS5
TS12 TS12
Void
1.0
1.0
1.0
1.0
EC
EC
EC
EC
D
TS6
TS6
TS6
TS6
TS12 TS12
Void0.50 0.50 0.50
0.50
EC
EC
EC
EC
E
TS7
TS7
TS7
TS7
TS13 TS13
Void0.25 0.25 0.25
0.25
F NSC NSC NSC
NSC
TS8
TS8
TS8
TS8
TS13 TS13
Void
G TS12 TS1
TS1
TS1
TS9
TS9
TS9
TS9
TS14 TS14
Void
H
TS2 TS2
TS2
TS2
TS10
TS10
TS10
TS10
TS14 TS14
Void
Abbreviations: EC = Endotoxin control; NSC = Negative saline control; TS = Test substance1
EC value (e.g., EC 5.0) represents the endotoxin concentration in EU/mL.
2
TS number (e.g., TS1) represents an arbitrary sequence for individual test substances.
3
Columns 11 and 12 are reserved for the IL-1β standard curve on the ELISA plate (see Table 6-3).
A

343
344
345
346
347
348

12
Void
Void
Void
Void
Void
Void
Void
Void

349

6.2

ELISA to Measure IL-1β Release

350

6.2.1

IL-1β Standard Curve

351

An IL-1β standard, supplied with the ELISA kit, is used. IL-1β standards are typically352

supplied in lyophilized form and should be reconstituted according to the manufacturer's

353

instructions. The stock solution should be diluted in RPMI to the following concentrations: 0,354

62.5, 125, 250, 500, 1000, 2000, and 4000 pg/mL. Each well on the ELISA plate will receive355

100 µL of an IL-1β blank or standard.

356

6.2.2

357

The manufacturer's instructions provided with the ELISA kit should be followed and a358

typical experimental design is outlined below. If the user prefers to prepare an in-house

359

ELISA, then appropriate modification and validation of these changes would be necessary.

360

The ELISA should be carried out at room temperature (RT) and therefore all components

361

must be at RT prior to use. Do not thaw frozen specimens by heating them in a water bath. A

362suggested ELISA plate template is shown in Table 6-3, which includes a five-point EC

363

standard curve, a NSC, an eight-point IL-1β standard curve (0 to 4000 pg/mL), and 14 test

ELISA

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364

substances in quadruplicate. The EC standard curve, the NSC, and the test sample

365

supernatants are transferred directly from the incubation plate. The IL-1β standard curve is366

prepared as described in Section 6.2.1. An overview of the ELISA plate preparation is shown367

in Table 6-4.

368

Step 1. Add 100 µL of enzyme-labeled detection antibody to each well.

369

Step 2. After pipetting up and down three times to mix the supernatant, transfer

370

100 µL from each well of the Incubation Plate (A1-10; H1-10) to the ELISA plate.

371

Step 3. Add 100 µL of each IL-1β standard (0 to 4000 pg/mL) into the respective372

wells on the ELISA plate.

373

Step 4. Cover the microtiter plate(s) with adhesive film and incubate for 90 min on374

a microplate mixer at 350-400 rpm at 20 to 25°C.

375

Step 5. Decant and wash each well three times with 300 µL Buffered Wash

376

Solution and then rinse three times with deionized water. Place the plates upside

377

down and tap to remove water.

378

Step 6. Add 200 µL of TMB/Substrate Solution to each well and incubate at RT in379

the dark for 15 min. If necessary, decrease the incubation time.

380

Step 7. Add 50 µL of Stop Solution to each well.

381

Step 8. Tap the plate gently after the addition of Stop Solution to aid in mixing.

382

Step 9. Read the OD450 within 15 min of adding the Stop Solution. Measurement383

with a reference wavelength of 600-690 nm is recommended.

384

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384
385

Table 6-3

ELISA Plate - Sample and Control Template

1

2

3

4

EC1
5.0
EC
2.5
EC
1.0
EC
0.50
EC
0.25

EC
5.0
EC
2.5
EC
1.0
EC
0.50
EC
0.25

EC
5.0
EC
2.5
EC
1.0
EC
0.50
EC
0.25

EC
5.0
EC
2.5
EC
1.0
EC
0.50
EC
0.25

F

NSC

NSC

NSC

G

TS12

TS1

H

TS2

TS2

A
B
C
D
E

5

6

7

8

9

10

TS3

TS3

TS3

TS3

TS11

TS11

TS4

TS4

TS4

TS4

TS11

TS11

TS5

TS5

TS5

TS5

TS12

TS12

TS6

TS6

TS6

TS6

TS12

TS12

TS7

TS7

TS7

TS7

TS13

TS13

NSC

TS8

TS8

TS8

TS8

TS13

TS13

TS1

TS1

TS9

TS9

TS9

TS9

TS14

TS14

TS2

TS2

TS10

TS10

TS10

TS10

TS14

TS14

386
387
388
389
390
391

Abbreviations: EC = Endotoxin control; NSC = Negative saline control; TS = Test substance1
EC value (e.g., EC 5.0) represents the endotoxin concentration in EU/mL.
2
TS number (e.g., TS1) represents an arbitrary sequence for individual test substances.
3
IL-1β values in columns 11 and 12 are in pg/mL.

392

Table 6-4

Overview of ELISA Procedure

Enzymelabeled
Antibody(µL)

MaterialtransferfromIncubation
Plate (µ L)

100

100

Incubate 90
min on aplate mixer
at 350-400rpm.

TMB/Substrate
Solution(µL)

200

Incubate 15
min at RT in
dark.

Stop
Solution
(µL)

11

12

IL-1β3
0
IL-1β
62.5
IL-1β
125
IL-1β
250
IL-1β
500
IL-1β
1000
IL-1β
2000
IL-1β
4000

IL-1β
0
IL-1β
62.5
IL-1β
125
IL-1β
250
IL-1β
500
IL-1β
1000
IL-1β
2000
IL-1β
4000

Read opticaldensity at 450
nm with a 600690 nm
wavelengthreference filter.

50

393
394

Abbreviations: RT = Room temperature

395

7.0

EVALUATION OF TEST RESULTS

396

7.1

OD Measurements

397

The OD of each well is obtained by reading the samples in a standard microplate398

spectrophotometer (i.e., plate reader) using a visible light wavelength of 450 nm (OD450) with
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399

a 600 to 690 nm reference filter (recommended). OD450 values are used to determine assay

400

acceptability and in the decision criteria for the detection of endotoxin in a test substance (see401

Sections 8.0 and 9.0).

402

8.0

403

Obtain the PPC and the corresponding NPC by interference testing of a test substance in the404

presence and absence of a fixed quantity of endotoxin (i.e., 1.0 EU/mL) in quadruplicate. An

405

EC (five-point standard curve) and a NSC should be included in each experiment. An IL-6

406

standard curve should be included in each ELISA as shown in the template presented in

407

Table 6-3. An assay is considered acceptable only if the following minimum criteria are met:

408

CRITERIA FOR AN ACCEPTABLE TEST•409

The quadratic function of the IL-1β standard curve produces an r ≥ 0.95 andthe OD450 of the blank control is below 0.15.

410

•

411

The endotoxin standard curve produces OD450 values that ascend in asigmoidal concentration response.

412

9.0

DATA INTERPRETATION/DECISION CRITERIA

413

9.1

Decision Criteria for Determination of Pyrogenicity

414

The t-test is used to compare the data of a test sample against the data of the EC (0.5 EU/mL)

415

that is performed in parallel. If this test results in a significant p-value (i.e., smaller than 1%),416

then the sample is considered to be non-pyrogenic, and as pyrogenic if otherwise (Hoffmann417

et al., 2005), as long as the assay acceptability criteria in Section 8.0 has been met.

418

10.0

419

The test report should include the following information:

420

Test Substances and Control Substances

STUDY REPORT

421•

Name of test substance422•

Purity and composition of the substance or preparation

423•

Physicochemical properties (e.g., physical state, water solubility)

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424•

Treatment of the test/control substances prior to testing (e.g., vortexing,

425

sonication, warming, resuspension solvent)426

Justification of the In Vitro Test Method and Protocol Used

427

Test Method Integrity

428

•

429

The procedure used to ensure the integrity (i.e., accuracy and reliability) of thetest method over time

430

•

431

If the test method employs proprietary components, documentation on theprocedure used to ensure their integrity from “lot-to-lot” and over time

432

•

433
434

01 Dec 2006

The procedures that the user may employ to verify the integrity of the
proprietary components

Criteria for an Acceptable Test

435•

Acceptable concurrent positive control ranges based on historical data

436•

Acceptable negative control data

437

Test Conditions

438•

Cell system used

439•

Calibration information for the spectrophotometer used to read the ELISA

440•

Details of test procedure

441•

Description of any modifications of the test procedure

442•

Reference to historical data of the model

443•

Description of evaluation criteria used•

Tabulation of data from individual test samples

444
445

Results

446

Description of Other Effects Observed

447

Discussion of the Results

448

Conclusion
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449

01 Dec 2006

A Quality Assurance Statement for Good Laboratory Practice (GLP)-Compliant Studies

450•This statement should indicate all inspections made during the study and the451

dates any results were reported to the Study Director. This statement should452

also confirm that the final report reflects the raw data.

453

If GLP-compliant studies are performed, then additional reporting requirements provided in454

the relevant guidelines (e.g., OECD 1998; EPA 2003a, 2003b; FDA 2003) should be455

followed.

456

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456
457

11.0

REFERENCES

458

EPA. 2003a. Good Laboratory Practice Standards. Toxic Substances Control Act. 40 CFR

459

792.

460

EPA. 2003b. Good Laboratory Practice Standards. Federal Insecticide, Fungicide, and461

Rodenticide Act. 40 CFR 160.

462

FDA. 2003. Good Laboratory Practices for Nonclinical Laboratory Studies. 21 CFR 58.

463

Fennrich S, Wendel A, Hartung T. 1999a. New applications of the human whole blood464

pyrogen assay (PyroCheck). ALTEX 16:146-149.

465

Fennrich S, Fischer M, Hartung T, Lexa P, Montag-Lessing T, Sonntag H-G, Weigand M,

466

Wendel A. 1999b. Detection of endotoxins and other pyrogens using human whole blood.

467

Dev Biol Stand. 101:131-139.

468

Hartung T, Wendel A. 1999. Detection of pyrogens using human whole blood. In Vitro

469

Toxicol. 9(4):353-359.

470

Hoffmann S, Peterbauer A, Schindler S, Fennrich S, Poole S, Mistry Y, Montag-Lessing T,

471

Spreitzer I, Löschner B, van Aalderen M, Bos R, Gommer M, Nibbeling R, Werner-

472

Felmayer G, Loitzl P, Jungi T, Brcic M, Brügger P, Frey E, Bowe G, Casado J, Coecke S, de473

Lange J, Mogster B, Næss LM, Aaberge IS, Wendel A, Hartung T. 2005. International474

validation of novel pyrogen tests based on human monocytoid cells. J Immunol Methods

475

298:161-173.

476

Organization for Economic Cooperation and Development (OECD). 1998. OECD Series on

477

Principle of Good Laboratory Practice and Compliance Monitoring. No. 1. OECD Principles

478

of Good Laboratory Practice (as revised in 1997). Organisation for Economic Co-operation

479

and Development (OECD), ENV/MC/CHEM (98)17. Paris: OECD.

480

Schindler S, Asmus S, von Aulock S, Wendel A, Hartung T, Fennrich S. 2004.

481

Cryopreservation of human whole blood for pyrogenicity testing. J Immunol Methods

482

294:89-100.

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483

Schindler S, Spreitzer I, Löschner B, Hoffmann S, Hennes K, Halder M, Brügger P, Frey E,

484

Hartung T, Montag T. 2006. International validation of pyrogen tests based on cryopreserved

485

human primary blood cells. J Immunol Methods 316:42-51.

486

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486
487

12.0

TERMINOLOGY AND FORMULA

488

12.1

Assay Sensitivity (λ)

489

For an in vitro cell-based assay, the variable λ is defined as the lowest statistically significant

490

point on the standard endotoxin concentration-response curve and represents the relative491

sensitivity of the test method for the detection of endotoxin (i.e., level of detection).

492

12.2

493

The EC is incubated with Cryo WB and serves as the positive control for the experiment. The

494

results should be compared to historical values to insure that it provides a known level of495

cytokine release relative to the NSC.

496

12.3

497

The ELC is the maximum allowable concentration of endotoxin for a particular product and498

is expressed in EU per volume (mL) or weight (mg). The ELC is defined by the FDA or

499

specified in the USP2. It is calculated as the product of K/M, where:500

K is the threshold pyrogen dose for parenteral use in rabbits or humans (5.0 EU/kg). At an

501

injection volume of 10 mL/kg, K is equal to 0.5 EU/mL.

502

M is the larger of the rabbit dose or the maximum human dose administered in one hour as

503

defined below and varies with test substance3.

504

12.4

505

The MVD is the maximum dilution of a test substance that can be tolerated in a test system506

without exceeding the ELC, if the test substance must be diluted as a result of assay

507

interference. Dilutions beyond the MVD would not be valid for endotoxin detection in the2

Endotoxin Control (EC)Endotoxin Limit Concentration (ELC)

Maximum Valid Dilution (MVD)

ELC values for most marketed pharmaceutical products are provided in the USP or in other pharmacopoeial or
regulatory publications (e.g., European Pharmacopoeia, Japanese Pharmacopoeia, Pharmacopoeial guidelines,
FDA publications).
3
Values for most marketed pharmaceutical products are provided in the USP or in other pharmacopoeial or
regulatory publications (e.g., European Pharmacopoeia, Japanese Pharmacopoeia, pharmacopoeial guidelines,
FDA publications).
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508

test system. Calculation of the MVD is dependent on whether or not the ELC for a test

509

substance is published. When the ELC is known, the MVD is:

510

MVD = (ELC x Product Potency [PP])/λ511

As an example, for "Cyclophosphamide Injection," the ELC is 0.17 EU/mg, PP is 20

512

mg/mL, and the assay sensitivity is 0.1 EU/mL. The calculated MVD would be 34. The test

513

substance can be diluted no more than 1:34 prior to testing.

514

If the ELC is not known, the MVD is:515

MVD = PP/Minimum Valid Concentration (MVC)516

where, MVC = (λ x M)/K

517

where, M is the maximum human dose518

As an example, for "Cylophosphamide Injection," the PP is 20 mg/mL, M is 30

519

mg/kg, and assay sensitivity is 0.1 EU/mL. The calculated MVC is 0.6 mg/mL and the MVD

520

is 33.3. The test substance can be diluted no more than 1:33 in the assay prior to testing.

521

12.5

522

The NPC is a test sample to which PFS is added. The NPC is the baseline for determination523

of cytokine release relative to the endotoxin-spiked PPC.

524

12.6

525

The NSC is Cryo WB (in RPMI) incubated with PFS (used for dilution of test substance) and526

is used as the blank.

527

12.7

528

The value K represents the threshold pyrogen dose for parenteral products for rabbits and529

humans. Based on experimental data, K is fixed at 5.0 EU/kg. For intrathecal products, K is

530

0.2 EU/kg.

531

12.8

532

The PPC is a test substance spiked with the control standard endotoxin (i.e., 0.5 EU/mL or an

533

amount of endotoxin equal to that which produces ½ the maximal increase in OD from the

Negative Product Control (NPC)

Negative Saline Control (NSC)

Parenteral Threshold Pyrogen Dose (K)Positive Product Control (PPC)

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534

endotoxin standard curve) to insure that the test system is capable of endotoxin detection in535

the product as diluted in the assay.

536

12.9

537

The concentration for a test substance is the PP typically expressed as µg/mL or mg/mL.

538

12.10

539

The variable M represents the rabbit test dose or the maximum human dose in 1 hr. The

540

variable M is expressed in mg/kg and varies with the test substance. For

541

radiopharmaceuticals, M should be adjusted to account for product activity (radioactive

542

decay) at time administration. An average human standard weight of 70 kg is used for the543

calculation. If a pediatric dose should be used and it is higher than the adult dose, then it

544

should be used in the formula.

Product Potency (PP)

Rabbit Pyrogen Test Dose or Maximum Human Dose (M)

545
546

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Appendix B2

Draft ICCVAM Recommended Protocol for Future Studies Using the Mono Mac 6(MM6)/Interleukin-6 (IL-6) Test Method

Draft ICCVAM Recommended Test Method Protocol: MM6/IL-6 Pyrogen Test

[This Page Intentionally Left Blank]

01 Dec 2006

Draft ICCVAM Recommended Test Method Protocol: MM6/IL-6 Pyrogen Test

01 Dec 2006

1

Draft ICCVAM Recommended Protocol for Future Studies Using the Mono Mac 62(MM6)/Interleukin-6 (IL-6) Test Method

3
4

PREFACE

5
6This proposed protocol for the detection of pyrogenicity is based on information obtained

7

from 1) The European Centre for the Validation of Alternative Methods (ECVAM) MM6/IL-

8

6 Background Review Document (BRD) presented in Appendix A of the draft Interagency

9

Coordinating Committee on the Validation of Alternative Methods (ICCVAM) Pyrogenicity

10

Test Method BRD, which includes ECVAM Standard Operating Procedures (SOPs) for the11

MM6/IL-6 test method, and 2) Information provided to the National Toxicology Program

12(NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods

13(NICEATM) by Dr. Thomas Hartung, Head of ECVAM. The ECVAM SOPs are based on

14

the method published by Taktak et al. (1991). A table of comparison between the draft

15

ICCVAM recommended protocol and the ECVAM SOPs is provided in Table 1. Future

16

studies using the MM6/IL-6 test method may include further characterization of the17

usefulness or limitations of the assay for regulatory decision-making. Users should be aware

18

that the proposed test method protocol might be revised based on additional optimization

19

and/or validation studies. ICCVAM recommends that test method users routinely consult the20

ICCVAM/NICEATM website (http://iccvam.niehs.nih.gov/) to ensure that the most current

21

test method protocol is used.

22

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22
23
24
25
26
27
28
29
30
31
32
33
34
35

[This Page Intentionally Left Blank]

36
37
38

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01 Dec 2006

Draft ICCVAM Recommended Test Method Protocol: MM6/IL-6 Pyrogen Test

39
40

Table 101 Dec 2006

Comparison of Draft ICCVAM Recommended Test Method Protocol with the ECVAM SOP for the Mono Mac
6 (MM6)/Interleukin-6 (IL-6) Pyrogen Test Method

Protocol Component
Test Substance

Incubation Plate(number of control or test groups
at n=4 each)

ELISA Plate
Decision Criteria for Interference

Draft ICCVAM Protocol
Test neat or at minimal dilution that
produces no interference
NSC (1)EC (5)
TS (14)
PPC1 (0)
NPC1 (0)PC (0)NC (0)
Includes seven point IL-6 SC and blankin duplicate
0.5 x Median OD4502 of 1 EU/mL EC<2x Median OD450 of 1 EU/mL EC
Mean OD4502 of PPC is 50% to 200% of
0.5 EU/mL EC
Mean OD450 of NSC ≤0.15
Not included

Assay Acceptability Criteria

Not included
Quadratic function of IL-6 SC r2 ≥0.95

Decision Criteria for Pyrogenicity

ECVAM SOP

ECVAM Validation SOPSame as ICCVAM protocol

Test at MVD

Same as ICCVAM protocol
Same as ICCVAM protocol
Same as ICCVAM protocol
Same as ICCVAM protocol
Same as ICCVAM protocol
Same as ICCVAM protocol
Same as ICCVAM protocol

Same as ICCVAM protocol
Same as ICCVAM protocol
TS (2) x EC (5) spikes
PPC (2)NPC (2)
PC (1)NC (1)

Same as ICCVAM protocol

Same as ICCVAM protocol

Same as ICCVAM protocol

Not applicable (tested at MVD)

Same as ICCVAM protocol

Same as ICCVAM protocol

Same as ICCVAM protocol
Outliers rejected using Dixon's text(p≥0.5)
Mean OD450 EC > NSC (2SD with n1 weighting)Same as ICCVAM protocol

Mean OD450 of NSC ≤0.20
Outliers rejected using Dixon's text(p≥0.5)EC SC produces OD450 values thatascend in a sigmoidal concentration
response

Same as ICCVAM protocol

Mean OD4502 of TS > Mean OD450 of
0.5 EU/mL EC

EC SC data transformed to 4parameter logistical model by an inhouse program and the linear meansquare is calculated. TS pyrogencontent is compared with the ELC4
using confidence limits for
significance. Dixon's test is used toreject outliers.

B2-3

Mean OD450 PC>LOQ3
Same as ICCVAM protocol
Mean OD450 of each EC > Mean OD450
of next lower EC concentration(minimum of 4 data points needed forvalid SC)EC SC data transformed to 4parameter logistical model by an inhouse program and the linear meansquare is calculated. TS pyrogencontent is compared with the ELC4
using confidence limits for
significance. Dixon's test is used toreject outliers.

Draft ICCVAM Recommended Test Method Protocol: MM6/IL-6 Pyrogen Test

41
42
43
44
45
46
47
48
49
50

01 Dec 2006

Abbreviations: EC = Endotoxin control; ELC = Endotoxin Limit Concentration; LOQ = Limit of Quantification; MVD = Maximum Valid Dilution; NC =Negative Control; NPC = Negative Product Control; NSC = Normal saline control; PC = Positive control; PPC = Positive Product Control; SC = Standard curve;
SD = Standard Deviation; TS = Test substance1
PPC and NPC are evaluated during the interference test.
2
Median or mean OD450 values are corrected (i.e., reference filter reading, if applicable, and NSC are subtracted).
3
LOQ is the mean OD450 of the NSC + 10xSD mean OD450 of the NSC.
4
Where unknown, the ELC is calculated (e.g., Based on a rabbit sensitivity of 5 EU/kg, for a product injected at 10 mL/kg, the detection limit is 5 EU/10 mL/kg
or 0.5 EU/mL/kg, or an ELC of 0.5 EU/mL).

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51

1.0

52

The purpose of this protocol is to describe the procedures used to evaluate the presence of a53

pyrogen (i.e., Gram-negative endotoxin) in a test substance. The presence of Gram-negative

54

endotoxin is detected by its ability to induce cytokine IL-6 release from MM6 cells. The

55

quantity of IL-6 released is obtained using an enzyme-linked immunosorbent assay (ELISA)

56

that includes monoclonal or polyclonal antibodies specific for IL-6. Release of this cytokine

57

is measured by incubation of the MM6 cells with test substances or controls (i.e., positive,

58

negative). The amount of pyrogen present is determined by comparing the values of59

endotoxin equivalents produced by MM6 cells exposed to the test substance to those exposed60

to an internationally-harmonized Reference Standard Endotoxin (RSE)1 or an equivalent

61

standard expressed in Endotoxin Units (EU)/mL. Based on a rabbit threshold pyrogen dose of62

0.5 EU/mL, which was established in a retrospective evaluation of rabbit pyrogen test (RPT)

63

data, a test substance is considered pyrogenic if it induces a level of IL-6 release equal to or

64

greater than 0.5 EU/mL.

65

The focus of this protocol is on the use of the MM6/IL-6 test method specifically for the66

detection of Gram-negative endotoxin in parenteral pharmaceuticals. The relevance and67

reliability for non-endotoxin pyrogens (e.g., lipoteichoic acid) has not been demonstrated in a68

formal validation study.

69

2.0

70

All procedures for procurement of eligible blood donors and blood donations should follow

71

the regulations and procedures set forth by institutional guidelines for utilization of human72

substances, which include but are not limited to blood, tissues, and tissue fluids. Standard

73

laboratory precautions are recommended, including the use of laboratory coats, eye

1

PURPOSE AND APPLICABILITY

SAFETY AND OPERATING PROCEDURES

RSEs are internationally-harmonized reference standards (e.g., WHO E. coli Lipopolysaccharide [LPS] 94/580[0113:H10:K-]; USP RSE Lot G3E069; FDA Lot EC-6). Equivalent endotoxins include commercially available
E. coli-derived LPS Control Standard Endotoxin (CSE) or other E. coli LPS preparations that have beencalibrated with an appropriate RSE.
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protection, and gloves. If necessary, additional precautions required for specific study

75

substances or hazardous chemicals will be identified in the Material Safety Data Sheet

76(MSDS).

77

The stop solution used in the ELISA kit is acidic and corrosive and should be handled with78

the proper personal protective devices. If this reagent comes into contact with skin or eyes,

79

wash thoroughly with water. Seek medical attention, if necessary.

80

Tetramethylbenzidine (TMB) solution contains a hydrogen peroxide substrate and 3, 3’, 5,81

5’-TMB. This reagent is a strong oxidizing agent and a suspected mutagen. Appropriate

82

personal protection should be used to prevent bodily contact.

83

Bacterial endotoxin is a toxic agent (i.e., can induce sepsis, shock, vascular damage,84

antigenic response) and should be handled with care. Skin cuts should be covered and85

appropriate personal protective devices should be worn. In case of contact with endotoxin,

86

immediately flush eyes or skin with water for at least 15 min. If inhaled, remove the affected87

individual from the area and provide oxygen and/or artificial respiration as needed. Skin88

absorption, ingestion, or inhalation may produce fever, headache, and hypotension.

89

3.0

MATERIALS, EQUIPMENT AND SUPPLIES

90

3.1

Source of Cells

91

The MM6 cell line is a human monocytic cell line originally described by Professor H.W.L.

92

Ziegler-Heitbrock (Institute for Immunology, University of Munich, Munich, Germany). A

93

Master Cell Bank and a Working Cell Bank have been established at the National Institute94

for Biological Standards and Control (NIBSC), from which the MM6 cells can be purchased.

95

3.2

96

For all steps in the protocol, excluding the ELISA procedure, the materials that will be in

97

close contact with samples (e.g., pipet tips, containers, solutions) should be sterile and free98

from detectable pyrogens.

Equipment and Supplies

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99

3.2.1

Utilization of MM6 cells

100

3.2.1.1

Equipment

101•

Centrifuge

102•

Hemacytometer

103•

Hood; Bio-safety, laminar flow (recommended)

104•

Incubator; cell culture (37±1°C + 5% CO2)105•

Inverted Microscope106•

pH meter

107•

Pipetter; multichannel (8- or 12-channel)

108•

Pipetters; single-channel adjustable (20, 200, and 1000 µL)

109•

Repeating pipetter

110•

Vortex mixer

111•

Water bath112

3.2.1.2

01 Dec 2006

Consumables

113•

Centrifuge tubes; nonpyrogenic, polystyrene (15 and 50 mL)

114•

Cryotubes; screw-cap (2 mL)

115•

Combitips; repeating pipetter (1.0 and 2.5 mL)

116•

Filters; sterile, 0.22 µm

117•

Flasks; tissue culture

118•

Phosphate buffered saline (PBS); sterile

119•

Pipets; sterile, glass

120•

Plates; microtiter, nonpyrogenic, 96-well, polystyrene, tissue culture

121•

Reaction tubes; polystyrene (1.5 mL)

122•

Reservoirs; fluid
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123

•

124

01 Dec 2006

RPMI-1640 cell culture medium; supplemented as described in Section 4.3 toyield RPMI-Complete (RPMI-C)

125•

Tips; pipetter, sterile, pyrogen-free (20 and 200 µL)

126•

Tubes; polystyrene

127

3.2.2

ELISA

128

3.2.2.1

Equipment

129•

Microplate mixer

130•

Microplate reader (450 nm with an optional reference filter in the range of

131

540-590 nm)

132•

Microplate washer (optional)

133•

Multichannel pipetter

134

3.2.2.2

Consumables

135•

Container; storage, plastic

136•

Deionized water; nonsterile

137•

Plates; microtiter, nonpyrogenic, 96-well, polystyrene, tissue culture

138•

Pyrogen-free water (PFW)139•

Reservoirs; fluid

140•

Tips; pipetter, nonsterile

141•

Tubes; polystyrene (12 mL)

142

3.2.2.3

ELISA Kit

143

An ELISA that measures IL-6 release from MM6 cells is used. A variety of IL-6 ELISA kits144

are commercially available and the IL-6 ELISA procedure outlined in this protocol is145

intended to serve as an example for using an ELISA kit. If the user prefers to prepare an in-

146

house ELISA, then additional reagents would be required. The IL-6 ELISA should be

147

calibrated using an IL-6 international reference standard (e.g., WHO 89/548) prior to use.

148

The IL-6 cytokine assay kits do not provide the RSE or endotoxin equivalent; therefore, thisB2-8

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149

reagent must be purchased separately. Results obtained using these products are subject to150

the assay acceptability and decision criteria described in Sections 8.0 and 9.0. IL-6 ELISA

151

kit components may include the following:

152

•

153

ELISA plates coated with anti-human IL-6 capture antibody; monoclonal or
polyclonal

154•

Buffered wash solution

155•

Dilution buffer

156•

Enzyme-labeled detection antibody

157•

Human IL-6 reference standard

158•

Pyrogen-free saline (PFS)159•

Stop solution

160•

TMB/substrate solution

161

3.3

162

Chemicals
•

163
164

Endotoxin (e.g., WHO E. coli LPS 2nd International Standard 94/580; USP
RSE E. coli LPS Lot G3E069; USP RSE E. coli Lot G; FDA E. coli Lot EC6)

3.4

165

Solutions•

RPMI-C cell culture medium

166

4.0

ASSAY PREPARATION167

All test substances, endotoxin, and endotoxin-spiked solutions should be stored at 4°C.

168

4.1

169

An internationally harmonized RSE or equivalent is used to generate the endotoxin standard

170

curve. The use of any other E. coli LPS requires calibration against a RSE using the171

MM6/IL-6 test method.

172

A standard endotoxin curve consisting of a Normal Saline Control (NSC) and five RSE

173

concentrations (0.125, 0.25, 0.50, 1.0, and 2.0 EU/mL) are included in the incubation step

Endotoxin Standard Curve

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174

(refer to Table 4-1) and then transferred to the ELISA plate. To prepare the endotoxin

175

standard curve, first obtain a 2000 EU/mL stock solution by addition of PFW to the176lyophilized content of the stock vial by following the instructions provided by the177

manufacturer (e.g., for a vial containing 10,000 EU, 5 mL of PFW is added). To reconstitute178

the endotoxin, the stock vial should be vortexed vigorously for at least 30 min or sonicated in179

a bath sonicator for 5 min. The stock solution is stable for 14 days when stored at 2 to 8°C.

180

An endotoxin standard curve is prepared by making serial dilutions of the stock solution in

181

PFS as described in Table 4-1.

182

Table 4-1

Preparation of Endotoxin Standard Curve

Stock Endotoxin
EU/mL

183
184
185
186
187
188
189

µL of Stock
EndotoxinµL of PFS

Endotoxin
Concentration
EU/mL
203
2.0
1.0
0.50
0.25
0.125
0

20001,2
20
1980
20
100
900
2.0
500
500
1.0
500
500
0.50
500
500
0.25
500
500
0
0
1000
Abbreviations: EU = Endotoxin units; PFS = Pyrogen-free saline
Each stock tube should be vortexed vigorously prior to its use to make the subsequent dilution.
1
A 2000 EU/mL stock solution of endotoxin is prepared according to the manufacturer's instructions.
2
The stock solution of USP RSE may be stored in aliquots and kept at -20°C for up to 6 months. Do not store
the endotoxin at -80°C.
3This concentration is not used in the assay.

190

4.2

Test Substances

191

Liquid test substances should be tested neat or, if interference is detected (see Section 4.2.1),

192

diluted in PFS. Solid test substances should be prepared as solutions in PFS or, if insoluble in

193

saline, dissolved in DMSO then diluted up to 0.5% (v/v) with PFS provided that this194

concentration does not interfere with the assay. The test substances should be vortexed

195

vigorously for at least 30 min or sonicated in a bath sonicator for 5 min.

196

4.2.1

197

Interference testing must be carried out on any test sample for which no interference

198

information is available. The purpose of the interference test is to determine the lowest

199

dilution (i.e., highest concentration) of a test substance from which an endotoxin spike can be

Interference Testing

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detected (i.e., based on the decision criteria described in Section 4.2.1.2). However, to ensure

201

a valid test, a test substance should not be diluted beyond its Maximum Valid Dilution

202(MVD).

203

For many marketed products, values for the MVD and the Endotoxin Limit Concentration

204(ELC) are published in the U.S. Pharmacopeia, the European Pharmacopoeia, and/or Food

205

and Drug Administration (FDA) guidelines. However, if one or both of these values are not

206

available, then calculation of the MVD is dependent on the ELC (see Section 12.3). If

207

unknown, the ELC can be approximated by dividing the maximum hourly dose of the

208

product by the hourly dose received per patient. For example, if a product is used at an

209

hourly dose of 100 mg per patient, then the ELC would be 350 EU/100 mg or 3.5 EU/mg.

2104.2.1.1

211

The WHO-LPS 94/580 [E. coli O113:H10:K-] or equivalent international RSE (e.g., USP

212

G3E069, FDA EC-6) is recommended for preparation of the endotoxin control (EC). If a213

different E. coli LPS is used and the potency relative to the RSE is not provided, then each214

lot must be calibrated against the RSE in the MM6/IL-6 test method. For interference testing,215

an endotoxin standard curve (see Section 4.1) should be included on each plate.

216

4.2.1.2

217

For interference testing, non-spiked and endotoxin-spiked test substances are prepared in218

microplate wells (n=4 replicates) and an in vitro pyrogen test is performed. Either RPMI-C or219

a fixed concentration (a concentration selected from the middle of the EC standard curve) of

220

the RSE (i.e., 1 EU/mL) in RPMI-C is added to the test substance in serial two-fold dilutions.

221

An illustrative example of endotoxin spiking solutions is shown in Table 4-2. For non-spiked

222

solutions, 150 µL of RPMI-C and 50 µL of the test substance (neat or at serial dilution) are

223

added to a well. Then, 50 µL of MM6 cells are added and the well contents mixed are mixed.

224

Endotoxin-spiked solutions are prepared by adding 100 µL of RPMI-C, 50 µL of the test

225

substance (neat or at serial dilution), and 50 µL of an endotoxin spike solution (1.0 EU/mL).

226

MM6 cells (50 µL) are then added and the well contents are mixed (see example presented in

227

Table 4-2).

Reference Endotoxin for Spiking Test Substances

Spiking Test Substances with Endotoxin

228
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229

Table 4-2

01 Dec 2006

Preparation of Endotoxin Spiked and Non-Spiked Solutions for

230

Determination of Test Substance Interference in the Incubation and

231

ELISA Test Systems
SpikedSample Addition

232
233
234
235
236
237

RPMI-C (containing 2% FCS)
Endotoxin spike solution2
Test substance (neat and each serial dilution)
MM6 cells3
Total4Abbreviations: MM6 cells = Mono Mac 6 cells
1
n=4 replicates each2
Endotoxin concentration is 1.0 EU/mL in RPMI-C.
3
MM6 cells are resuspended in RPMI-C (2.5 x 106 cells/mL).
4
A total volume of 250 µL per well is used for the incubation.

µL/well1

Non-spiked

100
50
50
50
250

150
0
50
50
250

238

The lowest dilution of the test substance that yields an endotoxin spike recovery of 50% to239

200% in the pyrogen test is determined. The optical density (OD) values of the endotoxin-240

spiked and non-spiked test substances are calibrated against the endotoxin calibration curve.

241

The resulting EU value of the non-spiked test substance is subtracted from the corresponding

242

EU value of the endotoxin-spiked test substance at each dilution. The % recovery for each243

sample dilution is then determined from the endotoxin spike solution concentration set to244

100%. For example, consider the following interference test results in Table 4-3:

245

Table 4-3

246

Assay

Example of Interference Data Used to Determine Sample Dilution for% Recovery of Endotoxin
Control
25
49
90
110

Sample Dilution
None
1:2
1:4
1:8

247
248

Based on these results, the dilution of the test substance used in the in vitro pyrogen test

249

would be 1:4 (i.e., the lowest dilution between 50% and 200% of the 1.0 EU/mL EC).

250

4.2.2

251

If the data obtained from the experiment in Section 4.2.1 suggests the presence of252interference, then a subsequent experiment similar to that described in Section 4.2.1 wouldInterference with ELISA

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253

need to be performed to confirm that the test substance(s) does not directly interfere with the254

ELISA. For this experiment, an ELISA would be performed in the absence of MM6 cells.

255

4.3

256

MM6 cells are maintained in RPMI-C. For use in the ELISA procedure, the concentration of257

fetal calf serum (FCS) is reduced to 2%. Each medium is prepared and stored as described by258

the manufacturer.

259

4.3.1

Cell Culture Medium

RPMI-C260•

Bovine insulin; 0.23 IU/mL

261•

FCS; heat-inactivated (50 mL or a 10% final concentration)

262•

HEPES buffer; 20 mM

263•

L–Glutamine; 2 mM

264•

MEM non-essential amino acids; 0.1 mM

265•

Oxaloacetic acid; 1 mM

266•

Penicillin/streptomycin (10,000 IU/mL penicillin, 10 mg/mL streptomycin)

267•

RPMI-1640 medium (500 mL)

268•

Sodium pyruvate; 1 mM

269

4.3.2

Starting a Culture of MM6 Cells

270

To initiate a culture of MM6 cells, remove a vial of the primary stock from liquid nitrogen.

271

Thaw the vial on ice. Gently mix and transfer the cells to a 50 mL centrifuge tube and add 10272

mL of RPMI-C. Centrifuge at 100 x g for 5 min at room temperature (RT). Remove the273

supernatant and resuspend the cells in ice-cold RPMI-C. Centrifuge at 100 x g for 5 min at

274

RT. Remove the supernatant and resuspend the MM6 cells in 2 mL of RPMI-C. Add 8 mL of275

RPMI-C to a 25 cm2 tissue culture flask and transfer the cell suspension to the flask. Place276

the flasks in a cell culture incubator and maintain the cells at 37±1°C + 5% CO2.

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277

4.3.3

Propagation of MM6 Cells

278

To propagate the MM6 cells, transfer the MM6 cells to a 50 mL tube. Centrifuge at 100 x g

279

for 8 min at RT. Remove the supernatant, resuspend the cell pellet in 4 mL of RPMI-C, and280

gently pipet up and down to mix. Transfer an aliquot of the cell suspension to new tissue281

culture flasks and add fresh RPMI-C to obtain a final concentration of 2 x 105 cells/mL.

282

Place the flasks in a cell culture incubator and maintain the cells at 37±1°C + 5% CO2.

283

4.3.4

284

To initiate a bank of MM6 cells, centrifuge the cell culture(s) at 100 x g for 8 min at 4°C.

285

Remove the supernatant and resuspend the cells in FBS at 4°C. Adjust the cell concentration286

to 4 x 106 cells/mL and store on ice for 10 min. Add an equal volume of ice-cold FBS

287

containing 10% dimethylsulfoxide (DMSO) drop-wise to the cell suspension (final

288

concentration is 2 x 106 cells/mL with 5% DMSO). Transfer the cell suspension to sterile,289

pyrogen-free cryotubes (1 mL/tube). Place the tubes in a well-insulated polystyrene box and290

store at -80°C or below for greater than 48 hr and then transfer to a liquid nitrogen container.

291

5.0

CONTROLS

292

5.1

Negative Control

293

A negative control (e.g., RPMI-C) is included in each experiment in order to detect294

nonspecific changes in the test system, as well as to provide a baseline for the assay

295

endpoints.

296

5.2

297

Solvent controls are recommended to demonstrate that the solvent is not interfering with the298test system when solvents other than PFS are used to dissolve test substances.

299

5.3

300

An internationally standardized EC (e.g., WHO 94/580; USP G3E069; 0.5 EU/mL) is

301

included in each experiment to verify that an appropriate response is induced.

Preparation of a MM6 Cell Bank

Solvent Control

Positive Control

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302

5.4

303

Benchmark controls may be used to demonstrate that the test method is functioning properly,

304

or to evaluate the relative pyrogenic potential of chemicals (e.g., parenteral pharmaceuticals,

305

medical device eluates) of a specific class or a specific range of responses, or for evaluating306

the relative pyrogenic potential of a test substance. Appropriate benchmark controls should307

have the following properties:

308

Benchmark Control•309

consistent and reliable source(s) for the chemicals (e.g., parenteralpharmaceuticals, medical device eluates)

310•

structural and functional similarities to the class of the substance being tested

311•

known physical/chemical characteristics

312•

supporting data on known effects in animal models

313•

known potency in the range of response

314

5.5

Positive Product Control (PPC)315

The PPC is a test substance diluted to a level that does not interfere with the test method and316

does not exceed the MVD. The PPC is obtained by spiking a test substance with a known EC

317(e.g., 1 EU/mL) and demonstrating that 50% to 200% of the EC is recovered.

318

5.6

319

The NPC is the test substance diluted to the MVD and then spiked with PFS. It is the320

negative control for the PPC.

321

6.0

EXPERIMENTAL DESIGN

322

6.1

Incubation with Test Samples and Measurement of IL-6 Release

323

6.1.1

Preparation of MM6 Cells for the Incubation Plate

324

To perform an ELISA on the following day, obtain 30 to 50 mL of MM6 cell suspension

325

from propagation flasks and centrifuge at 100 x g for 8 min at RT. Remove the supernatant,326

resuspend the cell pellet in 3-5 mL of RPMI-C (containing 2% FCS for all ELISA

327

procedures), and gently pipet up and down to mix. Transfer aliquots of cells to new culture

Negative Product Control (NPC)

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328

flasks and add RPMI-C to obtain a concentration of 4 x105 cells/mL. Place the flasks in a cell

329

culture incubator and maintain the cells at 37±1°C + 5% CO2. In general, each 96-well assay

330

plate requires approximately 10 mL of cell suspension at 2.5 x 106 cells/mL.

331

6.1.2

332

Test substances are prepared at a level of dilution that did not show interference with the test

333

system or for which it is known that interference does not occur. Each incubation plate can334

accommodate an endotoxin standard curve, a NSC, and 14 test substances (see Table 6-1).

335

Table 6-1

Numberof Wells

Incubation Plate

Overview of Incubation Plate Preparation in the MM6/IL-6 Test Method

Sample

RPMI-CEC

Test
Sample

MM61

µL
202

EC

100

50

0

100

4

NSC

150

0

0

100

563

Test
samples

100

0

50

100

Mix thesamples;incubateovernight at
37±1°C in ahumidified
atmospherewith 5%CO2.

Mix thesamples;
immediatelytransfer to an
ELISA plate4
and runELISA orstore plate at-20°C or
-80°C.

(1-14)

336
337
338
339
340
341
342

Abbreviations: EC = Endotoxin control; NSC = Negative saline control; MM6 = Mono Mac 6 cell line
1
MM6 cell concentration is 2.5 x 106 cells/mL.
2
Five EC concentrations (0.125, 0.25, 0.50, 1.0, and 2.0 EU/mL) in quadruplicate.
3
14 test samples (n=4 each) per plate.
4
An IL-6 standard curve is prepared in Columns 11 and 12 on the ELISA plate. Therefore, 80 wells areavailable for test samples and controls on the incubation plate.

343

6.1.3

Incubation Assay for IL-6 Release

344

Step 1. Refer to the suggested incubation plate template presented in Table 6-2.

345

Step 2. Using a pipetter, transfer 100 µL of RPMI-C into each well.

346

Step 3. Transfer 50 µL of test sample into the appropriate wells as indicated in the347

template.

348

Step 4. Transfer 50 µL of the EC (standard curve) and the NSC controls in349

quadruplicate into the appropriate wells according to the template.

350

Step 5. Transfer 100 µL of a well-mixed MM6 cell suspension into each well.
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351

Step 6. Place the covered plate in a tissue culture incubator for 16 to 24 hr at

352

37±1°C in a humidified atmosphere containing 5% CO2.

353

Step 7. Remove 150 µL of the supernatant from each well, without disrupting the354cells, and transfer to a new 96-well ‘transfer’ plate for the IL-6 ELISA.

355

356
357
358
359
360
361

Table 6-2

Incubation Plate - Sample and Control Template

1

2

3

4

5

6

7

8

9

10

11

12

A

EC1
2.0

EC
2.0

EC
2.0

EC
2.0

TS3

TS3

TS3

TS3

TS11

TS11

Void3

Void

B

EC
1.0

EC
1.0

EC
1.0

EC
1.0

TS4

TS4

TS4

TS4

TS11

TS11

Void

Void

C

EC
0.50

EC
0.50

EC
0.50

EC
0.50

TS5

TS5

TS5

TS5

TS12

TS12

Void

Void

D

EC
0.25

EC
0.25

EC
0.25

EC
0.25

TS6

TS6

TS6

TS6

TS12

TS12

Void

Void

E

EC
0.125

EC
0.125

EC
0.125

EC
0.125

TS7

TS7

TS7

TS7

TS13

TS13

Void

Void

F

NSC

NSC

NSC

NSC

TS8

TS8

TS8

TS8

TS13

TS13

Void

Void

G

TS12

TS1

TS1

TS1

TS9

TS9

TS9

TS9

TS14

TS14

Void

Void

H

TS2

TS2

TS2

TS2

TS10

TS10

TS10

TS10

TS14

TS14

Void

Void

Abbreviations: EC = Endotoxin control; NSC = Negative saline control; TS = Test substance1
EC value (e.g., EC 2.0) represents the endotoxin concentration in EU/mL.
2
TS number (e.g., TS 1) represents an arbitrary sequence for individual test substances.
3
Columns 11 and 12 are reserved for the IL-6 standard curve on the ELISA plate (see Table 6-3).

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362

6.2

ELISA to Measure IL-6 Release

363

6.2.1

IL-6 Standard Curve

364

An IL-6 standard supplied with the ELISA kit is used. IL-6 standards are typically supplied365

in lyophilized form and should be reconstituted according to the manufacturer's instructions.

366

The stock solution should be diluted in RPMI-C to the following concentrations: 0, 62.5, 125,

367

250, 500, 1000, 2000, and 4000 pg/mL in volumes of at least 500 µL. Each well on the368

ELISA plate will receive 50 µL of an IL-6 blank or standard.

369

6.2.2

370

The manufacturer's instructions provided with the ELISA kit should be followed and a371

typical experimental design is outlined below. If the user prefers to prepare an in-house

372

ELISA, then appropriate modifications and validation of these changes would be necessary.

373

The ELISA should be carried out at RT and therefore all components must be at RT prior to374

use. Do not thaw frozen specimens by heating them in a water bath. A suggested ELISA

375

plate template is shown in Table 6-3, which includes a five-point EC standard curve, a NSC,

376

an eight-point IL-6 standard curve (0 to 4000 pg/mL), and 14 test substances in377

quadruplicate. The EC standard curve, the NSC, and the test sample supernatants are

378transferred directly from the incubation plate. The IL-6 standard curve is prepared as

379

described in Section 6.2.1. An overview of the ELISA plate preparation is shown in Table 6-

380

4.

ELISA

381

Step 1. After pipetting up and down very carefully three times (avoid detachment382

of the adherent MM6 cells) to mix the supernatant, transfer 50 µL from each well383

of the Incubation Plate (A1-10; H1-10) to the ELISA plate.

384

Step 2. Add 50 µL of each IL-6 standard (0 to 4000 pg/mL) into the respective

385

wells on the ELISA plate.

386

Step 3. Add 200 µL of the enzyme-labeled detection antibody (neat as supplied, or

387

diluted, if necessary) to each of the wells.

388

Step 4. Cover the microtiter plate(s) with adhesive film and incubate for 2 to 3 hr

389

at 20 to 25°C.
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Step 5. Decant and wash each well three times with 300 µL Buffered Wash

391

Solution and then rinse three times with deionized water. Place the plates upside

392

down and tap to remove water.

393

Step 6. Add 200 µL of TMB/Substrate Solution to each well and incubate at RT in394

the dark for 15 min. If necessary, decrease the incubation time.

395

Step 7. Add 50 µL of Stop Solution to each well.

396

Step 8. Tap the plate gently after the addition of Stop Solution to aid in mixing.

397

Step 9. Read the OD450 within 15 min of adding the Stop Solution. Measurement398with a reference wavelength of 540 to 590 nm is recommended.

399
400

401
402
403
404
405
406

Table 6-3

ELISA Plate - Sample and Control Template

1

2

3

4

5

6

7

8

9

10

11

12

A

EC1
2.0

EC
2.0

EC
2.0

EC
2.0

TS3

TS3

TS3

TS3

TS11

TS11

IL-63
0

IL-6
0

B

EC
1.0

EC
1.0

EC
1.0

EC
1.0

TS4

TS4

TS4

TS4

TS11

TS11

IL-6
62.5

IL-6
62.5

C

EC
0.50

EC
0.50

EC
0.50

EC
0.50

TS5

TS5

TS5

TS5

TS12

TS12

IL-6
125

IL-6
125

D

EC
0.25

EC
0.25

EC
0.25

EC
0.25

TS6

TS6

TS6

TS6

TS12

TS12

IL-6
250

IL-6
250

E

EC
0.125

EC
0.125

EC
0.125

EC
0.125

TS7

TS7

TS7

TS7

TS13

TS13

IL-6
500

IL-6
500

F

NSC

NSC

NSC

NSC

TS8

TS8

TS8

TS8

TS13

TS13

IL-6
1000

IL-6
1000

G

TS12

TS1

TS1

TS1

TS9

TS9

TS9

TS9

TS14

TS14

IL-6
2000

IL-6
2000

H

TS2

TS2

TS2

TS2

TS10

TS10

TS10

TS10

TS14

TS14

IL-6
4000

IL-6
4000

Abbreviations: EC = Endotoxin control; NSC = Negative saline control; TS = Test substance1
EC value (e.g., EC 2.0) represents the endotoxin concentration in EU/mL.
2
TS number (e.g., TS1) represents an arbitrary sequence for individual test substances.
3
IL-6 values in columns 11 and 12 are in pg /mL.

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Draft ICCVAM Recommended Test Method Protocol: MM6/IL-6 Pyrogen Test

407

408
409

Table 6-4

Overview of ELISA Procedure

Materialtransfer from
Incubation
Plate (µ L)

Enzymelabeled
Antibody
(µL)

50

200

Cover theIncubation
Plate andincubate for
2 to 3 hr at
20-25°C.

TMB/Substrate
Solution(µL)

Incubate 15
min at RT in
dark.

200

01 Dec 2006

Stop
Solution(µL)

50

Read opticaldensity at
450 nm witha 540-590
nm
wavelengthreference
filter.

RT = Room temperature

410
411

7.0

EVALUATION OF TEST RESULTS

412

7.1

OD Measurements

413

The OD of each well is obtained by reading the samples in a standard microplate414

spectrophotometer (i.e., plate reader) using a visible light wavelength of 450 nm (OD450) with415

a 540 to 590 nm reference filter (recommended). OD450 values are used to determine assay

416

acceptability and in the decision criteria for the detection of endotoxin in a test substance (see417Sections 8.0 and 9.0).

418

8.0

419

Obtain the PPC and the corresponding NPC by interference testing of a test substance in the420

presence and absence of a fixed quantity of endotoxin (i.e., 1 EU/mL) in quadruplicate. An

421

EC (five-point standard curve) and a NSC should be included in each experiment. An IL-6

422

standard curve should be included in each ELISA as shown in the template presented in

423

Table 6-3. An assay is considered acceptable only if the following minimum criteria are met:

424

CRITERIA FOR AN ACCEPTABLE TEST

•

425
426
427

The quadratic function of the IL-6 standard curve produces an r ≥0.95 and the
OD450 of the blank control is below 0.15.

•

The endotoxin standard curve produces OD450 values that ascend in asigmoidal concentration response.

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428

9.0

DATA INTERPRETATION/DECISION CRITERIA

429

9.1

Decision Criteria for Determination of Pyrogenicity

430

The t-test is used to compare the data of a test sample against the data of the EC (0.5 EU/mL)

431

that is performed in parallel. If this test results in a significant p-value (i.e., smaller than 1%),

432

then the sample is considered to be non-pyrogenic, and as pyrogenic if otherwise (Hoffmann433

et al., 2005), as long as the assay acceptability criteria in Section 8.0 has been met.

434

10.0

435

The test report should include the following information:

436

Test Substances and Control Substances

STUDY REPORT

437•

Name of test substance

438•

Purity and composition of the substance or preparation

439•

Physicochemical properties (e.g., physical state, water solubility)

440•

Treatment of the test/control substances prior to testing (e.g., vortexing,

441

sonication, warming, resuspension solvent)442

Justification of the In Vitro Test Method and Protocol Used

443

Test Method Integrity

444

•

445
446

test method over time
•

447
448

If the test method employs proprietary components, documentation on theprocedure used to ensure their integrity from “lot-to-lot” and over time

•

449
450

The procedure used to ensure the integrity (i.e., accuracy and reliability) of the

The procedures that the user may employ to verify the integrity of the
proprietary components

Criteria for an Acceptable Test

451•

Acceptable concurrent positive control ranges based on historical data

452•

Acceptable negative control data
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453

01 Dec 2006

Test Conditions

454•

Cell system used

455•

Calibration information for the spectrophotometer used to read the ELISA

456•

Details of test procedure used

457•

Description of any modifications of the test procedure

458•

Reference to historical data of the model

459•

Description of evaluation criteria used•

Tabulation of data from individual test samples

460

Results

461
462

Description of Other Effects Observed

463

Discussion of the Results

464

Conclusion

465

A Quality Assurance Statement for Good Laboratory Practice (GLP)-Compliant Studies

466•This statement should indicate all inspections made during the study and the467

dates any results were reported to the Study Director. This statement should

468

also confirm that the final report reflects the raw data.

469

If GLP-compliant studies are performed, then additional reporting requirements provided in470

the relevant guidelines (e.g., OECD 1998; EPA 2003a, 2003b; FDA 2003) should be471

followed.

472

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472

11.0

REFERENCES

473

EPA. 2003a. Good Laboratory Practice Standards. Toxic Substances Control Act. 40 CFR

474

792.

475

EPA. 2003b. Good Laboratory Practice Standards. Federal Insecticide, Fungicide, and476

Rodenticide Act. 40 CFR 160.

477

FDA. 2003. Good Laboratory Practices for Nonclinical Laboratory Studies. 21 CFR 58.

478

Hoffmann S, Peterbauer A, Schindler S, Fennrich S, Poole S, Mistry Y, Montag-Lessing T,

479

Spreitzer I, Löschner B, van Aalderen M, Bos R, Gommer M, Nibbeling R, Werner-

480

Felmayer G, Loitzl P, Jungi T, Brcic M, Brügger P, Frey E, Bowe G, Casado J, Coecke S, de481

Lange J, Mogster B, Næss LM, Aaberge IS, Wendel A, Hartung T. 2005. International482

validation of novel pyrogen tests based on human monocytoid cells. J Immunol Methods

483

298:161-173.

484

Organization for Economic Cooperation and Development (OECD). 1998. OECD Series on

485

Principle of Good Laboratory Practice and Compliance Monitoring. No. 1. OECD Principles

486

of Good Laboratory Practice (as revised in 1997). Organization for Economic Co-operation

487

and Development (OECD), ENV/MC/CHEM(98)17. Paris: OECD.

488

Taktak YS, Selkirk S, Bristow AF, Carpenter A, Ball C, Rafferty B, Poole S. 1991. Assay of489

pyrogens by interleukin-6 release from monocytic cell lines. J Pharm Pharmacol 43:578-582.

490

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490

12.0

TERMINOLOGY AND FORMULA

491

12.1

Assay Sensitivity (λ)

492

For an in vitro cell-based assay, the variable λ is defined as the lowest statistically significant

493

point on the standard endotoxin concentration-response curve and represents the relative494

sensitivity of the test method for the detection of endotoxin (i.e., level of detection).

495

12.2

496

The EC is incubated with MM6 cells and serves as the positive control for the experiment.

497

The results should be compared to historical values to insure that it provides a known level of498

cytokine release relative to the NSC.

499

12.3

500

The ELC is the maximum allowable concentration of endotoxin for a particular product and501

is expressed in EU per volume (mL) or weight (mg). The ELC is defined by the FDA or

502

specified in the USP2. It is calculated as the product of K/M, where:503

K is the threshold pyrogen dose for parenteral use in rabbits or humans (5.0 EU/kg). At an

504

injection volume of 10 mL/kg, K is equal to 0.5 EU/mL.

505

M is the larger of the rabbit dose or the maximum human dose administered in one hour as

506

defined below and varies with test substance3.

507

12.4

508

The MVD is the maximum dilution of a test substance that can be tolerated in a test system509

without exceeding the ELC, if the test substance must be diluted as a result of assay

510

interference. Dilutions beyond the MVD would not be valid for endotoxin detection in the

Endotoxin Control (EC)Endotoxin Limit Concentration (ELC)

Maximum Valid Dilution (MVD)2

ELC values for most marketed pharmaceutical products are provided in the USP or in other pharmacopoeial or
regulatory publications (e.g., European Pharmacopoeia, Japanese Pharmacopoeia, Pharmacopoeial guidelines,
FDA publications).
3
Values for most marketed pharmaceutical products are provided in the USP or in other pharmacopoeial or
regulatory publications (e.g., European Pharmacopoeia, Japanese Pharmacopoeia, pharmacopoeial guidelines,
FDA publications).
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511

test system. Calculation of the MVD is dependent on whether or not the ELC for a test

512

substance is published. When the ELC is known, the MVD is:513

MVD = (ELC x Product Potency [PP])/λ514

As an example, for "Cyclophosphamide Injection," the ELC is 0.17 EU/mg, PP is 20

515

mg/mL, and the assay sensitivity is 0.1 EU/mL. The calculated MVD would be 34. The test

516

substance can be diluted no more than 1:34 prior to testing.

517

If the ELC is not known, the MVD is:518

MVD = PP/Minimum Valid Concentration (MVC)519

where, MVC = (λ x M)/K

520

where, M is the maximm human dose521

As an example, for "Cylophosphamide Injection," the PP is 20 mg/mL, M is 30522

mg/kg, and assay sensitivity is 0.1 EU/mL. The calculated MVC is 0.6 mg/mL and the MVD

523

is 33.3. The test substance can be diluted no more than 1:33 in the assay prior to testing.

524

12.5

525

The NPC is a test sample to which PFS is added. The NPC is the baseline for determination526

of cytokine release relative to the endotoxin-spiked PPC.

527

12.6

528

The NSC is MM6 cells (in RPMI-C) incubated with PFS (used for dilution of test substance)529

and is used as the blank.

530

12.7

531

The value K represents the threshold pyrogen dose for parenteral products for rabbits and532

humans. Based on experimental data, K is fixed at 5.0 EU/kg. For intrathecal products, K is533

0.2 EU/kg.

534

12.8

535

The PPC is a test substance spiked with the control standard endotoxin (i.e., 0.5 EU/mL or an

536

amount of endotoxin equal to that which produces ½ the maximal increase in OD from the

Negative Product Control (NPC)

Negative Saline Control (NSC)

Parenteral Threshold Pyrogen Dose (K)Positive Product Control (PPC)

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537

endotoxin standard curve) to insure that the test system is capable of endotoxin detection in538

the product as diluted in the assay.

539

12.9

540

The concentration for a test substance is the PP typically expressed as µg/mL or mg/mL.

541

12.10

542

The variable M represents the rabbit test dose or the maximum human dose in 1 hr. The

543

variable M is expressed in mg/kg and varies with the test substance. For

544

radiopharmaceuticals, M should be adjusted to account for product activity (radioactive

545

decay) at time administration. An average human standard weight of 70 kg is used for the546

calculation. If a pediatric dose should be used and it is higher than the adult dose, then it

547

should be used in the formula.

Product Potency (PP)

Rabbit Pyrogen Test Dose or Maximum Human Dose (M)

548

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01 Dec 2006

Appendix B3

Draft ICCVAM Recommended Protocol for Future Studies Using the Peripheral
Blood Mononuclear Cell (PBMC)/Interleukin-6 (IL-6) Test Method

Draft ICCVAM Recommended Test Method Protocol: PBMC/IL-6 Pyrogen Test

[This Page Intentionally Left Blank]

01 Dec 2006

Draft ICCVAM Recommended Test Method Protocol: PBMC/IL-6 Pyrogen Test

01 Dec 2006

1

Draft ICCVAM Recommended Protocol for Future Studies Using the Peripheral Blood

2

Mononuclear Cell (PBMC)/Interleukin-6 (IL-6) Test Method

3
4

PREFACE

5
6This proposed protocol for the detection of pyrogenicity is based on information obtained

7

from 1) The European Centre for the Validation of Alternative Methods (ECVAM)

8PBMC/IL-6 Background Review Document (BRD) presented in Appendix A of the draft

9Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM)

10

Pyrogenicity Test Method BRD, which includes ECVAM Standard Operating Procedures

11(SOPs) for the PBMC/IL-6 test method, and 2) Information provided to the National

12

Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative

13

Toxicological Methods (NICEATM) by Dr. Thomas Hartung, Head of ECVAM. The

14

ECVAM SOPs are based on various test methods that use human PBMCs to detect cytokine

15

production as a measure of pyrogenicity (Bleeker et al., 1994; Dinarello et al., 1984; Poole et16al., 2003). A table of comparison between the draft ICCVAM recommended protocol and the17

ECVAM SOPs is provided in Table 1. Future studies using the PBMC/IL-6 test method may

18

include further characterization of the usefulness or limitations of the assay for regulatory

19

decision-making. Users should be aware that the proposed test method protocol might be20

revised based on additional optimization and/or validation studies. ICCVAM recommends

21

that test method users routinely consult the ICCVAM/NICEATM website

22(http://iccvam.niehs.nih.gov/) to ensure that the most current test method protocol is used.

23

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23
24
25
26
27
28
29
30
31
32
33
34
35
36

[This Page Intentionally Left Blank]

37
38

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01 Dec 2006

Draft ICCVAM Recommended Test Method Protocol: PBMC/IL-6 Pyrogen Test

39
40

Table 101 Dec 2006

Comparison of Draft Recommended Test Method Protocol with the ECVAM SOP for the Peripheral Blood
Mononuclear Cell (PBMC)/Interleukin-6 (IL-6) Pyrogen Test
Protocol Component
Test Substance

Incubation Plate(number of control or test groups at n=4
each)
ELISA Plate
Decision Criteria for Interference

Assay Acceptability Criteria

Decision Criteria for Pyrogenicity

Draft ICCVAM Protocol
Test neat or at minimal dilution that produces nointerference
NSC (1)EC (5)
TS (14)
Includes seven point IL-6 SC and blank in duplicate
0.5 x Median OD4501 of 1 EU/mL EC <2x Median
OD450 of 1 EU/mL EC
Mean OD450 of PPC is 50% to 200% of 0.5 EU/mL
EC
Mean OD450 of NSC ≤0.15
Quadratic function of IL-6 SC r2 ≥0.95
EC SC produces OD450 values that ascend in asigmoidal concentration responseHigh responder (i.e., > 200 pg/mL IL-6) or low
responder (i.e., Mean OD450 for 1 EU/mL EC issignficantly less than that for 1000 pg/mL IL-6)
blood donors may be excluded (limited to 1 of 4
donors). One of four donors may be excluded or thetest repeated with four additional donors.

Mean corrected OD450 of TS > Mean corrected
OD450 of 0.5 EU/mL EC

B3-3

ECVAM SOP/
ECVAM Catch-Up Validation SOPTest at MVD during validation - otherwise
similar to ICCVAM protocol
Same as ICCVAM protocol
Same as ICCVAM protocol
Same as ICCVAM protocol
Same as ICCVAM protocol
0.5 x Median OD450 of 0.25 EU/mL3 EC <2xMedian OD450 of 0.25 EU/mL EC
Mean OD450 of PPC is 50% to 200% of 0.25
EU/mL2 EC
Same as ICCVAM protocol
Same as ICCVAM protocol
Same as ICCVAM protocol

Same as ICCVAM protocol

EC SC data transformed to 4-parameter
logistical model by an in-house program and thelinear mean square is calculated. TS pyrogencontent is compared with the ELC3 using
confidence limits for significance. The
preparation being examined must pass the testwith blood from three separate donors. Dixon's
test is used to reject outliers.

Draft ICCVAM Recommended Test Method Protocol: PBMC/IL-6 Pyrogen Test

Protocol Component

Draft ICCVAM Protocol
Decision Level 1
Passes if all are negative
Fails if 2 or more are positive
If one is positive, repeat with 4 more donors
Decision Level 2
Passes if 1/8 are positive
Fails if 2 or more of 8 are positive

ECVAM SOP/
ECVAM Catch-Up Validation SOPSame as ICCVAM protocol

Limit test is run to determine whether or not a
TS after correction and dilution contains < 0.5
EU/mL of endotoxin
Abbreviations: EC = Endotoxin control; ELC = Endotoxin Limit Concentration; MVD = Maximum Valid Dilution; NSC = Normal saline control; PPC =Positive Product Control; SC = Standard curve; TS = Test substance1
Median or mean OD450 values are corrected (i.e., reference filter reading, if applicable, and NSC are subtracted).
2
Value of 0.25 EU/mL is arbitrary - should correspond to a concentration in the middle of the SC for the EC.
3
Where unknown, the ELC is calculated (e.g., Based on a rabbit sensitivity of 5 EU/kg, for a product injected at 10 mL/kg, the detection limit is 5 EU/10 mL/kg
or 0.5 EU/mL/kg, or an ELC of 0.5 EU/mL).
Not included

41
42
43
44
45
46
47
48

01 Dec 2006

49
50
51
52
53
54
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55
56

1.0

57

The purpose of this protocol is to describe the procedures used to evaluate the presence of a58

pyrogen (i.e., Gram-negative endotoxin) in a test substance. The presence of Gram-negative

59

endotoxin is detected by its ability to induce cytokine IL-6 release from human PBMCs. The

60

quantity of IL-6 released is obtained using an enzyme-linked immunosorbent assay (ELISA)

61

that includes monoclonal or polyclonal antibodies specific for IL-6. Release of this cytokine

62

is measured by incubation of PBMCs with test substances or controls (i.e., positive,

63

negative). The amount of pyrogen present is determined by comparing the values of64

endotoxin equivalents produced by PBMCs exposed to the test substance to those exposed to65

an internationally-harmonized Reference Standard Endotoxin (RSE)1 or an equivalent

66

standard expressed in Endotoxin Units (EU)/mL.

67

The focus of this protocol is on the use of the PBMC/IL-6 test method specifically for the68

detection of Gram-negative endotoxin in parenteral pharmaceuticals. The relevance and69

reliability for non-endotoxin pyrogens (e.g., lipoteichoic acid) has not been demonstrated in a

70

formal validation study.

71

2.0

72

All procedures for procurement of eligible blood donors and blood donations should follow

73

the regulations and procedures set forth by institutional guidelines for utilization of human74

substances, which include but are not limited to blood, tissues, and tissue fluids. Standard

75

laboratory precautions are recommended including the use of laboratory coats, eye76protection, and gloves. If necessary, additional precautions required for specific study

77

substances or hazardous chemicals will be identified in the Material Safety Data Sheet

78(MSDS).

1

PURPOSE AND APPLICABILITY

SAFETY AND OPERATING PRECAUTIONSRSEs are internationally-harmonized reference standards (e.g., WHO E. coli Lipopolysaccharide [LPS] 94/580[0113:H10:K-]; USP RSE Lot G3E069; FDA Lot EC-6). Equivalent endotoxins include commercially available
E. coli-derived LPS Control Standard Endotoxin (CSE) or other E. coli LPS preparations that have beencalibrated with an appropriate RSE.
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79

The stop solution used in the ELISA kit is acidic and corrosive and should be handled with80

the proper personal protective devices. If this reagent comes into contact with skin or eyes,

81

wash thoroughly with water. Seek medical attention, if necessary.

82

Tetramethylbenzidine (TMB) solution contains a hydrogen peroxide substrate and 3, 3’, 5,83

5’- TMB. This reagent is a strong oxidizing agent and a suspected mutagen. Appropriate

84

personal protection should be used to prevent bodily contact.

85

Bacterial endotoxin is a toxic agent (i.e., can induce sepsis, shock, vascular damage,86

antigenic response) and should be handled with care. Skin cuts should be covered and87

appropriate personal protective devices should be worn. In case of contact with endotoxin,

88

immediately flush eyes or skin with water for at least 15 min. If inhaled, remove the affected89

individual from the area and provide oxygen and/or artificial respiration as needed. Skin90

absorption, ingestion, or inhalation may produce fever, headache, and hypotension.

91

3.0

MATERIALS, EQUIPMENT, AND SUPPLIES

92

3.1

Source of Cells

93

PBMCs from fresh WB are the source of cells for cytokine production in the PBMC/IL-6 test

94

method (Poole et al., 2003). WB is obtained from healthy human volunteers who have95

provided their consent according to established institutional guidelines. Volunteers are96

expected not to have taken any drugs (e.g., prescription drugs, recreational drugs, herbal

97

drugs) and to have been free from illness for at least two weeks prior to donation.

98

PBMCs are isolated from WB using density gradient centrifugation (refer to Section 6.1.1.1).

99

The isolated PBMC suspension may be used directly in the PBMC/IL-6 test assay (Section

100

6.1.3) or frozen for later use (Section 6.1.1.3).

101

3.2

102

For all steps in the protocol, excluding the ELISA procedure, the materials that will be in103

close contact with samples and/or blood cells (e.g., pipet tips, containers, solutions) should be104

sterile and free from detectable pyrogens.

Equipment and Supplies

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105

3.2.1

Preparation of PBMCs

106

3.2.1.1

Equipment

107•

Centrifuge

108•

Hood; Bio-safety, laminar flow (recommended)109•

Incubator; cell culture (37±1°C + 5% CO2)

110•

Lymphoprep™

111•

Pipetter; multichannel (8- or 12-channel)

112•

Pipetters; single-channel adjustable (20, 200, and 1000 µL)

113•

Repeating pipetter

114•

Vortex mixer

115

3.2.1.2

01 Dec 2006

Consumables

116•

Centrifuge tubes; nonpyrogenic, polystyrene (15 and 50 mL)

117•

Combitips; repeating pipetter (2.5 and 5.0 mL)

118•

Cryotubes; screw-cap, 2 mL

119•

Filters; sterile, 0.22 µm

120•

Needle set; Sarstedt multifly, pyrogen-free, 19 mm, 21 gauge for S-Monovette

121•

Phosphate buffered saline (PBS); sterile

122•

Pipets; serological, sterile (5, 10, and 25 mL)

123•

Plates; microtiter, nonpyrogenic, 96-well, polystyrene, tissue culture

124•

Reaction tubes; polystyrene (1.5 mL)

125•

Reservoirs; fluid

126•

RPMI-1640 cell culture medium; supplemented with the following reagents to127yield RPMI-Complete (RPMI-C)

128

o

FCS; heat-inactivated (5 mL or a 1% final concentration)

129o

L–Glutamine; 2 mM
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130

o

131

01 Dec 2006

Penicillin/streptomycin (10,000 IU/mL penicillin, 10 mg/mL
streptomycin)

132•

Syringes; sterile (100 µL and 30 mL)

133•

Tips; pipetter, sterile, pyrogen-free (20, 200, and 1000 µL)

134•

Tubes; Sarstedt S-Monovette, 7.5 mL, heparinized for blood collection

135

3.2.2

ELISA

136

3.2.2.1

Equipment

137•

Microplate mixer

138•

Microplate reader (450 nm with an optional reference filter in the range of

139

540-590 nm)

140•

Microplate washer (optional)

141•

Multichannel pipetter

142

3.2.2.2

Consumables

143•

Container; storage, plastic

144•

Deionized water; nonsterile

145•

Plates; microtiter, nonpyrogenic, 96-well, polystyrene, tissue culture146•

Pyrogen-free water (PFW)147•

Reservoirs; fluid

148•

Tips; pipetter, nonsterile

149•

Tubes; polystyrene (12 mL)

150

3.2.2.3

ELISA Kit

151

An ELISA that measures IL-6 release from PBMCs is used. A variety of IL-6 ELISA kits are152

commercially available and the IL-6 ELISA procedure outlined in this protocol is intended to153

serve as an example for using an ELISA kit. If the user prefers to prepare an in-house

154

ELISA, then additional reagents would be required. The IL-6 ELISA should be calibrated

155

using an IL-6 international reference standard (e.g., WHO 89/548) prior to use. The IL-6
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156

cytokine assay kits do not provide the RSE or endotoxin equivalent; therefore, this reagent

157

must be purchased separately. Results obtained using these products are subject to the assay

158

acceptability and decision criteria described in Sections 8.0 and 9.0. IL-6 ELISA kit

159

components may include the following:

160

•

161

ELISA plates coated with anti-human IL-6 capture antibody; monoclonal or
polyclonal

162•

Buffered wash solution

163•

Dilution buffer

164•

Enzyme-labeled detection antibody

165•

Human IL-6 reference standard

166•

Pyrogen-free saline (PFS)167•

Stop solution

168•

TMB/substrate solution

169

3.3

170

Chemicals
•

171
172

Endotoxin (e.g., WHO E. coli LPS 2nd International Standard 94/580; USP
RSE E. coli LPS Lot G3E069; USP RSE E. Coli Lot G; FDA E. coli Lot EC6)

3.4

173

Solutions•

RPMI-C cell culture medium

174

4.0

ASSAY PREPARATION

175

All test substances, endotoxin, and endotoxin-spiked solutions should be stored at 4°C.

176

4.1

177

An internationally harmonized RSE or equivalent is used to generate the endotoxin standard

178

curve. The use of any other E. coli LPS requires calibration against a RSE using the179

PBMC/IL-6 test method.

Endotoxin Standard Curve

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180

A standard endotoxin curve consisting of a Normal Saline Control (NSC) and five RSE

181

concentrations (0.063, 0.125, 0.25, 0.50, and 1.0 EU/mL) are included in the incubation step

182

(refer to Table 4-1) and then transferred to the ELISA plate. To prepare the endotoxin

183

standard curve, first obtain a 2000 EU/mL stock solution by addition of PFW to the184lyophilized content of the stock vial by following the instructions provided by the185

manufacturer (e.g., for a vial containing 10,000 EU, 5 mL of PFW is added). To reconstitute186

the endotoxin, the stock vial should be vortexed vigorously for at least 30 min or sonicated in187

a bath sonicator for 5 min. The stock solution is stable for 14 days when stored at 2 to 8°C.

188

An endotoxin standard curve is prepared by making serial dilutions of the stock solution in

189

PFS as described in Table 4-1.

190

Table 4-1

Preparation of Endotoxin Standard Curve

Stock Endotoxin
EU/mL

191
192
193
194
195
196
197

µL of Stock
EndotoxinµL of PFS

Endotoxin
Concentration
EU/mL
203
1.0
0.50
0.25
0.125
0.063
0

20001,2
20
1980
20
100
1900
1.0
500
500
0.50
500
500
0.25
500
500
0.125
500
500
0
0
1000
Abbreviations: EU = Endotoxin units; PFS = Pyrogen-free saline
Each stock tube should be vortexed vigorously prior to its use to make the subsequent dilution.
1
A 2000 EU/mL stock solution of endotoxin is prepared according to the manufacturer's instructions.
2
The stock solution of USP RSE may be stored in aliquots and kept at -20°C for up to 6 months. Do not store
the endotoxin at -80°C.
3This concentration is not used in the assay.

198

4.2

Test Substances

199

Liquid test substances should be tested neat or, if interference is detected (see Section 4.2.1),

200

diluted in PFS. Solid test substances should be prepared as solutions in PFS or, if insoluble in201

saline, dissolved in dimethylsulfoxide (DMSO) then diluted up to 0.5% (v/v) with PFS

202

provided that this concentration does not interfere with the assay. The test substances should203

be vortexed vigorously for at least 30 min or sonicated in a bath sonicator for 5 min.

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204

4.2.1

Interference Testing

205

Interference testing must be carried out on any test sample for which no interference

206

information is available. The purpose of the interference test is to determine the lowest

207

dilution (i.e., highest concentration) of a test substance from which an endotoxin spike can be

208

detected (i.e., based on the decision criteria described in Section 4.2.1.2). However, to ensure

209

a valid test, a test substance should not be diluted beyond its Maximum Valid Dilution

210(MVD).

211

For many marketed products, values for the MVD and the Endotoxin Limit Concentration

212(ELC) are published in the U.S. Pharmacopeia, the European Pharmacopoeia, and/or Food

213

and Drug Administration (FDA) guidelines. However if one or both of these values are not214

available, then calculation of the MVD is dependent on the ELC (see Section 12.3). If

215

unknown, the ELC can be approximated by dividing the maximum hourly dose of the216

product by the hourly dose received per patient. For example, if a product is used at an

217hourly dose of 100 mg per patient, then the ELC would be 350 EU/100 mg or 3.5 EU/mg.

218

4.2.1.1

219

The WHO-LPS 94/580 [E. coli O113:H10:K-] or equivalent international RSE (e.g., USP

220

G3E069, FDA EC-6) is recommended for preparation of the endotoxin control (EC). If a221

different E. coli LPS is used and the potency relative to the RSE is not provided, then each222

lot must be calibrated against the RSE in the PBMC/IL-6 test method. For interference223

testing, an endotoxin standard curve (see Section 4.1) should be included on each plate.

224

4.2.1.2

225

For interference testing, non-spiked and endotoxin-spiked test substances are prepared in226

microplate wells (n=4 replicates) and an in vitro pyrogen test is performed. Either RPMI-C or

227

a fixed concentration (a concentration selected from the middle of the EC standard curve) of

228

the RSE (i.e., 0.25 EU/mL) in RPMI-C is added to the test substance in serial two-fold229

dilutions. An illustrative example of endotoxin spiking solutions is shown in Table 4-2. For

230

non-spiked solutions, 150 µl of RPMI-C is added to a well followed by 50 µl of the test

231

substance (neat or at serial dilution) and 50 µL of PBMCs and the well contents are mixed.

232

Endotoxin-spiked solutions are prepared by adding 100 µL of RPMI-C to each well followed

Reference Endotoxin for Spiking Test Substances

Spiking Test Substances with Endotoxin

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233

by 50 µL of the test substance (neat or at serial dilution) and 50 µL of an endotoxin spike234

solution (0.25 EU/mL). Finally, 50 µL of PBMCs are added and the well contents are mixed

235(see example presented in Table 4-2).

236

Table 4-2

Preparation of Endotoxin-Spiked and Non-spiked Solutions for

237

Determination of Test Substance Interference in the Incubation and

238

ELISA Test Systems
SpikedSample Addition

239
240
241
242
243
244
245

RPMI-C
Endotoxin spike solution2
Test substance (neat and each serial dilution)
PBMCs3
Total4Abbreviations: PBMC = Peripheral blood mononuclear cells
1
n=4 replicates each2
Endotoxin concentration is 0.25 EU/mL in RPMI-C.
3
PBMCs are resuspended in RPMI-C (1 x 106 cells/mL).
4
A total volume of 250 µL per well is used for the incubation.

µL/well1

100
50
50
50
250

Non-spiked
150
0
50
50
250

The lowest dilution of the test substance that yields an endotoxin spike recovery of 50% to246

200% in the pyrogen test is determined. The optical density (OD) values of the endotoxin-247

spiked and non-spiked test substances are calibrated against the endotoxin calibration curve.

248

The resulting EU value of the non-spiked test substance is subtracted from the corresponding

249

EU value of the endotoxin-spiked test substance at each dilution. The % recovery for each250

sample dilution is then determined from the endotoxin spike solution concentration set to251

100%. For example, consider the following interference test results in Table 4-3:

252

Table 4-3

253

Example of Interference Data Used to Determine Sample Dilution for
Assay
Sample Dilution
None
1:2
1:4
1:8

% Recovery of Endotoxin Control
25
49
90
110

254
255

Based on these results, the dilution of the test substance used in the in vitro pyrogen test

256

would be 1:4 (i.e., the lowest dilution between 50% and 200% of the 0.25 EU/mL EC).
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257

4.2.2

258

If the data obtained from the experiment in Section 4.2.1 suggests the presence of259

interference, then a subsequent experiment similar to that described in Section 4.2.1 would

260

need to be performed to confirm that the test substance(s) does not directly interfere with the261

ELISA. For this experiment, an ELISA would be performed in the absence of PBMCs.

262

5.0

CONTROLS

263

5.1

Negative Control

264

A negative control (e.g., RPMI-C) is included in each experiment in order to detect265

nonspecific changes in the test system, as well as to provide a baseline for the assay

266

endpoints.

267

5.2

268

Solvent controls are recommended to demonstrate that the solvent is not interfering with the269

test system when solvents other than PFS are used to dissolve test substances.

270

5.3

271

An internationally standardized EC (e.g., WHO 94/580; USP G3E069; 0.5 EU/mL) is

272

included in each experiment to verify that an appropriate response is induced.

273

5.4

274

Benchmark controls may be used to demonstrate that the test method is functioning properly,

275

or to evaluate the relative pyrogenic potential of chemicals (e.g., parenteral pharmaceuticals,276

medical device eluates) of a specific class or a specific range of responses, or for evaluating277

the relative pyrogenic potential of a test substance. Appropriate benchmark controls should

278

have the following properties:

279

Interference with ELISA

Solvent Control

Positive Control

Benchmark Control•

280

consistent and reliable source(s) for the chemicals (e.g., parenteralpharmaceuticals, medical device eluates)281•

structural and functional similarities to the class of the substance being tested

282•

known physical/chemical characteristics
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283•

supporting data on known effects in animal models

284•

known potency in the range of response

01 Dec 2006

285

5.5

Positive Product Control (PPC)286

The PPC is a test substance diluted to a level that does not interfere with the test method and287

does not exceed the MVD. The PPC is obtained by spiking a test substance with a known EC

288(e.g., 0.25 EU/mL) and demonstrating that 50% to 200% of the EC is recovered.

289

5.6

290

The NPC is the test substance diluted to the MVD and then spiked with PFS. It is the291

negative control for the PPC.

292

6.0

EXPERIMENTAL DESIGN

293

6.1

Incubation with Test Samples and Measurement of IL-6 Release

294

6.1.1

Collection of Human Blood295

WB is obtained from healthy human volunteers who have provided their consent according296

to established institutional guidelines. Volunteers are expected not to have taken any drugs

297

and to have been free from illness for at least two weeks prior to donation. The criteria for298

rejection of data from donors that are low responders or that are suspect due to veracity of299

health information is addressed in Section 8.0.

300

Fresh WB is drawn by venipuncture using a multifly needle set and collected in heparinized301

tubes (15 IU/mL lithium heparin). All components of the blood collection system (i.e.,

302

syringes, tubes, connecting lines) must be sterile and pyrogen-free. Blood should be stored at303

room temperature (RT) and must be used within 4 hr. Prior to use in the assay, the collection

304

tubes should be gently inverted 1 to 2 times. Do Not Vortex.

305

6.1.1.1

306

PBMCs must be isolated within two hours of WB collection using Lymphoprep™. The

307

isolation procedure described below is a modification of the manufacturer's instructions as308

outlined in the ECVAM SOPs for the PBMC/IL-6 test method.

Negative Product Control (NPC)

Isolation of PBMCs from WB

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To each 15 mL of heparinized WB, add 15 mL of PBS and a sterile 10 mL pipet into the310

tube. Add 20 mL of Lymphoprep™ to each of the two tubes, through the inserted pipet, to311

form a lower, denser, layer. The tubes are then centrifuged at 340 x g for 45 min at room RT.

312

After centrifugation, a white band of PBMCs should be visible at approximately the 25 mL

313

graduation mark on the tube. Carefully remove the uppermost 9 mL from each tube and314

discard. If cryopreservation of PBMCs is to be performed (see Section 6.1.1.3), then transfer

315

the layer to a new 50 mL tube for preparing a cryoprotective solution. The remaining

316

supernatant above the PBMC band should be aspirated and discarded. Using a 10 mL pipet,

317

transfer the PBMC layer to a new 50 mL centrifuge tube.

318

6.1.1.2

319

The PBMCs are resuspended in a total volume of 50 mL of PBS and centrifuged at 340 x g

320

for 15 min. The supernatant is poured off and the cellular sediment resuspended in 10 mL of321

PBS by pipetting up and down several times with a serological pipet. Adjust the total volume322

in each tube to 50 mL with PBS and centrifuge at 340 x g for 10 min for a second time. After

323

centrifugation, the PBMCs are resuspended with 15 mL of RPMI-C and the two aliquots of

324

PBMCs are pooled into a single tube. Cell counts (expressed per mL) are determined using a325

hemacytometer. If the cell count is above 1.2 x 106/mL, the cell suspension should be diluted

326

to 1 x 106 cell/mL in RPMI-C. This suspension must be used in the PBMC/IL-6 assay within

327

4 hr from the time of WB collection.

328

6.1.1.3

329

To freeze the PBMCs, prepare a cryoprotective solution by adding 2 mL of pyrogen-free330

DMSO to the 18 mL of supernatant collected in the centrifugation procedure outlined in

331

Section 6.1.1.1. Cool the cryoprotective solution to between 2 and 8°C. Centrifuge the332

isolated PBMCs as instructed in Section 6.1.1.2 and then add 6 mL of the chilled333

cryoprotective solution to the cell sediment. Pool the cell suspensions from the same donor

334

and transfer 1.0 mL aliquots to appropriately labeled screw-cap cryotubes. The cryotubes are335

placed in a styrofoam box for thermal insulation and slowly frozen to -80°C. After 72 hr, the336

tubes can be transferred to liquid nitrogen for prolonged storage.

337

To thaw the cryopreserved PBMCs, take two tubes (each at 1.0 mL) from a single donor and338

submerge in a water bath at 37±1°C. After thawing, the cell suspensions are pooled in a

Washing PBMCs

Procedure for Cryopreservation and Thawing of PBMCs

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single 50 mL centrifuge tube and RPMI-C is added to give a total volume of 40 mL. The

340

PMBCs are centrifuged at 340 x g for 10 min, the supernatant removed, and the cells

341

resuspended in 10 mL of RPMI-C. Viability may be tested and should yield greater than 95%342cell survival.

343

6.1.2

344

Test substances are prepared at a level of dilution that did not show interference with the test

345

system or for which it is known that interference does not occur. Each incubation plate can346

accommodate an endotoxin standard curve, a NSC, and 14 test samples (see Table 6-1).

347

Table 6-1

348

Incubation Plate

Overview of Incubation Plate Preparation in the PBMC/IL-6 Test
MethodNumberof Wells

Sample

RPMI-CEC

Test
Sample

PBMCs

µL
201

EC

100

50

0

100

4

NSC

150

0

0

100

562

Test
samples

100

0

50

100

Mix thesamples;incubateovernight at
37±1°C in ahumidified
atmospherewith 5%CO2.

Mix thesamples;
immediatelytransfer to an
ELISA plate3
and runELISA orstore plate at-20°C or
-80°C.

(1-14)

349
350
351
352
353
354
355

Abbreviations: EC = Endotoxin control; NSC = Negative saline control; PBMC = Peripheral bloodmononuclear cell
1
Five EC concentrations (0.063, 0.125, 0.25, 0.50, and 1.0 EU/mL) in quadruplicate.
2
14 test samples (n=4 each) per plate.
3
An IL-6 standard curve is prepared in Columns 11 and 12 on the ELISA plate. Therefore, 80 wells areavailable for test samples and controls on the incubation plate.

356

6.1.3

357

PBMC samples are prepared in a microtiter plate using a laminar flow hood. All

358

consumables and solutions must be sterile and pyrogen-free. Each plate should be labeled359

appropriately with a permanent marker. An overview of the incubation plate preparation is360

shown in Table 6-1. The incubation procedure is outlined below:

Incubation Assay for IL-6 Release

361

Step 1. Refer to the suggested incubation plate template presented in Table 6-2.

362

Step 2. Using a pipetter, transfer 100 µL of RPMI-C into each well.
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363

Step 3. Transfer 50 µL of test sample into the appropriate wells as indicated in the364

template.

365

Step 4. Transfer 50 µL of the EC (standard curve) and the NSC controls in

366

quadruplicate into the appropriate wells according to the template.

367

Step 5. Transfer 100 µL of a well-mixed PBMC suspension into each well and mix

368

by gently swirling the plate.

369

Step 6. Mix the contents of the wells thoroughly by pipetting up and down several

370

times using a multichannel pipetter, changing the tips between each row in order to371

avoid cross-contamination.

372

Step 7. Place the covered plate in a tissue culture incubator for 16 to 24 hr at

373

37±1°C in a humidified atmosphere containing 5% CO2.

374

Step 8. Prior to transferring the test samples onto the ELISA plate, mix the375

contents of the wells by pipetting up and down using a multichannel pipetter,376

changing the tips between each row in order to avoid cross-contamination.

377

378
379
380
381
382

Table 6-2

Incubation Plate - Sample and Control Template

1

2

3

4

5

6

7

8

9

10

11

12

A

EC1
1.0

EC
1.0

EC
1.0

EC
1.0

TS3

TS3

TS3

TS3

TS11

TS11

Void3

Void

B

EC
0.50

EC
0.50

EC
0.50

EC
0.50

TS4

TS4

TS4

TS4

TS11

TS11

Void

Void

C

EC
0.25

EC
0.25

EC
0.25

EC
0.25

TS5

TS5

TS5

TS5

TS12

TS12

Void

Void

D

EC
0.125

EC
0.125

EC
0.125

EC
0.125

TS6

TS6

TS6

TS6

TS12

TS12

Void

Void

E

EC
0.063

EC
0.063

EC
0.063

EC
0.063

TS7

TS7

TS7

TS7

TS13

TS13

Void

Void

F

NSC

NSC

NSC

NSC

TS8

TS8

TS8

TS8

TS13

TS13

Void

Void

G

TS1

2

TS1

TS1

TS1

TS9

TS9

TS9

TS9

TS14

TS14

Void

Void

H

TS2

TS2

TS2

TS2

TS10

TS10

TS10

TS10

TS14

TS14

Void

Void

Abbreviations: EC = Endotoxin control; NSC = Negative saline control; TS = Test substance1
EC value (e.g., EC 1.0) represents the endotoxin concentration in EU/mL.
2
TS number (e.g., TS1) represents an arbitrary sequence for individual test substances.
3
Columns 11 and 12 are reserved for the IL-6 standard curve on the ELISA plate (see Table 6-3).
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383
384

6.2

ELISA to Measure IL-6 Release

385

6.2.1

IL-6 Standard Curve

386

An IL-6 standard supplied with the ELISA kit is used. IL-6 standards are typically supplied387

in lyophilized form and should be reconstituted according to the manufacturer's instructions.

388

The stock solution should be diluted in RPMI-C to the following concentrations: 0, 62.5, 125,

389

250, 500, 1000, 2000, and 4000 pg/mL in volumes of at least 500 µL. Each well on the390

ELISA plate will receive 50 µL of an IL-6 blank or standard.

391

6.2.2

392

The manufacturer's instructions provided with the ELISA kit should be followed and a393

typical experimental design is outlined below. If the user prefers to prepare an in-house

394

ELISA, then appropriate modifications and validation of these changes would be necessary.

395

The ELISA should be carried out at RT and therefore all components must be at RT prior to396use. Do not thaw frozen specimens by heating them in a water bath. A suggested ELISA

397

plate template is shown in Table 6-3, which includes a five-point EC standard curve, a NSC,

398

an eight-point IL-6 standard curve (0 to 4000 pg/mL), and 14 test substances in399

quadruplicate. The EC standard curve, the NSC, and the test sample supernatants are

400

transferred directly from the incubation plate. The IL-6 standard curve is prepared as

401

described in Section 6.2.1. An overview of the ELISA plate preparation is shown in Table 6-

402

4.

ELISA

403

Step 1. After pipetting up and down very carefully three times (avoid detachment404

of the adherent PBMCs) to mix the supernatant, transfer 50 µL from each well of405

the Incubation Plate (A1-10; H1-10) to the ELISA plate.

406

Step 2. Add 50 µL of each IL-6 standard (0 to 4000 pg/mL) into the respective

407

wells on the ELISA plate.

408

Step 3. Add 200 µL of the enzyme-labeled detection antibody (neat as supplied, or

409

diluted, if necessary) to each of the wells.

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410

Step 4. Cover the microtiter plate(s) with adhesive film and incubate for 2 to 3 hr

411

at 20 to 25°C.

412

Step 5. Decant and wash each well three times with 300 µL Buffered Wash

413

Solution and then rinse three times with deionized water. Place the plates upside

414

down and tap to remove water.

415

Step 6. Add 200 µL of TMB/Substrate Solution to each well and incubate at RT in416

the dark for 15 min. If necessary, decrease the incubation time.

417

Step 7. Add 50 µL of Stop Solution to each well.

418

Step 8. Tap the plate gently after the addition of Stop Solution to aid in mixing.

419

Step 9. Read the OD450 within 15 min of adding the Stop Solution. Measurement420

with a reference wavelength of 540 to 590 nm is recommended.

421

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421
422

Table 6-3

ELISA Plate - Sample and Control Template

1

2

3

4

5

6

7

8

9

10

11

12

A

EC1
1.0

EC
1.0

EC
1.0

EC
1.0

TS3

TS3

TS3

TS3

TS11

TS11

IL-63
0

IL-6
0

B

EC
0.50

EC
0.50

EC
0.50

EC
0.50

TS4

TS4

TS4

TS4

TS11

TS11

IL-6
62.5

IL-6
62.5

C

EC
0.25

EC
0.25

EC
0.25

EC
0.25

TS5

TS5

TS5

TS5

TS12

TS12

IL-6
125

IL-6
125

D

EC
0.125

EC
0.125

EC
0.125

EC
0.125

TS6

TS6

TS6

TS6

TS12

TS12

IL-6
250

IL-6
250

E

EC
0.063

EC
0.063

EC
0.063

EC
0.063

TS7

TS7

TS7

TS7

TS13

TS13

IL-6
500

IL-6
500

F

NSC

NSC

NSC

NSC

TS8

TS8

TS8

TS8

TS13

TS13

IL-6
1000

IL-6
1000

G

TS12

TS1

TS1

TS1

TS9

TS9

TS9

TS9

TS14

TS14

IL-6
2000

IL-6
2000

H

TS2

TS2

TS2

TS2

TS10

TS10

TS10

TS10

TS14

TS14

IL-6
4000

IL-6
4000

423
424
425
426
427
428

Abbreviations: EC = Endotoxin control; NSC = Negative saline control; TS = Test substance1
EC value (e.g., EC 1.0) represents the endotoxin concentration in EU/mL.
2
TS number (e.g., TS1) represents an arbitrary sequence for individual test substances.
3
IL-6 values in columns 11 and 12 are in pg/mL.

429

Table 6-4

Overview of ELISA Procedure

Materialtransfer from
Incubation
Plate

Enzymelabeled
Antibody

(µL)
50

430
431
432

(µL)

Cover theIncubation
Plate andincubate for
2 to 3 hr at
20-25oC.

200

TMB/Substrate
Solution(µL)

200

Abbreviations: RT = Room temperature

B3-20

Incubate 15
min at RT in
dark.

Stop
Solution(µL)

50

Read opticaldensity at
450 nm witha 540-590
nm
wavelengthreference
filter.

Draft ICCVAM Recommended Test Method Protocol: PBMC/IL-6 Pyrogen Test

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432
433

7.0

434

7.1

435

The OD of each well is obtained by reading the samples in a standard microplate436

spectrophotometer (i.e., plate reader) using a visible light wavelength of 450 nm (OD450) with437

a 540 to 590 nm reference filter (recommended). OD450 values are used to determine assay

438

acceptability and in the decision criteria for the detection of endotoxin in a test substance (see439

Sections 8.0 and 9.0).

440

8.0

441

Obtain the PPC and the corresponding NPC by interference testing of a test substance in the442

presence and absence of a fixed quantity of endotoxin (i.e., 0.25 EU/mL) in quadruplicate.

443

An EC (five-point standard curve) and a NSC should be included in each experiment. An IL-444

6 standard curve should be included in each ELISA as shown in the template presented in

445

Table 6-3. An assay is considered acceptable only if the following minimum criteria are met:

446

EVALUATION OF TEST METHODSOD Measurements

CRITERIA FOR AN ACCEPTABLE TEST•447

The quadratic function of the IL-6 standard curve produces an r ≥0.95 and the
OD450 of the blank control is below 0.15.

448

•

449

The endotoxin standard curve produces OD450 values that ascend in asigmoidal concentration response.

450

Blood donors are considered to be low responders if their mean OD450 value obtained for 1

451

EU/mL EC is significantly below the mean OD450 value obtained for 1000 pg/mL IL-6.

452

Blood donors who produce a mean OD450 value for the NSC that is significantly above the453mean OD450 value at 500 pg/mL IL-6 are considered to be high responders. Low and high454

responders should be excluded from analysis. Each test must be performed with PBMCs

455

from four different donors. From a set of four donors, a maximum of one donor may be

456

excluded from the assessment; otherwise, the test must be repeated with four different

457

donors.

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458

9.0

DATA INTERPRETATION/DECISION CRITERIA

459

9.1

Decision Criteria for Determination of Pyrogenicity

460

The validity of the endotoxin standard curve should be calculated using a four-parameter

461

logistic model. If necessary to satisfy the model, endotoxin concentrations may be modified.

462

Calculate the mean OD450 values of all of the replicates in each experimental group. Calibrate463

the mean OD450 value for each test substance using the endotoxin standard curve and464

document the estimated endotoxin concentration. Multiply the estimated endotoxin

465

concentration by the dilution factor, if necessary. This value represents the pyrogenicity of466

the sample in terms of endotoxin equivalents for that particular donor. The t-test is used to467

compare the data of a test sample against the data of the EC (0.5 EU/mL) that is performed in468

parallel. If this test results in a significant p-value (i.e., smaller than 1%), then the sample is469

considered to be non-pyrogenic, and as pyrogenic if otherwise (Hoffmann et al., 2005), as

470

long as the assay acceptability criteria in Section 8.0 has been met.

471

9.1.1

Decision Level 1

472•

If all donors show a negative reaction, then the product passes.

473•

If two or more donors show a positive reaction, then the product fails.

474•

If one donor shows a positive reaction, then an additional test with four donors

475
476

has to be performed (go to Decision Level 2).
9.1.2

477

Decision Level 2•478

If out of 6 to 8 donors, only one donor shows a positive reaction, then the
product passes.

479•

In any other case, the product fails.

480

10.0

481

The test report should include the following information:

482

Test Substances and Control Substances

483

STUDY REPORT•Name of test substance
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484•

Purity and composition of the substance or preparation

485•

Physicochemical properties (e.g., physical state, water solubility)

486•

Treatment of the test/control substances prior to testing (e.g., vortexing,

487

sonication, warming, resuspension solvent)488

Justification of the In Vitro Test Method and Protocol Used

489

Test Method Integrity

490

•

491

test method over time

492

•

493

If the test method employs proprietary components, documentation on theprocedure used to ensure their integrity from “lot-to-lot” and over time

494

•

495
496

The procedure used to ensure the integrity (i.e., accuracy and reliability) of the

The procedures that the user may employ to verify the integrity of the
proprietary components

Criteria for an Acceptable Test

497•

Acceptable concurrent positive control ranges based on historical data

498•

Acceptable negative control data

499

Test Conditions

500•

Cell system used

501•

Calibration information for the spectrophotometer used to read the ELISA

502•

Details of test procedure used

503•

Description of any modifications of the test procedure

504•

Reference to historical data of the model

505•

Description of evaluation criteria used•

Tabulation of data from individual test samples

506
507
508

Results

Description of Other Effects Observed
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509

Discussion of the Results

510

Conclusion

511

A Quality Assurance Statement for Good Laboratory Practice (GLP)-Compliant Studies

512•This statement should indicate all inspections made during the study and the513

dates any results were reported to the Study Director. This statement should514

also confirm that the final report reflects the raw data.

515

If GLP-compliant studies are performed, then additional reporting requirements provided in516

the relevant guidelines (e.g., OECD 1998; EPA 2003a, 2003b; FDA 2003) should be517

followed.

518

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518

11.0

REFERENCES

519

Bleeker WK, de Groot EM, den Boer PJ, Biessels PT, Aarden LA, Bakker JC. 1994.

520

Measurement of interleukin-6 production by monocytes for in vitro safety testing of

521

hemoglobin solutions. Artif Cells Blood Substit Immob Biotechnol 22:835-840.

522

Dinarello CA, O'Connor JV, LoPreste G, Swift RL. 1984. Human leukocyte pyrogen test for

523

detection of pyrogenic material in growth hormone produced by recombinant Escherichia

524

coli. J Clin Microbiol 20:323-329.

525

EPA. 2003a. Good Laboratory Practice Standards. Toxic Substances Control Act. 40 CFR

526

792.

527

EPA. 2003b. Good Laboratory Practice Standards. Federal Insecticide, Fungicide, and528

Rodenticide Act. 40 CFR 160.

529

FDA. 2003. Good Laboratory Practices for Nonclinical Laboratory Studies. 21 CFR 58.

530

Hoffmann S, Peterbauer A, Schindler S, Fennrich S, Poole S, Mistry Y, Montag-Lessing T,

531

Spreitzer I, Löschner B, van Aalderen M, Bos R, Gommer M, Nibbeling R, Werner-

532

Felmayer G, Loitzl P, Jungi T, Brcic M, Brügger P, Frey E, Bowe G, Casado J, Coecke S, de533

Lange J, Mogster B, Næss LM, Aaberge IS, Wendel A, Hartung T. 2005. International534

validation of novel pyrogen tests based on human monocytoid cells. J Immunol Methods

535

298:161-173.

536

Organization for Economic Co-operation and Development (OECD). 1998. OECD Series on

537

Principle of Good Laboratory Practice and Compliance Monitoring. No. 1. OECD Principles

538

of Good Laboratory Practice (as revised in 1997). OECD, ENV/MC/CHEM (98)17. Paris:

539

OECD.

540

Poole S, Mistry Y, Ball C, Gaines Das RE, Opie LP, Tucker G, Patel M. 2003. A rapid 'one-

541

plate' in vitro test for pyrogens. J Immunol Methods 274:209-220.

542

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542

12.0

TERMINOLOGY AND FORMULA

543

12.1

Assay Sensitivity (λ)

544

For an in vitro cell-based assay, the variable λ is defined as the lowest statistically significant

545

point on the standard endotoxin concentration-response curve and represents the relative546

sensitivity of the test method for the detection of endotoxin (i.e., level of detection).

547

12.2

548

The EC is incubated with PBMCs and serves as the positive control for the experiment. The

549

results should be compared to historical values to insure that it provides a known level of550

cytokine release relative to the NSC.

551

12.3

552

The ELC is the maximum allowable concentration of endotoxin for a particular product and553

is expressed in EU per volume (mL) or weight (mg). The ELC is defined by the FDA or

554

specified in the USP2. It is calculated as the product of K/M, where:

555

K is the threshold pyrogen dose for parenteral use in rabbits or humans (5.0 EU/kg). At an

556

injection volume of 10 mL/kg, K is equal to 0.5 EU/mL.

557

M is the larger of the rabbit dose or the maximum human dose administered in one hour as

558

defined below and varies with test substance3.

559

12.4

560

The MVD is the maximum dilution of a test substance that can be tolerated in a test system561

without exceeding the ELC, if the test substance must be diluted as a result of assay

562

interference. Dilutions beyond the MVD would not be valid for endotoxin detection in the

Endotoxin Control (EC)Endotoxin Limit Concentration (ELC)

Maximum Valid Dilution (MVD)2

ELC values for most marketed pharmaceutical products are provided in the USP or in other pharmacopoeial or
regulatory publications (e.g., European Pharmacopoeia, Japanese Pharmacopoeia, Pharmacopoeial guidelines,
FDA publications).
3
Values for most marketed pharmaceutical products are provided in the USP or in other pharmacopoeial or
regulatory publications (e.g., European Pharmacopoeia, Japanese Pharmacopoeia, pharmacopoeial guidelines,
FDA publications).
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563

test system. Calculation of the MVD is dependent on whether or not the ELC for a test

564

substance is published. When the ELC is known, the MVD is:565

MVD = (ELC x Product Potency [PP])/λ566

As an example, for "Cyclophosphamide Injection," the ELC is 0.17 EU/mg, PP is 20567

mg/mL, and the assay sensitivity is 0.1 EU/mL. The calculated MVD would be 34. The test

568

substance can be diluted no more than 1:34 prior to testing.

569

If the ELC is not known, the MVD is:570

MVD = PP/Minimum Valid Concentration (MVC)571

where, MVC = (λ x M)/K

572

where, M is the maximum human dose573

As an example, for "Cylophosphamide Injection," the PP is 20 mg/mL, M is 30574

mg/kg, and assay sensitivity is 0.1 EU/mL. The calculated MVC is 0.6 mg/mL and the MVD

575

is 33.3. The test substance can be diluted no more than 1:33 in the assay prior to testing.

576

12.5

577

The NPC is a test sample to which PFS is added. The NPC is the baseline for determination578

of cytokine release relative to the endotoxin-spiked PPC.

579

12.6

580

The NSC is PBMCs (in RPMI-C) incubated with PFS (used for dilution of test substance)581

and is used as the blank.

582

12.7

583

The value K represents the threshold pyrogen dose for parenteral products for rabbits and584

humans. Based on experimental data, K is fixed at 5.0 EU/kg. For intrathecal products, K is585

0.2 EU/kg.

586

12.8

587

The PPC is a test substance spiked with the control standard endotoxin (i.e., 0.5 EU/mL or an

588

amount of endotoxin equal to that which produces ½ the maximal increase in OD from the

Negative Product Control (NPC)

Negative Saline Control (NSC)

Parenteral Threshold Pyrogen Dose (K)Positive Product Control (PPC)

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589

endotoxin standard curve) to insure that the test system is capable of endotoxin detection in590

the product as diluted in the assay.

591

12.9

592

The concentration for a test substance is the PP typically expressed as µg/mL or mg/mL.

593

12.10

594

The variable M represents the rabbit test dose or the maximum human dose in 1 hr. The

595

variable M is expressed in mg/kg and varies with the test substance. For

596

radiopharmaceuticals, M should be adjusted to account for product activity (radioactive

597

decay) at time administration. An average human standard weight of 70 kg is used for the598

calculation. If a pediatric dose should be used and it is higher than the adult dose, then it

599

should be used in the formula.

Product Potency (PP)

Rabbit Pyrogen Test (RPT) Dose or Maximum Human Dose (M)

600
601

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Appendix B4

Draft ICCVAM Recommended Protocol for Future Studies Using the Whole Blood(WB)/Interleukin-1β (IL-1β) Test Method

Draft ICCVAM Recommended Test Method Protocol: WB/IL-1β Pyrogen Test

[This Page Intentionally Left Blank]

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Draft ICCVAM Recommended Test Method Protocol: WB/IL-1β Pyrogen Test

01 Dec 2006

1

Draft ICCVAM Recommended Protocol for Future Studies Using the Whole Blood2(WB)/Interleukin-1β (IL-1β) Test Method

3
4

PREFACE

5
6This proposed protocol for the detection of pyrogenicity is based on information obtained

7

from 1) The European Centre for the Validation of Alternative Methods (ECVAM) WB/IL-

8

1β Background Review Document (BRD) presented in Appendix A of the draft Interagency

9

Coordinating Committee on the Validation of Alternative Methods (ICCVAM) Pyrogenicity

10

Test Method BRD, which includes ECVAM Standard Operating Procedures (SOPs) for the11

WB/IL-1β test method, and 2) Information provided to the National Toxicology Program

12(NTP) Interagency Center for the Validation of Alternative Toxicological Methods

13(NICEATM) by Dr. Thomas Hartung, Head of ECVAM. The ECVAM SOPs are based on

14

the WB/IL-1β methodology first described by Hartung and Wendel (1996). A table of15comparison between the draft ICCVAM recommended protocol and the ECVAM SOPs is

16

provided. Future studies using the WB/IL-1β test method may include further

17

characterization of the usefulness or limitations of the assay for regulatory decision-making.

18

Users should be aware that the proposed test method protocol might be revised based on

19

additional optimization and/or validation studies. ICCVAM recommends that test method

20

users routinely consult the ICCVAM/NICEATM website (http://iccvam.niehs.nih.gov/) to21

ensure that the most current test method protocol is used.

22

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Draft ICCVAM Recommended Test Method Protocol: WB/IL-1β Pyrogen Test

22
23
24
25
26
27
28
29
30
31
32
33
34
35

[This Page Intentionally Left Blank]

36
37

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Draft ICCVAM Recommended Test Method Protocol: WB/IL-1β Pyrogen Test

37
38

Table 101 Dec 2006

Comparison of Draft ICCVAM Recommended Test Method Protocol with the ECVAM SOP for the Whole
Blood (WB)/Interleukin-1β (IL-1β) Pyrogen Test Method

Protocol Component

39
40
41
42
43
44
45

Draft ICCVAM Protocol
ECVAM SOP
ECVAM Validation SOPTest neat or at minimal dilution that
Test SubstanceSame as ICCVAM protocol
Test at MVD
produces no interference
NSC (1)Same as ICCVAM protocol
Same as ICCVAM protocol
EC (5)
Same as ICCVAM protocol
EC (2)
Incubation Plate
TS (14)
TS (25)EC spikes (3 x 5)
(number of control or test
PPC1 (0)Same as ICCVAM protocol
PPC (3)groups at n=4 each)
NPC1 (0)Same as ICCVAM protocol
NPC (3)
LTAC (0)LTAC (1)Same as ICCVAM protocol
Includes seven point IL-1β SC and
ELISA Plate
Same as ICCVAM protocol
Same as ICCVAM protocol
blank in duplicate
2
Decision Criteria for
0.5 x Median OD450 of 1 EU/mL EC
0.5 x Median OD450 of 0.5 EU/mL EC
Not applicable (tested at MVD)
Interference<2x Median OD450 of 1 EU/mL EC<2x Median OD450 of 0.5 EU/mL EC
Mean OD450 of 0.5 EU/mL EC ≥Same as ICCVAM protocol
Same as ICCVAM protocol
1.6X Mean OD450 of NSC
Mean OD450 of PPC is 50% to 200%
Same as ICCVAM protocol
Not specified
of 0.5 EU/mL EC
Assay Acceptability
Mean OD450 of NSC ≤0.15
Same as ICCVAM protocol
Same as ICCVAM protocol
Criteria
Quadratic function of IL-1β SC
Same as ICCVAM protocol
Same as ICCVAM protocol
r2 ≥0.95
EC SC produces OD450 values thatascend in a sigmoidal concentration
Same as ICCVAM protocol
Same as ICCVAM protocol
response
Decision Criteria for
OD450 Test sample ≥ OD450 0.5
Same as ICCVAM protocol
Same as ICCVAM protocol
Pyrogenicity
EU/mL
Abbreviations: EC = Endotoxin control; LTAC = Lipoteichoic acid (LTA) control; MVD = Maximum Valid Dilution; NPC = Negative Product Control; NSC =Normal saline control; PPC = Positive Product Control; SC = Standard curve; TS = Test substance1
PPC and NPC are evaluated during the interference test.
2
Median or mean OD450 values are corrected (i.e., reference filter reading, if applicable, and NSC are subtracted).

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46
47
48
49
50
51
52
53
54
55
56
57
58

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59
60
61
62
63

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64

1.0

65

The purpose of this protocol is to describe the procedures used to evaluate the presence of a66

pyrogen (i.e., Gram-negative endotoxin) in a test substance. The presence of Gram-negative

67

endotoxin is detected by its ability to induce cytokine IL-1β release from monocytoid cells in68

human WB. The quantity of IL-1β released is obtained using an enzyme-linked

69

immunosorbent assay (ELISA) that includes monoclonal or polyclonal antibodies specific for70

IL-1β. Release of IL-1β is measured by incubation of WB with test substances or controls

71(i.e., positive, negative). The amount of pyrogen present is determined by comparing the72

values of endotoxin equivalents produced by WB cells exposed to the test substance to those73

exposed to an internationally-harmonized Reference Standard Endotoxin (RSE)1 or an74

equivalent standard expressed in Endotoxin Units (EU)/mL. Based on a rabbit threshold

75

pyrogen dose of 0.5 EU/mL, which was established in a retrospective evaluation of rabbit

76

pyrogen test (RPT) data, a test substance is considered pyrogenic if it induces a level of IL-

77

1β release equal to or greater than 0.5 EU/mL.

78

The focus of this protocol is on the use of the WB/IL-1β test method, specifically for the79

detection of Gram-negative endotoxin in parenteral pharmaceuticals. The relevance and80

reliability for non-endotoxin pyrogens (e.g., lipoteichoic acid) has not been demonstrated in a81

formal validation study.

82

2.0

83

All procedures for procurement of eligible blood donors and blood donations should follow

84

the regulations and procedures set forth by institutional guidelines for utilization of human85

substances, which include but are not limited to blood, tissues, and tissue fluids. Standard

86

laboratory precautions are recommended including the use of laboratory coats, eye87protection, and gloves. If necessary, additional precautions required for specific study

88

substances or hazardous chemicals will be identified in the Material Safety Data Sheet

89(MSDS).
1

PURPOSE AND APPLICABILITY

SAFETY AND OPERATING PRECAUTIONSRSEs are internationally-harmonized reference standards (e.g., WHO E. coli Lipopolysaccharide [LPS] 94/580[0113:H10:K-]; USP RSE Lot G3E069; FDA Lot EC-6). Equivalent endotoxins include commercially available
E. coli-derived LPS Control Standard Endotoxin (CSE) or other E. coli LPS preparations that have beencalibrated with an appropriate RSE.
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90

The stop solution used in the ELISA kit is acidic and corrosive and should be handled with91

the proper personal protective devices. If this reagent comes into contact with skin or eyes,

92

wash thoroughly with water. Seek medical attention, if necessary.

93

Tetramethylbenzidine (TMB) solution contains a hydrogen peroxide substrate and 3, 3', 5, 5'-

94

TMB. This reagent is a strong oxidizing agent and a suspected mutagen. Appropriate

95

personal protection should be used to prevent bodily contact.

96

Bacterial endotoxin is a toxic agent (i.e., can induce sepsis, shock, vascular damage,97

antigenic response) and should be handled with care. Skin cuts should be covered and98

appropriate personal protective devices should be worn. In case of contact with endotoxin,99

immediately flush eyes or skin with water for at least 15 min. If inhaled, remove the affected100

individual from the area and provide oxygen and/or artificial respiration as needed. Skin101

absorption, ingestion, or inhalation may produce fever, headache, and hypotension.

102

3.0

MATERIALS, EQUIPMENT, AND SUPPLIES

103

3.1

Source of Cells

104

Leukocytes from WB are the source of cells for cytokine production in the WB/IL-1β test

105

method (Hartung and Wendel, 1996; Schindler et al., 2006). WB is obtained from healthy106

human volunteers who have provided their consent according to established institutional

107

guidelines. Volunteers are expected not to have taken any drugs (e.g., prescription drugs,

108

recreational drugs, herbal drugs) and to have been free from illness for at least two weeks

109prior to donation.

110

3.2

111

For all steps in the protocol, excluding the ELISA procedure, the materials that will be in

112

close contact with samples and/or blood cells (e.g., pipet tips, containers, solutions) should be

113

sterile and free from detectable pyrogens.

114

3.2.1

Blood Incubation

115

3.2.1.1

Equipment

116

Equipment and Supplies•

Centrifuge
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117•

Hood; Bio-safety, laminar flow (recommended)118•

Incubator; cell culture (37±1°C + 5% CO2)

119•

Pipetter; multichannel (8- or 12-channel)

120•

Pipetters; single-channel adjustable (20 and 200 µL)

121•

Repeating pipetter

122•

Vortex mixer

123

3.2.1.2

01 Dec 2006

Consumables

124•

Centrifuge tubes; nonpyrogenic, polypropylene (15 and 50 mL)

125•

Combitips; repeating pipetter (1.0 and 2.5 mL)

126•

Needle set; Sarstedt multifly, pyrogen-free, 19 mm, 21 gauge for S-Monovette

127•

Plates; microtiter, nonpyrogenic, 96-well, polystyrene, tissue culture

128•

Reaction tubes; polypropylene (1.5 mL)

129•

Reservoirs; fluid

130•

Tips; pipetter, sterile, pyrogen-free (20 and 200 µL)

131•

Tubes; Sarstedt S-Monovette, 7.5 mL, heparinized for blood collection

132

3.2.2

ELISA

133

3.2.2.1

Equipment

134•

Microplate mixer

135•

Microplate reader (450 nm with an optional reference filter in the range of

136

600-690 nm)

137•

Microplate washer (optional)

138•

Multichannel pipetter

139
140

3.2.2.2

Consumables•

Container; storage, plastic
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141•

Deionized water; nonsterile

142•

Plates; microtiter, nonpyrogenic, 96-well, polystyrene, tissue culture

143•

Pyrogen-free water (PFW)

144•

Reservoirs; fluid

145•

Tips; pipetter, nonsterile

146•

Tubes; polystyrene (12mL)

147

3.2.2.3

148

An ELISA that measures IL-1β release from Cryo WB is used. A variety of IL-1β ELISA

149

kits are commercially available and the IL-1β ELISA procedure outlined in this protocol is150

intended to serve as an example for using an ELISA kit. If the user prefers to prepare an in-151

house ELISA, then additional reagents would be required. The IL-1β ELISA should be

152

calibrated using an IL-1β international reference standard (e.g., WHO 86/680) prior to use.

153

The IL-1β cytokine assay kits do not provide the RSE or endotoxin equivalent; therefore, this154

reagent must be purchased separately. Results obtained using these products are subject to155

the assay acceptability and decision criteria described in Sections 8.0 and 9.0. IL-1β ELISA

156

kit components may include the following:

157

ELISA Kit•

158

ELISA plates coated with anti-human IL-1β capture antibody; monoclonal or
polyclonal

159•

Buffered wash solution

160•

Dilution buffer

161•

Enzyme-labeled detection antibody

162•

Human IL-1β reference standard

163•

Pyrogen-free saline (PFS)164•

Stop solution

165•

TMB/substrate solution

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166

3.3

167

01 Dec 2006

Chemicals•

168

Endotoxin (e.g., WHO E. coli LPS 2nd International Standard 94/580; USP
RSE E. coli LPS Lot G3E069; USP RSE E. coli Lot G; FDA E. coli Lot EC6)

169

3.4

Solutions

170

ELISA solutions are listed in Section 3.2..

171

4.0

172

All test substances, endotoxin, and endotoxin-spiked solutions should be stored at 4°C.

173

4.1

174

An internationally harmonized RSE or equivalent is used to generate the endotoxin standard

175

curve. The use of any other E. coli LPS requires calibration against a RSE using the WB/IL-

176

1β test method.

177

A standard endotoxin curve consisting of a Normal Saline Control (NSC) and five RSE

178

concentrations (0.25, 0.50, 1.0, 2.5, and 5.0 EU/mL) are included in the incubation step (refer179

to Table 4-1) and then transferred to the ELISA plate. To prepare the endotoxin standard

180

curve, first obtain a 2000 EU/mL stock solution by addition of PFW to the lyophilized

181

content of the stock vial by following the instructions provided by the manufacturer (e.g., for182

a vial containing 10,000 EU, 5 mL of PFW is added). To reconstitute the endotoxin, the stock

183

vial should be vortexed vigorously for at least 30 min or sonicated in a bath sonicator for 5

184

min. The stock solution is stable for 14 days when stored at 2 to 8°C. An endotoxin standard

185

curve is prepared by making serial dilutions of the stock solution in PFS as described in

186

Table 4-1.

ASSAY PREPARATION

Endotoxin Standard Curve

187

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187
188

Table 4-1

Preparation of Endotoxin Standard Curve

Stock Endotoxin
EU/mL

189
190
191
192
193
194
195

µL of Stock
EndotoxinµL of PFS

Endotoxin
Concentration
EU/mL
503
5.0
2.5
1.0
0.50
0.25
0

20001,2
50
1950
50
100
900
5.0
500
500
2.5
500
500
1.0
500
500
0.50
500
500
0
0
1000
Abbreviations: EU = Endotoxin units; PFS = Pyrogen-free saline
Each stock tube should be vortexed prior to its use to make the subsequent dilution.
1
A 2000 EU/mL stock solution of endotoxin is prepared according to the manufacturer's instructions.
2
The stock solution of USP RSE may be stored in aliquots and kept at -20°C for up to 6 months. Do not store
the endotoxin at -80°C.
3This concentration is not used in the assay.

196

4.2

Test Substances

197

Liquid test substances should be tested neat or, if interference is detected (see Section 4.2.1),

198

diluted in PFS. Solid test substances should be prepared as solutions in PFS or, if insoluble in199

saline, dissolved in dimethylsulfoxide (DMSO) then diluted up to 0.5% (v/v) with PFS,

200

provided that this concentration of DMSO does not interfere with the assay. The test

201

substances should be vortexed vigorously for at least 30 min or sonicated in a bath sonicator202

for 5 min.

203

4.2.1

204

Interference testing must be carried out on any test sample for which no interference

205

information is available. The purpose of the interference test is to determine the lowest

206

dilution (i.e., highest concentration) of a test substance from which an endotoxin spike can be

207

detected (i.e., based on the decision criteria described in Section 4.2.1.2. However, to ensure

208

a valid test, a test substance should not be diluted beyond its Maximum Valid Dilution

209(MVD).

210

For many marketed products, values for the MVD and the Endotoxin Limit Concentration

211(ELC) are published in the U.S. Pharmacopeia, the European Pharmacopoeia, and/or Food212

and Drug Administration (FDA) guidelines. However if one or both of these values are not

Interference Testing

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213

available, then calculation of the MVD is dependent on the ELC (see Section 12.3). If

214

unknown, the ELC can be approximated by dividing the maximum hourly dose of the215

product by the hourly dose received per patient. For example, if a product is used at an

216

hourly dose of 100 mg per patient, then the ELC would be 350 EU/100 mg or 3.5 EU/mg.

217

4.2.1.1

218

The WHO-LPS 94/580 [E. coli O113:H10:K-] or equivalent international RSE (e.g., USP

219

G3E069, FDA EC-6) is recommended for preparation of the endotoxin control (EC). If a220

different E. coli LPS is used and the potency relative to the RSE is not provided, then each221

lot must be calibrated against the RSE in the WB/IL-1β test method. For interference testing,222

an endotoxin standard curve (see Section 4.1) should be included on each plate.

223

4.2.1.2

224

For interference testing, non-spiked and endotoxin-spiked test substances are prepared in225

microplate wells (n=4 replicates) and an in vitro pyrogen test is performed. Either PFS or a226

fixed concentration (a concentration selected from the middle of the EC standard curve) of

227

the RSE (i.e., 1 EU/mL) in PFS is added to the test substance in serial two-fold dilutions. An

228

illustrative example of endotoxin-spiking solutions is shown in Table 4-2. For non-spiked

229

solutions, 200 µL of PFS is added to a well followed by 20 µL each of the test substance230(neat or at serial dilution) and 20 µL of WB. Endotoxin-spiked solutions are prepared by231

adding 180 µL of PFS to each well followed by 20 µL of the test substance (neat or at serial232

dilution) and 20 µL of WB. Then, 20 µL of a 1 EU/mL solution of endotoxin in PFS is added

233

and the well contents are mixed (see example presented in Table 4-2).

Reference Endotoxin for Spiking Test Substances

Spiking Test Substances with Endotoxin

234

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234
235

Table 4-2

Preparation of Endotoxin-spiked and Non-spiked Solutions for

236

Determination of Test Substance Interference in the Incubation and

237

ELISA Test Systems
SpikedSample Addition

Non-spikedµL/well

Pyrogen-free saline
Endotoxin spike solution2
Test substance (neat and each serial dilution)
WB
Total3

180
20
20
20
240

1

200
0
20
20
240

238
239
240
241
242
243
244

Abbreviations: WB = Whole blood1
n=4 replicates each2
Endotoxin concentration is 1 EU/mL in PFS.
3
A total volume of 240 µL per well is used for the incubation.

245

200% in the pyrogen test is determined. The optical density (OD) values of the endotoxin-

246

spiked and non-spiked test substances are calibrated against the endotoxin calibration curve.

247

The resulting EU value of the non-spiked test substance is subtracted from the corresponding

248

EU value of the endotoxin-spiked test substance at each dilution. The % recovery for each249

sample dilution is then determined from the endotoxin spike solution concentration set to250

100%. For example, consider the following interference test results in Table 4-3:

251

Table 4-3

252

The lowest dilution of the test substance that yields an endotoxin spike recovery of 50% to

Example of Interference Data Used to Determine Sample Dilution for
Assay

Sample Dilution
None
1:2
1:4
1:8

% Recovery of Endotoxin Control
25
49
90
110

253
254

Based on these results, the dilution of the test substance used in the in vitro pyrogen test

255

would be 1:4 (i.e., the lowest dilution between 50% and 200% of the 1 EU/mL EC).

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256

4.2.2

257

If the data obtained from the experiment in Section 4.2.1 suggests the presence of258interference, then a subsequent experiment similar to that described in Section 4.2.1 would

259

need to be performed to confirm that the test substance(s) does not directly interfere with the260

ELISA. For this experiment, an ELISA would be performed in the absence of PBMCs.

261

5.0

CONTROLS

262

5.1

Negative Control

263

A negative control (e.g., PFS is added instead of the test sample) is included in each264

experiment in order to detect nonspecific changes in the test system, as well as to provide a265baseline for the assay endpoints.

266

5.2

267

Solvent controls are recommended to demonstrate that the solvent is not interfering with the268test system when solvents other than PFS are used to dissolve test substances.

269

5.3

270

An internationally standardized EC (e.g., WHO 94/580; USP G3E069; 0.5 EU/mL) is

271

included in each experiment to verify that an appropriate response is induced.

272

5.4

273

Benchmark controls may be used to demonstrate that the test method is functioning properly,

274

or to evaluate the relative pyrogenic potential of chemicals (e.g., parenteral pharmaceuticals,

275

medical device eluates) of a specific class or a specific range of responses, or for evaluating276

the relative pyrogenic potential of a test substance. Appropriate benchmark controls should

277

have the following properties:

278

Interference with ELISA

Solvent Control

Positive Control

Benchmark Controls•279

consistent and reliable source(s) for the chemicals (e.g., parenteralpharmaceuticals, medical device eluates)

280•

structural and functional similarities to the class of substance being tested

281•

known physical/chemical characteristics
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supporting data on known effects in animal models

283•

known potency in the range of response

01 Dec 2006

284

5.5

285

The PPC is a test substance diluted to a level that does not interfere with the test method and286

does not exceed the MVD. The PPC is obtained by spiking a test substance with a known EC

287(e.g., 1 EU/mL) and demonstrating that 50% to 200% of the EC is recovered.

288

5.6

289

The NPC is the test substance diluted to the MVD and then spiked with PFS. It is the290

negative control for the PPC.

291

6.0

EXPERIMENTAL DESIGN

292

6.1

Incubation with Test Samples and Measurement of IL-1β Release

293

6.1.1

Collection of Human Blood294

WB is obtained from healthy human volunteers who have provided their consent according295

to established institutional guidelines. Volunteers are expected not to have taken any drugs

296

and to have been free from illness for at least two weeks prior to donation. The criteria for297

rejection of data from donors that are low responders or that are suspect due to veracity of298

health information is addressed in Section 8.0.

299

Fresh WB is drawn by venipuncture using a multifly needle set and collected in heparinized300

tubes (15 IU/mL lithium heparin). All components of the blood collection system (i.e.,

301

syringes, tubes, connecting lines) must be sterile and pyrogen-free. Blood should be stored at302

room temperature (RT) and must be used within 4 hr. Prior to use in the assay, the collection

303

tubes should be gently inverted 1 to 2 times. Do Not Vortex.

304

6.1.2

305

Test substances are prepared at a level of dilution that did not show interference with the test

306

system or for which it is known that interference does not occur. Each incubation plate can

307

accommodate an endotoxin standard curve, a NSC, and 14 test samples (see Table 6-1).

308

Positive Product Control (PPC)

Negative Product Control (NPC)

Incubation Plate

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308
309

Table 6-1

Overview of Incubation Plate Preparation in the WB/IL-1β Test Method

Mix thesamples;
PFS
EC
WB
Numberimmediately
Sampleof Wells
transfer to an
ELISA plate3µL
1
and run
20
EC
200
20
0
20
ELISA or
4
NSC
220
0
0
20
store plate atTest
2-20°C or 56
samples
200
0
20
20
80°C.
(1-14)
Abbreviations: EC = Endotoxin control; NSC = Negative saline control; PFS = Pyrogen-free saline; WB =Whole blood
1
Five EC concentrations (0.25, 0.50, 1.0, 2.5, and 5.0 EU/mL) in quadruplicate.
2
14 test samples (n=4) per plate.
3
An IL-1β standard curve is prepared in Columns 11 and 12 on the ELISA plate. Therefore, 80 wells areavailable for test samples and controls on the incubation plate.
Test
Sample

310
311
312
313
314
315
316
317

Mix thesamples;incubateovernight at
37±1°C in ahumidified
atmospherewith 5%
CO2.

318

6.1.3

Incubation Assay for IL-1β Release

319

Blood samples are prepared in a microtiter plate using a laminar flow hood. All consumables

320

and solutions must be sterile and pyrogen-free. Each plate should be labeled appropriately

321

with a permanent marker. An overview of the incubation plate preparation is shown in Table

322

6-1. The incubation procedure is outlined below:

323

Step 1. Refer to the incubation plate template presented in Table 6-2.

324

Step 2. Using a pipetter, transfer 200 µL of PFS into each well.

325

Step 3. Transfer 20 µL of test sample into the appropriate wells as indicated in the326

template.

327

Step 4. Transfer 20 µL of the EC (standard curve) and the NSC controls in

328

quadruplicate into the appropriate wells according to the template.

329

Step 5. Transfer 20 µL of WB into each well and mix by gently swirling the plate.

330

Step 6. Mix the contents of the wells thoroughly by gently pipetting up and down

331

five times using a multichannel pipetter, changing the tips between each row to332

avoid cross-contamination.
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333

Step 7. Place the covered plate in a tissue culture incubator for 10 to 24 hr at

334

37±1°C in a humidified atmosphere containing 5% CO2.

335

Step 8. Prior to transferring the test samples onto the ELISA plate, mix the336

contents of the wells by pipetting up and down three times using a multichannel

337

pipetter, changing the tips between each row to avoid cross-contamination.

338

Note: The aliquots may be tested immediately in the ELISA or stored at -20°C or

339-80°C for testing at a later time. After transfer to the ELISA plate, freeze the340

remaining aliquots at -20°C or -80°C for subsequent experiments, if necessary (see341

Assay Acceptability and Decision Criteria in Sections 8.0 and 9.0).

342

Table 6-2
1

Incubation Plate - Sample and Control Template
2

3

4

5

6

7

8

9

10

11

12

1

EC
EC
EC
EC
TS3
TS3
TS3
TS3
TS11 TS11
Void3
5.0
5.0
5.0
5.0
EC
EC
EC
EC
B
TS4
TS4
TS4
TS4
TS11 TS11
Void
2.5
2.5
2.5
2.5
EC
EC
EC
EC
C
TS5
TS5
TS5
TS5
TS12 TS12
Void
1.0
1.0
1.0
1.0
EC
EC
EC
EC
D
TS6
TS6
TS6
TS6
TS12 TS12
Void0.50 0.50
0.50
0.50
EC
EC
EC
EC
E
TS7
TS7
TS7
TS7
TS13 TS13
Void0.25 0.25
0.25
0.25
F NSC NSC NSC
NSC
TS8
TS8
TS8
TS8
TS13 TS13
Void
G TS12 TS1
TS1
TS1
TS9
TS9
TS9
TS9
TS14 TS14
Void
H
TS2 TS2
TS2
TS2
TS10
TS10
TS10
TS10
TS14 TS14
Void
Abbreviations: EC = Endotoxin control; NSC = Negative saline control; TS = Test substance1
EC value (e.g., EC 5.0) represents the endotoxin concentration in EU/mL.
2
TS number (e.g., TS1) represents an arbitrary sequence for individual test substances.
3
Columns 11 and 12 are reserved for the IL-1β standard curve on the ELISA plate (see Table 6-3).
A

343
344
345
346
347
348

Void
Void
Void
Void
Void
Void
Void
Void

349

6.2

ELISA to Measure IL-1β Release

350

6.2.1

IL-1β Standard Curve

351

An IL-1β standard, supplied with the ELISA kit, is used. IL-1β standards are typically352

supplied in lyophilized form and should be reconstituted according to the manufacturer's

353

instructions. The stock solution should be diluted in PFS to the following concentrations: 0,

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354

62.5, 125, 250, 500, 1000, 2000, and 4000 pg/mL. Each well on the ELISA plate will receive355

100 µL of an IL-1β blank or standard.

356

6.2.2

357

The manufacturer's instructions provided with the ELISA kit should be followed and a358

typical experimental design is outlined below. If the user prefers to prepare an in-house

359

ELISA, then appropriate modifications and validation of these changes would be necessary.

360

The ELISA should be carried out at RT and therefore all components must be at RT prior to361

use. Do not thaw frozen specimens by heating them in a water bath. A sample ELISA plate362

template is shown in Table 6-3, which includes a five-point EC standard curve, a NSC, an

363

eight-point IL-1β standard curve (0 to 4000 pg/mL), and 14 test substances in quadruplicate.

364

The EC standard curve, the NSC, and the test sample supernatants are transferred directly

365from the incubation plate. The IL-1β standard curve is prepared as described in Section366

6.2.1. An overview of the ELISA plate preparation is shown in Table 6-4.

367

Immediately prior to the ELISA procedure, dilute or mix any assay components according to368

the manufacturer’s instructions.

ELISA

369

Step 1. Add 100 µL of enzyme-labeled detection antibody to each well.

370

Step 2. After pipetting up and down three times to mix the supernatant, transfer

371

100 µL from each well of the Incubation Plate (A1-10; H1-10) to the ELISA plate.

372

Step 3. Add 100 µL of each IL-1β standard (0 to 4000 pg/mL) into the respective

373

wells on the ELISA plate.

374

Step 4. Cover the microtiter plate(s) with adhesive film and incubate for 90 min on

375

a microplate mixer at 350-400 rpm at 20 to 25°C.

376

Step 5. Decant and wash each well five to six times with 300 µL Buffered Wash

377

Solution per well and then rinse three times with deionized water. Place the plates

378

upside down and tap to remove the wash solution.

379

Step 6. Add 200 µL of TMB/Substrate Solution to each well and incubate at RT in380

the dark for 10 to 15 min. If necessary, decrease the incubation time.

381

Step 7. Add 50 µL of Stop Solution to each well.
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382

Step 8. Tap the plate gently after the addition of Stop Solution to aid in mixing.

383

Step 9. Read the OD450 within 15 min of adding the Stop Solution. Measurement384

with a reference wavelength of 600-690 nm is recommended.

385

Table 6-3

ELISA Plate - Sample and Control Template

1

2

3

4

EC1
5.0
EC
2.5
EC
1.0
EC
0.50
EC
0.25

EC
5.0
EC
2.5
EC
1.0
EC
0.50
EC
0.25

EC
5.0
EC
2.5
EC
1.0
EC
0.50
EC
0.25

EC
5.0
EC
2.5
EC
1.0
EC
0.50
EC
0.25

F

NSC

NSC

NSC

G

TS12

TS1

H

TS2

TS2

A
B
C
D
E

5

6

7

8

9

10

TS3

TS3

TS3

TS3

TS11

TS11

TS4

TS4

TS4

TS4

TS11

TS11

TS5

TS5

TS5

TS5

TS12

TS12

TS6

TS6

TS6

TS6

TS12

TS12

TS7

TS7

TS7

TS7

TS13

TS13

NSC

TS8

TS8

TS8

TS8

TS13

TS13

TS1

TS1

TS9

TS9

TS9

TS9

TS14

TS14

TS2

TS2

TS10

TS10

TS10

TS10

TS14

TS14

386
387
388
389
390
391

Abbreviations: EC = Endotoxin control; NSC = Negative saline control; TS = Test substance1
EC value (e.g., EC 5.0) represents the endotoxin concentration in EU/mL.
2
TS number (e.g., TS1) represents an arbitrary sequence for individual test substances.
3
IL-1β values in columns 11 and 12 are in pg/mL.

392

Table 6-4

393
394
395

11

12

IL-1β3
0
IL-1β
62.5
IL-1β
125
IL-1β
250
IL-1β
500
IL-1β
1000
IL-1β
2000
IL-1β
4000

IL-1β
0
IL-1β
62.5
IL-1β
125
IL-1β
250
IL-1β
500
IL-1β
1000
IL-1β
2000
IL-1β
4000

Overview of ELISA Procedure

Enzymelabeled
Antibody(µL)

MaterialtransferfromIncubation
Plate (µ L)

100

100

Incubate 90
min on aplate mixer
at 350-400rpm.

TMB/Substrate
Solution(µL)

200

Abbreviations: RT = Room temperature

B4-18

Incubate 15
min at RT in
the dark.

Stop
Solution(µL)

50

Read opticaldensity at
450 nm witha 600-690
nm
wavelengthreference
filter.

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396

7.0

EVALUATION OF TEST RESULTS

397

7.1

OD Measurements

398

The OD of each well is obtained by reading the samples in a standard microplate399

spectrophotometer (i.e., plate reader) using a visible light wavelength of 450 nm (OD450) with400

a 600 to 690 nm reference filter (recommended). OD450 values are used to determine assay

401

acceptability and in the decision criteria for the detection of endotoxin in a test substance (see402

Sections 8.0 and 9.0).

403

8.0

404

Obtain the PPC and the corresponding NPC by interference testing of a test substance in the405

presence and absence of a fixed quantity of endotoxin (i.e., 1 EU/mL) in quadruplicate. An

406

EC (five-point standard curve) and a NSC should be included in each experiment. An IL-1β

407standard curve should be included in each ELISA as shown in the template presented in

408

Table 6-3. An assay is considered acceptable only if the following minimum criteria are met:

409

CRITERIA FOR AN ACCEPTABLE TEST•410

The quadratic function of the IL-1β standard curve produces an r2 ≥ 0.95 andthe OD450 of the blank control is below 0.15.

411

•

412

The endotoxin standard curve produces OD450 values that ascend in asigmoidal concentration response.

413

9.0

DATA INTERPRETATION/DECISION CRITERIA

414

9.1

Decision Criteria for Determination of Pyrogenicity

415

The t-test is used to compare the data of a test sample against the data of the EC (0.5 EU/mL)

416

that is performed in parallel. If this test results in a significant p-value (i.e., smaller than 1%),

417

then the sample is considered to be non-pyrogenic, and as pyrogenic if otherwise (Hoffmann418

et al., 2005), as long as the assay acceptability criteria in Section 8.0 has been met.

419

10.0

420

The test report should include the following information:

421

Test Substances and Control Substances

STUDY REPORT

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422•

Name of test substance423•

Purity and composition of the substance or preparation

424•

Physicochemical properties (e.g., physical state, water solubility)

425•

Treatment of the test/control substances prior to testing (e.g., vortexing,426

sonication, warming, resuspension solvent)427

Justification of the In Vitro Test Method and Protocol Used

428

Test Method Integrity

429

•

430

test method over time

431

•

432

If the test method employs proprietary components, documentation on theprocedure used to ensure their integrity from “lot-to-lot” and over time

433

•

434
435

The procedure used to ensure the integrity (i.e., accuracy and reliability) of the

The procedures that the user may employ to verify the integrity of the
proprietary components

Criteria for an Acceptable Test

436•

Acceptable concurrent positive control ranges based on historical data

437•

Acceptable negative control data

438

Test Conditions

439•

Cell system used

440•

Calibration information for the spectrophotometer used to read the ELISA

441•

Details of test procedure

442•

Description of any modifications of the test procedure

443•

Reference to historical data of the model

444•

Description of evaluation criteria used•

Tabulation of data from individual test samples

445
446

Results

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447

Description of Other Effects Observed

448

Discussion of the Results

449

Conclusion

450

A Quality Assurance Statement for Good Laboratory Practice (GLP)-Compliant Studies

451•

This statement should indicate all inspections made during the study and the452dates any results were reported to the Study Director. This statement should453

also confirm that the final report reflects the raw data.

454

If GLP-compliant studies are performed, then additional reporting requirements provided in455

the relevant guidelines (e.g., OECD 1998; EPA 2003a, 2003b; FDA 2003) should be456

followed.

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457

11.0

REFERENCES

458

EPA. 2003a. Good Laboratory Practice Standards. Toxic Substances Control Act. 40 CFR

459

792.

460

EPA. 2003b. Good Laboratory Practice Standards. Federal Insecticide, Fungicide, and461

Rodenticide Act. 40 CFR 160.

462

FDA. 2003. Good Laboratory Practices for Nonclinical Laboratory Studies. 21 CFR 58.

463

Fennrich S, Wendel A, Hartung T. 1999a. New applications of the human whole blood464

pyrogen assay (PyroCheck). ALTEX 16:146-149.

465

Fennrich S, Fischer M, Hartung T, Lexa P, Montag-Lessing T, Sonntag H-G, Weigand M,

466

Wendel A. 1999b. Detection of endotoxins and other pyrogens using human whole blood.

467

Dev Biol Stand. 101:131-139.

468

Hartung T, Wendel A. 1996. Detection of pyrogens using human whole blood. In Vitro

469

Toxicol. 9(4):353-359.

470

Hoffmann S, Peterbauer A, Schindler S, Fennrich S, Poole S, Mistry Y, Montag-Lessing T,

471

Spreitzer I, Löschner B, van Aalderen M, Bos R, Gommer M, Nibbeling R, Werner-

472

Felmayer G, Loitzl P, Jungi T, Brcic M, Brügger P, Frey E, Bowe G, Casado J, Coecke S, de473

Lange J, Mogster B, Næss LM, Aaberge IS, Wendel A, Hartung T. 2005. International474

validation of novel pyrogen tests based on human monocytoid cells. J Immunol Methods

475

298:161-173.

476

Organization for Economic Cooperation and Development (OECD). 1998. OECD Series on

477

Principle of Good Laboratory Practice and Compliance Monitoring. No. 1. OECD Principles

478

of Good Laboratory Practice (as revised in 1997). Organization for Economic Co-operation

479

and Development (OECD), ENV/MC/CHEM (98)17. Paris: OECD.

480

Schindler S, Spreitzer I, Löschner B, Hoffmann S, Hennes K, Halder M, Brügger P, Frey E,

481

Hartung T, Montag T. 2006. International validation of pyrogen tests based on cryopreserved

482

human primary blood cells. J Immunol Methods 316:42-51.

483
484
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484

12.0

TERMINOLOGY AND FORMULA

485

12.1

Assay Sensitivity (λ)

486

For an in vitro cell-based assay, the variable λ is defined as the lowest statistically significant

487

point on the standard endotoxin concentration-response curve and represents the relative

488

sensitivity of the test method for the detection of endotoxin (i.e., level of detection).

489

12.2

490

The EC is incubated with WB and serves as the positive control for the experiment. The

491

results should be compared to historical values to insure that it provides a known level of492

cytokine release relative to the NSC.

493

12.3

494

The ELC is the maximum allowable concentration of endotoxin for a particular product and495

is expressed in EU per volume (mL) or weight (mg). The ELC is defined by the FDA or

496

specified in the USP2. It is calculated as the product of K/M, where:

497

K is the threshold pyrogen dose for parenteral use in rabbits or humans (5.0 EU/kg). At an

498

injection volume of 10 mL/kg, K is equal to 0.5 EU/mL.

499

M is the larger of the rabbit dose or the maximum human dose administered in one hour as

500

defined below and varies with test substance3.

501

12.4

502

The MVD is the maximum dilution of a test substance that can be tolerated in a test system503

without exceeding the ELC, if the test substance must be diluted as a result of assay

504interference. Dilutions beyond the MVD would not be valid for endotoxin detection in the2

Endotoxin Control (EC)Endotoxin Limit Concentration (ELC)

Maximum Valid Dilution (MVD)

ELC values for most marketed pharmaceutical products are provided in the USP or in other pharmacopoeial or
regulatory publications (e.g., European Pharmacopoeia, Japanese Pharmacopoeia, Pharmacopoeial guidelines,
FDA publications).
3
Values for most marketed pharmaceutical products are provided in the USP or in other pharmacopoeial or
regulatory publications (e.g., European Pharmacopoeia, Japanese Pharmacopoeia, Pharmacopoeial guidelines,
FDA publications).
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505

test system. Calculation of the MVD is dependent on whether or not the ELC for a test

506

substance is published. When the ELC is known, the MVD is:507

MVD = (ELC x Product Potency [PP])/λ508

As an example, for "Cyclophosphamide Injection," the ELC is 0.17 EU/mg, PP is 20

509

mg/mL, and the assay sensitivity is 0.1 EU/mL. The calculated MVD would be 34. The test

510

substance can be diluted no more than 1:34 prior to testing.

511

If the ELC is not known, the MVD is:

512

MVD = PP/Minimum Valid Concentration (MVC)513

where, MVC = (λ x M)/K

514

where, M is the maximum human dose

515

As an example, for "Cylophosphamide Injection," the PP is 20 mg/mL, M is 30

516

mg/kg, and assay sensitivity is 0.1 EU/mL. The calculated MVC is 0.6 mg/mL and the MVD

517

is 33.3. The test substance can be diluted no more than 1:33 in the assay prior to testing.

518

12.5

519

The NPC is a test sample to which PFS is added. The NPC is the baseline for determination

520

of cytokine release relative to the endotoxin-spiked Positive Product Control (PPC).

521

12.6

522

The NSC is WB and incubated with PFS (used for dilution of test substance) and is used as523

the blank.

524

12.7

525

The value K represents the threshold pyrogen dose for parenteral products for rabbits and526

humans. Based on experimental data, K is fixed at 5.0 EU/kg. For intrathecal products, K is527

0.2 EU/kg.

528

12.8

529

The PPC is a test substance spiked with the control standard endotoxin (i.e., 0.5 EU/mL or an

530

amount of endotoxin equal to that which produces ½ the maximal increase in OD from the

Negative Product Control (NPC)

Negative Saline Control (NSC)

Non-intrathecal Threshold Pyrogen Dose (K)Positive Product Control (PPC)

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531

endotoxin standard curve) to insure that the test system is capable of endotoxin detection in532

the product as diluted in the assay.

533

12.9

534

The concentration for a test substance is the PP typically expressed as µg/mL or mg/mL.

535

12.10

536

The variable M represents the rabbit test dose or the maximum human dose in 1 hr. The

537

variable M is expressed in mg/kg and varies with the test substance. For

538

radiopharmaceuticals, M should be adjusted to account for product activity (radioactive539

decay) at time administration. An average human standard weight of 70 kg is used for the540

calculation. If a pediatric dose should be used and it is higher than the adult dose, then it

541

should be used in the formula.

Product Potency (PP)

Rabbit Pyrogen Test Dose or Maximum Human Dose (M)

542

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542
543
544
545
546
547
548
549
550
551
552
553
554
555

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556
557

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Appendix B5

Draft ICCVAM Recommended Protocol for Future Studies Using the Whole Blood(WB)/Interleukin-6 (IL-6) Test Method

Draft ICCVAM Recommended Test Method Protocol: WB/IL-6 Pyrogen Test

[This Page Intentionally Left Blank]

01 Dec 2006

Draft ICCVAM Recommended Test Method Protocol: WB/IL-6 Pyrogen Test

01 Dec 2006

1

Draft ICCVAM Recommended Protocol for Future Studies Using the Whole Blood2(WB)/Interleukin-6 (IL-6) Test Method

3
4

PREFACE

5
6This proposed protocol for the detection of pyrogenicity is based on information obtained

7

from 1) The European Centre for the Validation of Alternative Methods (ECVAM) WB/IL-6

8Background Review Document (BRD) presented in Appendix A of the draft Interagency

9

Coordinating Committee on the Validation of Alternative Methods (ICCVAM) Pyrogenicity

10

Test Method BRD, which includes ECVAM Standard Operating Procedures (SOPs) for the11

WB/IL-6 test method, and 2) Information provided to the National Toxicology Program

12(NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods

13(NICEATM) by Dr. Thomas Hartung, Head of ECVAM. The ECVAM SOPs are based on

14

the WB/IL-6 methodology first described by Pool et al. (1998). A table of comparison

15

between the draft ICCVAM recommended protocol and the ECVAM SOPs is provided in

16

Table 1. Future studies using the WB/IL-6 test method may include further characterization

17of the usefulness or limitations of the assay for regulatory decision-making. Users should be

18

aware that the proposed test method protocol might be revised based on any additional

19

optimization and/or validation studies. ICCVAM recommends that test method users

20

routinely consult the ICCVAM/NICEATM website (http://iccvam.niehs.nih.gov/) to ensure

21

that the most current test method protocol is used.

22
23

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23
24
25
26
27
28
29
30
31
32
33
34
35
36

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37
38

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38
39

Table 101 Dec 2006

Comparison of Draft ICCVAM Recommended Test Method Protocol with the ECVAM SOP for the Whole
Blood (WB)/Interleukin-6 (IL-6) Pyrogen Test Method
Protocol Component
Test Substance

Incubation Plate(number of control or test groups at n=4
each)
ELISA Plate
Decision Criteria for Interference

Assay Acceptability Criteria

Draft ICCVAM Protocol
Test neat or at minimal dilution that produces nointerference
NSC (1)EC (5)
TS (14)
Includes seven point IL-6 SC and blank in
duplicate
0.5 x Median OD4501 of 1 EU/mL EC <2x Median
OD450 of 1 EU/mL EC
Mean OD450 of 0.5 EU/mL EC ≥1.6X Mean OD450
of NSC
Mean OD450 of PPC is 50% to 200% of 0.5 EU/mL
EC
Mean OD450 of NSC ≤0.15
Quadratic function of IL-6 SC r2 ≥0.95
Not included
EC SC produces OD450 values that ascend in asigmoidal concentration responseHigh responder blood donors (i.e., >200 pg/mL IL6) may be excluded (need at least 3 donors)
Not included

Decision Criteria for Pyrogenicity

Mean OD450 of TS > Mean corrected OD450 of 0.5
EU/mL EC
Not included

B5-3

ECVAM SOP
Same as ICCVAM protocol
Same as ICCVAM protocol
Same as ICCVAM protocol
Same as ICCVAM protocol
Same as ICCVAM protocol
0.5 x Median OD450 of 0.5 EU/mL EC <2xMedian OD450 of 0.5 EU/mL EC
Same as ICCVAM protocol
Same as ICCVAM protocol
Same as ICCVAM protocol
Same as ICCVAM protocol
EC SC satisfies ICH Harmonized Tripartite
Guideline: Validation of Analytical Procedures
Methodology; ICH Q2B, Nov 1996
Same as ICCVAM protocol
High responder blood donors (i.e., >200 pg/mLIL-6) may be excluded (need at least 3 donors)
Wilcoxon rank-sum test used to show that atleast 3 of 4 replicates at each increasing EC
concentration are higher relative to the next
lowest concentration
EC SC data transformed to logit responses by inhouse program and the linear mean squarecalculated. TS pyrogen content is compared with
the ELC2 using confidence limits. The
preparation being examined must pass the testwith blood from three separate donors
Limit test is run to determine whether or not aDraft ICCVAM Recommended Test Method Protocol: WB/IL-6 Pyrogen Test

Protocol Component

40
41
42
43
44
45
46

01 Dec 2006

Draft ICCVAM Protocol

ECVAM SOP
TS after correction and dilution contains < 0.5
EU/mL of endotoxin
Abbreviations: EC = Endotoxin control; ELC = Endotoxin Limit Concentration; MVD = Maximum Valid Dilution; NSC = Normal saline control; PPC =Positive Product Control; SC = Standard curve; TS = Test substance1
Median or mean OD450 values are corrected (i.e., reference filter reading, if applicable, and NSC are subtracted).
2
Where unknown, the ELC is calculated (e.g., Based on a rabbit sensitivity of 5 EU/kg, for a product injected at 10 mL/kg, the detection limit is 5 EU/10 mL/kg
or 0.5 EU/mL/kg, or an ELC of 0.5 EU/mL).

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47

1.0

48

The purpose of this protocol is to describe the procedures used to evaluate the presence of a49

pyrogen (i.e., Gram-negative endotoxin) in a test substance. The presence of Gram-negative50

endotoxin is detected by its ability to induce cytokine IL-6 release from monocytoid cells in

51

WB. The quantity of IL-6 released is obtained using an enzyme-linked immunosorbent assay

52(ELISA) that includes monoclonal or polyclonal antibodies specific for IL-6. Release of this53

cytokine is measured by incubation of WB with test substances or controls (i.e., positive,

54

negative). The amount of pyrogen present is determined by comparing the values of55

endotoxin equivalents produced by cells exposed to the test substance to those exposed to an

56

internationally-harmonized Reference Standard Endotoxin (RSE)1 or an equivalent standard

57

expressed in Endotoxin Units (EU)/mL. Based on a rabbit threshold pyrogen dose of 0.5

58

EU/mL, which was established in a retrospective evaluation of rabbit pyrogen test (RPT)

59

data, a test substance is considered pyrogenic if it induces a level of IL-6 release equal to or

60

greater than 0.5 EU/mL.

61

The focus of this protocol is on the use of the WB/IL-6 test method, specifically for the62

detection of Gram-negative endotoxin in parenteral pharmaceuticals. The relevance and63

reliability for non-endotoxin pyrogens (e.g., lipoteichoic acid) has not been demonstrated in a64

formal validation study.

65

2.0

66

All procedures for procurement of eligible blood donors and blood donations should follow

67

the regulations and procedures set forth by institutional guidelines for utilization of human68

substances, which include but are not limited to blood, tissues, and tissue fluids. Standard

69

laboratory precautions are recommended including the use of laboratory coats, eye70protection, and gloves. If necessary, additional precautions required for specific study

71

substances or hazardous chemicals will be identified in the Material Safety Data Sheet

72(MSDS).

1

PURPOSE AND APPLICABILITY

SAFETY AND OPERATING PRECAUTIONSRSEs are internationally-harmonized reference standards (e.g., WHO E. coli Lipopolysaccharide [LPS] 94/580[0113:H10:K-]; USP RSE Lot G3E069; FDA Lot EC-6). Equivalent endotoxins include commercially available
E. coli-derived LPS Control Standard Endotoxin (CSE) or other E. coli LPS preparations that have beencalibrated with an appropriate RSE.
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73

The stop solution used in the ELISA kit is acidic and corrosive and should be handled with74

the proper personal protective devices. If this reagent comes into contact with skin or eyes,

75

wash thoroughly with water. Seek medical attention, if necessary.

76

Tetramethylbenzidine (TMB) solution contains a hydrogen peroxide substrate and 3, 3', 5, 5'-

77

TMB. This reagent is a strong oxidizing agent and a suspected mutagen. Appropriate

78

personal protection should be used to prevent bodily contact.

79

Bacterial endotoxin is a toxic agent (i.e., can induce sepsis, shock, vascular damage,80

antigenic response) and should be handled with care. Skin cuts should be covered and81

appropriate personal protective devices should be worn. In case of contact with endotoxin,82

immediately flush eyes or skin with water for at least 15 min. If inhaled, remove the affected83

individual from the area and provide oxygen and/or artificial respiration as needed. Skin

84

absorption, ingestion, or inhalation may produce fever, headache, and hypotension.

85

3.0

MATERIALS, EQUIPMENT, AND SUPPLIES

86

3.1

Source of Cells

87

Leukocytes from fresh WB are the source of cells for cytokine production in the WB/IL-6

88

test method (Hartung and Wendel, 1996; Pool et al., 1998; Schindler et al., 2006). WB is

89

obtained from healthy human volunteers who have provided their consent according to90established institutional guidelines. Volunteers are expected not to have taken any drugs

91(e.g., prescription drugs, recreational drugs, herbal drugs) and to have been free from illness

92

for at least two weeks prior to donation.

93

3.2

94

For all steps in the protocol, excluding the ELISA procedure, the materials that will be in95

close contact with samples and/or blood cells (e.g., pipet tips, containers, solutions) should be

96

sterile and free from detectable pyrogens.

Equipment and Supplies

97

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97
98

3.2.1

99

3.2.1.1

Blood Incubation
Equipment

100•

Centrifuge

101•

Hood; Bio-safety, laminar flow (recommended)102•

Incubator; cell culture (37±1°C + 5% CO2)

103•

Pipetter, multichannel (8- or 12-channel)

104•

Pipetters, single-channel adjustable (20 and 200 µL)

105•

Repeating pipetter

106•

Vortex mixer

107

3.2.1.2

Consumables

108•

Centrifuge tubes; nonpyrogenic, polypropylene (15 and 50 mL)

109•

Combitips; repeating pipetter (1.0 and 2.5 mL)

110•

Needle set; Sarstedt multifly, pyrogen-free, 19 mm, 21 gauge for S-Monovette

111•

Plates; microtiter, nonpyrogenic, 96-well, polystyrene, tissue culture

112•

Reaction tubes; polypropylene (1.5 mL)

113•

Reservoirs; fluid

114•

Tips; pipetter, sterile, pyrogen-free (20 and 200 µL)

115•

Tubes; Sarstedt S-Monovette, 7.5 mL, heparinized for blood collection

116

3.2.2

ELISA

117

3.2.2.1

Equipment

118•

Microplate mixer

119•

Microplate reader (450 nm with an optional reference filter in the range of120

540-590 nm)
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121•

Microplate washer (optional)

122•

Multichannel pipetter

123

3.2.2.2

01 Dec 2006

Consumables

124•

Container; storage, plastic

125•

Deionized water; nonsterile

126•

Plates; microtiter, nonpyrogenic, 96-well, polystyrene, tissue culture

127•

Pyrogen-free water (PFW)128•

Reservoirs; fluid

129•

Tips; pipetter, nonsterile

130•

Tubes; polypropylene (12 mL)

131

3.2.2.3

132

An ELISA that measures IL-6 release from WB is used. A variety of IL-6 ELISA kits are133

commercially available and the IL-6 ELISA procedure outlined in this protocol is intended to134

serve as an example for using an ELISA kit. If the user prefers to prepare an in-house

135

ELISA, then additional reagents would be required. The IL-6 ELISA should be calibrated

136

using an IL-6 international reference standard (e.g., WHO 89/548) prior to use. The IL-6

137

cytokine assay kits do not provide the RSE or endotoxin equivalent; therefore this reagent

138

must be purchased separately. Results obtained using these products are subject to the assay

139

acceptability and decision criteria described in Sections 8.0 and 9.0. IL-6 ELISA kit

140

components may include the following:

141

ELISA Kit•

142

ELISA plates coated with anti-human IL-6 capture antibody; monoclonal or
polyclonal

143•

Buffered wash solution

144•

Dilution buffer

145•

Enzyme-labeled detection antibody

146•

Human IL-6 reference standard
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147•

Pyrogen-free saline (PFS)148•

Stop solution

149•

TMB/substrate solution

150

3.3

151

01 Dec 2006

Chemicals•

152

Endotoxin (e.g., WHO E. coli LPS 2nd International Standard 94/580; USP
RSE E. coli LPS Lot G3E069; USP RSE E. coli Lot G; FDA E. coli Lot EC6)

153

3.4

Solutions154

ELISA solutions are listed in Section 3.2.

155

4.0

156

All test substances and endotoxin-spiked solutions should be stored at 4°C.

157

4.1

158

An internationally harmonized RSE or equivalent is used to generate the endotoxin standard

159

curve. The use of any other E. coli LPS requires calibration against a RSE using the WB/IL-6

160

test method.

161

A standard endotoxin curve consisting of a Normal Saline Control (NSC) and five RSE

162

concentrations (0.125, 0.25, 0.50, 1.0, and 2.0 EU/mL) are included in the incubation step163

(refer to Table 4-1) and then transferred to the ELISA plate. To prepare the endotoxin

164

standard curve, first obtain a 2000 EU/mL stock solution by addition of PFW to the165lyophilized content of the stock vial by following the instructions provided by the166

manufacturer (e.g., for a vial containing 10,000 EU, 5 mL of PFW is added). To reconstitute167

the endotoxin, the stock vial should be vortexed vigorously for at least 30 min or sonicated in168

a bath sonicator for 5 min. The stock solution is stable for 14 days when stored at 2 to 8°C.

169

An endotoxin standard curve is prepared by making serial dilutions of the stock solution in

170

PFS as described in Table 4-1.

ASSAY PREPARATION

Endotoxin Standard Curve

171

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171
172

Table 4-1

Preparation of Endotoxin Standard Curve

Stock Endotoxin
EU/mL

173
174
175
176
177
178
179

µL of Stock
EndotoxinµL of PFS

Endotoxin
Concentrationin Tube
EU/mL
203
2.0
1.0
0.50
0.25
0.125
0

20001,2
20
1980
20
100
900
2.0
500
500
1.0
500
500
0.50
500
500
0.25
500
500
0
0
1000
Abbreviations: EU = Endotoxin units; PFS = Pyrogen-free saline
Each stock tube should be vortexed prior to its use to make the subsequent dilution.
1
A 2000 EU/mL stock solution of endotoxin is prepared according to the manufacturer's instructions.
2
The stock solution of USP RSE may be stored in aliquots and kept at -20°C for up to 6 months. Do not store
the endotoxin at -80°C.
3This concentration is not used in the assay.

180

4.2

Test Substances

181

Liquid test substances should be tested neat or, if interference is detected (see Section 4.2.1),

182

diluted in PFS. Solid test substances should be prepared as solutions in PFS or, if insoluble in183

saline, dissolved in dimethylsulfoxide (DMSO) then diluted up to 0.5% (v/v) with PFS

184

provided that this concentration of DMSO does not interfere with the assay. The test

185

substances should be vortexed vigorously for at least 30 min or sonicated in a bath sonicator186

for 5 min.

187

4.2.1

188

Interference testing must be carried out on any test sample for which no interference

189

information is available. The purpose of the interference test is to determine the lowest

190

dilution (i.e., highest concentration) of a test substance from which an endotoxin spike can be

191

detected (i.e., based on the decision criteria in Section 4.2.1.2). However, to ensure a valid

192

test, a test substance should not be diluted beyond its Maximum Valid Dilution (MVD).

193

For many marketed products, values for the MVD and the Endotoxin Limit Concentration

194(ELC) are published in the U.S. Pharmacopeia, the European Pharmacopoeia, and/or Food

195

and Drug Administration (FDA) guidelines. However if one or both of these values are not

Interference Testing

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196

available, then calculation of the MVD is dependent on the ELC (see Section 12.3). If

197

unknown, the ELC can be approximated by dividing the maximum hourly dose of the198

product by the hourly dose received per patient. For example, if a product were used at an

199hourly dose of 100 mg per patient, then the ELC would be 350 EU/100 mg or 3.5 EU/mg.

200

4.2.1.1

201

The WHO-LPS 94/580 [E. coli O113:H10:K-] or equivalent international RSE (e.g., USP

202

G3E069, FDA EC-6) is recommended for preparation of the endotoxin control (EC). If a203

different E. coli LPS is used and the potency relative to the RSE is not provided, then each204

lot must be calibrated against the RSE in the WB/IL-6 test method. For interference testing,205

an endotoxin standard curve (see Section 4.1) should be included on each plate.

206

4.2.1.2

207

For interference testing, non-spiked and endotoxin-spiked test substances are prepared in

208

microplate wells (n=4 replicates) and an in vitro pyrogen test is performed. Either PFS or a209

fixed concentration (a concentration selected from the middle of the EC standard curve) of

210

the RSE (i.e., 1 EU/mL) in PFS is added to the test substance in serial two-fold dilutions. An

211

illustrative example of endotoxin spiking dilutions is shown in Table 4-2. For non-spiked

212

solutions, 50 µL of PFS is added to a well followed by 50 µL of WB and mixed by inversion.

213

Then, 50 µL of the test substance (neat or at serial dilution) is added followed by 100 µL of

214

PFS and the well contents are mixed. Endotoxin-spiked solutions are prepared by adding 50

215µL of PFS to each well followed by 50 µL of WB and mixed by inversion. Then, 50 µL of216

the test substance (neat or at serial dilution), 50 µL of an endotoxin spike solution (1.0217

EU/mL), and 50 µL of PFS are added and the well contents are mixed (see example

218

presented in Table 4-2).

Reference Endotoxin for Spiking Test Substances

Spiking Test Substances with Endotoxin

219

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219
220

Table 4-2

Preparation of Endotoxin-spiked and Non-spiked Solutions for

221

Determination of Test Substance Interference in the Incubation and

222

ELISA Test Systems
SpikedSample Addition

223
224
225
226
227
228
229
230
231

Unspiked1µL/well
Pyrogen-free saline (total volume added)
1002
1502
3
Endotoxin spike solution
50
0
Test substance (neat and each serial dilution)
50
50
WB
50
50
Total4250
250
Abbreviations: WB = Whole blood1
n=4 replicates each2
50 µL of WB and 50 µL of PFS are added to each well and mixed by inversion prior to the addition of theremaining components and volume of PFS.
3
Endotoxin concentration is 1.0 EU/mL in PFS.
4
A total volume of 250 µL per well is used for the incubation.

232

The lowest dilution of the test substance that yields an endotoxin spike recovery of 50% to

233

200% in the pyrogen test is determined. The optical density (OD) values of the endotoxin-234

spiked and non-spiked test substances are calibrated against the endotoxin calibration curve.

235

The resulting EU value of the non-spiked test substance is subtracted from the corresponding

236

EU value of the endotoxin-spiked test substance at each dilution. The % recovery for each237

sample dilution is then determined from the endotoxin spike solution concentration set to238

100%. For example, consider the following interference test results in Table 4-3:

239

Table 4-3

240

Example of Interference Data Used to Determine Sample Dilution for
Assay
Sample Dilution
None
1:2
1:4
1:8

% Recovery of Endotoxin Control
25
49
90
110

241
242
243

Based on these results, the dilution of the test substance used in the in vitro pyrogen test

244

would be 1:4 (i.e., the lowest dilution between 50% and 200% of the 1 EU/mL EC).
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245

4.2.2

246

If the data obtained from the experiment in Section 4.2.1 suggests the presence of

247interference, then a subsequent experiment similar to that described in Section 4.2.1 would

248

need to be performed to confirm that the test substance(s) does not directly interfere with the249

ELISA. For this experiment, an ELISA would be performed in the absence of PBMCs.

250

5.0

CONTROLS251

5.1

Negative Control

252

A negative control (e.g., PFS) is included in each experiment in order to detect nonspecific253

changes in the test system, as well as to provide a baseline for the assay endpoints.

254

5.2

255

Solvent controls are recommended to demonstrate that the solvent is not interfering with the256

test system when solvents other than PFS are used to dissolve test substances.

257

5.3

258

An internationally standardized EC (e.g., WHO 94/580; USP G3E069; 0.5 EU/mL) is

259

included in each experiment to verify that an appropriate response is induced.

260

5.4

261

Benchmark controls may be used to demonstrate that the test method is functioning properly,

262

or to evaluate the relative pyrogenic potential of chemicals (e.g., parenteral pharmaceuticals,

263

medical device eluates) of a specific class or a specific range of responses, or for evaluating264

the relative pyrogenic potential of a test substance. Appropriate benchmark controls should265

have the following properties:

266

Interference with ELISA

Solvent Control

Positive Control

Benchmark Controls•

267

consistent and reliable source(s) for the chemicals (e.g., parenteralpharmaceuticals, medical device eluates)268•

structural and functional similarities to the class of the substance being tested

269•

known physical/chemical characteristics

270•

supporting data on known effects in animal models
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01 Dec 2006

known potency in the range of response

272

5.5

Positive Product Control (PPC)273

The PPC is a test substance diluted to a level that does not interfere with the test method and274

does not exceed the MVD. The PPC is obtained by spiking a test substance with a known EC

275(e.g., 1 EU/mL) and demonstrating that 50% to 200% of the EC is recovered.

276

5.6

277

The NPC is the test substance diluted to the MVD and then spiked with PFS. It is the278

negative control for the PPC.

279

6.0

EXPERIMENTAL DESIGN

280

6.1

Incubation with Test Samples and Measurement of IL-6 Release

281

6.1.1

Collection of Human Blood282

WB is obtained from healthy human volunteers who have provided their consent according283

to established institutional guidelines. Volunteers are expected not to have taken any drugs

284

and to have been free from illness for at least two weeks prior to donation. The criteria for285

rejection of data from donors that are low responders or that are suspect due to veracity of286

health information is addressed in Section 8.0.

287

Fresh WB is drawn by venipuncture using a multifly needle set and collected in heparinized288

tubes (15 IU/mL lithium heparin). All components of the blood collection system (i.e.,289

syringes, tubes, connecting lines) must be sterile and pyrogen-free. Blood should be stored at

290

room temperature (RT) and must be used within 4 hr. Prior to use in the assay, the collection

291

tubes should be gently inverted 1-2 times. Do Not Vortex.

292

6.1.2

293

Test substances are prepared at a level of dilution that did not show interference with the test

294

system or for which it is known that interference does not occur. Each incubation plate can295

accommodate an endotoxin standard curve, a NSC, and 14 test substances (see Table 6-1).

Negative Product Control (NPC)

Incubation Plate

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Table 6.1

Overview of Incubation Plate Preparation in the WB/IL-6 Test Method

Mix thesamples;
PFS
EC
WB
NumberSampleimmediatelyof Wells
transfer to an
ELISA plate3µL
and run
201
EC
200
20
0
20
ELISA
or
4
NSC
220
0
0
20
store
plate
at
Test
2
-20°C
or
56
samples200
020
20-80°C.
(1-14)
Abbreviations: EC = Endotoxin control; NSC = Negative saline control; PFS = Pyrogen-free saline WB =Whole blood
1
Five EC concentrations (0.125, 0.25, 0.50, 1.0, and 2.0 EU/mL) in quadruplicate.
2
14 test samples (n=4 each) per plate.
3
An IL-6 standard curve is prepared in Columns 11 and 12 on the ELISA plate. Therefore, 80 wells areavailable for test samples and controls on the incubation plate.
Test
Sample

297
298
299
300
301
302
303

01 Dec 2006

Mix thesamples;incubateovernight at
37±1°C in ahumidified
atmospherewith 5%
CO2.

304

6.1.3

Incubation Assay for IL-6 Release

305

Blood samples are prepared in a microtiter plate using a laminar flow hood. All consumables306

and solutions must be sterile and pyrogen-free. Each plate should be labeled appropriately

307

with a permanent marker. An overview of the incubation plate preparation is shown in Table

308

6-1. The incubation procedure is outlined below:

309•

Step 1. Refer to the incubation plate template presented in Table 6-2.

310•

Step 2. Using a pipetter, transfer 150 µL of PFS into each well.

311•

Step 3. Transfer 50 µL of test sample into the appropriate wells as indicated in

312
313

the template.
•

314
315

quadruplicate into the appropriate wells according to the template.
•

316
317

Step 4. Transfer 50 µL of the EC (standard curve) and the NSC controls in

Step 5. Transfer 50 µL of WB into each well and mix by gently swirling theplate.

•

Step 6. Mix the contents of the wells thoroughly by gently pipetting up and318

down several times using a multichannel pipetter, changing the tips between

319

each row in order to avoid cross-contamination.

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320

•

321
322

01 Dec 2006

Step 7. Place the covered plate in a tissue culture incubator for 10 to 24 hr at
37±1°C in a humidified atmosphere containing 5% CO2.

•

Step 8. Prior to transferring the test samples onto the ELISA plate, mix the323

contents of the wells by pipetting up and down using a multichannel pipetter,324

changing the tips between each row in order to avoid cross-contamination.

325

Note: The aliquots may be tested immediately in the ELISA or stored at -20°C or

326-80°C for testing at a later time. After transfer to the ELISA plate, freeze the327

remaining aliquots at -20°C or -80°C for subsequent experiments, if necessary (see328

Assay Acceptability and Decision Criteria in Sections 8.0 and 9.0).

329

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329
330

Table 6-2
1

Incubation Plate - Sample and Control Template
2

3

4

5

6

7

8

9

10

11

EC1
EC
EC
EC
TS3
TS3
TS3
TS3
TS11 TS11
Void3
2.0
2.0
2.0
2.0
EC
EC
EC
EC
B
TS4
TS4
TS4
TS4
TS11 TS11
Void
1.0
1.0
1.0
1.0
EC
EC
EC
EC
C
TS5
TS5
TS5
TS5
TS12 TS12
Void
0.50
0.50
0.50
0.50
EC
EC
EC
EC
D
TS6
TS6
TS6
TS6
TS12 TS12
Void
0.25
0.25
0.25
0.25
EC
EC
EC
EC
E
TS7
TS7
TS7
TS7
TS13 TS13
Void
0.125 0.125 0.125
0.125
F
NSC NSC
NSC
NSC
TS8
TS8
TS8
TS8
TS13 TS13
Void
G TS12
TS1
TS1
TS1
TS9
TS9
TS9
TS9
TS14 TS14
Void
H
TS2
TS2
TS2
TS2
TS10
TS10
TS10
TS10
TS14 TS14
Void
Abbreviations: EC = Endotoxin control; NSC = Negative saline control; TS = Test substance1
EC value (e.g., EC 2.0) represents the endotoxin concentration in EU/mL.
2
TS number (e.g., TS 1) represents an arbitrary sequence for individual test substances.
3
Columns 11 and 12 are reserved for the IL-6 standard curve on the ELISA plate (see Table 6-3).
A

331
332
333
334
335
336

12
Void
Void
Void
Void
Void
Void
Void
Void

337

6.2

ELISA to Measure IL-6 Release

338

6.2.1

IL-6 Standard Curve

339

An IL-6 standard, supplied with the ELISA kit, is used. IL-6 standards are typically supplied340

in lyophilized form and should be reconstituted according to the manufacturer's instructions.

341

The stock solution should be diluted in PFS to the following concentrations: 0, 62.5, 125,

342

250, 500, 1000, 2000, and 4000 pg/mL in volumes of at least 500 µL. Each well on the343

ELISA plate will receive 100 µL of an IL-6 blank or standard.

344

6.2.2

345

The manufacturer's instructions provided with the ELISA kit should be followed and a346

typical experimental design is outlined below. If the user prefers to prepare an in-house

347

ELISA, then appropriate modifications and validation of these changes would be necessary.

348

The ELISA should be carried out at RT and therefore all components must be at RT prior to349

use. Do not thaw frozen specimens by heating them in a water bath. A suggested ELISA

350

plate template is shown in Table 6-3, which includes a five-point EC standard curve, a NSC,

ELISA

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an eight-point IL-6 standard curve (0 to 4000 pg/mL), and 14 test substances in352

quadruplicate. The EC standard curve, the NSC, and the test sample supernatants are

353

transferred directly from the incubation plate. The IL-6 standard curve is prepared as

354

described in Section 6.2.1. An overview of the ELISA plate preparation is shown in Table 6-

355

4.

356

Immediately prior to the ELISA procedure, dilute or mix any assay components according to357

the manufacturer's instructions.

358

Step 1. After pipetting up and down three times to mix the supernatant, transfer 50

359µL from each well of the Incubation Plate (A1-10; H1-10) to the ELISA plate.

360

Step 2. Add 50 µL of each IL-6 standard (0 to 4000 pg/mL) into the respective

361

wells on the ELISA plate.

362

Step 3. Add 200 µL of the enzyme-labeled detection antibody (neat as supplied, or

363

diluted, if necessary) to each of the wells.

364

Step 4. Cover the microtiter plate(s) with adhesive film and incubate for 2 to 3 hr

365

at 20 to 25°C.

366

Step 5. Decant and wash each well five to six times with 300 mL Buffered Wash

367

Solution and then rinse three times with deionized water. Place the plates upside

368

down and tap to remove water.

369

Step 6. Add 200 µL of TMB/Substrate Solution to each well and incubate at RT in370

the dark for 15 min. If necessary, decrease the incubation time.

371

Step 7. Add 50 µL of Stop Solution to each well.

372

Step 8. Tap the plate gently after the addition of Stop Solution to aid in mixing.

373

Step 9. Read the OD450 within 15 min of adding the Stop Solution. Measurement374

with a reference wavelength of 540 to 590 nm is recommended.

375

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Table 6-3

ELISA Plate - Sample and Control Template

1

2
1

3

4

EC
2.0
EC
1.0
EC
0.50
EC
0.25
EC
0.125

EC
2.0
EC
1.0
EC
0.50
EC
0.25
EC
0.125

EC
2.0
EC
1.0
EC
0.50
EC
0.25
EC
0.125

EC
2.0
EC
1.0
EC
0.50
EC
0.25
EC
0.125

F

NSC

NSC

NSC

G

TS12

TS1

H

TS2

TS2

A
B
C
D
E

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5

6

7

8

9

10

11

12
3

TS3

TS3

TS3

TS3

TS11

TS11

TS4

TS4

TS4

TS4

TS11

TS11

TS5

TS5

TS5

TS5

TS12

TS12

TS6

TS6

TS6

TS6

TS12

TS12

TS7

TS7

TS7

TS7

TS13

TS13

NSC

TS8

TS8

TS8

TS8

TS13

TS13

TS1

TS1

TS9

TS9

TS9

TS9

TS14

TS14

TS2

TS2

TS10

TS10

TS10

TS10

TS14

TS14

376
377
378
379
380
381

Abbreviations: EC = Endotoxin control; NSC = Negative saline control; TS = Test substance1
EC value (e.g., EC 2.0) represents the endotoxin concentration in EU/mL.
2
TS number (e.g., TS1) represents an arbitrary sequence for individual test substances.
3
IL-6 values in columns 11 and 12 are in pg/mL.

382

Table 6-4

Overview of ELISA Procedure

Materialtransfer from
Incubation
Plate
(µL)

Enzymelabeled
Antibody
(µL)

50

200

Cover theIncubation
Plate andincubate for
2 to 3 hr at
20 to 25°C.

TMB/Substrate
Solution(µL)

200

Incubate 15
min at RT in
the dark.

Stop
Solution
(µL)

50

IL-6
0
IL-6
62.5
IL-6
125
IL-6
250
IL-6
500
IL-6
1000
IL-6
2000
IL-6
4000

IL-6
0
IL-6
62.5
IL-6
125
IL-6
250
IL-6
500
IL-6
1000
IL-6
2000
IL-6
4000

Read opticaldensity at
450 nm witha 540-590
nm
wavelengthreference
filter.

383
384
385

Abbreviations: RT = Room Temperature

386

7.0

EVALUATION OF TEST METHODS

387

7.1

OD Measurements

388

The OD of each well is obtained by reading the samples in a standard microplate389

spectrophotometer (i.e., plate reader) using a visible light wavelength of 450 nm (OD450) with
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a 540 to 590 nm reference filter (recommended). OD450 values are used to determine assay

391

acceptability and in the decision criteria for detection of endotoxin in a test sample (see392Sections 8.0 and 9.0).

393

8.0

394

Obtain the PPC and the corresponding NPC by interference testing of a test substance in the395

presence and absence of a fixed quantity of endotoxin (i.e., 1 EU/mL) in quadruplicate. An

396

EC (five-point standard curve) and a NSC should be included in each experiment. An IL-6

397

standard curve should be included in each ELISA plate as shown in the template presented in

398

Table 6-3. An assay is considered acceptable only if the following minimum criteria are met:

399

CRITERIA FOR AN ACCEPTABLE TEST•400

The quadratic function of the IL-6 standard curve produces an r2 ≥0.95 and the
OD450 of the blank control is below 0.15.

401

•

402

The endotoxin standard curve produces OD450 values that ascend in asigmoidal concentration response.

403

Blood donors are considered to be high responders if their concentration of IL-6 is greater404

than 200 pg/mL. High responders should be excluded from analysis. From a set of four405

donors, a maximum of one donor may be excluded from the test. Furthermore, the406

preparation being examined is required to pass the test with blood donations from at least407

three different donors.

408

9.0

DATA INTERPRETATION/DECEISION CRITERIA

409

9.1

Decision Criteria for Determination of Pyrogenicity

410

The validity of the endotoxin standard curve should be calculated using a four-parameter

411

logistic model. If necessary to satisfy the model, endotoxin concentrations may be modified.

412

Calculate the mean OD450 values of all of the replicates in each experimental group. Calibrate413

the mean OD450 value for each test substance using the endotoxin standard curve and414

document the estimated endotoxin concentration. Multiply the estimated endotoxin

415

concentration by the dilution factor, if necessary. This value represents the pyrogenicity of416

the sample in terms of endotoxin equivalents for that particular donor. The t-test is used to417

compare the data of a test sample against the data of the EC (0.5 EU/mL) that is performed in
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parallel. If this test results in a significant p-value (i.e., smaller than 1%), then the sample is419

considered to be non-pyrogenic, and as pyrogenic if otherwise (Hoffmann et al., 2005), as

420

long as the assay acceptability criteria in Section 8.0 has been met.

421

9.1.1

Decision Level 1422•

If all donors show a negative reaction, then the product passes.

423•

If two or more donors show a positive reaction, then the product fails.

424•

If one donor shows a positive reaction, then an additional test with four donors

425
426

has to be performed (go to Decision Level 2).
9.1.2

427

Decision Level 2•428

If out of 6 to 8 donors, only one donor shows a positive reaction, then the
product passes.

429•

In any other case, the product fails.

430

10.0

STUDY REPORT431

The test report should include the following information:

432

Test Substances and Control Substances

433•

Name of test substance

434•

Purity and composition of the substance or preparation

435•

Physicochemical properties (e.g., physical state, water solubility)

436•

Treatment of the test/control substances prior to testing (e.g., vortexing,

437

sonication, warming, resuspension solvent)438

Justification of the Test Method and the Protocol Used

439

Test Method Integrity

440

•

441
442
443

The procedure used to ensure the integrity (i.e., accuracy and reliability) of thetest method over time•

If the test method employs proprietary components, documentation on theprocedure used to ensure their integrity from “lot-to-lot” and over timeB5-21

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444

•

445
446

01 Dec 2006

The procedures that the user may employ to verify the integrity of the
proprietary components

Criteria for an Acceptable Test

447•

Acceptable concurrent positive control ranges based on historical data

448•

Acceptable negative control data

449

Test Conditions

450•

Cell system used

451•

Calibration information for the spectrophotometer used to read the ELISA

452•

Details of test procedure used

453•

Description of any modification to the test procedure

454•

Reference to historical data of the model

455•

Description of the evaluation criteria used•

Tabulation of data from individual test samples

456

Results

457
458

Description of Other Effects Observed

459

Discussion of the Results

460

Conclusion

461

A Quality Assurance Statement for Good Laboratory Practice (GLP)-Compliant Studies

462•

This statement should indicate all inspections made during the study and the463

dates any results were reported to the Study Director. This statement should464

also confirm that the final report reflects the raw data.

465

If GLP-compliant studies are performed, then additional reporting requirements provided in466

the relevant guidelines (e.g., OECD 1998; EPA 2003a, 2003b; FDA 2003) should be467

followed.

468
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11.0

REFERENCES

469

EPA. 2003a. Good Laboratory Practice Standards. Toxic Substances Control Act. 40 CFR

470

792.

471

EPA. 2003b. Good Laboratory Practice Standards. Federal Insecticide, Fungicide, and472

Rodenticide Act. 40 CFR 160.

473

FDA. 2003. Good Laboratory Practices for Nonclinical Laboratory Studies. 21 CFR 58.

474

Hartung T, Wendel A. 1996. Detection of pyrogens using human whole blood. In vitro

475

Toxicol 9(4):353-359.

476

Hoffmann S, Peterbauer A, Schindler S, Fennrich S, Poole S, Mistry Y, Montag-Lessing T,

477

Spreitzer I, Löschner B, van Aalderen M, Bos R, Gommer M, Nibbeling R, Werner-

478

Felmayer G, Loitzl P, Jungi T, Brcic M, Brügger P, Frey E, Bowe G, Casado J, Coecke S, de479

Lange J, Mogster B, Næss LM, Aaberge IS, Wendel A, Hartung T. 2005. International

480

validation of novel pyrogen tests based on human monocytoid cells. J Immunol Methods

481

298:161-173.

482

Organisation for Economic Co-operation and Development (OECD). 1998. OECD Series on

483

Principle of Good Laboratory Practice and Compliance Monitoring. No. 1. OECD Principles

484

of Good Laboratory Practice (as revised in 1997). OECD, ENV/MC/CHEM (98)17. Paris:

485

OECD.

486

Pool EJ, Johaar G, James S, Petersen I, Bouic P. 1998. The detection of pyrogens in blood

487

products using an ex vivo whole blood culture assay. J Immunoassay 19:95-111.

488

Schindler S, Spreitzer I, Löschner B, Hoffmann S, Hennes K, Halder M, Brügger P, Frey E,

489

Hartung T, Montag T. 2006. International validation of pyrogen tests based on cryopreserved

490

human primary blood cells. J Immunol Methods 316:42-51.

491
492

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492

12.0

TERMINOLOGY AND FORMULA

493

12.1

Assay Sensitivity (λ)

494

For an in vitro cell-based assay, the variable λ is defined as the lowest statistically significant

495

point on the standard endotoxin concentration-response curve and represents the relative496

sensitivity of the test method for the detection of endotoxin (i.e., level of detection).

497

12.2

498

The EC is incubated with WB and serves as the positive control for the experiment. The

499

results should be compared to historical values to insure that it provides a known level of500

cytokine release relative to the NSC.

501

12.3

502

The ELC is the maximum allowable concentration of endotoxin for a particular product and503

is expressed in EU per volume (mL) or weight (mg). The ELC is defined by the FDA or

504

specified in the USP2. It is calculated as the product of K/M, where:

505

K is the threshold pyrogen dose for parenteral use in rabbits or humans (5.0 EU/kg). At an

506

injection volume of 10 mL/kg, K is equal to 0.5 EU/mL.

507

M is the larger of the rabbit dose or maximum human dose administered in one hour as

508

defined below and varies with test substance3

509

12.4

510

The MVD is the maximum dilution of a test substance that can be tolerated in a test system

511

without exceeding the ELC, if the test substance must be diluted as a result of assay

512

interference. Dilutions beyond the MVD would not be valid for endotoxin detection in the

Endotoxin Control

Endotoxin Limit Concentration (ELC)

Maximum Valid Dilution (MVD)2

ELC values for most marketed pharmaceutical products are provided in the USP or in other pharmacopoeial or
regulatory publications (e.g., European Pharmacopoeia, Japanese Pharmacopoeia, Pharmacopoeial guidelines,
FDA publications).
3
Values for most marketed pharmaceutical products are provided in the USP or in other pharmacopoeial or
regulatory publications (e.g., European Pharmacopoeia, Japanese Pharmacopoeia, Pharmacopoeial guidelines,
FDA publications).
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513

test system. Calculation of the MVD is dependent on whether or not the ELC for a test

514

substance is published. When the ELC is known, the MVD is:515

MVD = (ELC x Product Potency [PP])/λ516

As an example, for "Cyclophosphamide Injection," the ELC is 0.17 EU/mg, PP is 20

517

mg/mL, and the assay sensitivity is 0.1 EU/mL. The calculated MVD would be 34. The test

518

substance can be diluted no more than 1:34 prior to testing.

519

If the ELC is not known, the MVD is:520

MVD = PP/Minimum Valid Concentration (MVC)521

where, MVC = (λ x M)/K

522

where, M is the maximum human dose523

As an example, for "Cylophosphamide Injection," the PP is 20 mg/mL, M is 30524

mg/kg, and assay sensitivity is 0.1 EU/mL. The calculated MVC is 0.6 mg/mL and the MVD

525

is 33.3. The test substance can be diluted no more than 1:33 prior to testing.

526

12.5

527

The NPC is a test sample to which PFS is added. The NPC is the baseline for determination528

of cytokine release relative to the endotoxin-spiked PPC.

529

12.6

530

The NSC is WB incubated with PFS (used for dilution of test substance) and is used as the531

blank.

532

12.7

533

The value K represents the threshold pyrogen dose for parenteral products for rabbits and534

humans. Based on experimental data, K is fixed at 5.0 EU/kg. For intrathecal products, K is535

0.2 EU/kg.

536

12.8

537

The PPC is a test substance spiked with the control standard endotoxin (i.e., 0.5 EU/mL or an

538

amount of endotoxin equal to that which produces ½ the maximal increase in OD from the

Negative Product Control (PPC)

Negative Saline Control (NSC)

Non-intrathecal Threshold Pyrogen Dose (K)Positive Product Control (PPC)

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endotoxin standard curve) to insure that the test system is capable of endotoxin detection in540

the product as diluted in the assay.

541

12.9

542

The concentration for a test substance is the PP typically expressed as µg/mL or mg/mL.

543

12.10

544

The variable M represents the rabbit test dose or the maximum human dose in 1 hr. The

545

variable M is expressed in mg/kg and varies with the test substance. For

546

radiopharmaceuticals, M should be adjusted to account for product activity (radioactive

547

decay) at time administration. An average human standard weight of 70 kg is used for the548

calculation. If a pediatric dose should be used and it is higher than the adult dose, then it

549

should be used in the formula.

Product Potency (PP)

Rabbit Pyrogen Test Dose of Maximum Human Dose (M)

550
551
552
553
554

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